Journal articles: 'Hexameric protein'

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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.

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de Haas, Felix, Jan F. L. van Breemen, Martha M. C. Bijlholt, and Ernst F. J. van Bruggen. "Analysis of Interhexameric Contact Regions in Two Dodecameric Hemocyanins at Subdohain Resolution by Combination of Data from Electron Microscopy, X-Ray Diffraction and Amino-Acid Sequence Studies." Proceedings, annual meeting, Electron Microscopy Society of America 48, no. 1 (August 12, 1990): 266–67. http://dx.doi.org/10.1017/s0424820100180082.

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Hemocyanin is a biological macromolecule which occurs freely dissolved in the hemolymph of certain invertebrates. The function of this copper containing protein is the transport of oxygen through the organism. In fulfilling this task hemocyanin has developed a similar mechanism as hemoglobin in binding oxygen reversibly and cooperatively. The hemocyanin of arthropods consists of one or more hexamers of subunits with a molecular weight of approx. 75 000. Depending on the species 3-15 types of monomeric subunits occur, which differ in amino-acid composition and in their oxygen binding properties. Each type of subunit fulfills a specific role in the architecture of that hemocyanin. In nature arthropodan hemocyanin is found as a one-hexameric, two-hexameric (dodecameric), four-hexameric or eight-hexameric molecular assembly depending on the species. In this work we focus on the difference in organization of the hexamers in the dodecamer of two different species i.e. the tarantula Eurypelma californicum (a chelicerate) and the crab Cancer pagurus (a crustacean). Eurypelma hemocyanin is made from 7 different subunits called a - g, whereas Cancer hemocyanin consists of 3 subunit types termed α, β, and γ .By image analysis of electron micrographs of the two-hexameric half hemocyanin molecules from Eurypelma and the two-hexameric whole hemocyanin molecules from Cancer, computer averaged projections of these dodecamers were obtained as shown in fig. 1. They differ clearly in their interhexameric contacts. To analyse this difference in more detail these projections were used as a reference in a simulation procedure.

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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.

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Lu, Jinghua, Terry Du Clos, Carolyn Mold, and Peter Sun. "The structural mechanism of Serum Amyloid A -mediated Secondary amyloid formation (135.34)." Journal of Immunology 184, no. 1_Supplement (April 1, 2010): 135.34. http://dx.doi.org/10.4049/jimmunol.184.supp.135.34.

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Abstract Secondary amyloidosis is a severe complication of many chronic inflammatory diseases, such as rheumatoid arthritis and atherosclerosis, when the C-terminally cleaved fragments of acute phase protein Serum Amyloid A (SAA) are deposited in tissues as amyloid fibrils. The pathologic process of secondary amyloidosis involves a conversion of the structure of native SAA into a predominantly antiparallel β-sheet secondary structure. We have now determined the X-ray structure of SAA in a monomeric and a hexameric form at 2.2Å and 2.7Å resolution, respectively. The hexameric SAA structure revealed a dimeric architecture arranged as two trimers. Strong interactions between the C-terminal loop region and four-helix-bundle core of the SAA monomers create a stable structure to prevent secondary structure refolding during amyloidogenesis. The presence of multimeric forms in human serum was confirmed by size-exclusion experiments. Recombinant monomeric, trimeric and hexameric SAA protein were purified from Escherichia coli and characterized. Binding studies show that the monomeric and trimeric but not the hexameric SAA bind to Toll-like receptor 2 and 4. In line with these experiments, SAA may undergo a transition between hexamers and monomers, which would activate Toll-like receptors to further complicate the chronic inflammation in disease situation.

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Hankins, J. S., H. Denroche, and G. A. Mackie. "Interactions of the RNA-Binding Protein Hfq with cspA mRNA, Encoding the Major Cold Shock Protein." Journal of Bacteriology 192, no. 10 (March 16, 2010): 2482–90. http://dx.doi.org/10.1128/jb.01619-09.

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ABSTRACT CspA, a small protein that is highly induced by cold shock, is encoded by a monocistronic mRNA of 428 nucleotides (nt) whose half-life and abundance are greatly increased following cold shock. We show here that in vitro cspA mRNA can bind multiple copies of Hfq, a hexameric Sm-like protein which promotes a variety of RNA-RNA interactions. Binding of the first Hfq hexamer occurs with an apparent Kd (dissociation constant) of <40 nM; up to seven additional hexamers can bind sequentially at higher concentrations. Known ligands of Hfq, including the small regulatory RNA, RyhB, compete with cspA mRNA. Several experiments suggest that the first binding site to be occupied by Hfq is located at or near the 3′ end of cspA mRNA. The consequences of limited Hfq binding in vitro include nearly total inhibition of RNase E cleavage at a site ∼35 nt from the 3′ end of the mRNA, stimulation of polyadenylation by poly(A) polymerase 1, and subsequent exonucleolytic degradation by polynucleotide phosphorylase. We propose that Hfq may play a facilitating role in the metabolism of cspA mRNA.

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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.

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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.

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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.

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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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Oohora, Koji, Shota Hirayama, Tsuyoshi Mashima, and Takashi Hayashi. "Supramolecular dimerization of a hexameric hemoprotein via multiple pyrene-pyrene interactions." Journal of Porphyrins and Phthalocyanines 24, no. 01n03 (January 2020): 259–67. http://dx.doi.org/10.1142/s1088424619500949.

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Protein assemblies are being investigated as a new-class of biomaterials. A supramolecular assembly of a mutant hexameric tyrosine coordinated hemoprotein (HTHP) modified with a pyrene derivative is described. Cysteine was first introduced as a site-specific reaction point at position V44 which is located at the bottom surface of the cylindrical structure of HTHP. [Formula: see text]-(1-pyrenyl)maleimide was then reacted with the mutant. The modification was confirmed by MALDI-TOF mass spectrometry and UV-vis absorption spectroscopy, indicating that approximately 90% cysteine residues are attached via the pyrene derivative. Size exclusion chromatography (SEC) measurements for pyrene-attached HTHP include a single peak which elutes earlier than the unmodified HTHP. Further investigation by SEC and dynamic light scattering (DLS) measurements indicate the desired size corresponding to the dimer of the hemoprotein hexamers. The multivalent effect of pyrene–pyrene interactions including hydrophobic and [Formula: see text]–[Formula: see text] stacking interactions appears to be responsible for including formation of the stable dimer of the hexamers. Interestingly, the assembly dissociates to the hexamer by removal of heme. In the case of the apo-form of pyrene-attached HTHP, the pyrene moiety appears to be incorporated into the heme pocket because the modification point is located at the adjacent residue of the Tyr45 coordinating to heme in the holo-form of HTHP. Subsequent addition of heme into the apo-form of pyrene-attached HTHP regenerates the dimer of the hexamers. The present study demonstrates a unique heme-dependent system in which HTHP is assembled to form a dimer of hexamers in the presence of heme and disassembled by removal of heme.

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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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Mitchell, Alison H., and Stephen C. West. "Hexameric Rings of Escherichia coli RuvB Protein." Journal of Molecular Biology 243, no. 2 (October 1994): 208–15. http://dx.doi.org/10.1006/jmbi.1994.1648.

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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.

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Muller, David A., Michael J. Landsberg, Cheryl Bletchly, Rosalba Rothnagel, Lynne Waddington, Ben Hankamer, and Paul R. Young. "Structure of the dengue virus glycoprotein non-structural protein 1 by electron microscopy and single-particle analysis." Journal of General Virology 93, no. 4 (April 1, 2012): 771–79. http://dx.doi.org/10.1099/vir.0.039321-0.

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The flavivirus non-structural protein 1 (NS1) is a glycoprotein that is secreted as a soluble hexameric complex during the course of natural infection. Growing evidence indicates that this secreted form of NS1 (sNS1) plays a significant role in immune evasion and modulation during infection. Attempts to determine the crystal structure of NS1 have been unsuccessful to date and relatively little is known about the macromolecular organization of the sNS1 hexamer. Here, we have applied single-particle analysis to images of baculovirus-derived recombinant dengue 2 virus NS1 obtained by electron microscopy to determine its 3D structure to a resolution of 23 Å. This structure reveals a barrel-like organization of the three dimeric units that comprise the hexamer and provides further insights into the overall organization of oligomeric sNS1.

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Redick, S. D., and J. E. Schwarzbauer. "Rapid intracellular assembly of tenascin hexabrachions suggests a novel cotranslational process." Journal of Cell Science 108, no. 4 (April 1, 1995): 1761–69. http://dx.doi.org/10.1242/jcs.108.4.1761.

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Tenascin, an extracellular matrix protein that modulates cell adhesion, exists as a unique six-armed structure called a hexabrachion. The human hexabrachion is composed of six identical 320 kDa subunits and the structure is stabilized by inter-subunit disulfide bonds between amino-terminal segments. We have examined the biosynthesis of tenascin and its assembly into hexabrachions using pulsechase labeling of U-138 MG human glioma cells. Newly synthesized tenascin hexamers are secreted within 60 minutes of translation initiation. Intracellularly, as early as full length tenascin can be detected in pulse-labeled cell lysates, it is already in hexameric form. No precursors, such as monomers, dimers, or trimers, were identified that could be chased into hexamers. This lack of assembly intermediates suggests that nascent tenascin polypeptides associate prior to completion of translation. In contrast, fibronectin monomers in the same lysates are gradually formed into disulfide-bonded dimers. Although hexamer assembly is rapid, the rate-limiting step in secretion appears to be transport to the medial Golgi as endoglycosidase H-resistance was not detected until after a 30 minute chase. These results provide evidence for a novel co-translational mechanism of tenascin assembly which would be facilitated by its length and by the amino-terminal location of the assembly domain.

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Pizzo, Federica, Maria Rosalia Mangione, Fabio Librizzi, Mauro Manno, Vincenzo Martorana, Rosina Noto, and Silvia Vilasi. "The Possible Role of the Type I Chaperonins in Human Insulin Self-Association." Life 12, no. 3 (March 18, 2022): 448. http://dx.doi.org/10.3390/life12030448.

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Insulin is a hormone that attends to energy metabolism by regulating glucose levels in the bloodstream. It is synthesised within pancreas beta-cells where, before being released into the serum, it is stored in granules as hexamers coordinated by Zn2+ and further packaged in microcrystalline structures. The group I chaperonin cpn60, known for its assembly-assisting function, is present, together with its cochaperonin cpn10, at each step of the insulin secretory pathway. However, the exact function of the heat shock protein in insulin biosynthesis and processing is still far from being understood. Here we explore the possibility that the molecular machine cpn60/cpn10 could have a role in insulin hexameric assembly and its further crystallization. Moreover, we also evaluate their potential protective effect in pathological insulin aggregation. The experiments performed with the cpn60 bacterial homologue, GroEL, in complex with its cochaperonin GroES, by using spectroscopic methods, microscopy and hydrodynamic techniques, reveal that the chaperonins in vitro favour insulin hexameric organisation and inhibit its aberrant aggregation. These results provide new details in the field of insulin assembly and its related disorders.

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Gomez Barroso, Juan Arturo, Mariana Reneé Miranda, Claudio Alejandro Pereira, Richard Charles Garratt, and Carlos Fernando Aguilar. "X-ray diffraction and in vivo studies reveal the quinary structure of Trypanosoma cruzi nucleoside diphosphate kinase 1: a novel helical oligomer structure." Acta Crystallographica Section D Structural Biology 78, no. 1 (January 1, 2022): 30–42. http://dx.doi.org/10.1107/s2059798321011219.

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Trypanosoma cruzi is a flagellated protozoan parasite that causes Chagas disease, which represents a serious health problem in the Americas. Nucleoside diphosphate kinases (NDPKs) are key enzymes that are implicated in cellular energy management. TcNDPK1 is the canonical isoform in the T. cruzi parasite. TcNDPK1 has a cytosolic, perinuclear and nuclear distribution. It is also found in non-membrane-bound filaments adjacent to the nucleus. In the present work, X-ray diffraction and in vivo studies of TcNDPK1 are described. The structure reveals a novel, multi-hexameric, left-handed helical oligomer structure. The results of directed mutagenesis studies led to the conclusion that the microscopic TcNDPK1 granules observed in vivo in T. cruzi parasites are made up by the association of TcNDPK1 oligomers. In the absence of experimental data, analysis of the interactions in the X-ray structure of the TcNDPK1 oligomer suggests the probable assembly and disassembly steps: dimerization, assembly of the hexamer as a trimer of dimers, hexamer association to generate the left-handed helical oligomer structure and finally oligomer association in a parallel manner to form the microscopic TcNDPK1 filaments that are observed in vivo in T. cruzi parasites. Oligomer disassembly takes place on the binding of substrate in the active site of TcNDPK1, leading to dissociation of the hexamers. This study constitutes the first report of such a protein arrangement, which has never previously been seen for any protein or NDPK. Further studies are needed to determine its physiological role. However, it may suggest a paradigm for protein storage reflecting the complex mechanism of action of TcNDPK1.

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Dostálková, Alžběta, Kryštof Škach, Filip Kaufman, Ivana Křížová, Romana Hadravová, Martin Flegel, Tomáš Ruml, Richard Hrabal, and Michaela Rumlová. "PF74 and Its Novel Derivatives Stabilize Hexameric Lattice of HIV-1 Mature-Like Particles." Molecules 25, no. 8 (April 20, 2020): 1895. http://dx.doi.org/10.3390/molecules25081895.

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A major structural retroviral protein, capsid protein (CA), is able to oligomerize into two different hexameric lattices, which makes this protein a key component for both the early and late stages of HIV-1 replication. During the late stage, the CA protein, as part of the Gag polyprotein precursor, facilitates protein–protein interactions that lead to the assembly of immature particles. Following protease activation and Gag polyprotein processing, CA also drives the assembly of the mature viral core. In the early stage of infection, the role of the CA protein is distinct. It controls the disassembly of the mature CA hexameric lattice i.e., uncoating, which is critical for the reverse transcription of the single-stranded RNA genome into double stranded DNA. These properties make CA a very attractive target for small molecule functioning as inhibitors of HIV-1 particle assembly and/or disassembly. Of these, inhibitors containing the PF74 scaffold have been extensively studied. In this study, we reported a series of modifications of the PF74 molecule and its characterization through a combination of biochemical and structural approaches. Our data supported the hypothesis that PF74 stabilizes the mature HIV-1 CA hexameric lattice. We identified derivatives with a higher in vitro stabilization activity in comparison to the original PF74 molecule.

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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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Bragg, D. Cristopher, Kotchaphorn Mangkalaphiban, Christine A. Vaine, Nichita J. Kulkarni, David Shin, Rachita Yadav, Jyotsna Dhakal, et al. "Disease onset in X-linked dystonia-parkinsonism correlates with expansion of a hexameric repeat within an SVA retrotransposon in TAF1." Proceedings of the National Academy of Sciences 114, no. 51 (December 11, 2017): E11020—E11028. http://dx.doi.org/10.1073/pnas.1712526114.

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X-linked dystonia-parkinsonism (XDP) is a neurodegenerative disease associated with an antisense insertion of a SINE-VNTR-Alu (SVA)-type retrotransposon within an intron of TAF1. This unique insertion coincides with six additional noncoding sequence changes in TAF1, the gene that encodes TATA-binding protein–associated factor-1, which appear to be inherited together as an identical haplotype in all reported cases. Here we examined the sequence of this SVA in XDP patients (n = 140) and detected polymorphic variation in the length of a hexanucleotide repeat domain, (CCCTCT)n. The number of repeats in these cases ranged from 35 to 52 and showed a highly significant inverse correlation with age at disease onset. Because other SVAs exhibit intrinsic promoter activity that depends in part on the hexameric domain, we assayed the transcriptional regulatory effects of varying hexameric lengths found in the unique XDP SVA retrotransposon using luciferase reporter constructs. When inserted sense or antisense to the luciferase reading frame, the XDP variants repressed or enhanced transcription, respectively, to an extent that appeared to vary with length of the hexamer. Further in silico analysis of this SVA sequence revealed multiple motifs predicted to form G-quadruplexes, with the greatest potential detected for the hexameric repeat domain. These data directly link sequence variation within the XDP-specific SVA sequence to phenotypic variability in clinical disease manifestation and provide insight into potential mechanisms by which this intronic retroelement may induce transcriptional interference in TAF1 expression.

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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.

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Ruiz-Ferrer, Virginia, Jasminka Boskovic, Carlos Alfonso, Germán Rivas, Oscar Llorca, Dionisio López-Abella, and Juan José López-Moya. "Structural Analysis of Tobacco Etch Potyvirus HC-Pro Oligomers Involved in Aphid Transmission." Journal of Virology 79, no. 6 (March 15, 2005): 3758–65. http://dx.doi.org/10.1128/jvi.79.6.3758-3765.2005.

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ABSTRACT Oligomeric forms of the HC-Pro protein of the tobacco etch potyvirus (TEV) have been analyzed by analytical ultracentrifugation and single-particle electron microscopy combined with three-dimensional (3D) reconstruction. Highly purified HC-Pro protein was obtained from plants infected with TEV by using a modified version of the virus that incorporates a histidine tag at the HC-Pro N terminus (hisHC-Pro). The purified protein retained a high biological activity in solution when tested for aphid transmission. Sedimentation equilibrium showed that the hisHC-Pro preparations were heterogenous in size. Sedimentation velocity confirmed the previous observation and revealed that the active protein solution contained several sedimenting species compatible with dimers, tetramers, hexamers, and octamers of the protein. Electron microscopy fields of purified protein showed particles of different sizes and shapes. The reconstructed 3D structures suggested that the observed particles could correspond to dimeric, tetrameric, and hexameric forms of the protein. A model of the interactions required for oligomerization of the HC-Pro of potyviruses is proposed.

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Lee, Cheng-Chung, Ta-Wei Lin, Tzu-Ping Ko, and Andrew H. J. Wang. "The Hexameric Structures of Human Heat Shock Protein 90." PLoS ONE 6, no. 5 (May 25, 2011): e19961. http://dx.doi.org/10.1371/journal.pone.0019961.

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Arthur, Christopher P., and Michael H. B. Stowell. "Structure of Synaptophysin: A Hexameric MARVEL-Domain Channel Protein." Structure 15, no. 6 (June 2007): 707–14. http://dx.doi.org/10.1016/j.str.2007.04.011.

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Hoskins, Joel R., Shannon M. Doyle, and Sue Wickner. "Coupling ATP utilization to protein remodeling by ClpB, a hexameric AAA+ protein." Proceedings of the National Academy of Sciences 106, no. 52 (November 25, 2009): 22233–38. http://dx.doi.org/10.1073/pnas.0911937106.

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Zhang, Shuwen, and Youdong Mao. "AAA+ ATPases in Protein Degradation: Structures, Functions and Mechanisms." Biomolecules 10, no. 4 (April 18, 2020): 629. http://dx.doi.org/10.3390/biom10040629.

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Adenosine triphosphatases (ATPases) associated with a variety of cellular activities (AAA+), the hexameric ring-shaped motor complexes located in all ATP-driven proteolytic machines, are involved in many cellular processes. Powered by cycles of ATP binding and hydrolysis, conformational changes in AAA+ ATPases can generate mechanical work that unfolds a substrate protein inside the central axial channel of ATPase ring for degradation. Three-dimensional visualizations of several AAA+ ATPase complexes in the act of substrate processing for protein degradation have been resolved at the atomic level thanks to recent technical advances in cryogenic electron microscopy (cryo-EM). Here, we summarize the resulting advances in structural and biochemical studies of AAA+ proteases in the process of proteolysis reactions, with an emphasis on cryo-EM structural analyses of the 26S proteasome, Cdc48/p97 and FtsH-like mitochondrial proteases. These studies reveal three highly conserved patterns in the structure–function relationship of AAA+ ATPase hexamers that were observed in the human 26S proteasome, thus suggesting common dynamic models of mechanochemical coupling during force generation and substrate translocation.

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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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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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Singharoy, Abhishek, Christophe Chipot, Mahmoud Moradi, and Klaus Schulten. "Chemomechanical Coupling in Hexameric Protein–Protein Interfaces Harnesses Energy within V-Type ATPases." Journal of the American Chemical Society 139, no. 1 (December 23, 2016): 293–310. http://dx.doi.org/10.1021/jacs.6b10744.

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Ji, Zhejian, Hao Li, Daniele Peterle, Joao A. Paulo, Scott B. Ficarro, Thomas E. Wales, Jarrod A. Marto, Steven P. Gygi, John R. Engen, and Tom A. Rapoport. "Translocation of polyubiquitinated protein substrates by the hexameric Cdc48 ATPase." Molecular Cell 82, no. 3 (February 2022): 570–84. http://dx.doi.org/10.1016/j.molcel.2021.11.033.

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Kielb, Patrycja, Tillmann Utesch, Jacek Kozuch, Jae-Hun Jeoung, Holger Dobbek, Maria Andrea Mroginski, Peter Hildebrandt, and Inez Weidinger. "Switchable Redox Chemistry of the Hexameric Tyrosine-Coordinated Heme Protein." Journal of Physical Chemistry B 121, no. 16 (April 19, 2017): 3955–64. http://dx.doi.org/10.1021/acs.jpcb.7b01286.

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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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McDuff, F. O., A. Doucet, and M. Beauregard. "Low concentration of guanidine hydrochloride induces the formation of an aggregation-prone state in α-urease." Biochemistry and Cell Biology 82, no. 2 (April 1, 2004): 305–13. http://dx.doi.org/10.1139/o03-072.

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Canavalia ensiformis (jack bean) α-urease is a hexameric protein characterized by a complex denaturation mechanism. In previous papers, we have shown that a hydrophobic 8-anilino-1-naphthalenesulfonic acid (ANSA) binding conformer could be populated in a moderate concentration of denaturant. This state was obtained under conditions that had no detectable impact on its tertiary structure, as indicated by fluorescence measurements. In the present study, we further characterized this ANSA-binding state in an attempt to understand urease behavior. Evidence presented here shows that the presence of ANSA was not required for the generation of the conformer and that its affinity for ANSA came from an increase in hydrophobicity leading to aggregation. Circular dichroism investigation of urease revealed that it had periodical secondary structure content similar to Klebsiella aerogenes urease (secondary structures calculated on the basis of crystallographic data). The impact of 0.9 M guanidine hydrochloride (GuHCl) on soluble urease secondary structures was minimal but is compatible with a slight increase in beta-sheet structures. Such modification may indicates that aggregation involves amyloid-like fibril formation. Electron microscopy analysis of urease in the absence of GuHCl revealed the presence of urease hexamers (round shape 13 nm in diameter). These particles disappeared in the presence of moderate denaturant concentration owing to the formation of aggregates and fibril-like structures. The fibrils obtained in 1.5 M GuHCl had an average diameter of 6.5 nm, suggesting that urease hexamers dissociated into smaller oligomeric forms when forming such fibrils.Key words: protein structure, protein folding, denaturation, aggregation, multimeric proteins, protein fibrils, hydrophobicity, molten globule state.

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Bazin, Alexandre, Mickaël Cherrier, and Laurent Terradot. "Structural insights into DNA replication initiation in Helicobacter pylori." Acta Crystallographica Section A Foundations and Advances 70, a1 (August 5, 2014): C1632. http://dx.doi.org/10.1107/s2053273314083673.

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In Gram-negative bacteria, opening of DNA double strand during replication is performed by the replicative helicase DnaB. This protein allows for replication fork elongation by unwinding DNA and interacting with DnaG primase. DnaB is composed of two domains: an N-terminal domain (NTD) and a C-terminal domain (CTD) connected by a flexible linker. The protein forms two-tiered hexamers composed of a NTD-ring and a CTD-ring. In Escherichia coli, the initiator protein DnaA binds to the origin of replication oriC and induces the opening of a AT-rich region. The replicative helicase DnaB is then loaded onto single stranded DNA by interacting with DnaA and with the AAA+ helicase loader DnaC. However, AAA+ loaders are absent in 80% of the bacterial genome, raising the question of how helicases are loaded in these bacteria [1]. In the genome of human pathogen Helicobacter pylori, no AAA+ loader has been identified. Moreover H. pylori DnaB (HpDnaB) has the ability to support replication of an otherwise unviable E. coli strain that bears a defective copy of DnaC by complementation [2]. In order to better understand the properties of HpDnaB we have first shown that HpDnaB forms double hexamers by negative stain electron microscopy [3]. Then, we have then solved the crystal structure of HpDnaB at a resolution of 6.7Å by X-ray crystallography with Rfree/Rfactor of 0.29/0.25. The structure reveals that the protein adopts a new dodecameric arrangement generated by crystallographic three fold symmetry. When compared to hexameric DnaBs, the hexamer of HpDnaB displays an original combination of NTD-ring and CTD-ring symmetries, intermediate between apo and ADP-bound structure. Biochemistry studies of HpDnaB interaction with HpDnaG-CTD and ssDNA provides mechanistic insights into the initial steps of DNA replication in H. pylori. Our results offer an alternative solution of helicase loading and DNA replication initiation in H. pylori and possibly other bacteria that do not employ helicase loaders.

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Xu, Yongbin, Arne Moeller, So-Young Jun, Minho Le, Bo-Young Yoon, Jin-Sik Kim, Kangseok Lee, and Nam-Chul Ha. "Assembly and Channel Opening of Outer Membrane Protein in Tripartite Drug Efflux Pumps of Gram-negative Bacteria." Journal of Biological Chemistry 287, no. 15 (February 3, 2012): 11740–50. http://dx.doi.org/10.1074/jbc.m111.329375.

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Gram-negative bacteria are capable of expelling diverse xenobiotic substances from within the cell by use of three-component efflux pumps in which the energy-activated inner membrane transporter is connected to the outer membrane channel protein via the membrane fusion protein. In this work, we describe the crystal structure of the membrane fusion protein MexA from the Pseudomonas aeruginosa MexAB-OprM pump in the hexameric ring arrangement. Electron microscopy study on the chimeric complex of MexA and the outer membrane protein OprM reveals that MexA makes a tip-to-tip interaction with OprM, which suggests a docking model for MexA and OprM. This docking model agrees well with genetic results and depicts detailed interactions. Opening of the OprM channel is accompanied by the simultaneous exposure of a protein structure resembling a six-bladed cogwheel, which intermeshes with the complementary cogwheel structure in the MexA hexamer. Taken together, we suggest an assembly and channel opening model for the MexAB-OprM pump. This study provides a better understanding of multidrug resistance in Gram-negative bacteria.

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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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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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Shorter, James. "Engineering therapeutic protein disaggregases." Molecular Biology of the Cell 27, no. 10 (May 15, 2016): 1556–60. http://dx.doi.org/10.1091/mbc.e15-10-0693.

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Therapeutic agents are urgently required to cure several common and fatal neurodegenerative disorders caused by protein misfolding and aggregation, including amyotrophic lateral sclerosis (ALS), Parkinson’s disease (PD), and Alzheimer’s disease (AD). Protein disaggregases that reverse protein misfolding and restore proteins to native structure, function, and localization could mitigate neurodegeneration by simultaneously reversing 1) any toxic gain of function of the misfolded form and 2) any loss of function due to misfolding. Potentiated variants of Hsp104, a hexameric AAA+ ATPase and protein disaggregase from yeast, have been engineered to robustly disaggregate misfolded proteins connected with ALS (e.g., TDP-43 and FUS) and PD (e.g., α-synuclein). However, Hsp104 has no metazoan homologue. Metazoa possess protein disaggregase systems distinct from Hsp104, including Hsp110, Hsp70, and Hsp40, as well as HtrA1, which might be harnessed to reverse deleterious protein misfolding. Nevertheless, vicissitudes of aging, environment, or genetics conspire to negate these disaggregase systems in neurodegenerative disease. Thus, engineering potentiated human protein disaggregases or isolating small-molecule enhancers of their activity could yield transformative therapeutics for ALS, PD, and AD.

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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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Lawson, Michael R., Kevin Dyer, and James M. Berger. "Ligand-induced and small-molecule control of substrate loading in a hexameric helicase." Proceedings of the National Academy of Sciences 113, no. 48 (November 7, 2016): 13714–19. http://dx.doi.org/10.1073/pnas.1616749113.

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Processive, ring-shaped protein and nucleic acid protein translocases control essential biochemical processes throughout biology and are considered high-prospect therapeutic targets. TheEscherichia coliRho factor is an exemplar hexameric RNA translocase that terminates transcription in bacteria. As with many ring-shaped motor proteins, Rho activity is modulated by a variety of poorly understood mechanisms, including small-molecule therapeutics, protein–protein interactions, and the sequence of its translocation substrate. Here, we establish the mechanism of action of two Rho effectors, the antibiotic bicyclomycin and nucleic acids that bind to Rho’s primary RNA recruitment site. Using small-angle X-ray scattering and a fluorescence-based assay to monitor the ability of Rho to switch between open-ring (RNA-loading) and closed-ring (RNA-translocation) states, we found bicyclomycin to be a direct antagonist of ring closure. Reciprocally, the binding of nucleic acids to its N-terminal RNA recruitment domains is shown to promote the formation of a closed-ring Rho state, with increasing primary-site occupancy providing additive stimulatory effects. This study establishes bicyclomycin as a conformational inhibitor of Rho ring dynamics, highlighting the utility of developing assays that read out protein conformation as a prospective screening tool for ring-ATPase inhibitors. Our findings further show that the RNA sequence specificity used for guiding Rho-dependent termination derives in part from an intrinsic ability of the motor to couple the recognition of pyrimidine patterns in nascent transcripts to RNA loading and activity.

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Pape, Tillmann, Hedije Meka, Shaoxia Chen, Giorgia Vicentini, Marin van Heel, and Silvia Onesti. "Hexameric ring structure of the full‐length archaeal MCM protein complex." EMBO reports 4, no. 11 (October 2003): 1079–83. http://dx.doi.org/10.1038/sj.embor.7400010.

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Pape, Tillmann, Hedije Meka, Shaoxia Chen, Giorgia Vicentini, Marin van Heel, and Silvia Onesti. "Hexameric ring structure of the full-length archaeal MCM protein complex." EMBO reports 4, no. 11 (October 2003): 1079–83. http://dx.doi.org/10.1038/sj.embor.embor7400010.

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Alcala-Torano, Rafael, Mathieu Walther, Dayn J. Sommer, Chad K. Park, and Giovanna Ghirlanda. "Rational design of a hexameric protein assembly stabilized by metal chelation." Biopolymers 109, no. 10 (September 6, 2018): e23233. http://dx.doi.org/10.1002/bip.23233.

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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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Price, Amanda, David Jacques, Sebastian Essig, Tom Elliott, Upul Halambage, Jason Chin, Christopher Aiken, and Leo James. "HIV-1 capsid binds host cofactors to mediate nuclear import." Acta Crystallographica Section A Foundations and Advances 70, a1 (August 5, 2014): C120. http://dx.doi.org/10.1107/s2053273314098799.

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The capsid (CA) protein of HIV-1, which forms the core of the virus, has been shown to have an increasingly important role in the early stages of the virus lifecycle, in particular during reverse transcription and nuclear import. We recently solved the structure of a fragment of the human cofactor CPSF6 in complex with the N-terminal domain of HIV-1 CA, revealing a previously unknown interface used by the virus to recruit CPSF6, which is required for the virus to successfully complete the early stages of its lifecycle. Using a recently developed hexameric unit of CA, we have solved the structure of the CPSF6 peptide with CA in a context that more closely resembles an intact CA lattice. This has revealed that CPSF6 contacts HIV-1 CA using an additional second site only present in the hexameric form of CA. Furthermore, we have now solved the structure of a fragment of NUP153 (an HIV-1 cofactor that is integral to the nuclear pore) in complex with hexameric CA and discovered that this also forms contacts specific to hexameric CA. Moreover, the binding sites for CPSF6 and NUP153 on CA overlap at one crucial residue, which is remarkably mimicked by two drugs independently discovered to bind at this same site. Together, these data provide evidence for an essential role for CA in HIV-1 infection, and highlights CA as an important target for antiretroviral drugs.

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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.

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Marchenkov, Victor, Natalia Lekontseva, Natalia Marchenko, Ivan Kashparov, Victoriia Murina, Alexey Nikulin, Vladimir Filimonov, and Gennady Semisotnov. "The Denaturant- and Mutation-Induced Disassembly of Pseudomonas aeruginosa Hexameric Hfq Y55W Mutant." Molecules 27, no. 12 (June 14, 2022): 3821. http://dx.doi.org/10.3390/molecules27123821.

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Although oligomeric proteins are predominant in cells, their folding is poorly studied at present. This work is focused on the denaturant- and mutation-induced disassembly of the hexameric mutant Y55W of the Qβ host factor (Hfq) from mesophilic Pseudomonas aeruginosa (Pae). Using intrinsic tryptophan fluorescence, dynamic light scattering (DLS), and high-performance liquid chromatography (HPLC), we show that the dissociation of Hfq Y55W occurs either under the effect of GuHCl or during the pre-denaturing transition, when the protein concentration is decreased, with both events proceeding through the accumulation of stable intermediate states. With an extremely low pH of 1.4, a low ionic strength, and decreasing protein concentration, the accumulated trimers and dimers turn into monomers. Also, we report on the structural features of monomeric Hfq resulting from a triple mutation (D9A/V43R/Y55W) within the inter-subunit surface of the protein. This globular and rigidly packed monomer displays a high thermostability and an oligomer-like content of the secondary structure, although its urea resistance is much lower.

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Nagata, Koji, Akitoshi Okada, Jun Ohtsuka, Takatoshi Ohkuri, Yusuke Akama, Yukari Sakiyama, Erika Miyazaki, et al. "Crystal structure of the complex of the interaction domains of Escherichia coli DnaB helicase and DnaC helicase loader: structural basis implying a distortion-accumulation mechanism for the DnaB ring opening caused by DnaC binding." Journal of Biochemistry 167, no. 1 (October 30, 2019): 1–14. http://dx.doi.org/10.1093/jb/mvz087.

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Abstract Loading the bacterial replicative helicase DnaB onto DNA requires a specific loader protein, DnaC/DnaI, which creates the loading-competent state by opening the DnaB hexameric ring. To understand the molecular mechanism by which DnaC/DnaI opens the DnaB ring, we solved 3.1-Å co-crystal structure of the interaction domains of Escherichia coli DnaB–DnaC. The structure reveals that one N-terminal domain (NTD) of DnaC interacts with both the linker helix of a DnaB molecule and the C-terminal domain (CTD) of the adjacent DnaB molecule by forming a three α-helix bundle, which fixes the relative orientation of the two adjacent DnaB CTDs. The importance of the intermolecular interface in the crystal structure was supported by the mutational data of DnaB and DnaC. Based on the crystal structure and other available information on DnaB–DnaC structures, we constructed a molecular model of the hexameric DnaB CTDs bound by six DnaC NTDs. This model suggested that the binding of a DnaC would cause a distortion in the hexameric ring of DnaB. This distortion of the DnaB ring might accumulate by the binding of up to six DnaC molecules, resulting in the DnaB ring to open.

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Journal articles on the topic 'Hexameric protein'

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