Ligand-specific conformational change drives interdomain allostery in Pin1

Abstract Pin1 is a two-domain cell regulator that isomerizes peptidyl-prolines. The catalytic domain (PPIase) and the other ligand-binding domain (WW) sample extended and compact conformations. Ligand binding changes the equilibrium of the interdomain conformations, but the conformational changes that lead to the altered domain sampling were unknown. Prior evidence has supported an interdomain allosteric mechanism. We recently introduced a magnetic resonance-based protocol that allowed us to determine the coupling of intra- and interdomain structural sampling in apo Pin1. Here, we describe ligand-specific conformational changes that occur upon binding of pCDC25c and FFpSPR. pCDC25c binding doubles the population of the extended states compared to the virtually identical populations of the apo and FFpSPR-bound forms. pCDC25c binding to the WW domain triggers conformational changes to propagate via the interdomain interface to the catalytic site, while FFpSPR binding displaces a helix in the PPIase that leads to repositioning of the PPIase catalytic loop.

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Positive co-operative binding at two weak lysine-binding sites governs the Glu-plasminogen conformational change

Biochemical Journal ◽

10.1042/bj2850419 ◽

1992 ◽

Vol 285 (2) ◽

pp. 419-425 ◽

Cited By ~ 34

Author(s):

U Christensen ◽

L Mølgaard

Keyword(s):

Ligand Binding ◽

Binding Site ◽

Conformational Change ◽

Conformational Changes ◽

Binding Sites ◽

High Specificity ◽

Stopped Flow ◽

Protein Fluorescence ◽

Lysine Residues ◽

Kinetics Of

The kinetics of a series of Glu-plasminogen ligand-binding processes were investigated at pH 7.8 and 25 degrees C (in 0.1 M-NaCl). The ligands include compounds analogous to C-terminal lysine residues and to normal lysine residues. Changes of the Glu-plasminogen protein fluorescence were measured in a stopped-flow instrument as a function of time after rapid mixing of Glu-plasminogen and ligand at various concentrations. Large positive fluorescence changes (approximately 10%) accompany the ligand-induced conformational changes of Glu-plasminogen resulting from binding at weak lysine-binding sites. Detailed studies of the concentration-dependencies of the equilibrium signals and the rate constants of the process induced by various ligands showed the conformational change to involve two sites in a concerted positive co-operative process with three steps: (i) binding of a ligand at a very weak lysine-binding site that preferentially, but not exclusively, binds C-terminal-type lysine ligands, (ii) the rate-determining actual-conformational-change step and (iii) binding of one more lysine ligand at a second weak lysine-binding site that then binds the ligand more tightly. Further, totally independent initial small negative fluorescence changes (approximately 2-4%) corresponding to binding at the strong lysine-binding site of kringle 1 [Sottrup-Jensen, Claeys, Zajdel, Petersen & Magnusson (1978) Prog. Chem. Fibrinolysis Thrombolysis 3, 191-209] were observed for the C-terminal-type ligands. The finding that the conformational change in Glu-plasminogen involves two weak lysine-binding sites indicates that the effect cannot be assigned to any single kringle and that the problem of whether kringle 4 or kringle 5 is responsible for the process resolves itself. Probably kringle 4 and 5 are both participating. The involvement of two lysine binding-sites further makes the high specificity of Glu-plasminogen effectors more conceivable.

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Chemical Footprinting Reveals Conformational Changes Following Activation of Factor XI

Thrombosis and Haemostasis ◽

10.1160/th17-09-0676 ◽

2018 ◽

Vol 118 (02) ◽

pp. 340-350 ◽

Cited By ~ 3

Author(s):

Ingrid Stroo ◽

J. Marquart ◽

Kamran Bakhtiari ◽

Tom Plug ◽

Alexander Meijer ◽

...

Keyword(s):

Conformational Change ◽

Conformational Changes ◽

Catalytic Domain ◽

Active Form ◽

Coagulation Factor ◽

Lysine Residue ◽

Factor Ix ◽

Factor Xi ◽

Lysine Residues ◽

Factor Xia

AbstractCoagulation factor XI is activated by thrombin or factor XIIa resulting in a conformational change that converts the catalytic domain into its active form and exposing exosites for factor IX on the apple domains. Although crystal structures of the zymogen factor XI and the catalytic domain of the protease are available, the structure of the apple domains and hence the interactions with the catalytic domain in factor XIa are unknown. We now used chemical footprinting to identify lysine residue containing regions that undergo a conformational change following activation of factor XI. To this end, we employed tandem mass tag in conjunction with mass spectrometry. Fifty-two unique peptides were identified, covering 37 of the 41 lysine residues present in factor XI. Two identified lysine residues that showed altered flexibility upon activation were mutated to study their contribution in factor XI stability or enzymatic activity. Lys357, part of the connecting loop between A4 and the catalytic domain, was more reactive in factor XIa but mutation of this lysine residue did not impact on factor XIa activity. Lys516 and its possible interactor Glu380 are located in the catalytic domain and are covered by the activation loop of factor XIa. Mutating Glu380 enhanced Arg369 cleavage and thrombin generation in plasma. In conclusion, we have identified novel regions that undergo a conformational change following activation. This information improves knowledge about factor XI and will contribute to development of novel inhibitors or activators for this coagulation protein.

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Conformational changes induced by binding of bivalent cations to oncomodulin, a paravalbumin-like tumour protein

Biochemical Journal ◽

10.1042/bj2200261 ◽

1984 ◽

Vol 220 (1) ◽

pp. 261-268 ◽

Cited By ~ 44

Author(s):

J P MacManus ◽

A G Szabo ◽

R E Williams

Keyword(s):

Conformational Change ◽

Conformational Changes ◽

The Other ◽

Excitation Spectra ◽

Fluorescence Excitation ◽

Protein Protein Interaction ◽

Tyrosine Fluorescence ◽

Bivalent Cations ◽

Fluorescence Excitation Spectra ◽

Dependent Protein

When Mg2+ was added to rat oncomodulin, a paravalbumin-like tumour protein, changes in the c.d. spectrum and tyrosine fluorescence intensity were observed. The addition of Ca2+ resulted in even greater changes in these spectra. The fluorescence excitation spectra of apo- and Mg-oncomodulin were superimposable, whereas that of Ca-oncomodulin was markedly different. The u.v.-absorption spectrum of the Ca2+ form also showed major differences from those of the other two forms. These observations indicate that Ca2+ induced a significant and specific conformational change in the protein that was not observed on binding Mg2+. In contrast, the conformational change induced by either Mg2+ or Ca2+ was identical in the homologous rat parvalbumin. This Ca2+-specific conformational change may be the basis for oncomodulin's Ca2+-dependent protein/protein interaction.

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Activation of the first component of human complement (C1) by antibody—antigen aggregates

Biochemical Journal ◽

10.1042/bj1750383 ◽

1978 ◽

Vol 175 (2) ◽

pp. 383-390 ◽

Cited By ~ 80

Author(s):

A W Dodds ◽

R B Sim ◽

R R Porter ◽

M A Kerr

Keyword(s):

Conformational Change ◽

Proteolytic Activity ◽

Maximum Rate ◽

Catalytic Site ◽

The Other ◽

Peptide Chain ◽

Human Complement ◽

Rate Of Activation

The activation of subcomponents C1r and C1s in the first component of complement, C1, when bound to antibody-antigen complexes was investigated. Activation was followed both by the splitting of the peptide chains of subcomponents C1r and C1s and by the development of proteolytic activity. For the maximum rate of activation to occur, all components must be present in approximate molar proportions of antibody: C1q:C1r:C1s of 13:1:5:5. For activation of subcomponent C1s, subcomponents C1r or C1r, but not C1r inactivated with iPr2P-F (di-isopropyl phosphorofluorideate), are effective. For activation of subcomponent C1r, subcomponents C1s, C1s or C1s inactivated with iPr2P-F are effective. Subcomponent C1s is activated by C1r, and C1r is activated autocatalytically, probably through the formation of an intermediary C1r. in which the peptide chain is unsplit but a conformational change caused by interaction with the other components has led to the formation of a catalytic site able to split subcomponent C1r to C1r.

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Ligand Binding Mechanism and Its Relationship with Conformational Changes in Adenine Riboswitch

International Journal of Molecular Sciences ◽

10.3390/ijms21061926 ◽

2020 ◽

Vol 21 (6) ◽

pp. 1926

Author(s):

Guodong Hu ◽

Haiyan Li ◽

Shicai Xu ◽

Jihua Wang

Keyword(s):

Ligand Binding ◽

Conformational Change ◽

Conformational Changes ◽

Md Simulations ◽

Binding Pocket ◽

Dynamical Analysis ◽

Mass Center ◽

Free Energies ◽

Binding Model ◽

Binding Free Energies

Riboswitches are naturally occurring RNA aptamers that control the expression of essential bacterial genes by binding to specific small molecules. The binding with both high affinity and specificity induces conformational changes. Thus, riboswitches were proposed as a possible molecular target for developing antibiotics and chemical tools. The adenine riboswitch can bind not only to purine analogues but also to pyrimidine analogues. Here, long molecular dynamics (MD) simulations and molecular mechanics Poisson–Boltzmann surface area (MM-PBSA) computational methodologies were carried out to show the differences in the binding model and the conformational changes upon five ligands binding. The binding free energies of the guanine riboswitch aptamer with C74U mutation complexes were compared to the binding free energies of the adenine riboswitch (AR) aptamer complexes. The calculated results are in agreement with the experimental data. The differences for the same ligand binding to two different aptamers are related to the electrostatic contribution. Binding dynamical analysis suggests a flexible binding pocket for the pyrimidine ligand in comparison with the purine ligand. The 18 μs of MD simulations in total indicate that both ligand-unbound and ligand-bound aptamers transfer their conformation between open and closed states. The ligand binding obviously affects the conformational change. The conformational states of the aptamer are associated with the distance between the mass center of two key nucleotides (U51 and A52) and the mass center of the other two key nucleotides (C74 and C75). The results suggest that the dynamical character of the binding pocket would affect its biofunction. To design new ligands of the adenine riboswitch, it is recommended to consider the binding affinities of the ligand and the conformational change of the ligand binding pocket.

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Role of conformational change and K-path ligands in controlling cytochrome c oxidase activity

Biochemical Society Transactions ◽

10.1042/bst20160138 ◽

2017 ◽

Vol 45 (5) ◽

pp. 1087-1095 ◽

Cited By ~ 9

Author(s):

Jian Liu ◽

Carrie Hiser ◽

Shelagh Ferguson-Miller

Keyword(s):

Cytochrome C ◽

Ligand Binding ◽

Cytochrome C Oxidase ◽

Conformational Change ◽

Conformational Changes ◽

Active Site ◽

Mutant Form ◽

Gating Mechanism ◽

Proton Uptake

Given the central role of cytochrome c oxidase (CcO) in health and disease, it is an increasingly important question as to how the activity and efficiency of this key enzyme are regulated to respond to a variety of metabolic states. The present paper summarizes evidence for two modes of regulation of activity: first, by redox-induced conformational changes involving the K-proton uptake path; and secondly, by ligand binding to a conserved site immediately adjacent to the entrance of the K-path that leads to the active site. Both these phenomena highlight the importance of the K-path in control of CcO. The redox-induced structural changes are seen in both the two-subunit and a new four-subunit crystal structure of bacterial CcO and suggest a gating mechanism to control access of protons to the active site. A conserved ligand-binding site, first discovered as a bile salt/steroid site in bacterial and mammalian oxidases, is observed to bind an array of ligands, including nucleotides, detergents, and other amphipathic molecules. Highly variable effects on activity, seen for these ligands and mutations at the K-path entrance, can be explained by differing abilities to inhibit or stimulate K-path proton uptake by preventing or allowing water organization. A new mutant form in which the K-path is blocked by substituting the conserved carboxyl with a tryptophan clarifies the singularity of the K-path entrance site. Further study in eukaryotic systems will determine the physiological significance and pharmacological potential of ligand binding and conformational change in CcO.

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Conformational kinetics reveals affinities of protein conformational states

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1502084112 ◽

2015 ◽

Vol 112 (30) ◽

pp. 9352-9357 ◽

Cited By ~ 20

Author(s):

Kyle G. Daniels ◽

Yang Suo ◽

Terrence G. Oas

Keyword(s):

Ligand Binding ◽

Conformational Change ◽

Conformational Changes ◽

Rate Constants ◽

Conformational Dynamics ◽

Forward Rate ◽

Binding Affinities ◽

Conformational States ◽

Kinetics Of

Most biological reactions rely on interplay between binding and changes in both macromolecular structure and dynamics. Practical understanding of this interplay requires detection of critical intermediates and determination of their binding and conformational characteristics. However, many of these species are only transiently present and they have often been overlooked in mechanistic studies of reactions that couple binding to conformational change. We monitored the kinetics of ligand-induced conformational changes in a small protein using six different ligands. We analyzed the kinetic data to simultaneously determine both binding affinities for the conformational states and the rate constants of conformational change. The approach we used is sufficiently robust to determine the affinities of three conformational states and detect even modest differences in the protein’s affinities for relatively similar ligands. Ligand binding favors higher-affinity conformational states by increasing forward conformational rate constants and/or decreasing reverse conformational rate constants. The amounts by which forward rate constants increase and reverse rate constants decrease are proportional to the ratio of affinities of the conformational states. We also show that both the affinity ratio and another parameter, which quantifies the changes in conformational rate constants upon ligand binding, are strong determinants of the mechanism (conformational selection and/or induced fit) of molecular recognition. Our results highlight the utility of analyzing the kinetics of conformational changes to determine affinities that cannot be determined from equilibrium experiments. Most importantly, they demonstrate an inextricable link between conformational dynamics and the binding affinities of conformational states.

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Binding of phosphorothioate oligonucleotides with RNase H1 can cause conformational changes in the protein and alter the interactions of RNase H1 with other proteins

Nucleic Acids Research ◽

10.1093/nar/gkab078 ◽

2021 ◽

Author(s):

Lingdi Zhang ◽

Timothy A Vickers ◽

Hong Sun ◽

Xue-hai Liang ◽

Stanley T Crooke

Keyword(s):

Conformational Change ◽

Binding Affinity ◽

Conformational Changes ◽

Protein Interactions ◽

Molecular Basis ◽

Catalytic Domain ◽

Dependent Manner ◽

Protein Protein Interactions ◽

Rna Duplexes ◽

Underlying Mechanisms

Abstract We recently found that toxic PS-ASOs can cause P54nrb and PSF nucleolar mislocalization in an RNase H1-dependent manner. To better understand the underlying mechanisms of these observations, here we utilize different biochemical approaches to demonstrate that PS-ASO binding can alter the conformations of the bound proteins, as illustrated using recombinant RNase H1, P54nrb, PSF proteins and various isolated domains. While, in general, binding of PS-ASOs or ASO/RNA duplexes stabilizes the conformations of these proteins, PS-ASO binding may also cause the unfolding of RNase H1, including both the hybrid binding domain and the catalytic domain. The extent of conformational change correlates with the binding affinity of PS-ASOs to the proteins. Consequently, PS-ASO binding to RNase H1 induces the interaction of RNase H1 with P54nrb or PSF in a 2′-modification and sequence dependent manner, and toxic PS-ASOs tend to induce more interactions than non-toxic PS-ASOs. PS-ASO binding also enhances the interaction between P54nrb and PSF. However, the interaction between RNase H1 and P32 protein can be disrupted upon binding of PS-ASOs. Together, these results suggest that stronger binding of PS-ASOs can cause greater conformational changes of the bound proteins, subsequently affecting protein–protein interactions. These observations thus provide deeper understanding of the molecular basis of PS-ASO-induced protein mislocalization or degradation observed in cells and advance our understanding of why some PS-ASOs are cytotoxic.

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Ligand binding, conformational change and plasma elimination of human, mouse and rat α-macroglobulin proteinase inhibitors

Biochemical Journal ◽

10.1042/bj2090099 ◽

1983 ◽

Vol 209 (1) ◽

pp. 99-105 ◽

Cited By ~ 69

Author(s):

S L Gonias ◽

A E Balber ◽

W J Hubbard ◽

S V Pizzo

Keyword(s):

Ligand Binding ◽

Conformational Change ◽

Trypsin Inhibitor ◽

Proteinase Inhibitor ◽

Proteinase Inhibitors ◽

Soya Bean ◽

The Other ◽

Complex Reaction ◽

Bean Trypsin Inhibitor ◽

Esterolytic Activity

Rat alpha 1-macroglobulin (alpha 1M), rat alpha 2-macroglobulin (alpha 2M) migrated as single bands on non-denaturing gels when purified by the methods described. All three proteins demonstrated increased mobility after reaction with trypsin. A single saturable pathway rapidly cleared complexes of trypsin and the alpha-macroglobulins of mouse, rat and human from the circulation of mice. None of the native alpha-macroglobulins competed for clearance with the trypsin complexes. [14C]Methylamine incorporation was 4.1, 3.9, 2.6 and 3.2 mol/mol of proteinase inhibitor for human alpha 2M, rat alpha 1M, rat alpha 2M and mouse alpha 2M, respectively. Only rat alpha 2M, the acute-phase alpha-macroglobulin studied, showed no evidence of conformational change when subjected to electrophoresis after reaction with methylamine. The clearance of rat alpha 2M-methylamine was comparable with that of the native molecule. The other alpha-macroglobulin-methylamine complexes cleared faster than the inhibitors that had not reacted. Rat alpha 2M and rat alpha 2M-methylamine bound equivalent quantities of 1251-labelled trypsin (1.01 and 0.96 mol/mol respectively). The soya-bean trypsin inhibitor-resistant esterolytic activity of trypsin bound to rat alpha 2M-methylamine was approx. 90% suppressed compared with proteinase bound to native rat alpha 2M. This suppression was not due to a change in the affinity of soya-bean trypsin inhibitor for the complex. Reaction of rat alpha 2M-methylamine with trypsin resulted in a ‘slow’ to ‘fast’ electrophoretic conversion of the proteinase inhibitor, and exposure of the signal on the alpha 2M that causes the complex to clear from the murine circulation.

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CarR, a MarR-family regulator from Corynebacterium glutamicum, modulated antibiotic and aromatic compound resistance

Biochemical Journal ◽

10.1042/bcj20190320 ◽

2019 ◽

Vol 476 (21) ◽

pp. 3141-3159 ◽

Cited By ~ 2

Author(s):

Meiru Si ◽

Can Chen ◽

Zengfan Wei ◽

Zhijin Gong ◽

GuiZhi Li ◽

...

Keyword(s):

Stress Response ◽

Ligand Binding ◽

Corynebacterium Glutamicum ◽

Conformational Changes ◽

Cysteine Oxidation ◽

Transcriptional Regulatory ◽

Ligand Free ◽

Redox Sensing

Abstract MarR (multiple antibiotic resistance regulator) proteins are a family of transcriptional regulators that is prevalent in Corynebacterium glutamicum. Understanding the physiological and biochemical function of MarR homologs in C. glutamicum has focused on cysteine oxidation-based redox-sensing and substrate metabolism-involving regulators. In this study, we characterized the stress-related ligand-binding functions of the C. glutamicum MarR-type regulator CarR (C. glutamicum antibiotic-responding regulator). We demonstrate that CarR negatively regulates the expression of the carR (ncgl2886)–uspA (ncgl2887) operon and the adjacent, oppositely oriented gene ncgl2885, encoding the hypothetical deacylase DecE. We also show that CarR directly activates transcription of the ncgl2882–ncgl2884 operon, encoding the peptidoglycan synthesis operon (PSO) located upstream of carR in the opposite orientation. The addition of stress-associated ligands such as penicillin and streptomycin induced carR, uspA, decE, and PSO expression in vivo, as well as attenuated binding of CarR to operator DNA in vitro. Importantly, stress response-induced up-regulation of carR, uspA, and PSO gene expression correlated with cell resistance to β-lactam antibiotics and aromatic compounds. Six highly conserved residues in CarR were found to strongly influence its ligand binding and transcriptional regulatory properties. Collectively, the results indicate that the ligand binding of CarR induces its dissociation from the carR–uspA promoter to derepress carR and uspA transcription. Ligand-free CarR also activates PSO expression, which in turn contributes to C. glutamicum stress resistance. The outcomes indicate that the stress response mechanism of CarR in C. glutamicum occurs via ligand-induced conformational changes to the protein, not via cysteine oxidation-based thiol modifications.

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