Both protein dynamics and ligand concentration can shift the binding mechanism between conformational selection and induced fit

Greives, Nicholas; Zhou, Huan‐Xiang

doi:10.1073/pnas.1407545111

Cited by 80 publications

(114 citation statements)

References 41 publications

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“…We and others have previously shown that such mechanisms depend on ligand concentration (17,(25)(26)(27). We have shown here that the molecular origins of this ligand concentration dependence are in the intrinsic affinities of the conformational states and in the effects that ligand has on conformational kinetics.…”

Section: Discussionsupporting

confidence: 51%

Conformational kinetics reveals affinities of protein conformational states

Daniels

Suo

Oas

2015

Proc. Natl. Acad. Sci. U.S.A.

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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.T he ensemble nature of proteins and nucleic acids, and their abilities to bind other molecules, are critical to their function (1). Often, the conformational ensembles of these macromolecules can be subdivided into kinetically distinguishable conformational subensembles or states in the thermodynamic sense. Thanks in part to the highly cooperative nature of biopolymer conformational changes (2-4), these conformational states are distinguishable by the kinetic barriers that separate them; conformers in separate states interconvert much more slowly than conformers within the same state. Thus, the reactions of such a system can be described as the interconversion of a small number of conformational states without ignoring the ensemble nature of each state (5, 6). To determine a mechanism that involves these conformational states, one must determine the timescale of their interconversion and their affinities for ligand. The practical definition of a conformational state is one whose delimiting kinetic barriers match the timescale of the experiment being used to measure its kinetics. In the case of the stopped-flow method used in this study, that timescale is 1-2 ms or slower. Numerous studies have focused on biophysical cha...

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Section: Discussionsupporting

confidence: 51%

Conformational kinetics reveals affinities of protein conformational states

Daniels

Suo

Oas

2015

Proc. Natl. Acad. Sci. U.S.A.

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“…When considering all information available it is difficult (at these nanomolar-to-micromolar concentrations) (23,30) to propose, or even support, a mechanism where association is limited by the selection of a lowly populated, helical PUMA conformation, formed in the absence of MCL-1. Though conformational selection may play a larger role in more structurally complex systems (27), for the particular case of a short, disordered motif binding an already folded protein, this study adds to the mounting experimental evidence for induced-fit mechanisms (14,(16)(17)(18)(19)54).…”

Section: Discussionmentioning

confidence: 99%

“…In isolation an IDP could, perhaps only transiently, occupy a conformation that resembles the bound state. In the pure conformational selection mechanism, the IDP must be in this conformation at the start of the eventually successful encounter with the partner protein (23,24) (Fig. 1A).…”

mentioning

confidence: 99%

“…Complex mixtures of these two extreme mechanisms can also be imagined: e.g., perhaps only a proportion of the IDP needs to fold before the encounter, i.e., conformational selection followed by induced fit of the remaining peptide chain (29). To add to the potential complexity, flux through different pathways could occur simultaneously, and may depend on the concentrations of protein involved (23,30). Further, confirming the degree of induced fit and conformational selection is only one aspect of the binding mechanism.…”

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confidence: 99%

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Interplay between partner and ligand facilitates the folding and binding of an intrinsically disordered protein

Rogers

Oleinikovas

Shammas

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

155

181

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Protein-protein interactions are at the heart of regulatory and signaling processes in the cell. In many interactions, one or both proteins are disordered before association. However, this disorder in the unbound state does not prevent many of these proteins folding to a well-defined, ordered structure in the bound state. Here we examine a typical system, where a small disordered protein (PUMA, p53 upregulated modulator of apoptosis) folds to an α-helix when bound to a groove on the surface of a folded protein (MCL-1, induced myeloid leukemia cell differentiation protein). We follow the association of these proteins using rapid-mixing stopped flow, and examine how the kinetic behavior is perturbed by denaturant and carefully chosen mutations. We demonstrate the utility of methods developed for the study of monomeric protein folding, including β-Tanford values, Leffler α, Φ-value analysis, and coarse-grained simulations, and propose a self-consistent mechanism for binding. Folding of the disordered protein before binding does not appear to be required and few, if any, specific interactions are required to commit to association. The majority of PUMA folding occurs after the transition state, in the presence of MCL-1. We also examine the role of the side chains of folded MCL-1 that make up the binding groove and find that many favor equilibrium binding but, surprisingly, inhibit the association process.Protein folding | stopped flow | coarse-grained simulation | protein-protein interactions | BCL-2

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“…Notably, several well-studied examples of mesophile–thermophile pairs are enzymes with small-molecule substrates where differences in activity can be correlated with differences in conformational dynamics of a loop involved in substrate binding [61–63], whereas RNase H has a large substrate-binding surface and thus can accommodate multiple forms of adaptation through subtle changes in the relative energies of local conformational states. Proteins with large, dynamic binding surfaces and whose substrates exhibit complex internal dynamics of their own complicate the emerging picture of flux through conformational selection and induced-fit binding pathways as determined primarily by conformational exchange kinetics and ligand concentration [64]. The behavior observed in the RNase H family, in which partially binding-competent conformations are sampled in the apo state but require additional induced-fit accommodation after binding substrate, is consistent with experimental observations of interdomain protein–protein interactions [65] and with simulations of RNA-binding U1A protein [66].…”

Section: Resultsmentioning

confidence: 99%

Conformational Preferences Underlying Reduced Activity of a Thermophilic Ribonuclease H

Stafford¹,

Trbovic²,

Butterwick³

et al. 2015

Journal of Molecular Biology

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The conformational basis for reduced activity of the thermophilic ribonuclease HI enzyme from Thermus thermophilus, compared to its mesophilic homolog from Escherichia coli, is elucidated using a combination of NMR spectroscopy and molecular dynamics (MD) simulations. Explicit-solvent all-atom MD simulations of the two wild-type proteins and an E. coli mutant in which a glycine residue is inserted after position 80 to mimic the T. thermophilus protein reproduce the differences in conformational dynamics determined from 15N spin-relaxation NMR spectroscopy of three loop regions that surround the active site and contain functionally important residues: the glycine-rich region, the handle region, and the β5/αE loop. Examination of the MD trajectories indicates that the thermophilic protein samples conformations productive for substrate binding and activity less frequently than the mesophilic enzyme, although these differences may manifest as either increased or decreased relative flexibility of the different regions. Additional MD simulations indicate that mutations increasing activity of the T. thermophilus enzyme at mesophilic temperatures do so by reconfiguring the local environments of the mutated sites to more closely resemble active conformations. Taken together, the results show that both locally increased and decreased flexibility contribute to an overall reduction in activity of T. thermophilus ribonuclease H compared to its mesophilic E. coli homolog.

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Both protein dynamics and ligand concentration can shift the binding mechanism between conformational selection and induced fit

Cited by 80 publications

References 41 publications

Conformational kinetics reveals affinities of protein conformational states

Conformational kinetics reveals affinities of protein conformational states

Interplay between partner and ligand facilitates the folding and binding of an intrinsically disordered protein

Conformational Preferences Underlying Reduced Activity of a Thermophilic Ribonuclease H

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