Topology-based modeling of intrinsically disordered proteins: balancing intrinsic folding and intermolecular interactions

Coupled binding and folding is frequently involved in specific recognition of so-called intrinsically disordered proteins (IDPs), a newly recognized class of proteins that rely on a lack of stable tertiary fold for function. Here, we exploit topology-based Gō-like modeling as an effective tool for the mechanism of IDP recognition within the theoretical framework of minimally frustrated energy landscape. Importantly, substantial differences exist between IDPs and globular proteins in both amino acid sequence and binding interface characteristics. We demonstrate that established Gō-like models designed for folded proteins tend to over-estimate the level of residual structures in unbound IDPs, whereas under-estimating the strength of intermolecular interactions. Such systematic biases have important consequences in the predicted mechanism of interaction. A strategy is proposed to recalibrate topology-derived models to balance intrinsic folding propensities and intermolecular interactions, based on experimental knowledge of the overall residual structure level and binding affinity. Applied to pKID/KIX, the calibrated Gō-like model predicts a dominant multistep sequential pathway for binding-induced folding of pKID that is initiated by KIX binding via the C-terminus in disordered conformations, followed by binding and folding of the rest of C-terminal helix and finally the N-terminal helix. This novel mechanism is consistent with key observations derived from a recent NMR titration and relaxation dispersion study and provides a molecular-level interpretation of kinetic rates derived from dispersion curve analysis. These case studies provide important insight into the applicability and potential pitfalls of topology-based modeling for studying IDP folding and interaction in general.     

The ensemble folding kinetics of the FBP28 WW domain revealed by an all-atom Monte Carlo simulation in a knowledge-based potential

In this work, we apply a detailed all‐atom model with a transferable knowledge‐based potential to study the folding kinetics of Formin‐Binding protein, FBP28, which is a canonical three‐stranded β‐sheet WW domain. Replica exchange Monte Carlo simulations starting from random coils find native‐like (Cα RMSD of 2.68 Å) lowest energy structure. We also study the folding kinetics of FBP28 WW domain by performing a large number of ab initio Monte Carlo folding simulations. Using these trajectories, we examine the order of formation of two β‐hairpins, the folding mechanism of each individual β‐hairpin, and transition state ensemble (TSE) of FBP28 WW domain and compare our results with experimental data and previous computational studies. To obtain detailed structural information on the folding dynamics viewed as an ensemble process, we perform a clustering analysis procedure based on graph theory. Further, a rigorous P_fold analysis is used to obtain representative samples of the TSEs showing good quantitative agreement between experimental and simulated Φ values. Our analysis shows that the turn structure between first and second β strands is a partially stable structural motif that gets formed before entering the TSE in FBP28 WW domain and there exist two major pathways for the folding of FBP28 WW domain, which differ in the order and mechanism of hairpin formation. Proteins 2011. © 2011 Wiley‐Liss, Inc.     

Transient exposure of a buried phosphorylation site in an autoinhibited protein

Autoinhibition is a mechanism used to regulate protein function, often by making functional sites inaccessible through the interaction with a cis-acting inhibitory domain. Such autoinhibitory domains often display a substantial degree of structural disorder when unbound, and only become structured in the inhibited state. These conformational dynamics make it difficult to study the structural origin of regulation, including effects of regulatory post-translational modifications. Here, we study the autoinhibition of the Dbl Homology domain in the protein Vav1 by the so-called acidic inhibitory domain. We use molecular simulations to study the process by which a mostly unstructured inhibitory domain folds upon binding and how transient exposure of a key buried tyrosine residue makes it accessible for phosphorylation. We show that the inhibitory domain, which forms a helix in the bound and inhibited stated, samples helical structures already before binding and that binding occurs via a molten-globule-like intermediate state. Together, our results shed light on key interactions that enable the inhibitory domain to sample a finely tuned equilibrium between an inhibited and a kinase-accessible state.     

Protein conformational transitions coupled to binding in molecular recognition of unstructured proteins: deciphering the effect of intermolecular interactions on computational structure prediction of the p27Kip1 protein bound to the cyclin A-cyclin-dependent kinase 2 complex

The relationship between folding mechanism coupled to binding and structure prediction of the tertiary complexes is studied for the p27(Kip) (1) protein which has an intrinsically disordered unbound form and undergoes a functional folding transition during complex formation with the phosphorylated cyclin A-cyclin-dependent kinase 2 (Cdk2) binary complex. Hierarchy of p27(Kip1) structural loss determined in our earlier studies from temperature-induced Monte Carlo simulations and subsequent characterization of the transition state ensemble (TSE) for the folding reaction have shown that simultaneous ordering of the p27(Kip1) native intermolecular interface for the beta-hairpin and beta-strand secondary structure elements is critical for nucleating a rapid kinetic transition to the native tertiary complex. In the present study, we investigate the effect of forming specific intermolecular interactions on structure prediction of the p27(Kip1) tertiary complex. By constraining different secondary structure elements of p27(Kip1) in their native bound conformations and conducting multiple simulated annealing simulations, we analyze differences in the success rate of predicting the native structure of p27(Kip1) in the tertiary complex. In accordance with the nucleation-condensation mechanism, we have found that further stabilization of the native intermolecular interface for the beta-hairpin and beta-strand elements of p27(Kip1), that become ordered in the TSE, but are hardly populated in the unbound state, results in a consistent acquisition of the native bound structure. Conversely, the excessive stablization of the local secondary structure elements, which are rarely detected in the TSE, has a detrimental effect on convergence to the native bound structure.     

Experiment and theory highlight role of native state topology in SH3 folding.

We use a combination of experiments, computer simulations and simple model calculations to characterize, first, the folding transition state ensemble of the src SH3 domain, and second, the features of the protein that determine its folding mechanism. Kinetic analysis of mutations at 52 of the 57 residues in the src SH3 domain revealed that the transition state ensemble is even more polarized than suspected earlier: no single alanine substitution in the N-terminal 15 residues or the C-terminal 9 residues has more than a two-fold effect on the folding rate, while such substitutions at 15 sites in the central three-stranded beta-sheet cause significant decreases in the folding rate. Molecular dynamics (MD) unfolding simulations and ab initio folding simulations on the src SH3 domain exhibit a hierarchy of folding similar to that observed in the experiments. The similarity in folding mechanism of different SH3 domains and the similar hierarchy of structure formation observed in the experiments and the simulations can be largely accounted for by a simple native state topology-based model of protein folding energy landscapes.     

Smoothing molecular interactions: the "kinetic buffer" effect of intrinsically disordered proteins

Intrinsically disordered proteins (IDPs) widely participate in molecular recognition and signaling processes in cells by interacting with other molecules. Compared with ordered proteins, IDPs usually possess stronger intermolecular interactions in binding. As a result, the interface structure of IDPs in complexes is distinct from that of ordered-protein complexes, and this difference may have essential effect on the response to various perturbations in a cell. In this study, we examined the perturbations of intermolecular interactions and temperature on the coupled folding and binding processes of pKID to KIX domains by performing molecular dynamics simulations. By comparing a series of virtual pKID systems with various degree of disorder, we found that the complex stability and the binding kinetics of the disordered systems were less sensitive to the perturbations than the ordered systems. The origin of the lower response sensitivity of IDPs was attributed to their higher flexibility in the complex interface, which was further supported by an analysis on protein complex structures. On the basis of our simulations and results from the literature, we speculate IDPs may not only interact with their biological partners with high specificity and low affinity but also may be resistant to the perturbations in the environment and transmit signals fast and smooth. We proposed to name it the "kinetic buffer" effect.     

Enhanced Sampling of Coarse-Grained Transmembrane-Peptide Structure Formation from Hydrogen-Bond Replica Exchange

Protein structure formation in the membrane highlights a grand challenge of sampling in computer simulations, because kinetic traps and slow dynamics make it difficult to find the native state. Exploiting increased fluctuations at higher temperatures can help overcome free-energy barriers, provided the membrane’s structure remains stable. In this work, we apply Hamiltonian replica-exchange molecular dynamics, where we only tune the backbone hydrogen-bond strength to help reduce the propensity of long-lived misfolded states. Using a recently developed coarse-grained model, we illustrate the robustness of the method by folding different WALP transmembrane helical peptides starting from stretched, unstructured conformations. We show the efficiency of the method by comparing to simulations without enhanced sampling, achieving folding in one example after significantly longer simulation times. Analysis of the bilayer structure during folding provides insight into the local membrane deformation during helix formation as a function of chain length (from 16 to 23 residues). Finally, we apply our method to fold the 50-residue-long major pVIII coat protein (fd coat) of the filamentous fd bacteriophage. Our results agree well with experimental structures and atomistic simulations based on implicit membrane models, suggesting that our explicit CG folding protocol can serve as a starting point for better-refined atomistic simulations in a multiscale framework.     

A preformed compact ribosome-binding domain in the cricket paralysis-like virus IRES RNAs

The internal ribosome site RNA of the cricket paralysis-like viruses (CrPV-like) binds directly to the ribosome, assembling the translation machinery without initiation factors. This mechanism does not require initiator tRNA, and translation starts from a non-AUG codon. A wealth of biochemical data has yielded a working model for this process, but the three-dimensional structure and biophysical characteristics of the unbound CrPV-like IRES RNAs are largely unexplored. Here, we demonstrate that the CrPV-like IRESes prefold into a two-part structure in the presence of magnesium ions. The largest part is a prefolded compact RNA domain that shares folding and structural characteristics with other compactly folded RNAs such as group I intron RNAs and RNase P RNA. Chemical probing reveals that the CrPV-like IRES' compact domain contains RNA helices that are packed tightly enough to exclude solvent, and analytical ultracentrifugation indicates a large change in the shape of the IRES upon folding. Formation of this compact domain is necessary for binding of the 40S subunit, and the structural organization of the unbound IRES RNA is consistent with the hypothesis that the IRES is functionally and structurally preorganized before ribosome binding.     

Slow folding of a three-helix protein via a compact intermediate

The KIX domain of CREB binding protein (CBP) forms a small three-helix bundle which folds autonomously. Previous equilibrium unfolding experiments led to the suggestion that folding may not be strictly two-state. To investigate the folding mechanism in more detail, the folding kinetics of KIX have been studied by urea jump fluorescence-detected stopped-flow experiments. Clear evidence for an intermediate is obtained from the plot of the natural log of the observed rate constant versus denaturant concentration, the chevron plot, and from analysis of the initial fluorescence amplitudes of the stopped-flow experiments. The chevron plot exhibits a change in shape, rollover, at low denaturant concentrations, characteristic of the formation of an intermediate. The kinetic data can be fit to a three-state model involving a compact intermediate. An on-pathway model predicts that the position of the intermediate lies close to the native state. The folding rate in the absence of denaturant is 260 s(-)(1) at pH 7.5 and 25 degrees C. This is significantly slower than the rates of other helical proteins similar in size. The slow folding may be due to the necessity of forming a buried polar interaction in the native state. The potential functional significance of the folding intermediate is discussed.     

Comparison of the transition states for folding of two Ig-like proteins from different superfamilies

In the "fold approach" proteins with a similar fold but different sequences are compared in order to investigate the relationship between native state structure and folding behaviour. Here we compare the properties of the transition states for folding of TI I27, the 27th immunoglobulin domain from human cardiac titin, and that of TNfn3, the third fibronectin type III domain from human tenascin. Experimental phi-values were used as restraints in molecular dynamics simulations to determine the structures that make up the transition state ensembles (TSEs) for folding of the two proteins. The restrained simulations that we present allow a detailed structural comparison of the two TSEs to be made. Further calculations show explicitly that for both proteins the formation of the interactions involving the residues in the folding nucleus is sufficient for the establishment of the topology of the Ig-like fold. We found that, although the folding nuclei of the two proteins are similar, the packing of the folding nucleus of TI I27 is much tighter than that of TNfn3, reflecting the higher experimental phi-values and beta(T) (Tanford Beta) of TI I27. These results suggest that the folding nucleus can be significantly deformed to accommodate extensive sequence variation while conserving the same folding mechanism.