Amino acid divergence in the ligand binding pocket of Vibrio LuxR/HapR proteins determines the efficacy of thiophenesulfonamide inhibitors

Motile bacteria follow gradients of nutrients or other environmental cues. Many bacterial chemoreceptors that sense exogenous amino acids contain a double Cache (dCache; calcium channels and chemotaxis receptors) ligand-binding domain (LBD). A growing number of studies suggest that broad-specificity dCache-type receptors that sense more than one amino acid are common. Here, we present an investigation into the mechanism by which the dCache LBD of the chemoreceptor CtaA from a plant growth–promoting rhizobacterium, Pseudomonas fluorescens, recognizes several chemically distinct amino acids. We established that amino acids that signal by directly binding to the CtaA LBD include ones with aliphatic (l-alanine, l-proline, l-leucine, l-isoleucine, l-valine), small polar (l-serine), and large charged (l-arginine) side chains. We determined the structure of CtaA LBD in complex with different amino acids, revealing that its ability to recognize a range of structurally and chemically distinct amino acids is afforded by its easily accessible plastic pocket, which can expand or contract according to the size of the ligand side chain. The amphipathic character of the pocket enables promiscuous interactions with both polar and nonpolar amino acids. The results not only clarify the means by which various amino acids are recognized by CtaA but also reveal that a conserved mobile lid over the ligand-binding pocket adopts the same conformation in all complexes, consistent with this being an important and invariant part of the signaling mechanism.

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Four Amino Acid Changes in HIV-2 Protease Confer Class-Wide Sensitivity to Protease Inhibitors

Journal of Virology ◽

10.1128/jvi.01772-15 ◽

2015 ◽

Vol 90 (2) ◽

pp. 1062-1069 ◽

Cited By ~ 14

Author(s):

Dana N. Raugi ◽

Robert A. Smith ◽

Geoffrey S. Gottlieb ◽

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Ligand Binding ◽

Protease Inhibitors ◽

Binding Pocket ◽

Wild Type ◽

Ligand Binding Pocket ◽

Crystallographic Structures ◽

Retroviral Replication ◽

Hiv 1

ABSTRACTProtease is essential for retroviral replication, and protease inhibitors (PI) are important for treating HIV infection. HIV-2 exhibits intrinsic resistance to most FDA-approved HIV-1 PI, retaining clinically useful susceptibility only to lopinavir, darunavir, and saquinavir. The mechanisms for this resistance are unclear; although HIV-1 and HIV-2 proteases share just 38 to 49% sequence identity, all critical structural features of proteases are conserved. Structural studies have implicated four amino acids in the ligand-binding pocket (positions 32, 47, 76, and 82). We constructed HIV-2ROD9molecular clones encoding the corresponding wild-type HIV-1 amino acids (I32V, V47I, M76L, and I82V) either individually or together (clone PRΔ4) and compared the phenotypic sensitivities (50% effective concentration [EC50]) of mutant and wild-type viruses to nine FDA-approved PI. Single amino acid replacements I32V, V47I, and M76L increased the susceptibility of HIV-2 to multiple PI, but no single change conferred class-wide sensitivity. In contrast, clone PRΔ4 showed PI susceptibility equivalent to or greater than that of HIV-1 for all PI. We also compared crystallographic structures of wild-type HIV-1 and HIV-2 proteases complexed with amprenavir and darunavir to models of the PRΔ4 enzyme. These models suggest that the amprenavir sensitivity of PRΔ4 is attributable to stabilizing enzyme-inhibitor interactions in the P2 and P2′ pockets of the protease dimer. Together, our results show that the combination of four amino acid changes in HIV-2 protease confer a pattern of PI susceptibility comparable to that of HIV-1, providing a structural rationale for intrinsic HIV-2 PI resistance and resolving long-standing questions regarding the determinants of differential PI susceptibility in HIV-1 and HIV-2.IMPORTANCEProteases are essential for retroviral replication, and HIV-1 and HIV-2 proteases share a great deal of structural similarity. However, only three of nine FDA-approved HIV-1 protease inhibitors (PI) are active against HIV-2. The underlying reasons for intrinsic PI resistance in HIV-2 are not known. We examined the contributions of four amino acids in the ligand-binding pocket of the enzyme that differ between HIV-1 and HIV-2 by constructing HIV-2 clones encoding the corresponding HIV-1 amino acids and testing the PI susceptibilities of the resulting viruses. We found that the HIV-2 clone containing all four changes (PRΔ4) was as susceptible as HIV-1 to all nine PI. We also modeled the PRΔ4 enzyme structure and compared it to existing crystallographic structures of HIV-1 and HIV-2 proteases complexed with amprenavir and darunavir. Our findings demonstrate that four positions in the ligand-binding cleft of protease are the primary cause of HIV-2 PI resistance.

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Identification of Amino Acid Residues That Form Part of the Ligand-binding Pocket of Integrin α5β1

Journal of Biological Chemistry ◽

10.1074/jbc.273.40.25664 ◽

1998 ◽

Vol 273 (40) ◽

pp. 25664-25672 ◽

Cited By ~ 37

Author(s):

A. Paul Mould ◽

Louise Burrows ◽

Martin J. Humphries

Keyword(s):

Amino Acid ◽

Ligand Binding ◽

Binding Pocket ◽

Amino Acid Residues ◽

Integrin Α5β1 ◽

Ligand Binding Pocket ◽

Form Part

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Suramin Targets the Conserved Ligand-Binding Pocket of Human Raf1 Kinase Inhibitory Protein

Molecules ◽

10.3390/molecules26041151 ◽

2021 ◽

Vol 26 (4) ◽

pp. 1151

Author(s):

Chenyun Guo ◽

Zhihua Wu ◽

Weiliang Lin ◽

Hao Xu ◽

Ting Chang ◽

...

Keyword(s):

Side Effects ◽

Ligand Binding ◽

Mapk Pathway ◽

Regulatory Protein ◽

Therapeutic Index ◽

Binding Pocket ◽

Biological Macromolecules ◽

Inhibitory Protein ◽

Ligand Binding Pocket ◽

Raf1 Kinase

Suramin was initially used to treat African sleeping sickness and has been clinically tested to treat human cancers and HIV infection in the recent years. However, the therapeutic index is low with numerous clinical side-effects, attributed to its diverse interactions with multiple biological macromolecules. Here, we report a novel binding target of suramin, human Raf1 kinase inhibitory protein (hRKIP), which is an important regulatory protein involved in the Ras/Raf1/MEK/ERK (MAPK) signal pathway. Biolayer interference technology showed that suramin had an intermediate affinity for binding hRKIP with a dissociation constant of 23.8 µM. Both nuclear magnetic resonance technology and molecular docking analysis revealed that suramin bound to the conserved ligand-binding pocket of hRKIP, and that residues K113, W173, and Y181 play crucial roles in hRKIP binding suramin. Furthermore, suramin treatment at 160 µM could profoundly increase the ERK phosphorylation level by around 3 times. Our results indicate that suramin binds to hRKIP and prevents hRKIP from binding with hRaf1, thus promoting the MAPK pathway. This work is beneficial to both mechanistically understanding the side-effects of suramin and efficiently improving the clinical applications of suramin.

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Ligands with poly-fluorophenyl moieties promote a local structural rearrangement in the Spinach2 and Broccoli aptamers that increases ligand affinities

10.1101/2021.10.05.463302 ◽

2021 ◽

Author(s):

Sharif Anisuzzaman ◽

Ivan M Geraskin ◽

Muslum Ilgu ◽

Lee Bendickson ◽

George A Kraus ◽

...

Keyword(s):

Nucleic Acids ◽

Ligand Binding ◽

Small Molecule ◽

Binding Pocket ◽

Structural Rearrangement ◽

Structural Reorganization ◽

Ligand Binding Pocket ◽

Rna Remodeling ◽

Small Molecule Ligands ◽

Target Molecules

The interaction of nucleic acids with their molecular targets often involves structural reorganization that may traverse a complex folding landscape. With the more recent recognition that many RNAs, both coding and noncoding, may regulate cellular activities by interacting with target molecules, it becomes increasingly important to understand the means by which nucleic acids interact with their targets and how drugs might be developed that can influence critical folding transitions. We have extensively investigated the interaction of the Spinach2 and Broccoli aptamers with a library of small molecule ligands modified by various extensions from the imido nitrogen of DFHBI (3,5-difluoro-4-hydroxybenzylidene imidazolinone) that reach out from the Spinach2 ligand binding pocket. Studies of the interaction of these compounds with the aptamers revealed that poly-fluorophenyl-modified ligands initiate a slow change in aptamer affinity that takes an extended time (half-life of ~40 min) to achieve. The change in affinity appears to involve an initial disruption of the entrance to the ligand binding pocket followed by a gradual lockdown for which the most likely driving force is an interaction of the gateway adenine with a nearby 2'OH group. These results suggest that poly-fluorophenyl modifications might increase the ability of small molecule drugs to disrupt local structure and promote RNA remodeling.

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Activation of transcription through the ligand-binding pocket of the orphan nuclear receptor ultraspiracle

European Journal of Biochemistry ◽

10.1046/j.1432-1033.2002.03293.x ◽

2002 ◽

Vol 269 (24) ◽

pp. 6026-6036 ◽

Cited By ~ 60

Author(s):

Yong Xu ◽

Fang Fang ◽

YanXia Chu ◽

Davy Jones ◽

Grace Jones

Keyword(s):

Ligand Binding ◽

Nuclear Receptor ◽

Binding Pocket ◽

Orphan Nuclear Receptor ◽

Ligand Binding Pocket ◽

Activation Of Transcription

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Ligand binding pocket function of Drosophila USP is necessary for metamorphosis

General and Comparative Endocrinology ◽

10.1016/j.ygcen.2012.11.009 ◽

2013 ◽

Vol 182 ◽

pp. 73-82 ◽

Cited By ~ 24

Author(s):

Grace Jones ◽

Peter Teal ◽

Vincent C. Henrich ◽

Anna Krzywonos ◽

Agnes Sapa ◽

...

Keyword(s):

Ligand Binding ◽

Binding Pocket ◽

Ligand Binding Pocket

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Agonist Activation of the Angiotensin II Type 1 Receptor Alters the Spatial Proximity of Transmembrane Domain 7 to the Ligand Binding Pocket

Understanding Biology Using Peptides ◽

10.1007/978-0-387-26575-9_144 ◽

2010 ◽

pp. 357-358

Author(s):

Dany Fillion ◽

Gaétan Guillemette ◽

Richard Leduc ◽

Emanuel Escher

Keyword(s):

Angiotensin Ii ◽

Ligand Binding ◽

Transmembrane Domain ◽

Binding Pocket ◽

Spatial Proximity ◽

Ligand Binding Pocket

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Computational redesign of the Escherichia coli ribose-binding protein ligand binding pocket for 1,3-cyclohexanediol and cyclohexanol

Scientific Reports ◽

10.1038/s41598-019-53507-5 ◽

2019 ◽

Vol 9 (1) ◽

Author(s):

Diogo Tavares ◽

Artur Reimer ◽

Shantanu Roy ◽

Aurélie Joublin ◽

Vladimir Sentchilo ◽

...

Keyword(s):

Escherichia Coli ◽

Ligand Binding ◽

Binding Proteins ◽

Binding Pocket ◽

Wild Type ◽

Ligand Binding Pocket ◽

Mutant Proteins ◽

Periplasmic Binding Proteins ◽

Natural Ligands ◽

Ribose Binding Protein

AbstractBacterial periplasmic-binding proteins have been acclaimed as general biosensing platform, but their range of natural ligands is too limited for optimal development of chemical compound detection. Computational redesign of the ligand-binding pocket of periplasmic-binding proteins may yield variants with new properties, but, despite earlier claims, genuine changes of specificity to non-natural ligands have so far not been achieved. In order to better understand the reasons of such limited success, we revisited here the Escherichia coli RbsB ribose-binding protein, aiming to achieve perceptible transition from ribose to structurally related chemical ligands 1,3-cyclohexanediol and cyclohexanol. Combinations of mutations were computationally predicted for nine residues in the RbsB binding pocket, then synthesized and tested in an E. coli reporter chassis. Two million variants were screened in a microcolony-in-bead fluorescence-assisted sorting procedure, which yielded six mutants no longer responsive to ribose but with 1.2–1.5 times induction in presence of 1 mM 1,3-cyclohexanediol, one of which responded to cyclohexanol as well. Isothermal microcalorimetry confirmed 1,3-cyclohexanediol binding, although only two mutant proteins were sufficiently stable upon purification. Circular dichroism spectroscopy indicated discernable structural differences between these two mutant proteins and wild-type RbsB. This and further quantification of periplasmic-space abundance suggested most mutants to be prone to misfolding and/or with defects in translocation compared to wild-type. Our results thus affirm that computational design and library screening can yield RbsB mutants with recognition of non-natural but structurally similar ligands. The inherent arisal of protein instability or misfolding concomitant with designed altered ligand-binding pockets should be overcome by new experimental strategies or by improved future protein design algorithms.

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