Conformational Selection Model Research Articles

Structural dynamics of calmodulin-ryanodine receptor interactions: electron paramagnetic resonance using stereospecific spin labels

We have used electron paramagnetic resonance, with rigid and stereospecific spin labels, to resolve structural states in calmodulin (CaM), as affected by binding of Ca and a CaM-binding peptide (RyRp) derived from the ryanodine receptor (RyR), the Ca channel that triggers muscle contraction. CaM mutants containing a pair of cysteines in the N-lobe and/or C-lobe were engineered and labeled with a stereospecifically bound bifunctional spin label (BSL). RyRp was synthesized with and without TOAC (a stereospecifically attached spin-labeled amino acid) substituted for a single amino acid near the N-terminus. Intramolecular DEER distance measurements of doubly-labeled BSL-CaM revealed that CaM exists in dynamic equilibrium among multiple states, consistent with open, closed, and compact structural models. Addition of RyRp shifted the equilibrium partially toward the compact state in the absence of Ca, and completely toward the compact state in the presence of Ca, supporting a conformational selection model. Inter-protein distance measurements show that Ca stabilizes the compact state primarily by inducing ordered binding of the CaM N-lobe to RyRp, while only slightly affecting the C-lobe. The results provide insight into the structural mechanism of CaM-mediated RyR regulation, while demonstrating the power of using two types of rigidly and stereospecifically bound spin labels.

Previously Hidden Dynamics at the TCR–Peptide–MHC Interface Revealed

A structural characterization of the interaction between αβ TCRs and cognate peptide-MHC (pMHC) is central to understanding adaptive T cell-mediated immunity. X-ray crystallography, although the source of much structural data, traditionally provides only a static snapshot of the protein. Given the emerging evidence for the important role of conformational dynamics in protein function, we interrogated 309 crystallographic structures of pMHC complexes using ensemble refinement, a technique that can extract dynamic information from the x-ray data. Focusing on a subset of human pMHC class I systems, we found that in many cases, ensemble methods were able to uncover previously hidden evidence of significant conformational plasticity, thereby revealing additional information that can build upon and significantly enhance functional interpretations that are based on a single static structure. Notable examples include the interpretation of differences in the disease association of HLA subtypes, the relationship between peptide prominence and TCR recognition, the role of conformational flexibility in vaccine design, and the discrimination between induced fit and conformational selection models of TCR binding. We show that the currently widespread practice of analyzing pMHC interactions via the study of a single crystallographic structure does not make use of pertinent and easily accessible information from x-ray data concerning alternative protein conformations. This new analysis therefore not only highlights the capacity for ensemble methods to significantly enrich the interpretation of decades of structural data but also provides previously missing information concerning the dynamics of existing characterized TCR-pMHC interactions.

Allosteric mechanism of the V. vulnificus adenine riboswitch resolved by four-dimensional chemical mapping.

The structural interconversions that mediate the gene regulatory functions of RNA molecules may be different from classic models of allostery, but the relevant structural correlations have remained elusive in even intensively studied systems. Here, we present a four-dimensional expansion of chemical mapping called lock-mutate-map-rescue (LM2R), which integrates multiple layers of mutation with nucleotide-resolution chemical mapping. This technique resolves the core mechanism of the adenine-responsive V. vulnificus add riboswitch, a paradigmatic system for which both Monod-Wyman-Changeux (MWC) conformational selection models and non-MWC alternatives have been proposed. To discriminate amongst these models, we locked each functionally important helix through designed mutations and assessed formation or depletion of other helices via compensatory rescue evaluated by chemical mapping. These LM2R measurements give strong support to the pre-existing correlations predicted by MWC models, disfavor alternative models, and suggest additional structural heterogeneities that may be general across ligand-free riboswitches.

Allosteric Logic of the V. vulnificus Adenine Riboswitch Resolved by Four-Dimensional Chemical Mapping

The structural logic that define the functions of gene regulatory RNA molecules may be radically different from classic models of allostery, but the relevant structural correlations have remained elusive in even intensively studied RNA model systems. Here, we present a four-dimensional expansion of chemical mapping called lock-mutate-map-rescue (LM2R), which integrates multiple layers of mutation with nucleotide-resolution chemical mapping. This technique resolves the core mechanism of the adenine-responsive V. vulnificus add riboswitch including its gene expression platform, a paradigmatic system for which both Monod-Wyman-Changeux (MWC) conformational selection models and non-MWC alternatives have been proposed. To discriminate amongst these models, we locked each functionally important helix through designed mutations and assessed formation or depletion of other helices via high- throughput compensatory rescue experiments. These LM2R measurements give strong support to the pre-existing correlations predicted by MWC models, disfavor alternative models, and reveal new structural heterogeneities that may be general across ligand-free riboswitches.

Network of Conformational Transitions Revealed by Molecular Dynamics Simulations of the Carbonic Anhydrase II Apo-Enzyme.

Human carbonic anhydrase II (HCA II) is an enzyme that catalyzes the reversible hydration of CO2 into bicarbonate (HCO3–) and a proton (H+) as well as other reactions at an extremely high rate. This enzyme plays fundamental roles in human physiology/pathology, such as controlling the pH level in cells and so on. However, the binding mechanism between apo-HCA II and CO2 or other ligands as well as related conformational changes remains poorly understood, and atomic investigation into it could promote our understanding of related internal physiological/pathological mechanisms. In this study, long-time atomic molecular dynamics simulations as well as the clustering and free-energy analysis were performed to reveal the dynamics of apo-HCA II as well as the mechanism upon ligand binding. Our simulations indicate that the crystallographic B-factors considerably underestimate the loop dynamics: multiple conformations can be adopted by loops 1 and 2, especially for loop 1 because loop 1 is one side of the binding pocket, and its left-to-right movement can compress or extend the binding pocket, leading to one inactive (closed) state, three intermediate (semiopen) states, and one active (open) state; CO2 cannot get into the binding pocket of the inactive state but can get into those of intermediate and active states. The coexistence of multiple conformational states proposes a possible conformational selection model for the binding mechanism between apo-HCA II and CO2 or other ligands, revising our previous view of its functional mechanism of conformational change upon ligand binding and offering valuable structural insights into the workings of HCA II.

The molten-globule residual structure is critical for reflavination of glucose oxidase

Glucose oxidase (GOX) is a homodimeric glycoprotein with tightly bound one molecule of FAD cofactor per monomer of the protein. GOX has numerous applications, but the preparation of biotechnologically interesting GOX sensors requires a removal of the native FAD cofactor. This process often leads to unwanted irreversible deflavination and, as a consequence, to the low enzyme recovery. Molecular mechanisms of reversible reflavination are poorly understood; our current knowledge is based only on empiric rules, which is clearly insufficient for further development. To develop conceptual understanding of flavin-binding competent states, we studied the effect of deflavination protocols on conformational properties of GOX. After deflavination, the apoform assembles into soluble oligomers with nearly native-like holoform secondary structure but largely destabilized tertiary structure presumambly due to the packing density defects around the vacant flavin binding site. The reflavination is cooperative but not fully efficient; after the binding the flavin cofactor, the protein directly disassembles into native homodimers while the fraction of oligomers remains irreversibly inactivated. Importantly, the effect of Hofmeister salts on the conformational properties of GOX and reflavination efficiency indicates that the native-like residual tertiary structure in the molten-globule states favorably supports the reflavination and minimizes the inactivated oligomers. We interpret our results by combining the ligand-induced changes in quaternary structure with salt-sensitive, non-equilibrated conformational selection model. In summary, our work provides the very first steps toward molecular understanding the complexity of the GOX reflavination mechanism.

Substrate binding interferes with active site conformational dynamics in endoglucanase Cel5A from Thermobifida fusca

The role of protein dynamics in enzyme catalysis is one of the most active areas in current enzymological research. Here, using endoglucanase Cel5A from Thermobifida fusca (TfCel5A) as a model, we applied molecular dynamics simulations to explore the dynamic behavior of the enzyme upon substrate binding. The collective motions of the active site revealed that the mechanism of TfCel5A substrate binding can likely be described by the conformational-selection model; however, we observed that the conformations of active site residues changed differently along with substrate binding. Although most active site residues retained their native conformational ensemble, some (Tyr163 and Glu355) generated newly induced conformations, whereas others (Phe162 and Tyr189) exhibited shifts in the equilibration of their conformational distributions. These results showed that TfCel5A substrate binding relied on a hybrid mechanism involving induced fit and conformational selection. Interestingly, we found that TfCel5A active site could only partly rebalance its conformational dynamics upon substrate dissociation within the same simulation time, which implies that the conformational rebalance upon substrate dissociation is likely more difficult than the conformational selection upon substrate binding at least in the view of the time required. Our findings offer new insight into enzyme catalysis and potential applications for future protein engineering.

Structural basis for ligand binding to an enzyme by a conformational selection pathway

Proteins can bind target molecules through either induced fit or conformational selection pathways. In the conformational selection model, a protein samples a scarcely populated high-energy state that resembles a target-bound conformation. In enzymatic catalysis, such high-energy states have been identified as crucial entities for activity and the dynamic interconversion between ground states and high-energy states can constitute the rate-limiting step for catalytic turnover. The transient nature of these states has precluded direct observation of their properties. Here, we present a molecular description of a high-energy enzyme state in a conformational selection pathway by an experimental strategy centered on NMR spectroscopy, protein engineering, and X-ray crystallography. Through the introduction of a disulfide bond, we succeeded in arresting the enzyme adenylate kinase in a closed high-energy conformation that is on-pathway for catalysis. A 1.9-Å X-ray structure of the arrested enzyme in complex with a transition state analog shows that catalytic sidechains are properly aligned for catalysis. We discovered that the structural sampling of the substrate free enzyme corresponds to the complete amplitude that is associated with formation of the closed and catalytically active state. In addition, we found that the trapped high-energy state displayed improved ligand binding affinity, compared with the wild-type enzyme, demonstrating that substrate binding to the high-energy state is not occluded by steric hindrance. Finally, we show that quenching of fast time scale motions observed upon ligand binding to adenylate kinase is dominated by enzyme-substrate interactions and not by intramolecular interactions resulting from the conformational change.

A Network of Conformational Transitions in the Apo Form of NDM-1 Enzyme Revealed by MD Simulation and a Markov State Model

New Delhi metallo-β-lactamase-1 (NDM-1) is a novel β-lactamase enzyme that confers enteric bacteria with nearly complete resistance to all β-lactam antibiotics, so it raises a formidable and global threat to human health. However, the binding mechanism between apo-NDM-1 and antibiotics as well as related conformational changes remains poorly understood, which largely hinders the overcoming of its antibiotic resistance. In our study, long-time conventional molecular dynamics simulation and Markov state models were applied to reveal both the dynamical and conformational landscape of apo-NDM-1: the MD simulation demonstrates that loop L3, which is responsible for antibiotic binding, is the most flexible and undergoes dramatic conformational changes; moreover, the Markov state model built from the simulation maps four metastable states including open, semiopen, and closed conformations of loop L3 as well as frequent transitions between the states. Our findings propose a possible conformational selection model for the binding mechanism between apo-NDM-1 and antibiotics, which facilitates the design of novel inhibitors and antibiotics.

Crystal Structures of the Global Regulator DasR from Streptomyces coelicolor: Implications for the Allosteric Regulation of GntR/HutC Repressors.

Small molecule effectors regulate gene transcription in bacteria by altering the DNA-binding affinities of specific repressor proteins. Although the GntR proteins represent a large family of bacterial repressors, only little is known about the allosteric mechanism that enables their function. DasR from Streptomyces coelicolor belongs to the GntR/HutC subfamily and specifically recognises operators termed DasR-responsive elements (dre-sites). Its DNA-binding properties are modulated by phosphorylated sugars. Here, we present several crystal structures of DasR, namely of dimeric full-length DasR in the absence of any effector and of only the effector-binding domain (EBD) of DasR without effector or in complex with glucosamine-6-phosphate (GlcN-6-P) and N-acetylglucosamine-6-phosphate (GlcNAc-6-P). Together with molecular dynamics (MD) simulations and a comparison with other GntR/HutC family members these data allowed for a structural characterisation of the different functional states of DasR. Allostery in DasR and possibly in many other GntR/HutC family members is best described by a conformational selection model. In ligand-free DasR, an increased flexibility in the EBDs enables the attached DNA-binding domains (DBD) to sample a variety of different orientations and among these also a DNA-binding competent conformation. Effector binding to the EBDs of DasR significantly reorganises the atomic structure of the latter. However, rather than locking the orientation of the DBDs, the effector-induced formation of β-strand β* in the DBD-EBD-linker segment merely appears to take the DBDs ‘on a shorter leash’ thereby impeding the ‘downwards’ positioning of the DBDs that is necessary for a concerted binding of two DBDs of DasR to operator DNA.

Protein Binding Pocket Dynamics.

The dynamics of protein binding pockets are crucial for their interaction specificity. Structural flexibility allows proteins to adapt to their individual molecular binding partners and facilitates the binding process. This implies the necessity to consider protein internal motion in determining and predicting binding properties and in designing new binders. Although accounting for protein dynamics presents a challenge for computational approaches, it expands the structural and physicochemical space for compound design and thus offers the prospect of improved binding specificity and selectivity. A cavity on the surface or in the interior of a protein that possesses suitable properties for binding a ligand is usually referred to as a binding pocket. The set of amino acid residues around a binding pocket determines its physicochemical characteristics and, together with its shape and location in a protein, defines its functionality. Residues outside the binding site can also have a long-range effect on the properties of the binding pocket. Cavities with similar functionalities are often conserved across protein families. For example, enzyme active sites are usually concave surfaces that present amino acid residues in a suitable configuration for binding low molecular weight compounds. Macromolecular binding pockets, on the other hand, are located on the protein surface and are often shallower. The mobility of proteins allows the opening, closing, and adaptation of binding pockets to regulate binding processes and specific protein functionalities. For example, channels and tunnels can exist permanently or transiently to transport compounds to and from a binding site. The influence of protein flexibility on binding pockets can vary from small changes to an already existent pocket to the formation of a completely new pocket. Here, we review recent developments in computational methods to detect and define binding pockets and to study pocket dynamics. We introduce five different classes of protein pocket dynamics: (1) appearance/disappearance of a subpocket in an existing pocket; (2) appearance/disappearance of an adjacent pocket on the protein surface in the direct vicinity of an already existing pocket; (3) pocket breathing, which may be caused by side-chain fluctuations or backbone or interdomain vibrational motion; (4) opening/closing of a channel or tunnel, connecting a pocket inside the protein with solvent, including lid motion; and (5) the appearance/disappearance of an allosteric pocket at a site on a protein distinct from an already existing pocket with binding of a ligand to the allosteric binding site affecting the original pocket. We suggest that the class of pocket dynamics, as well as the type and extent of protein motion affecting the binding pocket, should be factors considered in choosing the most appropriate computational approach to study a given binding pocket. Furthermore, we examine the relationship between pocket dynamics classes and induced fit, conformational selection, and gating models of ligand binding on binding kinetics and thermodynamics. We discuss the implications of protein binding pocket dynamics for drug design and conclude with potential future directions for computational analysis of protein binding pocket dynamics.

Mapping the Free Energy Landscape of PKA Inhibition and Activation: A Double-Conformational Selection Model for the Tandem cAMP-Binding Domains of PKA RIα.

Protein Kinase A (PKA) is the major receptor for the cyclic adenosine monophosphate (cAMP) secondary messenger in eukaryotes. cAMP binds to two tandem cAMP-binding domains (CBD-A and -B) within the regulatory subunit of PKA (R), unleashing the activity of the catalytic subunit (C). While CBD-A in RIα is required for PKA inhibition and activation, CBD-B functions as a “gatekeeper” domain that modulates the control exerted by CBD-A. Preliminary evidence suggests that CBD-B dynamics are critical for its gatekeeper function. To test this hypothesis, here we investigate by Nuclear Magnetic Resonance (NMR) the two-domain construct RIα (91–379) in its apo, cAMP2, and C-bound forms. Our comparative NMR analyses lead to a double conformational selection model in which each apo CBD dynamically samples both active and inactive states independently of the adjacent CBD within a nearly degenerate free energy landscape. Such degeneracy is critical to explain the sensitivity of CBD-B to weak interactions with C and its high affinity for cAMP. Binding of cAMP eliminates this degeneracy, as it selectively stabilizes the active conformation within each CBD and inter-CBD contacts, which require both cAMP and W260. The latter is contributed by CBD-B and mediates capping of the cAMP bound to CBD-A. The inter-CBD interface is dispensable for intra-CBD conformational selection, but is indispensable for full activation of PKA as it occludes C-subunit recognition sites within CBD-A. In addition, the two structurally homologous cAMP-bound CBDs exhibit marked differences in their residual dynamics profiles, supporting the notion that conservation of structure does not necessarily imply conservation of dynamics.

Mechanism of cAMP Partial Agonism in Protein Kinase G (PKG)

Protein kinase G (PKG) is a major receptor of cGMP and controls signaling pathways often distinct from those regulated by cAMP. Hence, the selective activation of PKG by cGMP versus cAMP is critical. However, the mechanism of cGMP-versus-cAMP selectivity is only limitedly understood. Although the C-terminal cyclic nucleotide-binding domain B of PKG binds cGMP with higher affinity than cAMP, the intracellular concentrations of cAMP are typically higher than those of cGMP, suggesting that the cGMP-versus-cAMP selectivity of PKG is not controlled uniquely through affinities. Here, we show that cAMP is a partial agonist for PKG, and we elucidate the mechanism for cAMP partial agonism through the comparative NMR analysis of the apo, cGMP-, and cAMP-bound forms of the PKG cyclic nucleotide-binding domain B. We show that although cGMP activation is adequately explained by a two-state conformational selection model, the partial agonism of cAMP arises from the sampling of a third, partially autoinhibited state.

On the link between conformational changes, ligand binding and heat capacity

BackgroundConformational changes coupled to ligand binding constitute the structural and energetics basis underlying cooperativity, allostery and, in general, protein regulation. These conformational rearrangements are associated with heat capacity changes. ITC is a unique technique for studying binding interactions because of the simultaneous determination of the binding affinity and enthalpy, and for providing the best estimates of binding heat capacity changes. Scope of reviewStill controversial issues in ligand binding are the discrimination between the “conformational selection model” and the “induced fit model”, and whether or not conformational changes lead to temperature dependent apparent binding heat capacities. The assessment of conformational changes associated with ligand binding by ITC is discussed. In addition, the “conformational selection” and “induced fit” models are reconciled, and discussed within the context of intrinsically (partially) unstructured proteins. Major conclusionsConformational equilibrium is a major contribution to binding heat capacity changes. A simple model may explain both conformational selection and induced fit scenarios. A temperature-independent binding heat capacity does not necessarily indicate absence of conformational changes upon ligand binding. ITC provides information on the energetics of conformational changes associated with ligand binding (and other possible additional coupled equilibria). General significancePreferential ligand binding to certain protein states leads to an equilibrium shift that is reflected in the coupling between ligand binding and additional equilibria. This represents the structural/energetic basis of the widespread dependence of ligand binding parameters on temperature, as well as pH, ionic strength and the concentration of other chemical species. This article is part of a Special Issue entitled Microcalorimetry in the BioSciences — Principles and Applications, edited by Fadi Bou-Abdallah.

Microsecond simulations of mdm2 and its complex with p53 yield insight into force field accuracy and conformational dynamics.

The p53-MDM2 complex is both a major target for cancer drug development and a valuable model system for computational predictions of protein-ligand binding. To investigate the accuracy of molecular simulations of MDM2 and its complex with p53, we performed a number of long (200 ns to 1 µs) explicit-solvent simulations using a range of force fields. We systematically compared nine popular force fields (AMBER ff03, ff12sb, ff14sb, ff99sb, ff99sb-ildn, ff99sb-ildn-nmr, ff99sb-ildn-phi, CHARMM22*, and CHARMM36) against experimental chemical shift data, and found similarly accurate results, with microsecond simulations achieving better agreement compared to 200-ns trajectories. Although the experimentally determined apo structure has a closed binding cleft, simulations in all force fields suggest the apo state of MDM2 is highly flexible, and able to sample holo-like conformations, consistent with a conformational selection model. Initial structuring of the MDM2 lid region, known to competitively bind the binding cleft, is also observed in long simulations. Taken together, these results show molecular simulations can accurately sample conformations relevant for ligand binding. We expect this study to inform future computational work on folding and binding of MDM2 ligands.

CNMA: a framework of encounter complex-based normal mode analysis to model conformational changes in protein interactions.

Motivation: It remains both a fundamental and practical challenge to understand and anticipate motions and conformational changes of proteins during their associations. Conventional normal mode analysis (NMA) based on anisotropic network model (ANM) addresses the challenge by generating normal modes reflecting intrinsic flexibility of proteins, which follows a conformational selection model for protein–protein interactions. But earlier studies have also found cases where conformational selection alone could not adequately explain conformational changes and other models have been proposed. Moreover, there is a pressing demand of constructing a much reduced but still relevant subset of protein conformational space to improve computational efficiency and accuracy in protein docking, especially for the difficult cases with significant conformational changes.Method and results: With both conformational selection and induced fit models considered, we extend ANM to include concurrent but differentiated intra- and inter-molecular interactions and develop an encounter complex-based NMA (cNMA) framework. Theoretical analysis and empirical results over a large data set of significant conformational changes indicate that cNMA is capable of generating conformational vectors considerably better at approximating conformational changes with contributions from both intrinsic flexibility and inter-molecular interactions than conventional NMA only considering intrinsic flexibility does. The empirical results also indicate that a straightforward application of conventional NMA to an encounter complex often does not improve upon NMA for an individual protein under study and intra- and inter-molecular interactions need to be differentiated properly. Moreover, in addition to induced motions of a protein under study, the induced motions of its binding partner and the coupling between the two sets of protein motions present in a near-native encounter complex lead to the improved performance. A study to isolate and assess the sole contribution of intermolecular interactions toward improvements against conventional NMA further validates the additional benefit from induced-fit effects. Taken together, these results provide new insights into molecular mechanisms underlying protein interactions and new tools for dimensionality reduction for flexible protein docking.Availability and implementation: Source codes are available upon request.Contact: yshen@tamu.edu

Impact of stable protein-protein interaction on protein conformational space

Backbone dihedral angle based clustering approach was applied to investigate the effect of protein complexation on backbone conformational space and the effect on protein dynamics. Three representative enzyme-inhibitor complexes and their comprised proteins were used as models for small- and moderate-sized globular proteins to compare available backbone conformational space before and after complexation. Microsecond time scale molecular dynamic simulations were generated to ensure sufficient statistics. The result suggests that stable protein-protein interactions lead to redistribution of protein backbone mobility and restriction of the protein backbone conformational space, especially for short time scale motions. Surprisingly, these effects are found to be uncorrelated with protein-protein interaction surface. Consistent with many experimental and computational observations, our results indicate that both induced-fit and conformational selection models play roles in stable protein complexation process, with the dominant role being different for different protein complexes.

From Macroscopic to Molecular Interfaces: How do they Alter Protein Conformation?

Conformation of proteins and peptides are not solely determined by their sequence, but are also strongly dependent on the environmental conditions. In addition to factors like salt concentration or pH, the environment presented by the surrounding molecules also play a major role on the conformational behavior of proteins and peptides. In this study, by using molecular dynamics simulations, we perform a comparative study on the conformational behavior of LKalpha-14 peptides in bulk water vs. macroscopic and molecular interfaces. The molecule under study is designed to have an alpha-helical conformation with the sequence (LKKLLKL)2. Our replica exchange simulations show that, similar to intrinsically disordered proteins, this molecule lacks a unique conformation when isolated in bulk water. However, in the presence of an air/water interface the molecule uniquely adopts the alpha-helix conformation. Further more, such a stabilizing effect is not limited to macroscopic interfaces, but can also be seen in the presence of molecular interfaces presented by surrounding molecules. Even when they are in a disordered state, molecules in solution display temporary molecular interfaces as a result of segregation of their hydrophobic and hydrophilic residues. Our simulations show that, these temporary molecular interfaces act as molecular chaperons in both accelarating the folding of peptides and also stabilize the alpha helical conformation when it forms. In light of these results, LK peptide can be identified as a classic example for conformational selection model.

A New and Un-Conventional Ultrafast Binding Mechanism of Intrinsically Disordered Proteins to Structured Partners

Intrinsically disordered proteins (IDPs) mediate several and very diverse processes such as signaling, regulation and formation of entropic barriers. Such functions often involve the binding between an IDP and one or more structured partners. Several mechanisms to describe the binding of IDPs to structured proteins have been identified: the conformational selection model implies that, along the conformational ensemble explored by the IDP, binding-prone configurations are abundant and favor binding. In contrast, the induced-fit model describes binding as a series of conformational changes of the IDP that can form secondary structure elements upon binding. By investigating the association between the intrinsically disordered nucleoporin Nup153 and its structured binding partner Importin-β through means of single-molecule FRET experiments, molecular dynamics (MD) and Brownian dynamics (BD) simulations, we have identified a new mechanism that leads to the formation of complexes without the need of any structural re-arrangement or selection of a pre-configured conformation of the IDP. We found that binding can occur between Importin-β and highly diverse configurations of Nup153 in a globular-like state. Being conformational-independent, the association is solely regulated by the availability of nucleoporin's Phe-Gly dipeptide responsible for the interaction with Importin. More importantly, since conformational adaptations are absent, specific binding is rapid enough to be observed on the sub-microsecond time scale of the MD simulations. This ultrafast binding mechanism of nucleoporin to Importin-β can justify the observation of rapid, yet selective nucleo-cytoplasmatic transport. Our joint computational and experimental approach could help to explain if a similar binding scenario also applies to other repetitive IDPs.

Evidence of conformational selection driving the formation of ligand binding sites in protein-protein interfaces.

Many protein-protein interactions (PPIs) are compelling targets for drug discovery, and in a number of cases can be disrupted by small molecules. The main goal of this study is to examine the mechanism of binding site formation in the interface region of proteins that are PPI targets by comparing ligand-free and ligand-bound structures. To avoid any potential bias, we focus on ensembles of ligand-free protein conformations obtained by nuclear magnetic resonance (NMR) techniques and deposited in the Protein Data Bank, rather than on ensembles specifically generated for this study. The measures used for structure comparison are based on detecting binding hot spots, i.e., protein regions that are major contributors to the binding free energy. The main tool of the analysis is computational solvent mapping, which explores the surface of proteins by docking a large number of small “probe” molecules. Although we consider conformational ensembles obtained by NMR techniques, the analysis is independent of the method used for generating the structures. Finding the energetically most important regions, mapping can identify binding site residues using ligand-free models based on NMR data. In addition, the method selects conformations that are similar to some peptide-bound or ligand-bound structure in terms of the properties of the binding site. This agrees with the conformational selection model of molecular recognition, which assumes such pre-existing conformations. The analysis also shows the maximum level of similarity between unbound and bound states that is achieved without any influence from a ligand. Further shift toward the bound structure assumes protein-peptide or protein-ligand interactions, either selecting higher energy conformations that are not part of the NMR ensemble, or leading to induced fit. Thus, forming the sites in protein-protein interfaces that bind peptides and can be targeted by small ligands always includes conformational selection, although other recognition mechanisms may also be involved.