Статті в журналах: "Hexameric proteins"

1

Skálová, Tereza, Jan Bláha, Karl Harlos, Jarmila Dušková, Tomáš Koval', Jan Stránský, Jindřich Hašek, Ondřej Vaněk, and Jan Dohnálek. "Four crystal structures of human LLT1, a ligand of human NKR-P1, in varied glycosylation and oligomerization states." Acta Crystallographica Section D Biological Crystallography 71, no. 3 (February 26, 2015): 578–91. http://dx.doi.org/10.1107/s1399004714027928.

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Human LLT1 is a C-type lectin-like ligand of NKR-P1 (CD161, geneKLRB1), a C-type lectin-like receptor of natural killer cells. Using X-ray diffraction, the first experimental structures of human LLT1 were determined. Four structures of LLT1 under various conditions were determined: monomeric, dimeric deglycosylated after the firstN-acetylglucosamine unit in two forms and hexameric with homogeneous GlcNAc2Man5glycosylation. The dimeric form follows the classical dimerization mode of human CD69. The monomeric form keeps the same fold with the exception of the position of an outer part of the long loop region. The hexamer of glycosylated LLT1 consists of three classical dimers. The hexameric packing may indicate a possible mode of interaction of C-type lectin-like proteins in the glycosylated form.

2

Jomaa, Ahmad, Jack Iwanczyk, Julie Tran, and Joaquin Ortega. "Characterization of the Autocleavage Process of the Escherichia coli HtrA Protein: Implications for its Physiological Role." Journal of Bacteriology 191, no. 6 (December 19, 2008): 1924–32. http://dx.doi.org/10.1128/jb.01187-08.

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ABSTRACT The Escherichia coli HtrA protein is a periplasmic protease/chaperone that is upregulated under stress conditions. The protease and chaperone activities of HtrA eliminate or refold damaged and unfolded proteins in the bacterial periplasm that are generated upon stress conditions. In the absence of substrates, HtrA oligomerizes into a hexameric cage, but binding of misfolded proteins transforms the hexamers into bigger 12-mer and 24-mer cages that encapsulate the substrates for degradation or refolding. HtrA also undergoes partial degradation as a consequence of self-cleavage of the mature protein, producing short-HtrA protein (s-HtrA). The aim of this study was to examine the physiological role of this self-cleavage process. We found that the only requirement for self-cleavage of HtrA into s-HtrA in vitro was the hydrolysis of protein substrates. In fact, peptides resulting from the hydrolysis of the protein substrates were sufficient to induce autocleavage. However, the continuous presence of full-length substrate delayed the process. In addition, we observed that the hexameric cage structure is required for autocleavage and that s-HtrA accumulates only late in the degradation reaction. These results suggest that self-cleavage occurs when HtrA reassembles back into the resting hexameric structure and peptides resulting from substrate hydrolysis are allosterically stimulating the HtrA proteolytic activity. Our data support a model in which the physiological role of the self-cleavage process is to eliminate the excess of HtrA once the stress conditions cease.

3

Myasoedova, Ksenia N., and Natalia N. Magretova. "Cross-Linking Study of Cytochrome P450 1A2 in Proteoliposomes." Bioscience Reports 21, no. 1 (February 1, 2001): 63–72. http://dx.doi.org/10.1023/a:1010486118448.

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Proteoliposomes, containing cytochrome P450 1A2, were obtained by the cholate-dialysis technique. The effect of bifunctional cross-linking reagents on the purified hexameric cytochrome P450 1A2 in an aqueous medium and on the proteoliposomal P450 1A2 have been compared. Electrophoretic analysis of the modified proteins demonstrated the same oligomeric (hexameric) organization of the hemoprotein in each case.

4

Chaudhury, Paushali, Chris van der Does, and Sonja-Verena Albers. "Characterization of the ATPase FlaI of the motor complex of the Pyrococcus furiosus archaellum and its interactions between the ATP-binding protein FlaH." PeerJ 6 (June 18, 2018): e4984. http://dx.doi.org/10.7717/peerj.4984.

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The archaellum, the rotating motility structure of archaea, is best studied in the crenarchaeon Sulfolobus acidocaldarius. To better understand how assembly and rotation of this structure is driven, two ATP-binding proteins, FlaI and FlaH of the motor complex of the archaellum of the euryarchaeon Pyrococcus furiosus, were overexpressed, purified and studied. Contrary to the FlaI ATPase of S. acidocaldarius, which only forms a hexamer after binding of nucleotides, FlaI of P. furiosus formed a hexamer in a nucleotide independent manner. In this hexamer only 2 of the ATP binding sites were available for binding of the fluorescent ATP-analog MANT-ATP, suggesting a twofold symmetry in the hexamer. P. furiosus FlaI showed a 250-fold higher ATPase activity than S. acidocaldarius FlaI. Interaction studies between the isolated N- and C-terminal domains of FlaI showed interactions between the N- and C-terminal domains and strong interactions between the N-terminal domains not previously observed for ATPases involved in archaellum assembly. These interactions played a role in oligomerization and activity, suggesting a conformational state of the hexamer not observed before. Further interaction studies show that the C-terminal domain of PfFlaI interacts with the nucleotide binding protein FlaH. This interaction stimulates the ATPase activity of FlaI optimally at a 1:1 stoichiometry, suggesting that hexameric PfFlaI interacts with hexameric PfFlaH. These data help to further understand the complex interactions that are required to energize the archaellar motor.

5

Jomaa, Ahmad, Daniela Damjanovic, Vivian Leong, Rodolfo Ghirlando, Jack Iwanczyk, and Joaquin Ortega. "The Inner Cavity of Escherichia coli DegP Protein Is Not Essentialfor Molecular Chaperone and Proteolytic Activity." Journal of Bacteriology 189, no. 3 (November 22, 2006): 706–16. http://dx.doi.org/10.1128/jb.01334-06.

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ABSTRACT The Escherichia coli DegP protein is an essential periplasmic protein for bacterial survival at high temperatures. DegP has the unusual property of working as a chaperone below 28°C, but efficiently degrading unfolded proteins above 28°C. Monomeric DegP contains a protease domain and two PDZ domains. It oligomerizes into a hexameric cage through the staggered association of trimers. The active sites are located in a central cavity that is only accessible laterally, and the 12 PDZ domains act as mobile sidewalls that mediate opening and closing of the gates. As access to the active sites is restricted, DegP is an example of a self-compartmentalized protease. To determine the essential elements of DegP that maintain the integrity of the hexameric cage, we constructed several deletion mutants of DegP that formed trimers rather than hexamers. We found that residues 39 to 78 within the LA loops, as well as the PDZ2 domains are essential for the integrity of the DegP hexamer. In addition, we asked whether an enclosed cavity or cage of specific dimensions is required for the protease and chaperone activities in DegP. Both activities were maintained in the trimeric DegP mutants without an enclosed cavity and in deletion DegP mutants with significantly reduced dimensions of the cage. We conclude that the functional unit for the protease and chaperone activities of DegP is a trimer and that neither a cavity of specific dimensions nor the presence of an enclosed cavity appears to be essential for the protease and chaperone activities of DegP.

6

Miller, Justin M., and Eric J. Enemark. "Archaeal MCM Proteins as an Analog for the Eukaryotic Mcm2–7 Helicase to Reveal Essential Features of Structure and Function." Archaea 2015 (2015): 1–14. http://dx.doi.org/10.1155/2015/305497.

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In eukaryotes, the replicative helicase is the large multisubunit CMG complex consisting of the Mcm2–7 hexameric ring, Cdc45, and the tetrameric GINS complex. The Mcm2–7 ring assembles from six different, related proteins and forms the core of this complex. In archaea, a homologous MCM hexameric ring functions as the replicative helicase at the replication fork. Archaeal MCM proteins form thermostable homohexamers, facilitating their use as models of the eukaryotic Mcm2–7 helicase. Here we review archaeal MCM helicase structure and function and how the archaeal findings relate to the eukaryotic Mcm2–7 ring.

7

Sellin, Mikael E., Sonja Stenmark, and Martin Gullberg. "Mammalian SEPT9 isoforms direct microtubule-dependent arrangements of septin core heteromers." Molecular Biology of the Cell 23, no. 21 (November 2012): 4242–55. http://dx.doi.org/10.1091/mbc.e12-06-0486.

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Septin-family proteins assemble into rod-shaped heteromeric complexes that form higher-order arrangements at the cell cortex, where they serve apparently conserved functions as diffusion barriers and molecular scaffolds. There are 13 confirmed septin paralogues in mammals, which may be ubiquitous or tissue specific. Septin hetero-oligomerization appears homology subgroup directed, which in turn determines the subunit arrangement of six- to eight-subunit core heteromers. Here we address functional properties of human SEPT9, which, due to variable mRNA splicing, exists as multiple isoforms that differ between tissues. Myeloid K562 cells express three SEPT9 isoforms, all of which have an equal propensity to hetero-oligomerize with SEPT7-containing hexamers to generate octameric heteromers. However, due to limiting amounts of SEPT9, K562 cells contain both hexameric and octameric heteromers. To generate cell lines with controllable hexamer-to-octamer ratios and that express single SEPT9 isoforms, we developed a gene product replacement strategy. By this means we identified SEPT9 isoform–specific properties that either facilitate septin heteromer polymerization along microtubules or modulate the size range of submembranous septin disks—a prevalent septin structure in nonadhered cells. Our findings show that the SEPT9 expression level directs the hexamer-to-octamer ratio, and that the isoform composition and expression level together determine higher-order arrangements of septins.

8

Zhao, Li, Shuji Kanamaru, Chatree'chalerm Chaidirek, and Fumio Arisaka. "P15 and P3, the Tail Completion Proteins of Bacteriophage T4, Both Form Hexameric Rings." Journal of Bacteriology 185, no. 5 (March 1, 2003): 1693–700. http://dx.doi.org/10.1128/jb.185.5.1693-1700.2003.

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ABSTRACT Two proteins, gp15 and gp3 (gp for gene product), are required to complete the assembly of the T4 tail. gp15 forms the connector which enables the tail to bind to the head, whereas gp3 is involved in terminating the elongation of the tail tube. In this work, genes 15 and 3 were cloned and overexpressed, and the purified gene products were studied by analytical ultracentrifugation, electron microscopy, and circular dichroism. Determination of oligomerization state by sedimentation equilibrium revealed that both gp15 and gp3 are hexamers of the respective polypeptide chains. Electron microscopy of the negatively stained P15 and P3 (P denotes the oligomeric state of the gene product) revealed that both proteins form hexameric rings, the diameter of which is close to that of the tail tube. The differential roles between gp15 and gp3 upon completion of the tail are discussed.

9

Ochoa, Jessica M., Oscar Mijares, Andrea A. Acosta, Xavier Escoto, Nancy Leon-Rivera, Joanna D. Marshall, Michael R. Sawaya, and Todd O. Yeates. "Structural characterization of hexameric shell proteins from two types of choline-utilization bacterial microcompartments." Acta Crystallographica Section F Structural Biology Communications 77, no. 9 (August 24, 2021): 275–85. http://dx.doi.org/10.1107/s2053230x21007470.

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Bacterial microcompartments are large supramolecular structures comprising an outer proteinaceous shell that encapsulates various enzymes in order to optimize metabolic processes. The outer shells of bacterial microcompartments are made of several thousand protein subunits, generally forming hexameric building blocks based on the canonical bacterial microcompartment (BMC) domain. Among the diverse metabolic types of bacterial microcompartments, the structures of those that use glycyl radical enzymes to metabolize choline have not been adequately characterized. Here, six structures of hexameric shell proteins from type I and type II choline-utilization microcompartments are reported. Sequence and structure analysis reveals electrostatic surface properties that are shared between the four types of shell proteins described here.

10

Heinemann, Udo, Yvette Roske, Anup Arumughan, and Erich Wanker. "Remodeling of the AAA+ ATPase p97 by the UBX Adaptor Protein ASPL." Acta Crystallographica Section A Foundations and Advances 70, a1 (August 5, 2014): C430. http://dx.doi.org/10.1107/s2053273314095692.

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The hexameric mammalian AAA+ ATPase p97, also known as VCP (valosin-containing protein; CDC48 in yeast), is a very abundant cytosolic protein and serves a wide variety of cellular functions. p97 is a central component in endoplasmic reticulum-associated degradation (ERAD) of proteins where it delivers ubiquitinated ERAD substrates to the proteasome. In addition, cellular roles of p97 in organelle membrane fusion, mitosis, DNA repair and suppression of apoptosis have been described. These different functions are linked to the binding of adaptor proteins to p97. Many of these adaptors contain ubiquitin regulatory X (UBX) domains. ASPL (alveolar soft part sarcoma locus, also known as TUG) was recenty identified as a p97 adaptor protein. As shown by crystal structure analysis, ASPL uses a substantially extended UBX domain for binding to the N domain of p97 where a lariat-like, mostly α-helical extension wraps around one subunit of p97. By this binding ASPL triggers the dissociation of functional p97 hexamers and the formation of p97:ASPL heterotetramers with 2:2 stoichiometry, leading to inactivation of the AAA+ ATPase. The p97-ASPL interaction in the heterotetramer is very tight, but p97 hexamer dissociation and heterotetramer formation may be suppressed by single-site mutations at p97-ASPL interfaces. p97 hexamer dissociation and p97-ASPL heterotetramer formation are linked to reduced ATPase activity of p97, cellular accumulation of ERAD substrates and apoptosis induction. To the best of our knowledge, this is the first time that the structural basis for adaptor protein-induced inactivation by hexamer dissociation of p97 and, indeed, any AAA+ ATPase has been demonstrated. This observation has far reaching implications for AAA+ ATPase-regulated processes.

11

Chen, Zhenguo, Lei Sun, Zhihong Zhang, Andrei Fokine, Victor Padilla-Sanchez, Dorit Hanein, Wen Jiang, Michael G. Rossmann, and Venigalla B. Rao. "Cryo-EM structure of the bacteriophage T4 isometric head at 3.3-Å resolution and its relevance to the assembly of icosahedral viruses." Proceedings of the National Academy of Sciences 114, no. 39 (September 11, 2017): E8184—E8193. http://dx.doi.org/10.1073/pnas.1708483114.

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The 3.3-Å cryo-EM structure of the 860-Å-diameter isometric mutant bacteriophage T4 capsid has been determined. WT T4 has a prolate capsid characterized by triangulation numbers (T numbers) Tend= 13 for end caps and Tmid= 20 for midsection. A mutation in the major capsid protein, gp23, produced T=13 icosahedral capsids. The capsid is stabilized by 660 copies of the outer capsid protein, Soc, which clamp adjacent gp23 hexamers. The occupancies of Soc molecules are proportional to the size of the angle between the planes of adjacent hexameric capsomers. The angle between adjacent hexameric capsomers is greatest around the fivefold vertices, where there is the largest deviation from a planar hexagonal array. Thus, the Soc molecules reinforce the structure where there is the greatest strain in the gp23 hexagonal lattice. Mutations that change the angles between adjacent capsomers affect the positions of the pentameric vertices, resulting in different triangulation numbers in bacteriophage T4. The analysis of the T4 mutant head assembly gives guidance to how other icosahedral viruses reproducibly assemble into capsids with a predetermined T number, although the influence of scaffolding proteins is also important.

12

Fu, Xinyi, Vladyslava Sokolova, Kristofor J. Webb, William Old, and Soyeon Park. "Ubiquitin-dependent switch during assembly of the proteasomal ATPases mediated by Not4 ubiquitin ligase." Proceedings of the National Academy of Sciences 115, no. 52 (December 10, 2018): 13246–51. http://dx.doi.org/10.1073/pnas.1805353115.

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In the proteasome holoenzyme, the hexameric ATPases (Rpt1-Rpt6) enable degradation of ubiquitinated proteins by unfolding and translocating them into the proteolytic core particle. During early-stage proteasome assembly, individual Rpt proteins assemble into the hexameric “Rpt ring” through binding to their cognate chaperones: Nas2, Hsm3, Nas6, and Rpn14. Here, we show that Rpt ring assembly employs a specific ubiquitination-mediated control. An E3 ligase, Not4, selectively ubiquitinates Rpt5 during Rpt ring assembly. To access Rpt5, Not4 competes with Nas2 until the penultimate step and then with Hsm3 at the final step of Rpt ring completion. Using the known Rpt–chaperone cocrystal structures, we show that Not4-mediated ubiquitination sites in Rpt5 are obstructed by Nas2 and Hsm3. Thus, Not4 can distinguish a Rpt ring that matures without these chaperones, based on its accessibility to Rpt5. Rpt5 ubiquitination does not destabilize the ring but hinders incorporation of incoming subunits—Rpn1 ubiquitin receptor and Ubp6 deubiquitinase—thereby blocking progression of proteasome assembly and ubiquitin regeneration from proteasome substrates. Our findings reveal an assembly checkpoint where Not4 monitors chaperone actions during hexameric ATPase ring assembly, thereby ensuring the accuracy of proteasome holoenzyme maturation.

13

Hoebe, E. K., S. H. Hutajulu, J. van Beek, S. J. Stevens, D. K. Paramita, A. E. Greijer, and J. M. Middeldorp. "Purified Hexameric Epstein-Barr Virus-Encoded BARF1 Protein for Measuring Anti-BARF1 Antibody Responses in Nasopharyngeal Carcinoma Patients." Clinical and Vaccine Immunology 18, no. 2 (December 1, 2010): 298–304. http://dx.doi.org/10.1128/cvi.00193-10.

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ABSTRACTWHO type III nasopharyngeal carcinoma (NPC) is highly prevalent in Indonesia and 100% associated with Epstein-Barr virus (EBV). NPC tumor cells express viral proteins, including BARF1, which is secreted and is considered to have oncogenic and immune-modulating properties. Recently, we found conserved mutations in the BARF1 gene in NPC isolates. This study describes the expression and purification of NPC-derived BARF1 and analyzes humoral immune responses against prototype BARF1 (B95-8) and purified native hexameric BARF1 in sera of Indonesian NPC patients (n= 155) compared to healthy EBV-positive (n= 56) and EBV-negative (n= 16) individuals. BARF1 (B95-8) expressed inEscherichia coliand baculovirus, as well as BARF1-derived peptides, did not react with IgG or IgA antibodies in NPC. Purified native hexameric BARF1 protein isolated from culture medium was used in enzyme-linked immunosorbent assay (ELISA) and revealed relatively weak IgG and IgA responses in human sera, although it had strong antibody responses to other EBV proteins. Higher IgG reactivity was found in NPC patients (P= 0.015) than in regional Indonesian controls or EBV-negative individuals (P< 0.001). IgA responses to native BARF1 were marginal. NPC sera with the highest IgG responses to hexameric BARF1 in ELISA showed detectable reactivity with denatured BARF1 by immunoblotting. In conclusion, BARF1 has low immunogenicity for humoral responses and requires native conformation for antibody binding. The presence of antibodies against native BARF1 in the blood of NPC patients provides evidence that the protein is expressed and secreted as a hexameric protein in NPC patients.

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Duprez, Kevin, Melissa A. Scranton, Linda L. Walling, and Li Fan. "Structure of tomato wound-induced leucine aminopeptidase sheds light on substrate specificity." Acta Crystallographica Section D Biological Crystallography 70, no. 6 (May 29, 2014): 1649–58. http://dx.doi.org/10.1107/s1399004714006245.

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The acidic leucine aminopeptidase (LAP-A) from tomato is induced in response to wounding and insect feeding. Although LAP-A showsin vitropeptidase activity towards peptides and peptide analogs, it is not clear what kind of substrates LAP-A hydrolyzesin vivo. In the current study, the crystal structure of LAP-A was determined to 2.20 Å resolution. Like other LAPs in the M17 peptidase family, LAP-A is a dimer of trimers containing six monomers of bilobal structure. Each monomer contains two metal ions bridged by a water or a hydroxyl ion at the active site. Modeling of different peptides or peptide analogs in the active site of LAP-A reveals a spacious substrate-binding channel that can bind peptides of five or fewer residues with few geometric restrictions. The sequence specificity of the bound peptide is likely to be selected by the structural and chemical restrictions on the amino acid at the P1 and P1′ positions because these two amino acids have to bind perfectly at the active site for hydrolysis of the first peptide bond to occur. The hexameric assembly results in the merger of the open ends of the six substrate-binding channels from the LAP-A monomers to form a spacious central cavity allowing the hexameric LAP-A enzyme to simultaneously hydrolyze six peptides containing up to six amino acids each. The hexameric LAP-A enzyme may also hydrolyze long peptides or proteins if only one such substrate is bound to the hexamer because the substrate can extend through the central cavity and the two major solvent channels between the two LAP-A trimers.

15

Riciluca, K. C. T., A. C. Borges, J. F. R. Mello, U. C. de Oliveira, D. C. Serdan, A. Florez-Ariza, E. Chaparro, et al. "Myriapod haemocyanin: the first three-dimensional reconstruction of Scolopendra subspinipes and preliminary structural analysis of S. viridicornis." Open Biology 10, no. 4 (April 2020): 190258. http://dx.doi.org/10.1098/rsob.190258.

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Haemocyanins (Hcs) are copper-containing, respiratory proteins that occur in the haemolymph of many arthropod species. Here, we report the presence of Hcs in the chilopode Myriapoda, demonstrating that these proteins are more widespread among the Arthropoda than previously thought. The analysis of transcriptome of S. subspinipes subpinipes reveals the presence of two distinct subunits of Hc, where the signal peptide is present, and six of prophenoloxidase (PPO), where the signal peptide is absent, in the 75 kDa range. Size exclusion chromatography profiles indicate different quaternary organization for Hc of both species, which was corroborated by TEM analysis: S. viridicornis Hc is a 6 × 6-mer and S. subspinipes Hc is a 3 × 6-mer, which resembles the half-structure of the 6 × 6-mer but also includes the presence of phenoloxidases, since the 1 × 6-mer quaternary organization is commonly associated with hexamers of PPO. Studies with Chelicerata showed that PPO activity are exclusively associated with the Hcs. This study indicates that Scolopendra may have different proteins playing oxygen transport (Hc) and PO function, both following the hexameric oligomerization observed in Hcs.

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Jeoung, Jae-Hun, Diana A. Pippig, Berta M. Martins, Nadine Wagener, and Holger Dobbek. "HTHP: A Novel Class of Hexameric, Tyrosine-coordinated Heme Proteins." Journal of Molecular Biology 368, no. 4 (May 2007): 1122–31. http://dx.doi.org/10.1016/j.jmb.2007.02.079.

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Gao, Forson, Amy E. Danson, Fuzhou Ye, Milija Jovanovic, Martin Buck, and Xiaodong Zhang. "Bacterial Enhancer Binding Proteins—AAA+ Proteins in Transcription Activation." Biomolecules 10, no. 3 (February 25, 2020): 351. http://dx.doi.org/10.3390/biom10030351.

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Bacterial enhancer-binding proteins (bEBPs) are specialised transcriptional activators. bEBPs are hexameric AAA+ ATPases and use ATPase activities to remodel RNA polymerase (RNAP) complexes that contain the major variant sigma factor, σ54 to convert the initial closed complex to the transcription competent open complex. Earlier crystal structures of AAA+ domains alone have led to proposals of how nucleotide-bound states are sensed and propagated to substrate interactions. Recently, the structure of the AAA+ domain of a bEBP bound to RNAP-σ54-promoter DNA was revealed. Together with structures of the closed complex, an intermediate state where DNA is partially loaded into the RNAP cleft and the open promoter complex, a mechanistic understanding of how bEBPs use ATP to activate transcription can now be proposed. This review summarises current structural models and the emerging understanding of how this special class of AAA+ proteins utilises ATPase activities to allow σ54-dependent transcription initiation.

18

Lin, Qing-Peng, Zeng-Qiang Gao, Zhi Geng, Heng Zhang, and Yu-Hui Dong. "Crystal structure of the putative cytoplasmic protein STM0279 (Hcp2) from Salmonella typhimurium." Acta Crystallographica Section F Structural Biology Communications 73, no. 8 (July 26, 2017): 463–68. http://dx.doi.org/10.1107/s2053230x17010512.

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STM0279 is a putative cytoplasmic protein from Salmonella typhimurium and was recently renamed haemolysin co-regulated protein 2 (Hcp2), with the neighbouring STM0276 being Hcp1. Both of them are encoded by the type VI secretion system (T6SS) of the Salmonella pathogenicity island 6 (SPI-6) locus and have high sequence identity. The Hcp proteins may function as a vital component of the T6SS nanotube and as a transporter and chaperone of diverse effectors from the bacterial T6SS. In this study, the crystal structure and the oligomeric state in solution of Hcp2 from S. typhimurium (StHcp2) were investigated. The crystal structure refined to 3.0 Å resolution showed that the protein is composed of a β-barrel domain with extended loops and can form hexameric rings as observed in known Hcp homologues. Mutation of the extended loop was found to partly destabilize the hexameric conformation into monomers or cause the production of inclusion bodies, suggesting it has an important role in hexameric ring formation.

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JAENICKE, Elmar, and Heinz DECKER. "Tyrosinases from crustaceans form hexamers." Biochemical Journal 371, no. 2 (April 15, 2003): 515–23. http://dx.doi.org/10.1042/bj20021058.

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Tyrosinases, which are widely distributed among animals, plants and fungi, are involved in many biologically essential functions, including pigmentation, sclerotization, primary immune response and host defence. In the present study, we present a structural and physicochemical characterization of two new tyrosinases from the crustaceans Palinurus elephas (European spiny lobster) and Astacus leptodactylus (freshwater crayfish). In vivo, the purified crustacean tyrosinases occur as hexamers composed of one subunit type with a molecular mass of approx. 71kDa. The tyrosinase hexamers appear to be similar to the haemocyanins, based on electron microscopy. Thus a careful purification protocol was developed to discriminate clearly between tyrosinases and the closely related haemocyanins. The physicochemical properties of haemocyanins and tyrosinases are different with respect to electronegativity and hydrophobicity. The hexameric nature of arthropod tyrosinases suggests that these proteins were the ideal predecessors from which to develop the oxygen-carrier protein haemocyanin, with its allosteric and co-operative properties, later on.

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Lu, Connie, Young-un Park, Konstantin Korotkov, Wei Mi, Stewart Turley, Veer Bhatt, Ripal Shah, and Wim Hol. "Multiple approaches towards understanding the type II secretion system." Acta Crystallographica Section A Foundations and Advances 70, a1 (August 5, 2014): C577. http://dx.doi.org/10.1107/s2053273314094224.

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Transport of folded proteins across membranes is a feat accomplished by few biomacromolecular machines. One of the machineries able to do so is the sophisticated type II secretion system (T2SS). It can translocate key virulence factors from the bacterial periplasm into the lumen of the gut of the human host. A prime example is the secretion of cholera toxin by Vibrio cholerae. The T2SS consists of ~12 different proteins, most of these present in multiple copies, organized into three subassemblies: (i) the Inner Membrane Platform; (ii) the Pseudopilus in the periplasm, which acts most likely as a piston pushing exoproteins through the outer membrane pore; (iii) the Outer Membrane Complex, allowing passage of ~100 kDa folded proteins. We have determined crystal structures from more than a dozen T2SS domains, yet, a full understanding of the architecture and mechanism of action of the T2SS remains a formidable challenge. Our approaches include the use of "assistant-multimers" to promote recalcitrant multimer formation and of nanobodies to overcome reluctant crystal formation. The Inner Membrane Platform is interacting with the secretion ATPase GspE which most likely needs to be hexameric for full activity. Full-length GspE co-crystallized with its major partner, the cytoplasmic domain of GspL, revealed a tremendous flexibility of this ATPase, and, most unexpectedly, also the organization of the same linear arrangement of cyto-GspL domains throughout three entirely different crystal forms. Two very different hexamers of GspE were elucidated by linking the GspE subunit to the subunit of Hcp1, which successfully acted as an "assistant hexamer", inducing hexamer formation by GspE. The dodecameric nature of the ~ 850 kDa GspD, the major component of the Outer Membrane Complex, evident in earlier electron microscopy studies, was observed in the dodecameric ring-like helix in crystals of its N-terminal domain. The contacts between GspD and the inner-membrane protein GspC will be discussed as well as the remarkably frequent occurrence of dimers of Inner Membrane Platform domains. How dimers are co-assembled with an ATPase hexamer with C6 symmetry and the Outer Membrane Complex dodecamer with C12 symmetry remains one of the many fascinating outstanding questions of the T2SS.

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Watanabe, Yo-hei, Yosuke Nakazaki, Ryoji Suno, and Masasuke Yoshida. "Stability of the two wings of the coiled-coil domain of ClpB chaperone is critical for its disaggregation activity." Biochemical Journal 421, no. 1 (June 12, 2009): 71–77. http://dx.doi.org/10.1042/bj20082238.

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The ClpB chaperone forms a hexamer ring and rescues aggregated proteins in co-operation with the DnaK system. Each subunit of ClpB has two nucleotide-binding modules, AAA (ATPase associated with various cellular activities)-1 and AAA-2, and an 85-Å (1 Å=0.1 nm)-long coiled-coil. The coiled-coil consists of two halves: wing-1, leaning toward AAA-1, and wing-2, leaning away from all the domains. The coiled-coil is stabilized by leucine zipper-like interactions between leucine and isoleucine residues of two amphipathic α-helices that twist around each other to form each wing. To destabilize the two wings, we developed a series of mutants by replacing these residues with alanine. As the number of replaced residues increased, the chaperone activity was lost and the hexamer became unstable. The mutants, which had a stable hexameric structure but lost the chaperone activities, were able to exert the threading of soluble denatured proteins through their central pore. The destabilization of wing-1, but not wing-2, resulted in a several-fold stimulation of ATPase activity. These results indicate that stability of both wings of the coiled-coil is critical for full functioning of ClpB, but not for the central-pore threading of substrate proteins, and that wing-1 is involved in the communication between AAA-1 and AAA-2.

22

Holler, Nils, Aubry Tardivel, Magdalena Kovacsovics-Bankowski, Sylvie Hertig, Olivier Gaide, Fabio Martinon, Antoine Tinel, et al. "Two Adjacent Trimeric Fas Ligands Are Required for Fas Signaling and Formation of a Death-Inducing Signaling Complex." Molecular and Cellular Biology 23, no. 4 (February 15, 2003): 1428–40. http://dx.doi.org/10.1128/mcb.23.4.1428-1440.2003.

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ABSTRACT The membrane-bound form of Fas ligand (FasL) signals apoptosis in target cells through engagement of the death receptor Fas, whereas the proteolytically processed, soluble form of FasL does not induce cell death. However, soluble FasL can be rendered active upon cross-linking. Since the minimal extent of oligomerization of FasL that exerts cytotoxicity is unknown, we engineered hexameric proteins containing two trimers of FasL within the same molecule. This was achieved by fusing FasL to the Fc portion of immunoglobulin G1 or to the collagen domain of ACRP30/adiponectin. Trimeric FasL and hexameric FasL both bound to Fas, but only the hexameric forms were highly cytotoxic and competent to signal apoptosis via formation of a death-inducing signaling complex. Three sequential early events in Fas-mediated apoptosis could be dissected, namely, receptor binding, receptor activation, and recruitment of intracellular signaling molecules, each of which occurred independently of the subsequent one. These results demonstrate that the limited oligomerization of FasL, and most likely of some other tumor necrosis factor family ligands such as CD40L, is required for triggering of the signaling pathways.

23

Payá, Gloria, Vanesa Bautista, Sandra Pastor-Soler, Mónica Camacho, Julia Esclapez, and María-José Bonete. "Analysis of Lsm Protein-Mediated Regulation in the Haloarchaeon Haloferax mediterranei." International Journal of Molecular Sciences 25, no. 1 (January 1, 2024): 580. http://dx.doi.org/10.3390/ijms25010580.

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The Sm protein superfamily includes Sm, like-Sm (Lsm), and Hfq found in the Eukarya, Archaea, and Bacteria domains. Archaeal Lsm proteins have been shown to bind sRNAs and are probably involved in various cellular processes, suggesting a similar function in regulating sRNAs by Hfq in bacteria. Moreover, archaeal Lsm proteins probably represent the ancestral Lsm domain from which eukaryotic Sm proteins have evolved. In this work, Haloferax mediterranei was used as a model organism because it has been widely used to investigate the nitrogen cycle and its regulation in Haloarchaea. Predicting this protein’s secondary and tertiary structures has resulted in a three-dimensional model like the solved Lsm protein structure of Archaeoglobus fulgidus. To obtain information on the oligomerization state of the protein, homologous overexpression and purification by means of molecular exclusion chromatography have been performed. The results show that this protein can form hexameric complexes, which can aggregate into 6 or 12 hexameric rings depending on the NaCl concentration and without RNA. In addition, the study of transcriptional expression via microarrays has allowed us to obtain the target genes regulated by the Lsm protein under nutritional stress conditions: nitrogen or carbon starvation. Microarray analysis has shown the first universal stress proteins (USP) in this microorganism that mediate survival in situations of nitrogen deficiency.

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Miller, Scott A., Sharon Tollefson, James E. Crowe, John V. Williams, and David W. Wright. "Examination of a Fusogenic Hexameric Core from Human Metapneumovirus and Identification of a Potent Synthetic Peptide Inhibitor from the Heptad Repeat 1 Region." Journal of Virology 81, no. 1 (October 11, 2006): 141–49. http://dx.doi.org/10.1128/jvi.01243-06.

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ABSTRACT Paramyxoviruses are a leading cause of childhood illness worldwide. A recently discovered paramyxovirus, human metapneumovirus (hMPV), has been studied by our group in order to determine the structural relevance of its fusion (F) protein to other well-characterized viruses utilizing type I integral membrane proteins as fusion aids. Sequence analysis and homology models suggested the presence of requisite heptad repeat (HR) regions. Synthetic peptides from HR regions 1 and 2 (HR-1 and -2, respectively) were induced to form a thermostable (melting temperature, ∼90°C) helical structure consistent in mass with a hexameric coiled coil. Inhibitory studies of hMPV HR-1 and -2 indicated that the synthetic HR-1 peptide was a significant fusion inhibitor with a 50% inhibitory concentration and a 50% effective concentration of ∼50 nM. Many viral fusion proteins are type I integral membrane proteins utilizing the formation of a hexameric coiled coil of HR peptides as a major driving force for fusion. Our studies provide evidence that hMPV also uses a coiled-coil structure as a major player in the fusion process. Additionally, viral HR-1 peptide sequences may need further investigation as potent fusion inhibitors.

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Song, Saemee, Seokho Hong, Jinyang Jang, Ji-Hyun Yeom, Nohra Park, Jaejin Lee, Yeri Lim, et al. "Functional implications of hexameric assembly of RraA proteins from Vibrio vulnificus." PLOS ONE 12, no. 12 (December 20, 2017): e0190064. http://dx.doi.org/10.1371/journal.pone.0190064.

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26

Gres, Anna, Karen Kirby, Atsuko Hachiya, Eleftherios Michailidis, Owen Pornillos, Wataru Sugiura, KyeongEun Lee, Vineet KewalRamani, John Tanner, and Stefan Sarafianos. "Native Hexameric Full-Length HIV-1 Capsid: Crystal Structure and Drug Targeting." Acta Crystallographica Section A Foundations and Advances 70, a1 (August 5, 2014): C696. http://dx.doi.org/10.1107/s2053273314093036.

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The HIV-1 full length capsid protein (CA-FL) is increasingly viewed as an attractive therapeutic target since proper capsid formation is required for viral infection. CA-FL is synthesized as a central domain of a structural Gag polyprotein that is involved in both early and late stages of the viral life cycle. During the HIV-1 maturation process, Gag is cleaved by a viral protease to produce several discrete new proteins that include matrix, capsid (CA-FL), and nucleocapsid. After proteolytic cleavage, CA-FL forms hexamers and pentamers that rearrange into a fullerene cone-shaped structure, which surrounds the viral genome at the center of the mature virus. Crystal structures of the native unassembled hexameric CA-FL (without cross-linked residues that might prevent changes in the inter- or intra-subunit interactions) are of great interest, as they may provide insights relevant to the development of drugs that prevent or impede the transition from the preassembled to the assembled capsid states. Recently, we crystallized and solved the crystal structure of the first hexameric HIV-1 CA-FL in its native form (without engineered cross-linking cysteines). There is one molecule per asymmetric unit, and the P6 space group generates the native hexameric assembly. We have also identified a small molecule, 18E8, which exhibits broad anti-HIV activity in cell-based assays, and targets CA-FL. This was demonstrated by experiments that selected for viruses with drug resistance and revealed that an A105T mutation in CA-FL confers resistance to the compound. Time-of-inhibitor addition experiments showed that 18E8 targets an early step in the HIV replication cycle, after reverse transcription and before integration. Electron microscopy experiments suggest that 18E8 does not impart significant morphological changes in CA-FL tubular assemblies. Our structure of CA-FL and our ongoing work with the CA-FL/18E8 complex will provide a system for the investigation of molecular interactions between CA-FL and small molecule antivirals that work with a novel mechanism of action.

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Song, Saemee, Seokho Hong, Jinyang Jang, Ji-Hyun Yeom, Nohra Park, Jaejin Lee, Yeri Lim, et al. "Correction: Functional implications of hexameric assembly of RraA proteins from Vibrio vulnificus." PLOS ONE 13, no. 1 (January 19, 2018): e0191775. http://dx.doi.org/10.1371/journal.pone.0191775.

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Wu, Ling, Sidharth S. Madhavan, Christopher Tan, and Bin Xu. "Hexameric Aggregation Nucleation Core Sequences and Diversity of Pathogenic Tau Strains." Pathogens 11, no. 12 (December 19, 2022): 1559. http://dx.doi.org/10.3390/pathogens11121559.

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Tau aggregation associates with multiple neurodegenerative diseases including Alzheimer’s disease and rare tauopathies such as Pick’s disease, progressive supranuclear palsy, and corticobasal degeneration. The molecular and structural basis of tau aggregation and related diverse misfolded tau strains are not fully understood. To further understand tau-protein aggregation mechanisms, we performed systematic truncation mutagenesis and mapped key segments of tau proteins that contribute to tau aggregation, where it was determined that microtubule binding domains R2 and R3 play critical roles. We validated that R2- or R3-related hexameric PHF6 and PHF6* peptide sequences are necessary sequences that render tau amyloidogenicity. We also determined that the consensus VQI peptide sequence is not sufficient for amyloidogenicity. We further proposed single- and dual-nucleation core-based strain classifications based on recent cryo-EM structures. We analyzed the structural environment of the hexameric peptide sequences in diverse tau strains in tauopathies that, in part, explains why the VQI consensus core sequence is not sufficient to induce tau aggregation. Our experimental work and complementary structural analysis highlighted the indispensible roles of the hexameric core sequences, and shed light on how the interaction environment of these core sequences contributes to diverse pathogenic tau-strains formation in various tauopathy brains.

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Castanzo, Dominic T., Benjamin LaFrance, and Andreas Martin. "The AAA+ ATPase Msp1 is a processive protein translocase with robust unfoldase activity." Proceedings of the National Academy of Sciences 117, no. 26 (June 15, 2020): 14970–77. http://dx.doi.org/10.1073/pnas.1920109117.

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Msp1 is a conserved eukaryotic AAA+ ATPase localized to the outer mitochondrial membrane, where it is thought to extract mislocalized tail-anchored proteins. Despite recent in vivo and in vitro studies supporting this function, a mechanistic understanding of how Msp1 extracts its substrates is still lacking. Msp1’s ATPase activity depends on its hexameric state, and previous characterizations of the cytosolic AAA+ domain in vitro had proved challenging due to its monomeric nature in the absence of the transmembrane domain. Here, we used a hexamerization scaffold to study the substrate-processing mechanism of the soluble Msp1 motor, the functional homo-hexameric state of which was confirmed by negative-stain electron microscopy. We demonstrate that Msp1 is a robust bidirectional protein translocase that is able to unfold diverse substrates by processive threading through its central pore. This unfoldase activity is inhibited by Pex3, a membrane protein proposed to regulate Msp1 at the peroxisome.

30

Lekontceva, Natalia, Alisa Mikhailina, Svetlana Tishchenko, and Alexey Nikulin. "Comparison of the uridine-binding site of hexameric and heptameric archaeal Lsm proteins." Acta Crystallographica Section A Foundations and Advances 74, a2 (August 22, 2018): e201-e201. http://dx.doi.org/10.1107/s2053273318092148.

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31

Hong, Lu, Bodhi P. Vani, Erik H. Thiede, Michael J. Rust, and Aaron R. Dinner. "Molecular dynamics simulations of nucleotide release from the circadian clock protein KaiC reveal atomic-resolution functional insights." Proceedings of the National Academy of Sciences 115, no. 49 (November 15, 2018): E11475—E11484. http://dx.doi.org/10.1073/pnas.1812555115.

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The cyanobacterial clock proteins KaiA, KaiB, and KaiC form a powerful system to study the biophysical basis of circadian rhythms, because an in vitro mixture of the three proteins is sufficient to generate a robust ∼24-h rhythm in the phosphorylation of KaiC. The nucleotide-bound states of KaiC critically affect both KaiB binding to the N-terminal domain (CI) and the phosphotransfer reactions that (de)phosphorylate the KaiC C-terminal domain (CII). However, the nucleotide exchange pathways associated with transitions among these states are poorly understood. In this study, we integrate recent advances in molecular dynamics methods to elucidate the structure and energetics of the pathway for Mg·ADP release from the CII domain. We find that nucleotide release is coupled to large-scale conformational changes in the KaiC hexamer. Solvating the nucleotide requires widening the subunit interface leading to the active site, which is linked to extension of the A-loop, a structure implicated in KaiA binding. These results provide a molecular hypothesis for how KaiA acts as a nucleotide exchange factor. In turn, structural parallels between the CI and CII domains suggest a mechanism for allosteric coupling between the domains. We relate our results to structures observed for other hexameric ATPases, which perform diverse functions.

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Sugimoto, Shinya, Hiroyuki Yoshida, Yoshimitsu Mizunoe, Keigo Tsuruno, Jiro Nakayama, and Kenji Sonomoto. "Structural and Functional Conversion of Molecular Chaperone ClpB from the Gram-Positive Halophilic Lactic Acid Bacterium Tetragenococcus halophilus Mediated by ATP and Stress." Journal of Bacteriology 188, no. 23 (September 22, 2006): 8070–78. http://dx.doi.org/10.1128/jb.00404-06.

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ABSTRACT In this study, we report the purification, initial structural characterization, and functional analysis of the molecular chaperone ClpB from the gram-positive, halophilic lactic acid bacterium Tetragenococcus halophilus. A recombinant T. halophilus ClpB (ClpB Tha ) was overexpressed in Escherichia coli and purified by affinity chromatography, hydroxyapatite chromatography, and gel filtration chromatography. As demonstrated by gel filtration chromatography, chemical cross-linking with glutaraldehyde, and electron microscopy, ClpB Tha forms a homohexameric single-ring structure in the presence of ATP under nonstress conditions. However, under stress conditions, such as high-temperature (>45°C) and high-salt concentrations (>1 M KCl), it dissociated into dimers and monomers, regardless of the presence of ATP. The hexameric ClpB Tha reactivated heat-aggregated proteins dependent upon the DnaK system from T. halophilus (KJE Tha ) and ATP. Interestingly, the mixture of dimer and monomer ClpB Tha , which was formed under stress conditions, protected substrate proteins from thermal inactivation and aggregation in a manner similar to those of general molecular chaperones. From these results, we hypothesize that ClpB Tha forms dimers and monomers to function as a holding chaperone under stress conditions, whereas it forms a hexamer ring to function as a disaggregating chaperone in cooperation with KJE Tha and ATP under poststress conditions.

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Müller, Daniel, Inga Benz, Damini Tapadar, Christian Buddenborg, Lilo Greune, and M. Alexander Schmidt. "Arrangement of the Translocator of the Autotransporter Adhesin Involved in Diffuse Adherence on the Bacterial Surface." Infection and Immunity 73, no. 7 (July 2005): 3851–59. http://dx.doi.org/10.1128/iai.73.7.3851-3859.2005.

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ABSTRACT Autotransporters of gram-negative bacteria are single-peptide secretion systems that consist of a functional N-terminal α-domain (“passenger”) fused to a C-terminal β-domain (“translocator”). How passenger proteins are translocated through the outer membrane has not been resolved, and at present essentially three different models are discussed. In the widely accepted “hairpin model” the passenger proteins are translocated through a channel formed by the β-barrel of the translocator that is integrated in the outer membrane. This model has been challenged by a recent proposal for a general autotransporter model suggesting that there is a hexameric translocation pore that is generated by the oligomerization of six β-domains. A third model suggests that conserved Omp85 participates in autotransporter integration and passenger protein translocation. To examine these models, in this study we investigated the presence of putative oligomeric structures of the translocator of the autotransporter adhesin involved in diffuse adherence (AIDA) in vivo by cross-linking techniques. Furthermore, the capacity of isolated AIDA fusion proteins to form oligomers was studied in vitro by several complementary analytical techniques, such as analytical gel filtration, electron microscopy, immunogold labeling, and cross-linking of recombinant autotransporter proteins in which different passenger proteins were fused to the AIDA translocator. Our results show that the AIDA translocator is mostly present as a monomer. Only a fraction of the AIDA autotransporter was found to form dimers on the bacterial surface and in solution. Higher-order structures, such as hexamers, were not detected either in vivo or in vitro and can therefore be excluded as functional moieties for the AIDA autotransporter.

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Sakwe, Amos M., Tin Nguyen, Vicki Athanasopoulos, Kathy Shire, and Lori Frappier. "Identification and Characterization of a Novel Component of the Human Minichromosome Maintenance Complex." Molecular and Cellular Biology 27, no. 8 (February 12, 2007): 3044–55. http://dx.doi.org/10.1128/mcb.02384-06.

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ABSTRACT Minichromosome maintenance (MCM) complex replicative helicase complexes play essential roles in DNA replication in all eukaryotes. Using a tandem affinity purification-tagging approach in human cells, we discovered a form of the MCM complex that contains a previously unstudied protein, MCM binding protein (MCM-BP). MCM-BP is conserved in multicellular eukaryotes and shares limited homology with MCM proteins. MCM-BP formed a complex with MCM3 to MCM7, which excluded MCM2; and, conversely, hexameric complexes of MCM2 to MCM7 lacked MCM-BP, indicating that MCM-BP can replace MCM2 in the MCM complex. MCM-BP-containing complexes exhibited increased stability under experimental conditions relative to those containing MCM2. MCM-BP also formed a complex with the MCM4/6/7 core helicase in vitro, but, unlike MCM2, did not inhibit this helicase activity. A proportion of MCM-BP bound to cellular chromatin in a cell cycle-dependent manner typical of MCM proteins, and, like other MCM subunits, preferentially associated with a cellular origin in G1 but not in S phase. In addition, down-regulation of MCM-BP decreased the association of MCM4 with chromatin, and the chromatin association of MCM-BP was at least partially dependent on MCM4 and cdc6. The results indicate that multicellular eukaryotes contain two types of hexameric MCM complexes with unique properties and functions.

35

Lu, Jinghua, Kristopher Marjon, Lorraine Marnell, Carolyn Mold, Terry Du Clos, and Peter Sun. "The Structure and Function of Acute Phase Proteins: C-Reactive Protein and Serum Amyloid A in Innate Immunity (110.21)." Journal of Immunology 186, no. 1_Supplement (April 1, 2011): 110.21. http://dx.doi.org/10.4049/jimmunol.186.supp.110.21.

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Abstract During an acute infection, serum levels of certain proteins will be increased dramatically at the early stage of the infection, which are so-called acute phase proteins (APPs). Among them, C-reactive protein (CRP) and Serum Amyloid A (SAA) are two major APPs in humans and therefore they have long been used as clinical biomarkers to monitor the inflammation and the outcome of therapy. However, the physiological function of CRP and SAA remains to be better characterized. In our study, we have shown that CRP can functionally activate Fc receptors pathways, particularly FcγRs and FcαRI (CD89), resulting in the phagocytosis of microbial pathogens and cytokine secretion, such as IL6, and IL8. The ability of CRP to activate FcγRs and FcαRI defines a novel function for acute phase proteins in inflammatory responses involving neutrophils and macrophages and also highlights the innate aspect of otherwise humoral immunity-associated antibody receptors. Furthermore, SAA was shown to be able to bind Gram-positive bacteria. We have now determined the X-ray structure of SAA at 2.7Å resolution. The natural hexameric SAA structure revealed a dimeric architecture arranged as two trimers. We also discovered that SAA hexamer could undergo a transition to monomers that are able to bind and activate Toll-like receptor 2 and 4. Taken together, our studies highlighted CRP and SAA as the powerful first lines of host immune defense during acute infection.

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Stoeva, Stanka, Krassimira Idakieva, Dessislava Nikolova Georgieva, Wolfgang Voelter, and Nicolay Genov. "Penaeus monodon (Tiger Shrimp) Hemocyanin: Subunit Composition and Thermostability." Zeitschrift für Naturforschung C 56, no. 5-6 (June 1, 2001): 416–22. http://dx.doi.org/10.1515/znc-2001-5-616.

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Penaeus monodon (class Crustacea, order Decapoda) is one of the largest shrimps of the Penaeidea family from the Indo - West Pacific region. The dioxygen-transporting protein hemocyanin, isolated from the hemolymph of this invertebrate, is composed of three 75 -76 kDa structural/functional subunits designated as Pm1, Pm2 and Pm3. The N-terminal sequences of the chains were determined and compared with those of other decapodan hemocyanin subunits. Pm2 and Pm3 are highly homologous and electrophoretically undistinguishable polypeptides. In comparison to Pm1, they have an extension of six residues. Pm1 is closely related to the subunit Pv2 of the Penaeus vannamei hemocyanin. Probably, subunits like Pm1 and Pv2 are family-specific for the Penaeidea hemocyanins and the other subunits are speciesspecific. Comparison of N-terminal sequences of respiratory proteins from the sub-orders Natantia and Reptantia demonstrated family- and sub-order-specific sequences. A melting point of 69 °C, lower than those for the di-hexameric decapodan hemocyanins, was determined from the temperature dependence of ellipticity of the mono-hexameric Penaeus monodon hemocyanin. Thermostability of decapodan hemocyanins depends on their aggregation state.

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Dreveny, I., V. E. Pye, F. Beuron, L. C. Briggs, R. L. Isaacson, S. J. Matthews, C. McKeown, X. Yuan, X. Zhang, and P. S. Freemont. "p97 and close encounters of every kind: a brief review." Biochemical Society Transactions 32, no. 5 (October 26, 2004): 715–20. http://dx.doi.org/10.1042/bst0320715.

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The AAA (ATPase associated with various cellular activities) ATPase, p97, is a hexameric protein of chaperone-like function, which has been reported to interact with a number of proteins of seemingly unrelated functions. For the first time, we report a classification of these proteins and aim to elucidate any common structural or functional features they may share. The interactors are grouped into those containing ubiquitin regulatory X domains, which presumably bind to p97 in the same way as the p47 adaptor, and into non-ubiquitin regulatory X domain proteins of different functional subgroups that may employ a different mode of interaction (assuming they also bind directly to p97 and are not experimental artifacts). Future studies will show whether interacting proteins direct p97 to different cellular pathways or a common one and structural elucidation of these interactions will be crucial in understanding these underlying functions.

38

Jewett, Travis J., and L. David Sibley. "The Toxoplasma Proteins MIC2 and M2AP Form a Hexameric Complex Necessary for Intracellular Survival." Journal of Biological Chemistry 279, no. 10 (December 10, 2003): 9362–69. http://dx.doi.org/10.1074/jbc.m312590200.

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Ovejero, César A., Silvia A. González та José L. Affranchino. "The Conserved Tyr176/Leu177 Motif in the α-Helix 9 of the Feline Immunodeficiency Virus Capsid Protein Is Critical for Gag Particle Assembly". Viruses 11, № 9 (4 вересня 2019): 816. http://dx.doi.org/10.3390/v11090816.

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The capsid domain (CA) of the lentiviral Gag polyproteins has two distinct roles during virion morphogenesis. As a domain of Gag, it mediates the Gag–Gag interactions that drive immature particle assembly, whereas as a mature protein, it self-assembles into the conical core of the mature virion. Lentiviral CA proteins are composed of an N-terminal region with seven α-helices and a C-terminal domain (CA-CTD) formed by four α-helices. Structural studies performed in HIV-1 indicate that the CA-CTD helix 9 establishes homodimeric interactions that contribute to the formation of the hexameric Gag lattice in immature virions. Interestingly, the mature CA core also shows inter-hexameric associations involving helix 9 residues W184 and M185. The CA proteins of feline immunodeficiency virus (FIV) and equine infectious anemia virus (EIAV) exhibit, at equivalent positions in helix 9, the motifs Y176/L177 and L169/F170, respectively. In this paper, we investigated the relevance of the Y176/L177 motif for FIV assembly by introducing a series of amino acid substitutions into this sequence and studying their effect on in vivo and in vitro Gag assembly, CA oligomerization, mature virion production, and viral infectivity. Our results demonstrate that the Y176/L177 motif in FIV CA helix 9 is essential for Gag assembly and CA oligomerization. Notably, mutations converting the FIV CA Y176/L177 motif into the HIV-1 WM and EIAV FL sequences allow substantial particle production and viral replication in feline cells.

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Onwubiko, Nichodemus O., Angela Borst, Suraya A. Diaz, Katharina Passkowski, Felicia Scheffel, Ingrid Tessmer, and Heinz P. Nasheuer. "SV40 T antigen interactions with ssDNA and replication protein A: a regulatory role of T antigen monomers in lagging strand DNA replication." Nucleic Acids Research 48, no. 7 (March 4, 2020): 3657–77. http://dx.doi.org/10.1093/nar/gkaa138.

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Abstract DNA replication is a central process in all living organisms. Polyomavirus DNA replication serves as a model system for eukaryotic DNA replication and has considerably contributed to our understanding of basic replication mechanisms. However, the details of the involved processes are still unclear, in particular regarding lagging strand synthesis. To delineate the complex mechanism of coordination of various cellular proteins binding simultaneously or consecutively to DNA to initiate replication, we investigated single-stranded DNA (ssDNA) interactions by the SV40 large T antigen (Tag). Using single molecule imaging by atomic force microscopy (AFM) combined with biochemical and spectroscopic analyses we reveal independent activity of monomeric and oligomeric Tag in high affinity binding to ssDNA. Depending on ssDNA length, we obtain dissociation constants for Tag-ssDNA interactions (KD values of 10–30 nM) that are in the same order of magnitude as ssDNA binding by human replication protein A (RPA). Furthermore, we observe the formation of RPA-Tag-ssDNA complexes containing hexameric as well as monomeric Tag forms. Importantly, our data clearly show stimulation of primase function in lagging strand Okazaki fragment synthesis by monomeric Tag whereas hexameric Tag inhibits the reaction, redefining DNA replication initiation on the lagging strand.

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Baker, Michael J., Chaille T. Webb, David A. Stroud, Catherine S. Palmer, Ann E. Frazier, Bernard Guiard, Agnieszka Chacinska, Jacqueline M. Gulbis, and Michael T. Ryan. "Structural and Functional Requirements for Activity of the Tim9–Tim10 Complex in Mitochondrial Protein Import." Molecular Biology of the Cell 20, no. 3 (February 2009): 769–79. http://dx.doi.org/10.1091/mbc.e08-09-0903.

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The Tim9–Tim10 complex plays an essential role in mitochondrial protein import by chaperoning select hydrophobic precursor proteins across the intermembrane space. How the complex interacts with precursors is not clear, although it has been proposed that Tim10 acts in substrate recognition, whereas Tim9 acts in complex stabilization. In this study, we report the structure of the yeast Tim9–Tim10 hexameric assembly determined to 2.5 Å and have performed mutational analysis in yeast to evaluate the specific roles of Tim9 and Tim10. Like the human counterparts, each Tim9 and Tim10 subunit contains a central loop flanked by disulfide bonds that separate two extended N- and C-terminal tentacle-like helices. Buried salt-bridges between highly conserved lysine and glutamate residues connect alternating subunits. Mutation of these residues destabilizes the complex, causes defective import of precursor substrates, and results in yeast growth defects. Truncation analysis revealed that in the absence of the N-terminal region of Tim9, the hexameric complex is no longer able to efficiently trap incoming substrates even though contacts with Tim10 are still made. We conclude that Tim9 plays an important functional role that includes facilitating the initial steps in translocating precursor substrates into the intermembrane space.

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Drennan, Amanda C., Shivaani Krishna, Mark A. Seeger, Michael P. Andreas, Jennifer M. Gardner, Emily K. R. Sether, Sue L. Jaspersen, and Ivan Rayment. "Structure and function of Spc42 coiled-coils in yeast centrosome assembly and duplication." Molecular Biology of the Cell 30, no. 12 (June 2019): 1505–22. http://dx.doi.org/10.1091/mbc.e19-03-0167.

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Centrosomes and spindle pole bodies (SPBs) are membraneless organelles whose duplication and assembly is necessary for bipolar mitotic spindle formation. The structural organization and functional roles of major proteins in these organelles can provide critical insights into cell division control. Spc42, a phosphoregulated protein with an N-terminal dimeric coiled-coil (DCC), assembles into a hexameric array at the budding yeast SPB core, where it functions as a scaffold for SPB assembly. Here, we present in vitro and in vivo data to elucidate the structural arrangement and biological roles of Spc42 elements. Crystal structures reveal details of two additional coiled-coils in Spc42: a central trimeric coiled-coil and a C-terminal antiparallel DCC. Contributions of the three Spc42 coiled-coils and adjacent undetermined regions to the formation of an ∼145 Å hexameric lattice in an in vitro lipid monolayer assay and to SPB duplication and assembly in vivo reveal structural and functional redundancy in Spc42 assembly. We propose an updated model that incorporates the inherent symmetry of these Spc42 elements into a lattice, and thereby establishes the observed sixfold symmetry. The implications of this model for the organization of the central SPB core layer are discussed.

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Blok, Neil B., Dongyan Tan, Ray Yu-Ruei Wang, Pawel A. Penczek, David Baker, Frank DiMaio, Tom A. Rapoport, and Thomas Walz. "Unique double-ring structure of the peroxisomal Pex1/Pex6 ATPase complex revealed by cryo-electron microscopy." Proceedings of the National Academy of Sciences 112, no. 30 (July 13, 2015): E4017—E4025. http://dx.doi.org/10.1073/pnas.1500257112.

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Members of the AAA family of ATPases assemble into hexameric double rings and perform vital functions, yet their molecular mechanisms remain poorly understood. Here, we report structures of the Pex1/Pex6 complex; mutations in these proteins frequently cause peroxisomal diseases. The structures were determined in the presence of different nucleotides by cryo-electron microscopy. Models were generated using a computational approach that combines Monte Carlo placement of structurally homologous domains into density maps with energy minimization and refinement protocols. Pex1 and Pex6 alternate in an unprecedented hexameric double ring. Each protein has two N-terminal domains, N1 and N2, structurally related to the single N domains in p97 and N-ethylmaleimide sensitive factor (NSF); N1 of Pex1 is mobile, but the others are packed against the double ring. The N-terminal ATPase domains are inactive, forming a symmetric D1 ring, whereas the C-terminal domains are active, likely in different nucleotide states, and form an asymmetric D2 ring. These results suggest how subunit activity is coordinated and indicate striking similarities between Pex1/Pex6 and p97, supporting the hypothesis that the Pex1/Pex6 complex has a role in peroxisomal protein import analogous to p97 in ER-associated protein degradation.

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Bochman, Matthew L., Stephen P. Bell, and Anthony Schwacha. "Subunit Organization of Mcm2-7 and the Unequal Role of Active Sites in ATP Hydrolysis and Viability." Molecular and Cellular Biology 28, no. 19 (July 28, 2008): 5865–73. http://dx.doi.org/10.1128/mcb.00161-08.

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ABSTRACT The Mcm2-7 (minichromosome maintenance) complex is a toroidal AAA+ ATPase and the putative eukaryotic replicative helicase. Unlike a typical homohexameric helicase, Mcm2-7 contains six distinct, essential, and evolutionarily conserved subunits. Precedence to other AAA+ proteins suggests that Mcm ATPase active sites are formed combinatorially, with Walker A and B motifs contributed by one subunit and a catalytically essential arginine (arginine finger) contributed by the adjacent subunit. To test this prediction, we used copurification experiments to identify five distinct and stable Mcm dimer combinations as potential active sites; these subunit associations predict the architecture of the Mcm2-7 complex. Through the use of mutant subunits, we establish that at least three sites are active for ATP hydrolysis and have a canonical AAA+ configuration. In isolation, these five active-site dimers have a wide range of ATPase activities. Using Walker B and arginine finger mutations in defined Mcm subunits, we demonstrate that these sites similarly make differential contributions toward viability and ATP hydrolysis within the intact hexamer. Our conclusions predict a structural discontinuity between Mcm2 and Mcm5 and demonstrate that in contrast to other hexameric helicases, the six Mcm2-7 active sites are functionally distinct.

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Wheeler, Diana E., Irina Tuchinskaya, Norman A. Buck, and Bruce E. Tabashnik. "Hexameric storage proteins during metamorphosis and egg production in the diamondback moth, Plutella xylostella (Lepidoptera)." Journal of Insect Physiology 46, no. 6 (June 2000): 951–58. http://dx.doi.org/10.1016/s0022-1910(99)00202-4.

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46

Dodge, Greg J., Ashay Patel, Kara L. Jaremko, J. Andrew McCammon, Janet L. Smith, and Michael D. Burkart. "Structural and dynamical rationale for fatty acid unsaturation inEscherichia coli." Proceedings of the National Academy of Sciences 116, no. 14 (March 14, 2019): 6775–83. http://dx.doi.org/10.1073/pnas.1818686116.

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Fatty acid biosynthesis in α- and γ-proteobacteria requires two functionally distinct dehydratases, FabA and FabZ. Here, mechanistic cross-linking facilitates the structural characterization of a stable hexameric complex of sixEscherichia coliFabZ dehydratase subunits with six AcpP acyl carrier proteins. The crystal structure sheds light on the divergent substrate selectivity of FabA and FabZ by revealing distinct architectures of the binding pocket. Molecular dynamics simulations demonstrate differential biasing of substrate orientations and conformations within the active sites of FabA and FabZ such that FabZ is preorganized to catalyze only dehydration, while FabA is primed for both dehydration and isomerization.

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Sweeny, Elizabeth A., Amber Tariq, Esin Gurpinar, Michelle S. Go, Matthew A. Sochor, Zhong-Yuan Kan, Leland Mayne, S. Walter Englander, and James Shorter. "Structural and mechanistic insights into Hsp104 function revealed by synchrotron X-ray footprinting." Journal of Biological Chemistry 295, no. 6 (December 27, 2019): 1517–38. http://dx.doi.org/10.1074/jbc.ra119.011577.

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Hsp104 is a hexameric AAA+ ring translocase, which drives protein disaggregation in nonmetazoan eukaryotes. Cryo-EM structures of Hsp104 have suggested potential mechanisms of substrate translocation, but precisely how Hsp104 hexamers disaggregate proteins remains incompletely understood. Here, we employed synchrotron X-ray footprinting to probe the solution-state structures of Hsp104 monomers in the absence of nucleotide and Hsp104 hexamers in the presence of ADP or ATPγS (adenosine 5′-O-(thiotriphosphate)). Comparing side-chain solvent accessibilities between these three states illuminated aspects of Hsp104 structure and guided design of Hsp104 variants to probe the disaggregase mechanism in vitro and in vivo. We established that Hsp104 hexamers switch from a more-solvated state in ADP to a less-solvated state in ATPγS, consistent with switching from an open spiral to a closed ring visualized by cryo-EM. We pinpointed critical N-terminal domain (NTD), NTD-nucleotide–binding domain 1 (NBD1) linker, NBD1, and middle domain (MD) residues that enable intrinsic disaggregase activity and Hsp70 collaboration. We uncovered NTD residues in the loop between helices A1 and A2 that can be substituted to enhance disaggregase activity. We elucidated a novel potentiated Hsp104 MD variant, Hsp104–RYD, which suppresses α-synuclein, fused in sarcoma (FUS), and TDP-43 toxicity. We disambiguated a secondary pore-loop in NBD1, which collaborates with the NTD and NBD1 tyrosine-bearing pore-loop to drive protein disaggregation. Finally, we defined Leu-601 in NBD2 as crucial for Hsp104 hexamerization. Collectively, our findings unveil new facets of Hsp104 structure and mechanism. They also connect regions undergoing large changes in solvation to functionality, which could have profound implications for protein engineering.

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Auerbach, Marcy R., Kristy R. Brown, Artem Kaplan, Denise de Las Nueces, and Ila R. Singh. "A Small Loop in the Capsid Protein of Moloney Murine Leukemia Virus Controls Assembly of Spherical Cores." Journal of Virology 80, no. 6 (March 15, 2006): 2884–93. http://dx.doi.org/10.1128/jvi.80.6.2884-2893.2006.

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ABSTRACT We report the identification of a novel domain in the Gag protein of Moloney murine leukemia virus (MoLV) that is important for the formation of spherical cores. Analysis of 18 insertional mutations in the N-terminal domain of the capsid protein (CA) identified 3 that were severely defective for viral assembly and release. Transmission electron microscopy of cells producing these mutants showed assembly of Gag proteins in large, flat or dome-shaped patches at the plasma membrane. Spherical cores were not formed, and viral particles were not released. This late assembly/release block was partially rescued by wild-type virus. All three mutations localized to the small loop between α-helices 4 and 5 of CA, analogous to the cyclophilin A-binding loop of human immunodeficiency virus type 1 CA. In the X-ray structure of the hexameric form of MLV CA, this loop is located at the periphery of the hexamer. The phenotypes of mutations in this loop suggest that formation of a planar lattice of Gag is unhindered by mutations in the loop. However, the lack of progression of these planar structures to spherical ones suggests that mutations in this loop may prevent formation of pentamers or of stable pentamer-hexamer interactions, which are essential for the formation of a closed, spherical core. This region in CA, focused to a few residues of a small loop, may offer a novel therapeutic target for retroviral diseases.

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Varikoti, Rohith Anand, Hewafonsekage Yasan Y. Fonseka, Maria S. Kelly, Alex Javidi, Mangesh Damre, Sarah Mullen, Jimmie L. Nugent, Christopher M. Gonzales, George Stan, and Ruxandra I. Dima. "Exploring the Effect of Mechanical Anisotropy of Protein Structures in the Unfoldase Mechanism of AAA+ Molecular Machines." Nanomaterials 12, no. 11 (May 28, 2022): 1849. http://dx.doi.org/10.3390/nano12111849.

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Essential cellular processes of microtubule disassembly and protein degradation, which span lengths from tens of μm to nm, are mediated by specialized molecular machines with similar hexameric structure and function. Our molecular simulations at atomistic and coarse-grained scales show that both the microtubule-severing protein spastin and the caseinolytic protease ClpY, accomplish spectacular unfolding of their diverse substrates, a microtubule lattice and dihydrofolate reductase (DHFR), by taking advantage of mechanical anisotropy in these proteins. Unfolding of wild-type DHFR requires disruption of mechanically strong β-sheet interfaces near each terminal, which yields branched pathways associated with unzipping along soft directions and shearing along strong directions. By contrast, unfolding of circular permutant DHFR variants involves single pathways due to softer mechanical interfaces near terminals, but translocation hindrance can arise from mechanical resistance of partially unfolded intermediates stabilized by β-sheets. For spastin, optimal severing action initiated by pulling on a tubulin subunit is achieved through specific orientation of the machine versus the substrate (microtubule lattice). Moreover, changes in the strength of the interactions between spastin and a microtubule filament, which can be driven by the tubulin code, lead to drastically different outcomes for the integrity of the hexameric structure of the machine.

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Zuromski, Kristin L., Robert T. Sauer, and Tania A. Baker. "Modular and coordinated activity of AAA+ active sites in the double-ring ClpA unfoldase of the ClpAP protease." Proceedings of the National Academy of Sciences 117, no. 41 (October 5, 2020): 25455–63. http://dx.doi.org/10.1073/pnas.2014407117.

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ClpA is a hexameric double-ring AAA+ unfoldase/translocase that functions with the ClpP peptidase to degrade proteins that are damaged or unneeded. How the 12 ATPase active sites of ClpA, 6 in the D1 ring and 6 in the D2 ring, work together to fuel ATP-dependent degradation is not understood. We use site-specific cross-linking to engineer ClpA hexamers with alternating ATPase-active and ATPase-inactive modules in the D1 ring, the D2 ring, or both rings to determine if these active sites function together. Our results demonstrate that D2 modules coordinate with D1 modules and ClpP during mechanical work. However, there is no requirement for adjacent modules in either ring to be active for efficient enzyme function. Notably, ClpAP variants with just three alternating active D2 modules are robust protein translocases and function with double the energetic efficiency of ClpAP variants with completely active D2 rings. Although D2 is the more powerful motor, three or six active D1 modules are important for high enzyme processivity, which depends on D1 and D2 acting coordinately. These results challenge sequential models of ATP hydrolysis and coupled mechanical work by ClpAP and provide an engineering strategy that will be useful in testing other aspects of ClpAP mechanism.

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