Electrostatically Accelerated Encounter and Folding for Facile Recognition of Intrinsically Disordered Proteins

Achieving facile specific recognition is essential for intrinsically disordered proteins (IDPs) that are involved in cellular signaling and regulation. Consideration of the physical time scales of protein folding and diffusion-limited protein-protein encounter has suggested that the frequent requirement of protein folding for specific IDP recognition could lead to kinetic bottlenecks. How IDPs overcome such potential kinetic bottlenecks to viably function in signaling and regulation in general is poorly understood. Our recent computational and experimental study of cell-cycle regulator p27 (Ganguly et al., J. Mol. Biol. (2012)) demonstrated that long-range electrostatic forces exerted on enriched charges of IDPs could accelerate protein-protein encounter via “electrostatic steering” and at the same time promote “folding-competent” encounter topologies to enhance the efficiency of IDP folding upon encounter. Here, we further investigated the coupled binding and folding mechanisms and the roles of electrostatic forces in the formation of three IDP complexes with more complex folded topologies. The surface electrostatic potentials of these complexes lack prominent features like those observed for the p27/Cdk2/cyclin A complex to directly suggest the ability of electrostatic forces to facilitate folding upon encounter. Nonetheless, similar electrostatically accelerated encounter and folding mechanisms were consistently predicted for all three complexes using topology-based coarse-grained simulations. Together with our previous analysis of charge distributions in known IDP complexes, our results support a prevalent role of electrostatic interactions in promoting efficient coupled binding and folding for facile specific recognition. These results also suggest that there is likely a co-evolution of IDP folded topology, charge characteristics, and coupled binding and folding mechanisms, driven at least partially by the need to achieve fast association kinetics for cellular signaling and regulation.     

Electrostatically accelerated coupled binding and folding of intrinsically disordered proteins

Intrinsically disordered proteins (IDPs) are now recognized to be prevalent in biology, and many potential functional benefits have been discussed. However, the frequent requirement of peptide folding in specific interactions of IDPs could impose a kinetic bottleneck, which could be overcome only by efficient folding upon encounter. Intriguingly, existing kinetic data suggest that specific binding of IDPs is generally no slower than that of globular proteins. Here, we exploited the cell cycle regulator p27(Kip1) (p27) as a model system to understand how IDPs might achieve efficient folding upon encounter for facile recognition. Combining experiments and coarse-grained modeling, we demonstrate that long-range electrostatic interactions between enriched charges on p27 and near its binding site on cyclin A not only enhance the encounter rate (i.e., electrostatic steering) but also promote folding-competent topologies in the encounter complexes, allowing rapid subsequent formation of short-range native interactions en route to the specific complex. In contrast, nonspecific hydrophobic interactions, while hardly affecting the encounter rate, can significantly reduce the efficiency of folding upon encounter and lead to slower binding kinetics. Further analysis of charge distributions in a set of known IDP complexes reveals that, although IDP binding sites tend to be more hydrophobic compared to the rest of the target surface, their vicinities are frequently enriched with charges to complement those on IDPs. This observation suggests that electrostatically accelerated encounter and induced folding might represent a prevalent mechanism for promoting facile IDP recognition.     

Modulation of Correlated Segment Fluctuations in IDPs upon Complex Formation as an Allosteric Regulatory Mechanism

Molecular recognition of and by intrinsically disordered proteins (IDPs) is an intriguing and still largely elusive phenomenon. Typically, protein recognition involving IDPs requires either folding upon binding or, alternatively, the formation of “fuzzy complexes.” Here we show via correlation analyses of paramagnetic relaxation enhancement data unprecedented and striking alterations of the concerted fluctuations within the conformational ensemble of IDPs upon ligand binding. We study the binding of α-synuclein to calmodulin, a ubiquitous calcium-binding protein, and the binding of the extracellular matrix IDP osteopontin to heparin, a mimic of the extracellular matrix ligand hyaluronic acid. In both cases, binding leads to reduction of correlated long-range motions in these two IDPs and thus indicates a loosening of structural compaction upon binding. Most importantly, however, the simultaneous presence of correlated and anti-correlated fluctuations in IDPs suggests the prevalence of “energetic frustration” and provides an explanation for the puzzling observation of disordered allostery in IDPs.     

Evolutionarily Conserved Network Properties of Intrinsically Disordered Proteins

BackgroundIntrinsically disordered proteins (IDPs) lack a stable tertiary structure in isolation. Remarkably, however, a substantial portion of IDPs undergo disorder-to-order transitions upon binding to their cognate partners. Structural flexibility and binding plasticity enable IDPs to interact with a broad range of partners. However, the broader network properties that could provide additional insights into the functional role of IDPs are not known.ResultsHere, we report the first comprehensive survey of network properties of IDP-induced sub-networks in multiple species from yeast to human. Our results show that IDPs exhibit greater-than-expected modularity and are connected to the rest of the protein interaction network (PIN) via proteins that exhibit the highest betweenness centrality and connect to fewer-than-expected IDP communities, suggesting that they form critical communication links from IDP modules to the rest of the PIN. Moreover, we found that IDPs are enriched at the top level of regulatory hierarchy.ConclusionOverall, our analyses reveal coherent and remarkably conserved IDP-centric network properties, namely, modularity in IDP-induced network and a layer of critical nodes connecting IDPs with the rest of the PIN.     

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.     

Intrinsic disorder mediates the diverse regulatory functions of the Cdk inhibitor p21

Traditionally, well-defined three-dimensional structure has been thought to be essential for protein function. However, myriad biological functions are performed by highly dynamic, intrinsically disordered proteins (IDPs). IDPs often fold upon binding their biological targets and frequently show 'binding diversity' by targeting multiple ligands. We sought to understand the physical basis of IDP binding diversity and report here that the cyclin-dependent kinase (Cdk) inhibitor p21(Cip1) adaptively binds to and inhibits the various Cdk-cyclin complexes that regulate eukaryotic cell division. Using results from NMR spectroscopy and biochemical and cellular assays, we show that structural adaptability of a helical subdomain within p21, termed LH, enables two other subdomains, D1 and D2, to specifically bind conserved surface features of the cyclin and Cdk subunits, respectively, within otherwise structurally distinct Cdk-cyclin complexes. Adaptive folding upon binding is likely to mediate the diverse biological functions of the thousands of IDPs present in eukaryotes.     

Unveiling induced folding of intrinsically disordered proteins – Protein engineering,frustration and emerging themes

Intrinsically disordered proteins (IDPs) can be generally described as a class of proteins that lack a well-defined ordered structure in isolation at physiological conditions. Upon binding to their physiological ligands, IDPs typically undergo a disorder-to-order transition, which may or may not lead to the complete folding of the IDP. In this short review, we focus on some of the key findings pertaining to the mechanisms of such induced folding. In particular, first we describe the general features of the reaction; then, we discuss some of the most remarkable findings obtained from applying protein engineering in synergy with kinetic studies to induced folding; and finally, we offer a critical view on some of the emerging themes when considering the structural heterogeneity of IDPs vis-à-vis to their inherent frustration.     

Residual Structure Accelerates Binding of Intrinsically Disordered ACTR by Promoting Efficient Folding upon Encounter

Intrinsically disordered proteins (IDPs) often fold into stable structures upon specific binding. The roles of residual structure of unbound IDPs in coupling binding and folding have been under much debate. While many studies emphasize the importance of conformational flexibility for IDP recognition, it was recently demonstrated that stabilization the N-terminal helix of intrinsically disordered ACTR accelerated its binding to another IDP, NCBD of the CREB-binding protein. To understand how enhancing ACTR helicity accelerates binding, we derived a series of topology-based coarse-grained models that mimicked various ACTR mutants with increasing helical contents and reproduced their NCBD binding affinities. Molecular dynamics simulations were then performed to sample hundreds of reversible coupled binding and folding transitions. The results show that increasing ACTR helicity does not alter the baseline mechanism of synergistic folding, which continues to follow “extended conformational selection” with multiple stages of selection and induced folding. Importantly, these coarse-grained models, while only calibrated based on binding thermodynamics, recapitulate the observed kinetic acceleration with increasing ACTR helicity. However, the residual helices do not enhance the association kinetics via more efficient seeding of productive collisions. Instead, they allow the nonspecific collision complexes to evolve more efficiently into the final bound and folded state, which is the primary source of accelerated association kinetics. Meanwhile, reduced dissociation kinetics with increasing ACTR helicity can be directly attributed to smaller entropic cost of forming the bound state. Altogether, this study provides important mechanistic insights into how residual structure may modulate thermodynamics and kinetics of IDP interactions.     

The Disordered Region of the HCV Protein NS5A: Conformational Dynamics,SH3 Binding,and Phosphorylation

Intrinsically disordered proteins (IDPs) perform their physiological role without possessing a well-defined three-dimensional structure. Still, residual structure and conformational dynamics of IDPs are crucial for the mechanisms underlying their functions. For example, regions of transient secondary structure are often involved in molecular recognition, with the structure being stabilized (or not) upon binding. Long-range interactions, on the other hand, determine the hydrodynamic radius of the IDP, and thus the distance over which the protein can catch binding partners via so-called fly-casting mechanisms. The modulation of long-range interactions also presents a convenient way of fine-tuning the protein’s interaction network, by making binding sites more or less accessible. Here we studied, mainly by nuclear magnetic resonance spectroscopy, residual secondary structure and long-range interactions in nonstructural protein 5A (NS5A) from hepatitis C virus (HCV), a typical viral IDP with multiple functions during the viral life cycle. NS5A comprises an N-terminal folded domain, followed by a large (∼250-residue) disordered C-terminal part. Comparing nuclear magnetic resonance spectra of full-length NS5A with those of a protein construct composed of only the C-terminal residues 191–447 (NS5A-D2D3) allowed us to conclude that there is no significant interaction between the globular and disordered parts of NS5A. NS5A-D2D3, despite its overall high flexibility, shows a large extent of local residual (α-helical and β-turn) structure, as well as a network of electrostatic long-range interactions. Furthermore, we could demonstrate that these long-range interactions become modulated upon binding to the host protein Bin1, as well as after NS5A phosphorylation by CK2. As the charged peptide regions involved in these interactions are well conserved among the different HCV genotypes, these transient long-range interactions may be important for some of the functions of NS5A over the course of the HCV life cycle.     

Features of molecular recognition of intrinsically disordered proteins via coupled folding and binding

The sequence–structure–function paradigm of proteins has been revolutionized by the discovery of intrinsically disordered proteins (IDPs) or intrinsically disordered regions (IDRs). In contrast to traditional ordered proteins, IDPs/IDRs are unstructured under physiological conditions. The absence of well‐defined three‐dimensional structures in the free state of IDPs/IDRs is fundamental to their function. Folding upon binding is an important mode of molecular recognition for IDPs/IDRs. While great efforts have been devoted to investigating the complex structures and binding kinetics and affinities, our knowledge on the binding mechanisms of IDPs/IDRs remains very limited. Here, we review recent advances on the binding mechanisms of IDPs/IDRs. The structures and kinetic parameters of IDPs/IDRs can vary greatly, and the binding mechanisms can be highly dependent on the structural properties of IDPs/IDRs. IDPs/IDRs can employ various combinations of conformational selection and induced fit in a binding process, which can be templated by the target and/or encoded by the IDP/IDR. Further studies should provide deeper insights into the molecular recognition of IDPs/IDRs and enable the rational design of IDP/IDR binding mechanisms in the future.     

Partner-Mediated Polymorphism of an Intrinsically Disordered Protein

Intrinsically disordered proteins (IDPs) recognize their partners through molecular recognition elements (MoREs). The MoRE of the C-terminal intrinsically disordered domain of the measles virus nucleoprotein (N_TAIL) is partly pre-configured as an α-helix in the free form and undergoes α-helical folding upon binding to the X domain (XD) of the viral phosphoprotein. Beyond XD, N_TAIL also binds the major inducible heat shock protein 70 (hsp70). So far, no structural information is available for the N_TAIL/hsp70 complex. Using mutational studies combined with a protein complementation assay based on green fluorescent protein reconstitution, we have investigated both N_TAIL/XD and N_TAIL/hsp70 interactions. Although the same N_TAIL region binds the two partners, the binding mechanisms are different. Hsp70 binding is much more tolerant of MoRE substitutions than XD, and the majority of substitutions lead to an increased N_TAIL/hsp70 interaction strength. Furthermore, while an increased and a decreased α-helicity of the MoRE lead to enhanced and reduced interaction strength with XD, respectively, the impact on hsp70 binding is negligible, suggesting that the MoRE does not adopt an α-helical conformation once bound to hsp70. Here, by showing that the α-helical conformation sampled by the free form of the MoRE does not systematically commit it to adopt an α-helical conformation in the bound form, we provide an example of partner-mediated polymorphism of an IDP and of the relative insensitiveness of the bound structure to the pre-recognition state. The present results therefore contribute to shed light on the molecular mechanisms by which IDPs recognize different partners.     

Intrinsically disordered proteins may escape unwanted interactions via functional misfolding

Intrinsically disordered proteins are highly abundant in nature and play a number of crucial roles in the living cells. They are commonly involved in a wide range of intermolecular interactions, and some of them possess remarkable binding promiscuity, being able to interact specifically with structurally unrelated partners. Although they do not have well-folded structure, some IDPs are known to fold at binding to their specific partners. IDPs are highly pliable and one IDP can form an array of unrelated structures being bound to different partners. It is believed that many IDPs, being mostly disordered, have transient elements of the preformed secondary structure which are highly interaction prone and is used by IDPs for binding to specific partners. The overall disordered nature of IDPs, their high conformational dynamics and flexibility, the presence of sticky preformed binding elements, and their ability to morph into differently-shaped bound configurations raised a very important question about the mechanisms preventing IDPs from unwanted interactions with non-native partners. In this review, a concept of functional misfolding is introduced. Accumulated to date data on the conformational behavior and fine structure of several IDPs suggest that the preformed binding elements might be involved in a set of non-native intramolecular interactions. In other words, there is a chance that a polypeptide chain misfolds to sequester the preformed elements inside the non-interactive or less-interactive cage, therefore preventing these elements from the unnecessary and unwanted interactions with non-native binding partners.     

How multisite phosphorylation impacts the conformations of intrinsically disordered proteins

Phosphorylation of intrinsically disordered proteins (IDPs) can produce changes in structural and dynamical properties and thereby mediate critical biological functions. How phosphorylation effects intrinsically disordered proteins has been studied for an increasing number of IDPs, but a systematic understanding is still lacking. Here, we compare the collapse propensity of four disordered proteins, Ash1, the C-terminal domain of RNA polymerase (CTD2’), the cytosolic domain of E-Cadherin, and a fragment of the p130Cas, in unphosphorylated and phosphorylated forms using extensive all-atom molecular dynamics (MD) simulations. We find all proteins to show V-shape changes in their collapse propensity upon multi-site phosphorylation according to their initial net charge: phosphorylation expands neutral or overall negatively charged IDPs and shrinks positively charged IDPs. However, force fields including those tailored towards and commonly used for IDPs overestimate these changes. We find quantitative agreement of MD results with SAXS and NMR data for Ash1 and CTD2’ only when attenuating protein electrostatic interactions by using a higher salt concentration (e.g. 350 mM), highlighting the overstabilization of salt bridges in current force fields. We show that phosphorylation of IDPs also has a strong impact on the solvation of the protein, a factor that in addition to the actual collapse or expansion of the IDP should be considered when analyzing SAXS data. Compared to the overall mild change in global IDP dimension, the exposure of active sites can change significantly upon phosphorylation, underlining the large susceptibility of IDP ensembles to regulation through post-translational modifications.     

Intrinsically Disordered Proteins and Their Environment: Effects of Strong Denaturants, Temperature, pH, Counter Ions, Membranes, Binding Partners, Osmolytes, and Macromolecular Crowding

Intrinsically disordered proteins (IDPs) differ from “normal” ordered proteins at several levels, structural, functional and conformational. Amino acid biases characteristic for IDPs determine their structural variability and lack of rigid well-folded structure. This structural plasticity is necessary for the unique functional repertoire of IDPs, which is complementary to the catalytic activities of ordered proteins. Amino acid biases also drive atypical responses of IDPs to changes in their environment. The conformational behavior of IDPs is characterized by the low cooperativity (or the complete lack thereof) of the denaturant-induced unfolding, lack of the measurable excess heat absorption peak(s) characteristic for the melting of ordered proteins, “turned out” response to heat and changes in pH, the ability to gain structure in the presence of various counter ions, osmolytes, membranes and binding partners, and by the unique response to macromolecular crowding. This review describes some of the most characteristic features of the IDP conformational behavior and the unique response of IDPs to changes in their environment.