Journal articles: 'Substrate-binding proteins'

1

Berntsson, Ronnie P. A., Sander H. J. Smits, Lutz Schmitt, Dirk-Jan Slotboom, and Bert Poolman. "A structural classification of substrate-binding proteins." FEBS Letters 584, no. 12 (April 20, 2010): 2606–17. http://dx.doi.org/10.1016/j.febslet.2010.04.043.

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Scheepers, Giel H., Jelger A. Lycklama a Nijeholt, and Bert Poolman. "An updated structural classification of substrate-binding proteins." FEBS Letters 590, no. 23 (October 23, 2016): 4393–401. http://dx.doi.org/10.1002/1873-3468.12445.

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Manjeet, Kaur, Pallinti Purushotham, Chilukoti Neeraja, and Appa Rao Podile. "Bacterial chitin binding proteins show differential substrate binding and synergy with chitinases." Microbiological Research 168, no. 7 (August 2013): 461–68. http://dx.doi.org/10.1016/j.micres.2013.01.006.

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M. Counago, Rafael, Christopher A. McDevitt, Miranda P. Ween, and Bostjan Kobe. "Prokaryotic Substrate-Binding Proteins as Targets for Antimicrobial Therapies." Current Drug Targets 13, no. 11 (August 1, 2012): 1400–1410. http://dx.doi.org/10.2174/138945012803530170.

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Richarme, Gilbert, and Teresa Dantas Caldas. "Chaperone Properties of the Bacterial Periplasmic Substrate-binding Proteins." Journal of Biological Chemistry 272, no. 25 (June 20, 1997): 15607–12. http://dx.doi.org/10.1074/jbc.272.25.15607.

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Spooner, Paul J. R., W. John O’Reilly, Steven W. Homans, Nicholas G. Rutherford, Peter J. F. Henderson, and Anthony Watts. "Weak Substrate Binding to Transport Proteins Studied by NMR." Biophysical Journal 75, no. 6 (December 1998): 2794–800. http://dx.doi.org/10.1016/s0006-3495(98)77722-7.

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7

Pratt, R. F. "Substrate specificity of bacterial DD-peptidases (penicillin-binding proteins)." Cellular and Molecular Life Sciences 65, no. 14 (April 14, 2008): 2138–55. http://dx.doi.org/10.1007/s00018-008-7591-7.

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8

Altenberg, Guillermo A. "The Engine of ABC Proteins." Physiology 18, no. 5 (October 2003): 191–95. http://dx.doi.org/10.1152/nips.01445.2003.

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Proteins that belong to the ATP-binding cassette superfamily span from bacteria to humans and comprise one of the largest protein families. These proteins are characterized by the presence of two nucleotide-binding domains, and recent studies suggest that association and dissociation of these domains is a common basic molecular mechanism of operation that couples ATP binding/hydrolysis to substrate transport across membranes.

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Schimpl, Marianne, Alexander W. Schüttelkopf, Vladimir S. Borodkin, and Daan M. F. van Aalten. "Human OGA binds substrates in a conserved peptide recognition groove." Biochemical Journal 432, no. 1 (October 25, 2010): 1–12. http://dx.doi.org/10.1042/bj20101338.

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Modification of cellular proteins with O-GlcNAc (O-linked N-acetylglucosamine) competes with protein phosphorylation and regulates a plethora of cellular processes. O-GlcNAcylation is orchestrated by two opposing enzymes, O-GlcNAc transferase and OGA (O-GlcNAcase or β-N-acetylglucosaminidase), which recognize their target proteins via as yet unidentified mechanisms. In the present study, we uncovered the first insights into the mechanism of substrate recognition by human OGA. The structure of a novel bacterial OGA orthologue reveals a putative substrate-binding groove, conserved in metazoan OGAs. Guided by this structure, conserved amino acids lining this groove in human OGA were mutated and the activity on three different substrate proteins [TAB1 (transforming growth factor-β-activated protein kinase 1-binding protein 1), FoxO1 (forkhead box O1) and CREB (cAMP-response-element-binding protein)] was tested in an in vitro deglycosylation assay. The results provide the first evidence that human OGA may possess a substrate-recognition mechanism that involves interactions with O-GlcNAcylated proteins beyond the GlcNAc-binding site, with possible implications for differential regulation of cycling of O-GlcNAc on different proteins.

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Moutsita, R., J. Botti, MA Doyennette-Moyne, M. Aubery, and P. Codogno. "Cell spreading on laminin substrate involves Con A-binding proteins." Reproduction Nutrition Développement 30, no. 3 (1990): 397–401. http://dx.doi.org/10.1051/rnd:19900312.

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11

Vuković, Lela, Hye Ran Koh, Sua Myong, and Klaus Schulten. "Substrate Recognition and Specificity of Double-Stranded RNA Binding Proteins." Biochemistry 53, no. 21 (May 21, 2014): 3457–66. http://dx.doi.org/10.1021/bi500352s.

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12

Farr, George W., Krystyna Furtak, Matthew B. Rowland, Neil A. Ranson, Helen R. Saibil, Tomas Kirchhausen, and Arthur L. Horwich. "Multivalent Binding of Nonnative Substrate Proteins by the Chaperonin GroEL." Cell 100, no. 5 (March 2000): 561–73. http://dx.doi.org/10.1016/s0092-8674(00)80692-3.

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13

Fischer, Marcus, Qian Yi Zhang, Roderick E. Hubbard, and Gavin H. Thomas. "Caught in a TRAP: substrate-binding proteins in secondary transport." Trends in Microbiology 18, no. 10 (October 2010): 471–78. http://dx.doi.org/10.1016/j.tim.2010.06.009.

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14

Vukovic, Lela, Hye Ran Koh, Sua Myong, and Klaus Schulten. "Substrate Recognition and Specificity of Double-Stranded RNA Binding Proteins." Biophysical Journal 106, no. 2 (January 2014): 22a. http://dx.doi.org/10.1016/j.bpj.2013.11.175.

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15

Khanra, Nandish, Paolo Rossi, Anastassios Economou, and Charalampos G. Kalodimos. "Recognition and targeting mechanisms by chaperones in flagellum assembly and operation." Proceedings of the National Academy of Sciences 113, no. 35 (August 15, 2016): 9798–803. http://dx.doi.org/10.1073/pnas.1607845113.

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The flagellum is a complex bacterial nanomachine that requires the proper assembly of several different proteins for its function. Dedicated chaperones are central in preventing aggregation or undesired interactions of flagellar proteins, including their targeting to the export gate. FliT is a key flagellar chaperone that binds to several flagellar proteins in the cytoplasm, including its cognate filament-capping protein FliD. We have determined the solution structure of the FliT chaperone in the free state and in complex with FliD and the flagellar ATPase FliI. FliT adopts a four-helix bundle and uses a hydrophobic surface formed by the first three helices to recognize its substrate proteins. We show that the fourth helix constitutes the binding site for FlhA, a membrane protein at the export gate. In the absence of a substrate protein FliT adopts an autoinhibited structure wherein both the binding sites for substrates and FlhA are occluded. Substrate binding to FliT activates the complex for FlhA binding and thus targeting of the chaperone–substrate complex to the export gate. The activation and targeting mechanisms reported for FliT appear to be shared among the other flagellar chaperones.

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16

Rosenzweig, Rina, Patrick Farber, Algirdas Velyvis, Enrico Rennella, Michael P. Latham, and Lewis E. Kay. "ClpB N-terminal domain plays a regulatory role in protein disaggregation." Proceedings of the National Academy of Sciences 112, no. 50 (November 30, 2015): E6872—E6881. http://dx.doi.org/10.1073/pnas.1512783112.

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ClpB/Hsp100 is an ATP-dependent disaggregase that solubilizes and reactivates protein aggregates in cooperation with the DnaK/Hsp70 chaperone system. The ClpB–substrate interaction is mediated by conserved tyrosine residues located in flexible loops in nucleotide-binding domain-1 that extend into the ClpB central pore. In addition to the tyrosines, the ClpB N-terminal domain (NTD) was suggested to provide a second substrate-binding site; however, the manner in which the NTD recognizes and binds substrate proteins has remained elusive. Herein, we present an NMR spectroscopy study to structurally characterize the NTD–substrate interaction. We show that the NTD includes a substrate-binding groove that specifically recognizes exposed hydrophobic stretches in unfolded or aggregated client proteins. Using an optimized segmental labeling technique in combination with methyl-transverse relaxation optimized spectroscopy (TROSY) NMR, the interaction of client proteins with both the NTD and the pore-loop tyrosines in the 580-kDa ClpB hexamer has been characterized. Unlike contacts with the tyrosines, the NTD–substrate interaction is independent of the ClpB nucleotide state and protein conformational changes that result from ATP hydrolysis. The NTD interaction destabilizes client proteins, priming them for subsequent unfolding and translocation. Mutations in the NTD substrate-binding groove are shown to have a dramatic effect on protein translocation through the ClpB central pore, suggesting that, before their interaction with substrates, the NTDs block the translocation channel. Together, our findings provide both a detailed characterization of the NTD–substrate complex and insight into the functional regulatory role of the ClpB NTD in protein disaggregation.

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17

RANSON, Neil A., Helen E. WHITE, and Helen R. SAIBIL. "Chaperonins." Biochemical Journal 333, no. 2 (July 15, 1998): 233–42. http://dx.doi.org/10.1042/bj3330233.

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The molecular chaperones are a diverse set of protein families required for the correct folding, transport and degradation of other proteins in vivo. There has been great progress in understanding the structure and mechanism of action of the chaperonin family, exemplified by Escherichia coli GroEL. The chaperonins are large, double-ring oligomeric proteins that act as containers for the folding of other protein subunits. Together with its co-protein GroES, GroEL binds non-native polypeptides and facilitates their refolding in an ATP-dependent manner. The action of the ATPase cycle causes the substrate-binding surface of GroEL to alternate in character between hydrophobic (binding/unfolding) and hydrophilic (release/folding). ATP binding initiates a series of dramatic conformational changes that bury the substrate-binding sites, lowering the affinity for non-native polypeptide. In the presence of ATP, GroES binds to GroEL, forming a large chamber that encapsulates substrate proteins for folding. For proteins whose folding is absolutely dependent on the full GroE system, ATP binding (but not hydrolysis) in the encapsulating ring is needed to initiate protein folding. Similarly, ATP binding, but not hydrolysis, in the opposite GroEL ring is needed to release GroES, thus opening the chamber. If the released substrate protein is still not correctly folded, it will go through another round of interaction with GroEL.

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18

Gebauer, Mathias, Matthias Zeiner, and Ulrich Gehring. "Interference between Proteins Hap46 and Hop/p60, Which Bind to Different Domains of the Molecular Chaperone hsp70/hsc70." Molecular and Cellular Biology 18, no. 11 (November 1, 1998): 6238–44. http://dx.doi.org/10.1128/mcb.18.11.6238.

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ABSTRACT Several structurally divergent proteins associate with molecular chaperones of the 70-kDa heat shock protein (hsp70) family and modulate their activities. We investigated the cofactors Hap46 and Hop/p60 and the effects of their binding to mammalian hsp70 and the cognate form hsc70. Hap46 associates with the amino-terminal ATP binding domain and stimulates ATP binding two- to threefold but inhibits binding of misfolded protein substrate to hsc70 and reactivation of thermally denatured luciferase in an hsc70-dependent refolding system. By contrast, Hop/p60 interacts with a portion of the carboxy-terminal domain of hsp70s, which is distinct from that involved in the binding of misfolded proteins. Thus, Hop/p60 and substrate proteins can form ternary complexes with hsc70. Hop/p60 exerts no effect on ATP and substrate binding but nevertheless interferes with protein refolding. Even though there is no direct interaction between these accessory proteins, Hap46 inhibits the binding of Hop/p60 to hsc70 but Hop/p60 does not inhibit the binding of Hap46 to hsc70. As judged from respective deletions, the amino-terminal portions of Hap46 and Hop/p60 are involved in this interference. These data suggest steric hindrance between Hap46 and Hop/p60 during interaction with distantly located binding sites on hsp70s. Thus, not only do the major domains of hsp70 chaperones communicate with each other, but cofactors interacting with these domains affect each other as well.

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19

Ibarra, Rebeca, Heather R. Borror, Bryce Hart, Richard G. Gardner, and Gary Kleiger. "The San1 Ubiquitin Ligase Avidly Recognizes Misfolded Proteins through Multiple Substrate Binding Sites." Biomolecules 11, no. 11 (November 2, 2021): 1619. http://dx.doi.org/10.3390/biom11111619.

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Cellular homeostasis depends on robust protein quality control (PQC) pathways that discern misfolded proteins from functional ones in the cell. One major branch of PQC involves the controlled degradation of misfolded proteins by the ubiquitin-proteasome system. Here ubiquitin ligases must recognize and bind to misfolded proteins with sufficient energy to form a complex and with an adequate half-life to achieve poly-ubiquitin chain formation, the signal for protein degradation, prior to its dissociation from the ligase. It is not well understood how PQC ubiquitin ligases accomplish these tasks. Employing a fully reconstituted enzyme and substrate system to perform quantitative biochemical experiments, we demonstrate that the yeast PQC ubiquitin ligase San1 contains multiple substrate binding sites along its polypeptide chain that appear to display specificity for unique misfolded proteins. The results are consistent with a model where these substrate binding sites enable San1 to bind to misfolded substrates avidly, resulting in high affinity ubiquitin ligase-substrate complexes.

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20

Wang, Xing-Guo, J. Michael Kidder, Joanna P. Scagliotti, Mark S. Klempner, Richard Noring, and Linden T. Hu. "Analysis of Differences in the Functional Properties of the Substrate Binding Proteins of the Borrelia burgdorferi Oligopeptide Permease (opp) Operon." Journal of Bacteriology 186, no. 1 (January 1, 2004): 51–60. http://dx.doi.org/10.1128/jb.186.1.51-60.2004.

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ABSTRACT The Borrelia burgdorferi genome encodes five orthologues of the substrate binding protein oligopeptide permease A (OppA). It was previously shown that these genes are under the control of separate promoters and are differentially expressed under various environmental conditions. We were interested in determining whether there are also differences in substrate specificities among the proteins. The substrate specificities of recombinant proteins were determined by screening for high-affinity peptides by use of a combinatorial phage display heptapeptide library. Different heptapeptides with high affinities for OppA-1, OppA-2, and OppA-3 were identified. No heptapeptide binding OppA-4 or OppA-5 could be identified. Competitive binding assays were performed under various conditions to determine the substrate preferences of the OppA proteins. OppA-1 retained maximal activity over a broad range of pHs (5.5 to 7.5), whereas OppA-2 and OppA-3 showed peak activities at pHs below 5.5. OppA-1 and OppA-2 showed preferences for tripeptides over dipeptides and longer-chain peptides. Although a wide variety of amino acyl side chains were tolerated by all three OppA proteins, OppA-1 showed the broadest substrate specificity and was able to accommodate peptides composed of bulky hydrophobic residues; OppA-2 and OppA-3 showed preferences for peptides composed of small nonpolar amino acids. All three OppA proteins showed preferences for peptides composed of l- rather than d-amino acids. OppA-3 showed the greatest tolerance for changes in stereochemistry. Substantial differences in the substrate specificities of the OppA proteins of B. burgdorferi suggest that they may have distinct functions in the organism.

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Khrenova, M. G., I. V. Polyakov, and A. V. Nemukhin. "Molecular Dynamics of Enzyme-Substrate Complexes in Guanosine Trifosphate-Binding Proteins." Russian Journal of Physical Chemistry B 16, no. 3 (June 2022): 455–60. http://dx.doi.org/10.1134/s1990793122030174.

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22

Durrani, Mehvish K., and Jonghoon Kang. "Thermodynamic analysis of the binding of p38 MAPK to substrate proteins." Journal of Biological Chemistry 295, no. 5 (January 2020): 1366. http://dx.doi.org/10.1016/s0021-9258(17)49892-9.

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23

Daleke, David. "Substrate Specificity of the Aminophospholipid Flippase and Other Phosphatidylserine Binding Proteins." Biophysical Journal 98, no. 3 (January 2010): 507a. http://dx.doi.org/10.1016/j.bpj.2009.12.2758.

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Durrani, Mehvish K., and Jonghoon Kang. "Thermodynamic analysis of the binding of p38 MAPK to substrate proteins." Journal of Biological Chemistry 295, no. 5 (January 31, 2020): 1366. http://dx.doi.org/10.1074/jbc.l119.011911.

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25

Kishimoto, Ayaka, Kenji Takagi, Tsunehiro Mizushima, Keisuke Sakurai, Katsuyoshi Harada, Takashi Hayashi, and Hideo Shimada. "1P095 Substrate access to slow substrate binding P450cam with mutation at the proposed gate for water egress/ingress from/to the active site(02. Heme proteins,Poster)." Seibutsu Butsuri 53, supplement1-2 (2013): S121. http://dx.doi.org/10.2142/biophys.53.s121_5.

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26

Houry, Walid A. "Mechanism of substrate recognition by the chaperonin GroEL." Biochemistry and Cell Biology 79, no. 5 (October 1, 2001): 569–77. http://dx.doi.org/10.1139/o01-131.

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The bacterial chaperonin GroEL functions with its cofactor GroES in assisting the folding of a wide range of proteins in an ATP-dependent manner. GroELGroES constitute one of the main chaperone systems in the Escherichia coli cytoplasm. The chaperonin facilitates protein folding by enclosing substrate proteins in a cage defined by the GroEL cylinder and the GroES cap where folding can take place in a protected environment. The in vivo role of GroEL has recently been elucidated. GroEL is found to interact with 1015% of newly synthesized proteins, with a strong preference for proteins in the molecular weight range of 2060 kDa. A large number of GroEL substrates have been identified and were found to preferentially contain proteins with multiple αβ domains that have α-helices and β-sheets with extensive hydrophobic surfaces. Based on the preferential binding of GroEL to these proteins and structural and biochemical data, a model of substrate recognition by GroEL is proposed. According to this model, binding takes place preferentially between the hydrophobic residues in the apical domains of GroEL and the hydrophobic faces exposed by the β-sheets or α-helices in the αβ domains of protein substrates.Key words: chaperone, folding, binding, hydrophobic interaction, structure.

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27

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.

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Liebl, Martina P., and Thorsten Hoppe. "It's all about talking: two-way communication between proteasomal and lysosomal degradation pathways via ubiquitin." American Journal of Physiology-Cell Physiology 311, no. 2 (August 1, 2016): C166—C178. http://dx.doi.org/10.1152/ajpcell.00074.2016.

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Selective degradation of proteins requires a fine-tuned coordination of the two major proteolytic pathways, the ubiquitin-proteasome system (UPS) and autophagy. Substrate selection and proteolytic activity are defined by a plethora of regulatory cofactors influencing each other. Both proteolytic pathways are initiated by ubiquitylation to mark substrate proteins for degradation, although the size and/or topology of the modification are different. In this context E3 ubiquitin ligases, ensuring the covalent attachment of activated ubiquitin to the substrate, are of special importance. The regulation of E3 ligase activity, competition between different E3 ligases for binding E2 conjugation enzymes and substrates, as well as their interplay with deubiquitylating enzymes (DUBs) represent key events in the cross talk between the UPS and autophagy. The coordination between both degradation routes is further influenced by heat shock factors and ubiquitin-binding proteins (UBPs) such as p97, p62, or optineurin. Mutations in enzymes and ubiquitin-binding proteins or a general decline of both proteolytic systems during aging result in accumulation of damaged and aggregated proteins. Thus further mechanistic understanding of how UPS and autophagy communicate might allow therapeutic intervention especially against age-related diseases.

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Roston, Rebecca L., Jinpeng Gao, Monika W. Murcha, James Whelan, and Christoph Benning. "TGD1, -2, and -3 Proteins Involved in Lipid Trafficking Form ATP-binding Cassette (ABC) Transporter with Multiple Substrate-binding Proteins." Journal of Biological Chemistry 287, no. 25 (April 27, 2012): 21406–15. http://dx.doi.org/10.1074/jbc.m112.370213.

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Fritz, Jutta, Alexander Strehblow, Andreas Taschner, Sandy Schopoff, Pawel Pasierbek, and Michael F. Jantsch. "RNA-Regulated Interaction of Transportin-1 and Exportin-5 with the Double-Stranded RNA-Binding Domain Regulates Nucleocytoplasmic Shuttling of ADAR1." Molecular and Cellular Biology 29, no. 6 (January 5, 2009): 1487–97. http://dx.doi.org/10.1128/mcb.01519-08.

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ABSTRACT Double-stranded RNA (dsRNA)-binding proteins interact with substrate RNAs via dsRNA-binding domains (dsRBDs). Several proteins harboring these domains exhibit nucleocytoplasmic shuttling and possibly remain associated with their substrate RNAs bound in the nucleus during nuclear export. In the human RNA-editing enzyme ADAR1-c, the nuclear localization signal overlaps the third dsRBD, while the corresponding import factor is unknown. The protein also lacks a clear nuclear export signal but shuttles between the nucleus and the cytoplasm. Here we identify transportin-1 as the import receptor for ADAR1. Interestingly, dsRNA binding interferes with transportin-1 binding. At the same time, each of the dsRBDs in ADAR1 interacts with the export factor exportin-5. RNA binding stimulates this interaction but is not a prerequisite. Thus, our data demonstrate a role for some dsRBDs as RNA-sensitive nucleocytoplasmic transport signals. dsRBD3 in ADAR1 can mediate nuclear import, while interaction of all dsRBDs might control nuclear export. This finding may have implications for other proteins containing dsRBDs and suggests a selective nuclear export mechanism for substrates interacting with these proteins.

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Raj, Nixon, Timothy H. Click, Haw Yang, and Jhih-Wei Chu. "Structure-mechanics statistical learning uncovers mechanical relay in proteins." Chemical Science 13, no. 13 (2022): 3688–96. http://dx.doi.org/10.1039/d1sc06184d.

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Protein residues exhibit specific routes of mechanical relay as the adaptive responses to substrate binding or dissociation. On such physically contiguous connections, residues experience prominent changes in their coupling strengths.

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Sen, Liu, and Xiao Hong Ma. "Binding as a Rate-Limiting Step for Substrate Recognition of ADAM17." Advanced Materials Research 717 (July 2013): 244–48. http://dx.doi.org/10.4028/www.scientific.net/amr.717.244.

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ADAM17 is an important membrane-bound proteinase, and it can release a lot of proteins from their membrane-bound forms, such as cytokines, cytokine receptors and adhesion proteins. ADAM17 has long been an interesting therapeutic target in a lot of diseases; however, the development of its inhibitors has been hurdled by our very limited knowledge on its substrate specificity and selectivity. To understand the substrate specificity of ADAM17, here in this paper, a rational complex model is computationally built for the catalytic domain of ADAM17 and its recognizing sequence from the TNF-alpha precursor (proTNF-alpha). With protein-peptide docking analysis, we found that the substrate binding step is indeed important for ADAM17 recognition and processing. The result in this paper could be useful for the understanding of the substrate specificity and selectivity, and the design of novel ADAM17 inhibitors in the future.

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Sekhar, Ashok, Rina Rosenzweig, Guillaume Bouvignies, and Lewis E. Kay. "Hsp70 biases the folding pathways of client proteins." Proceedings of the National Academy of Sciences 113, no. 20 (May 2, 2016): E2794—E2801. http://dx.doi.org/10.1073/pnas.1601846113.

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The 70-kDa heat shock protein (Hsp70) family of chaperones bind cognate substrates to perform a variety of different processes that are integral to cellular homeostasis. Although detailed structural information is available on the chaperone, the structural features of folding competent substrates in the bound form have not been well characterized. Here we use paramagnetic relaxation enhancement (PRE) NMR spectroscopy to probe the existence of long-range interactions in one such folding competent substrate, human telomere repeat binding factor (hTRF1), which is bound to DnaK in a globally unfolded conformation. We show that DnaK binding modifies the energy landscape of the substrate by removing long-range interactions that are otherwise present in the unbound, unfolded conformation of hTRF1. Because the unfolded state of hTRF1 is only marginally populated and transiently formed, it is inaccessible to standard NMR approaches. We therefore developed a 1H-based CEST experiment that allows measurement of PREs in sparse states, reporting on transiently sampled conformations. Our results suggest that DnaK binding can significantly bias the folding pathway of client substrates such that secondary structure forms first, followed by the development of longer-range contacts between more distal parts of the protein.

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IGARASHI, Kazuei, and Keiko KASHIWAGI. "Polyamine transport in bacteria and yeast." Biochemical Journal 344, no. 3 (December 8, 1999): 633–42. http://dx.doi.org/10.1042/bj3440633.

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The polyamine content of cells is regulated by biosynthesis, degradation and transport. In Escherichia coli, the genes for three different polyamine transport systems have been cloned and characterized. Two uptake systems (putrescine-specific and spermidine-preferential) were ABC transporters, each consisting of a periplasmic substrate-binding protein, two transmembrane proteins and a membrane-associated ATPase. The crystal structures of the substrate-binding proteins (PotD and PotF) have been solved. They consist of two domains with an alternating β-α-β topology, similar to other periplasmic binding proteins. The polyamine-binding site is in a cleft between the two domains, as determined by crystallography and site-directed mutagenesis. Polyamines are mainly recognized by aspartic acid and glutamic acid residues, which interact with the NH2- (or NH-) groups, and by tryptophan and tyrosine residues that have hydrophobic interactions with the methylene groups of polyamines. The precursor of one of the substrate binding proteins, PotD, negatively regulates transcription of the operon for the spermidine-preferential uptake system, thus providing another level of regulation of cellular polyamines. The third transport system, catalysed by PotE, mediates both uptake and excretion of putrescine. Uptake of putrescine is dependent on membrane potential, whereas excretion involves an exchange reaction between putrescine and ornithine. In Saccharomyces cerevisiae, the gene for a polyamine transport protein (TPO1) was identified. The properties of this protein are similar to those of PotE, and TPO1 is located on the vacuolar membrane.

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SAYED, Yasien, Judith A. T. HORNBY, Marimar LOPEZ, and Heini DIRR. "Thermodynamics of the ligandin function of human class Alpha glutathione transferase A1-1: energetics of organic anion ligand binding." Biochemical Journal 363, no. 2 (April 8, 2002): 341–46. http://dx.doi.org/10.1042/bj3630341.

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In addition to their catalytic functions, cytosolic glutathioneS-transferases (GSTs) are a major reserve of high-capacity binding proteins for a large variety of physiological and exogenous non-substrate compounds. This ligandin function has implicated GSTs in numerous ligand-uptake, -transport and -storage processes. The binding of non-substrate ligands to GSTs can inhibit catalysis. In the present study, the energetics of the binding of the non-substrate ligand 8-anilino-1-naphthalene sulphonate (ANS) to wild-type human class Alpha GST with two type-1 subunits (hGSTA1-1) and its ΔPhe-222 deletion mutant were studied by isothermal titration calorimetry. The stoichiometry of binding to both proteins is one ANS molecule per GST subunit with a greater affinity for the wild-type (Kd=65μM) than for the ΔPhe-222 mutant (Kd=105μM). ANS binding to the wild-type protein is enthalpically driven and it is characterized by a large negative heat-capacity change, ΔCp. The negative ΔCp value for ANS binding indicates a specific interface with a significant hydrophobic component in the protein—ligand complex. The negatively charged sulphonate group of the anionic ligand is apparently not a major determinant of its binding. Phe-222 contributes to the binding affinity for ANS and the hydrophobicity of the binding site.

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Yu, Qian, Hong Chen, Yanxia Zhang, Lin Yuan, Tieliang Zhao, Xin Li, and Hongwei Wang. "pH-Reversible, High-Capacity Binding of Proteins on a Substrate with Nanostructure." Langmuir 26, no. 23 (December 7, 2010): 17812–15. http://dx.doi.org/10.1021/la103647s.

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Shukla, Shantanu, Khushboo Bafna, Caeley Gullett, Dean A. A. Myles, Pratul K. Agarwal, and Matthew J. Cuneo. "Differential Substrate Recognition by Maltose Binding Proteins Influenced by Structure and Dynamics." Biochemistry 57, no. 40 (September 11, 2018): 5864–76. http://dx.doi.org/10.1021/acs.biochem.8b00783.

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Lebreton, B., P. V. Prasad, M. Jayaram, and P. Youderian. "Mutations that improve the binding of yeast FLP recombinase to its substrate." Genetics 118, no. 3 (March 1, 1988): 393–400. http://dx.doi.org/10.1093/genetics/118.3.393.

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Abstract When yeast FLP recombinase is expressed from the phage lambda PR promoter in a Salmonella host, it cannot efficiently repress an operon controlled by an operator/promoter region that includes a synthetic, target FLP site. On the basis of this phenotype, we have identified four mutant FLP proteins that function as more efficient repressors of such an operon. At least two of these mutant FLP proteins bind better to the FLP site in vivo and in vitro. One mutant changes the presumed active site tyrosine residue of FLP protein to phenylalanine, is blocked in recombination, and binds the FLP site about five-fold better than the wild-type protein. A second mutant protein that functions as a more efficient repressor retains catalytic activity. We conclude that the eukaryotic yeast FLP recombinase, when expressed in a heterologous prokaryotic host, can function as a repressor, and that mutant FLP proteins that bind DNA more tightly may be selected as more efficient repressors.

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Jaya, Nomalie, Victor Garcia, and Elizabeth Vierling. "Substrate binding site flexibility of the small heat shock protein molecular chaperones." Proceedings of the National Academy of Sciences 106, no. 37 (August 26, 2009): 15604–9. http://dx.doi.org/10.1073/pnas.0902177106.

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Small heat shock proteins (sHSPs) serve as a first line of defense against stress-induced cell damage by binding and maintaining denaturing proteins in a folding-competent state. In contrast to the well-defined substrate binding regions of ATP-dependent chaperones, interactions between sHSPs and substrates are poorly understood. Defining substrate-binding sites of sHSPs is key to understanding their cellular functions and to harnessing their aggregation-prevention properties for controlling damage due to stress and disease. We incorporated a photoactivatable cross-linker at 32 positions throughout a well-characterized sHSP, dodecameric PsHsp18.1 from pea, and identified direct interaction sites between sHSPs and substrates. Model substrates firefly luciferase and malate dehydrogenase form strong contacts with multiple residues in the sHSP N-terminal arm, demonstrating the importance of this flexible and evolutionary variable region in substrate binding. Within the conserved α-crystallin domain both substrates also bind the β-strand (β7) where mutations in human homologs result in inherited disease. Notably, these binding sites are poorly accessible in the sHSP atomic structure, consistent with major structural rearrangements being required for substrate binding. Detectable differences in the pattern of cross-linking intensity of the two substrates and the fact that substrates make contacts throughout the sHSP indicate that there is not a discrete substrate binding surface. Our results support a model in which the intrinsically-disordered N-terminal arm can present diverse geometries of interaction sites, which is likely critical for the ability of sHSPs to protect efficiently many different substrates.

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Graef, Martin, Georgeta Seewald, and Thomas Langer. "Substrate Recognition by AAA+ ATPases: Distinct Substrate Binding Modes in ATP-Dependent Protease Yme1 of the Mitochondrial Intermembrane Space." Molecular and Cellular Biology 27, no. 7 (January 29, 2007): 2476–85. http://dx.doi.org/10.1128/mcb.01721-06.

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ABSTRACT The energy-dependent proteolysis of cellular proteins is mediated by conserved proteolytic AAA+ complexes. Two such machines, the m- and i-AAA proteases, are present in the mitochondrial inner membrane. They exert chaperone-like properties and specifically degrade nonnative membrane proteins. However, molecular mechanisms of substrate engagement by AAA proteases remained elusive. Here, we define initial steps of substrate recognition and identify two distinct substrate binding sites in the i-AAA protease subunit Yme1. Misfolded polypeptides are recognized by conserved helices in proteolytic and AAA domains. Structural modeling reveals a lattice-like arrangement of these helices at the surface of hexameric AAA protease ring complexes. While helices within the AAA domain apparently play a general role for substrate binding, the requirement for binding to surface-exposed helices within the proteolytic domain is determined by the folding and membrane association of substrates. Moreover, an assembly factor of cytochrome c oxidase, Cox20, serves as a substrate-specific cofactor during proteolysis and modulates the initial interaction of nonassembled Cox2 with the protease. Our findings therefore reveal the existence of alternative substrate recognition pathways within AAA proteases and shed new light on molecular mechanisms ensuring the specificity of proteolysis by energy-dependent proteases.

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Swain, Joanna F., Renuka Sivendran, and Lila M. Gierasch. "Defining the structure of the substrate-free state of the DnaK molecular chaperone." Biochemical Society Symposia 68 (August 1, 2001): 69–82. http://dx.doi.org/10.1042/bss0680069.

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Members of the Hsp70 (heat-shock protein of 70 kDa) family of molecular chaperones bind to exposed hydrophobic stretches on substrate proteins in order to dissociate molecular complexes and prevent aggregation in the cell. Substrate affinity for the C-terminal domain of the Hsp70 is regulated by ATP binding to the N-terminal domain utilizing an allosteric mechanism. Our multi-dimensional NMR studies of a substrate-binding domain fragment (amino acids 387-552) from an Escherichia coli Hsp70, DnaK(387-552), have uncovered a pH-dependent conformational change, which we propose to be relevant for the full-length protein also. At pH 7, the C-terminus of DnaK(387-552) mimics substrate by binding to its own substrate-binding site, as has been observed previously for truncated Hsp70 constructs. At pH 5, the C-terminus is released from the binding site, such that DnaK is in the substrate-free state 10-20% of the time. We propose that the mechanism for the release of the tail is a loss of affinity for substrate at low pH. The pH-dependent fluorescence changes at a tryptophan residue near the substrate-binding pocket in full-length DnaK lead us to extend these conclusions to the full-length DnaK as well. In the context of the DnaK substrate-binding domain fragment, the release of the C-terminus from the substrate-binding site provides our first glimpse of the empty conformation of an Hsp70 substrate-binding domain containing a portion of the helical subdomain.

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Miyata, Hidefumi, Kazuhiro Yumoto, Kanako Itoh, Miki Sasahara, Hiroki Kawaura, Nobuyuki Oshima, Taiho Shuzuki, Shunsuke Takahashi, Masahiko Oshige, and Shinji Katsura. "Immobilization of His-Tagged Proteins through Interaction with L-Cysteine Electrodeposited on Modified Gold Surfaces." Key Engineering Materials 596 (December 2013): 219–23. http://dx.doi.org/10.4028/www.scientific.net/kem.596.219.

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A method for preparing a high-density His-tagged protein array was developed. The method is based on specific binding between His-tags and Ni ions chelated with the carboxyl groups of L-cysteine applied to the substrate surface by electrodeposition. About 13 ng/mm2 of His-tagged green fluorescent protein (His-GFP) could be immobilized on the substrate. The immobilized His-GFP could be subsequently released by washing with imidazole, suggesting that immobilization involves specific binding between the His-tag and the Ni ion complex.

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Arana, Maite Rocío, and Guillermo Alejandro Altenberg. "ATP-binding Cassette Exporters: Structure and Mechanism with a Focus on P-glycoprotein and MRP1." Current Medicinal Chemistry 26, no. 7 (May 14, 2019): 1062–78. http://dx.doi.org/10.2174/0929867324666171012105143.

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Background:Proteins that belong to the ATP-binding cassette superfamily include transporters that mediate the efflux of substrates from cells. Among these exporters, P-glycoprotein and MRP1 are involved in cancer multidrug resistance, protection from endo and xenobiotics, determination of drug pharmacokinetics, and the pathophysiology of a variety of disorders. Objective:To review the information available on ATP-binding cassette exporters, with a focus on Pglycoprotein, MRP1 and related proteins. We describe tissue localization and function of these transporters in health and disease, and discuss the mechanisms of substrate transport. We also correlate recent structural information with the function of the exporters, and discuss details of their molecular mechanism with a focus on the nucleotide-binding domains. Methods: Evaluation of selected publications on the structure and function of ATP-binding cassette proteins. Conclusions:Conformational changes on the nucleotide-binding domains side of the exporters switch the accessibility of the substrate-binding pocket between the inside and outside, which is coupled to substrate efflux. However, there is no agreement on the magnitude and nature of the changes at the nucleotide- binding domains side that drive the alternate-accessibility. Comparison of the structures of Pglycoprotein and MRP1 helps explain differences in substrate selectivity and the bases for polyspecificity. P-glycoprotein substrates are hydrophobic and/or weak bases, and polyspecificity is explained by a flexible hydrophobic multi-binding site that has a few acidic patches. MRP1 substrates are mostly organic acids, and its polyspecificity is due to a single bipartite binding site that is flexible and displays positive charge.

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Putman, Monique, Hendrik W. van Veen, and Wil N. Konings. "Molecular Properties of Bacterial Multidrug Transporters." Microbiology and Molecular Biology Reviews 64, no. 4 (December 1, 2000): 672–93. http://dx.doi.org/10.1128/mmbr.64.4.672-693.2000.

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SUMMARY One of the mechanisms that bacteria utilize to evade the toxic effects of antibiotics is the active extrusion of structurally unrelated drugs from the cell. Both intrinsic and acquired multidrug transporters play an important role in antibiotic resistance of several pathogens, including Neisseria gonorrhoeae, Mycobacterium tuberculosis, Staphylococcus aureus, Streptococcus pneumoniae, Pseudomonas aeruginosa, and Vibrio cholerae. Detailed knowledge of the molecular basis of drug recognition and transport by multidrug transport systems is required for the development of new antibiotics that are not extruded or of inhibitors which block the multidrug transporter and allow traditional antibiotics to be effective. This review gives an extensive overview of the currently known multidrug transporters in bacteria. Based on energetics and structural characteristics, the bacterial multidrug transporters can be classified into five distinct families. Functional reconstitution in liposomes of purified multidrug transport proteins from four families revealed that these proteins are capable of mediating the export of structurally unrelated drugs independent of accessory proteins or cytoplasmic components. On the basis of (i) mutations that affect the activity or the substrate specificity of multidrug transporters and (ii) the three-dimensional structure of the drug-binding domain of the regulatory protein BmrR, the substrate-binding site for cationic drugs is predicted to consist of a hydrophobic pocket with a buried negatively charged residue that interacts electrostatically with the positively charged substrate. The aromatic and hydrophobic amino acid residues which form the drug-binding pocket impose restrictions on the shape and size of the substrates. Kinetic analysis of drug transport by multidrug transporters provided evidence that these proteins may contain multiple substrate-binding sites.

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Maekawa, Masashi, and Shigeki Higashiyama. "The Roles of SPOP in DNA Damage Response and DNA Replication." International Journal of Molecular Sciences 21, no. 19 (October 2, 2020): 7293. http://dx.doi.org/10.3390/ijms21197293.

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Speckle-type BTB/POZ protein (SPOP) is a substrate recognition receptor of the cullin-3 (CUL3)/RING type ubiquitin E3 complex. To date, approximately 30 proteins have been identified as ubiquitinated substrates of the CUL3/SPOP complex. Pathologically, missense mutations in the substrate-binding domain of SPOP have been found in prostate and endometrial cancers. Prostate and endometrial cancer-associated SPOP mutations lose and increase substrate-binding ability, respectively. Expression of these SPOP mutants, thus, causes aberrant turnovers of the substrate proteins, leading to tumor formation. Although the molecular properties of SPOP and its cancer-associated mutants have been intensively elucidated, their cellular functions remain unclear. Recently, a number of studies have uncovered the critical role of SPOP and its mutants in DNA damage response and DNA replication. In this review article, we summarize the physiological functions of SPOP as a “gatekeeper” of genome stability.

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Harada, Ryuhei, Yu Takano, Takeshi Baba, and Yasuteru Shigeta. "Simple, yet powerful methodologies for conformational sampling of proteins." Physical Chemistry Chemical Physics 17, no. 9 (2015): 6155–73. http://dx.doi.org/10.1039/c4cp05262e.

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This feature article reviews four different conformational sampling methods for proteins recently developed by us. We here deal with protein folding of small proteins, large amplitude domain motion of T4 lysozyme, and induced-fit motion of a loop region after substrate binding using our methods.

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Bolduc, David M., Daniel R. Montagna, Yongli Gu, Dennis J. Selkoe, and Michael S. Wolfe. "Nicastrin functions to sterically hinder γ-secretase–substrate interactions driven by substrate transmembrane domain." Proceedings of the National Academy of Sciences 113, no. 5 (December 22, 2015): E509—E518. http://dx.doi.org/10.1073/pnas.1512952113.

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γ-Secretase is an intramembrane-cleaving protease that processes many type-I integral membrane proteins within the lipid bilayer, an event preceded by shedding of most of the substrate’s ectodomain by α- or β-secretases. The mechanism by which γ-secretase selectively recognizes and recruits ectodomain-shed substrates for catalysis remains unclear. In contrast to previous reports that substrate is actively recruited for catalysis when its remaining short ectodomain interacts with the nicastrin component of γ-secretase, we find that substrate ectodomain is entirely dispensable for cleavage. Instead, γ-secretase–substrate binding is driven by an apparent tight-binding interaction derived from substrate transmembrane domain, a mechanism in stark contrast to rhomboid—another family of intramembrane-cleaving proteases. Disruption of the nicastrin fold allows for more efficient cleavage of substrates retaining longer ectodomains, indicating that nicastrin actively excludes larger substrates through steric hindrance, thus serving as a molecular gatekeeper for substrate binding and catalysis.

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BRIX, Lulu A., Ronald G. DUGGLEBY, Andrea GAEDIGK, and Michael E. McMANUS. "Structural characterization of human aryl sulphotransferases." Biochemical Journal 337, no. 2 (January 8, 1999): 337–43. http://dx.doi.org/10.1042/bj3370337.

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Human aryl sulphotransferase (HAST) 1, HAST3, HAST4 and HAST4v share greater than 90% sequence identity, but vary markedly in their ability to catalyse the sulphonation of dopamine and p-nitrophenol. In order to investigate the amino acid(s) involved in determining differing substrate specificities of HASTs, a range of chimaeric HAST proteins were constructed. Analysis of chimaeric substrate specificities showed that enzyme affinities are mainly determined within the N-terminal end of each HAST protein, which includes two regions of high sequence divergence, termed Regions A (amino acids 44–107) and B (amino acids 132–164). To investigate the substrate-binding sites of HASTs further, site-directed mutagenesis was performed on HAST1 to change 13 individual residues within these two regions to the HAST3 equivalent. A single amino acid change in HAST1 (A146E) was able to change the specificity for p-nitrophenol to that of HAST3. The substrate specificity of HAST1 towards dopamine could not be converted into that of HAST3 with a single amino acid change. However, compared with wild-type HAST1, a number of the mutations resulted in interference with substrate binding, as shown by elevated Ki values towards the co-substrate 3´-phosphoadenosine 5´-phosphosulphate, and in some cases loss of activity towards dopamine. These findings suggest that a co-ordinated change of multiple amino acids in HAST proteins is needed to alter the substrate specificities of these enzymes towards dopamine, whereas a single amino acid at position 146 determines p-nitrophenol affinity. A HAST1 mutant was constructed to express a protein with four amino acids deleted (P87–P90). These amino acids were hypothesized to correspond to a loop region in close proximity to the substrate-binding pocket. Interestingly, the protein showed substrate specificities more similar to wild-type HAST3 than HAST1 and indicates an important role of these amino acids in substrate binding.

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Gorelik, Maryna, Stephen Orlicky, Maria A. Sartori, Xiaojing Tang, Edyta Marcon, Igor Kurinov, Jack F. Greenblatt, et al. "Inhibition of SCF ubiquitin ligases by engineered ubiquitin variants that target the Cul1 binding site on the Skp1–F-box interface." Proceedings of the National Academy of Sciences 113, no. 13 (March 14, 2016): 3527–32. http://dx.doi.org/10.1073/pnas.1519389113.

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Skp1–Cul1–F-box (SCF) E3 ligases play key roles in multiple cellular processes through ubiquitination and subsequent degradation of substrate proteins. Although Skp1 and Cul1 are invariant components of all SCF complexes, the 69 different human F-box proteins are variable substrate binding modules that determine specificity. SCF E3 ligases are activated in many cancers and inhibitors could have therapeutic potential. Here, we used phage display to develop specific ubiquitin-based inhibitors against two F-box proteins, Fbw7 and Fbw11. Unexpectedly, the ubiquitin variants bind at the interface of Skp1 and F-box proteins and inhibit ligase activity by preventing Cul1 binding to the same surface. Using structure-based design and phage display, we modified the initial inhibitors to generate broad-spectrum inhibitors that targeted many SCF ligases, or conversely, a highly specific inhibitor that discriminated between even the close homologs Fbw11 and Fbw1. We propose that most F-box proteins can be targeted by this approach for basic research and for potential cancer therapies.

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MORILLAS, Manuel, Colin E. McVEY, James A. BRANNIGAN, Andreas G. LADURNER, Larry J. FORNEY, and Richard VIRDEN. "Mutations of penicillin acylase residue B71 extend substrate specificity by decreasing steric constraints for substrate binding." Biochemical Journal 371, no. 1 (April 1, 2003): 143–50. http://dx.doi.org/10.1042/bj20021383.

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Two mutant forms of penicillin acylase from Escherichia coli strains, selected using directed evolution for the ability to use glutaryl-l-leucine for growth [Forney, Wong and Ferber (1989) Appl. Environ. Microbiol. 55, 2550—2555], are changed within one codon, replacing the B-chain residue PheB71 with either Cys or Leu. Increases of up to a factor of ten in kcat/Km values for substrates possessing a phenylacetyl leaving group are consistent with a decrease in Ks. Values of kcat/Km for glutaryl-l-leucine are increased at least 100-fold. A decrease in kcat/Km for the CysB71 mutant with increased pH is consistent with binding of the uncharged glutaryl group. The mutant proteins are more resistant to urea denaturation monitored by protein fluorescence, to inactivation in the presence of substrate either in the presence of urea or at high pH, and to heat inactivation. The crystal structure of the LeuB71 mutant protein, solved to 2Å resolution, shows a flip of the side chain of PheB256 into the periphery of the catalytic centre, associated with loss of the π-stacking interactions between PheB256 and PheB71. Molecular modelling demonstrates that glutaryl-l-leucine may bind with the uncharged glutaryl group in the S1 subsite of either the wild-type or the LeuB71 mutant but with greater potential freedom of rotation of the substrate leucine moiety in the complex with the mutant protein. This implies a smaller decrease in the conformational entropy of the substrate on binding to the mutant proteins and consequently greater catalytic activity.

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Journal articles on the topic 'Substrate-binding proteins'

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