Binding and folding in transcriptional complexes.

Transcription factors are among the classes of proteins with the highest levels of disorder. Investigation of these regulatory proteins is uncovering not just the mechanisms that underlie gene regulation, but relationships that apply to all intrinsically disordered proteins. Recent studies confirm that binding does not necessarily induce folding but that when it does, it tends to follow induced fit mechanisms. Other work emphasises the importance of electrostatics to interactions involving intrinsically disordered proteins, and roles of intrinsic disorder in phase transitions. All these features help direct transcription factors to target sites in the genome to upregulate or downregulate transcription.

Charge Interactions Modulate the Encounter Complex Ensemble of Two Differently Charged Disordered Protein Partners of KIX.

Disordered proteins play important roles in cell signaling and are frequently involved in protein-protein interactions. They also have a larger proportion of charged and polar residues than their folded counterparts. Here, we developed a structure-based model and applied molecular dynamics simulations to examine the presence and importance of electrostatic interactions in the binding processes of two differently charged intrinsically disordered ligands of the KIX domain of CBP. We observed non-native opposite-charged contacts in the encounter complexes for both ligands with KIX, and this may be a general feature of coupled folding and binding reactions. The ensemble of successful encounter complexes is a diverse set of structures, and in the case of the highly charged ligand, this ensemble was found to be malleable with respect to ionic strength. There are only minor differences between encounter complex ensembles for successful and unsuccessful collisions with no key interactions that appear to make the process far more productive. The energy landscape at this early stage in the process does not appear highly funneled. Strikingly we observed many native interactions that appear to reduce chances of an encounter complex being productive. Instead it appears that collectively non-native electrostatic interactions in the encounter complex increase the likelihood of productivity by holding the proteins together long enough for folding to take place. This mechanism is more effective for the more highly charged ligand.

Stopped-Flow Kinetic Techniques for Studying Binding Reactions of Intrinsically Disordered Proteins.

Intrinsically disordered proteins are abundant in signaling processes such as transcription. Suitable binding and unbinding rates of proteins with their partners are critical for allowing them to perform their biological roles. Understanding how these are achieved, and indeed designing strategies for intervening or modulating related biological processes, therefore requires kinetic studies. In this chapter, we describe stopped-flow-based methods for determining association and dissociation rate constants for pairs of macromolecular binding partners. We describe how to select the simplest appropriate model to describe the interaction, and highlight cases where it is possible to distinguish between induced fit and conformational selection binding mechanisms. Finally, we go on to describe methods for examining the role of electrostatic forces in binding processes, and for describing the transition state for binding processes that have folding associated with them.

Two Differential Binding Mechanisms of FG-Nucleoporins and Nuclear Transport Receptors.

Phenylalanine-glycine-rich nucleoporins (FG-Nups) are intrinsically disordered proteins, constituting the selective barrier of the nuclear pore complex (NPC). Previous studies showed that nuclear transport receptors (NTRs) were found to interact with FG-Nups by forming an "archetypal-fuzzy" complex through the rapid formation and breakage of interactions with many individual FG motifs. Here, we use single-molecule studies combined with atomistic simulations to show that, in sharp contrast, FG-Nup214 undergoes a coupled reconfiguration-binding mechanism when interacting with the export receptor CRM1. Association and dissociation rate constants are more than an order of magnitude lower than in the archetypal-fuzzy complex between FG-Nup153 and NTRs. Unexpectedly, this behavior appears not to be encoded selectively into CRM1 but rather into the FG-Nup214 sequence. The same distinct binding mechanisms are unperturbed in O-linked ß-N-acetylglucosamine-modified FG-Nups. Our results have implications for differential roles of distinctly spatially distributed FG-Nup⋅NTR interactions in the cell.

Hsp70 Inhibits the Nucleation and Elongation of Tau and Sequesters Tau Aggregates with High Affinity.

As a key player of the protein quality control network of the cell, the molecular chaperone Hsp70 inhibits the aggregation of the amyloid protein tau. To date, the mechanism of this inhibition and the tau species targeted by Hsp70 remain unknown. This is partly due to the inherent difficulty of studying amyloid aggregates because of their heterogeneous and transient nature. Here, we used ensemble and single-molecule fluorescence measurements to dissect how Hsp70 counteracts the self-assembly process of the K18 ΔK280 tau variant. We found that Hsp70 blocks the early stages of tau aggregation by suppressing the formation of tau nuclei. Additionally, Hsp70 sequesters oligomers and mature tau fibrils with nanomolar affinity into a protective complex, efficiently neutralizing their ability to damage membranes and seed further tau aggregation. Our results provide novel insights into the molecular mechanisms by which the chaperone Hsp70 counteracts the formation, propagation, and toxicity of tau aggregates.

Phosphorylation of the IDP KID Modulates Affinity for KIX by Increasing the Lifetime of the Complex.

Intrinsically disordered proteins (IDPs) are known to undergo a range of posttranslational modifications, but by what mechanism do such modifications affect the binding of an IDP to its partner protein? We investigate this question using one such IDP, the kinase inducible domain (KID) of the transcription factor CREB, which interacts with the KIX domain of CREB-binding protein upon phosphorylation. As with many other IDPs, KID undergoes coupled folding and binding to form α-helical structure upon interacting with KIX. This single site phosphorylation plays an important role in the control of transcriptional activation in vivo. Here we show that, contrary to expectation, phosphorylation has no effect on association rates-unphosphorylated KID binds just as rapidly as pKID, the phosphorylated form-but rather, acts by increasing the lifetime of the complex. We propose that by controlling the lifetime of the bound complex of pKID:KIX via altering the dissociation rate, phosphorylation can facilitate effective control of transcription regulation.

pKID Binds to KIX via an Unstructured Transition State with Nonnative Interactions.

Understanding the detailed mechanism of interaction of intrinsically disordered proteins with their partners is crucial to comprehend their functions in signaling and transcription. Through its interaction with KIX, the disordered pKID region of CREB protein is central in the transcription of cAMP responsive genes, including those involved in long-term memory. Numerous simulation studies have investigated these interactions. Combined with experimental results, these can provide valuable and comprehensive understanding of the mechanisms involved. Here, we probe the transition state of this interaction experimentally through analyzing the kinetic effect of mutating both interface and solvent exposed residues in pKID. We show that very few specific interactions between pKID and KIX are required in the initial binding process. Only a small number of weak interactions are formed at the transition state, including nonnative interactions, and most of the folding occurs after the initial binding event. These properties are consistent with computational results and also the majority of experimental studies of intrinsically disordered protein coupled folding and binding in other protein systems, suggesting that these may be common features.

Affinity of IDPs to their targets is modulated by ion-specific changes in kinetics and residual structure.

Intrinsically disordered proteins (IDPs) are characterized by a lack of defined structure. Instead, they populate ensembles of rapidly interconverting conformations with marginal structural stabilities. Changes in solution conditions such as temperature and crowding agents consequently affect IDPs more than their folded counterparts. Here we reveal that the residual structure content of IDPs is modulated both by ionic strength and by the type of ions present in solution. We show that these ion-specific structural changes result in binding affinity shifts of up to sixfold, which happen through alteration of both association and dissociation rates. These effects follow the Hofmeister series, but unlike the well-established effects on the stability of folded proteins, they already occur at low, hypotonic concentrations of salt. We attribute this sensitivity to the marginal stability of IDPs, which could have physiological implications given the role of IDPs in signaling, the asymmetric ion profiles of different cellular compartments, and the role of ions in biology.

Role of non-native electrostatic interactions in the coupled folding and binding of PUMA with Mcl-1.

PUMA, which belongs to the BH3-only protein family, is an intrinsically disordered protein (IDP). It binds to its cellular partner Mcl-1 through its BH3 motif, which folds upon binding into an α helix. We have applied a structure-based coarse-grained model, with an explicit Debye-Hückel charge model, to probe the importance of electrostatic interactions both in the early and the later stages of this model coupled folding and binding process. This model was carefully calibrated with the experimental data on helical content and affinity, and shown to be consistent with previously published experimental data on binding rate changes with respect to ionic strength. We find that intramolecular electrostatic interactions influence the unbound states of PUMA only marginally. Our results further suggest that intermolecular electrostatic interactions, and in particular non-native electrostatic interactions, are involved in formation of the initial encounter complex. We are able to reveal the binding mechanism in more detail than is possible using experimental data alone however, and in particular we uncover the role of non-native electrostatic interactions. We highlight the potential importance of such electrostatic interactions for describing the binding reactions of IDPs. Such approaches could be used to provide predictions for the results of mutational studies.

Conserved Helix-Flanking Prolines Modulate Intrinsically Disordered Protein:Target Affinity by Altering the Lifetime of the Bound Complex.

Appropriate integration of cellular signals requires a delicate balance of ligand-target binding affinities. Increasing the level of residual structure in intrinsically disordered proteins (IDPs), which are overrepresented in these cellular processes, has been shown previously to enhance binding affinities and alter cellular function. Conserved proline residues are commonly found flanking regions of IDPs that become helical upon interacting with a partner protein. Here, we mutate these helix-flanking prolines in p53 and MLL and find opposite effects on binding affinity upon an increase in free IDP helicity. In both cases, changes in affinity were due to alterations in dissociation, not association, rate constants, which is inconsistent with conformational selection mechanisms. We conclude that, contrary to previous suggestions, helix-flanking prolines do not regulate affinity by modulating the rate of complex formation. Instead, they influence binding affinities by controlling the lifetime of the bound complex.

Mechanistic roles of protein disorder within transcription.

Understanding the interactions of proteins involved in transcriptional regulation is critical to describing biological systems because they control the expression profile of the cell. Yet sadly they belong to a less well biophysically characterized subset of proteins; they frequently contain long disordered regions that are highly dynamic. A key question therefore is, why? What functional roles does protein disorder play in transcriptional regulation? Experimental data exemplifying these roles are starting to emerge, with common themes being enabling complexity within networks and quick responses. Most recently a role for disorder in mediating phase transitions of membrane-less organelles has been proposed.

GADIS: Algorithm for designing sequences to achieve target secondary structure profiles of intrinsically disordered proteins.

Many intrinsically disordered proteins (IDPs) participate in coupled folding and binding reactions and form alpha helical structures in their bound complexes. Alanine, glycine, or proline scanning mutagenesis approaches are often used to dissect the contributions of intrinsic helicities to coupled folding and binding. These experiments can yield confounding results because the mutagenesis strategy changes the amino acid compositions of IDPs. Therefore, an important next step in mutagenesis-based approaches to mechanistic studies of coupled folding and binding is the design of sequences that satisfy three major constraints. These are (i) achieving a target intrinsic alpha helicity profile; (ii) fixing the positions of residues corresponding to the binding interface; and (iii) maintaining the native amino acid composition. Here, we report the development of a G: enetic A: lgorithm for D: esign of I: ntrinsic secondary S: tructure (GADIS) for designing sequences that satisfy the specified constraints. We describe the algorithm and present results to demonstrate the applicability of GADIS by designing sequence variants of the intrinsically disordered PUMA system that undergoes coupled folding and binding to Mcl-1. Our sequence designs span a range of intrinsic helicity profiles. The predicted variations in sequence-encoded mean helicities are tested against experimental measurements.

Insights into Coupled Folding and Binding Mechanisms from Kinetic Studies.

Intrinsically disordered proteins (IDPs) are characterized by a lack of persistent structure. Since their identification more than a decade ago, many questions regarding their functional relevance and interaction mechanisms remain unanswered. Although most experiments have taken equilibrium and structural perspectives, fewer studies have investigated the kinetics of their interactions. Here we review and highlight the type of information that can be gained from kinetic studies. In particular, we show how kinetic studies of coupled folding and binding reactions, an important class of signaling event, are needed to determine mechanisms.

Plasticity of an ultrafast interaction between nucleoporins and nuclear transport receptors.

The mechanisms by which intrinsically disordered proteins engage in rapid and highly selective binding is a subject of considerable interest and represents a central paradigm to nuclear pore complex (NPC) function, where nuclear transport receptors (NTRs) move through the NPC by binding disordered phenylalanine-glycine-rich nucleoporins (FG-Nups). Combining single-molecule fluorescence, molecular simulations, and nuclear magnetic resonance, we show that a rapidly fluctuating FG-Nup populates an ensemble of conformations that are prone to bind NTRs with near diffusion-limited on rates, as shown by stopped-flow kinetic measurements. This is achieved using multiple, minimalistic, low-affinity binding motifs that are in rapid exchange when engaging with the NTR, allowing the FG-Nup to maintain an unexpectedly high plasticity in its bound state. We propose that these exceptional physical characteristics enable a rapid and specific transport mechanism in the physiological context, a notion supported by single molecule in-cell assays on intact NPCs.

A mechanistic model of tau amyloid aggregation based on direct observation of oligomers.

Protein aggregation plays a key role in neurodegenerative disease, giving rise to small oligomers that may become cytotoxic to cells. The fundamental microscopic reactions taking place during aggregation, and their rate constants, have been difficult to determine due to lack of suitable methods to identify and follow the low concentration of oligomers over time. Here we use single-molecule fluorescence to study the aggregation of the repeat domain of tau (K18), and two mutant forms linked with familial frontotemporal dementia, the deletion mutant ΔK280 and the point mutant P301L. Our kinetic analysis reveals that aggregation proceeds via monomeric assembly into small oligomers, and a subsequent slow structural conversion step before fibril formation. Using this approach, we have been able to quantitatively determine how these mutations alter the aggregation energy landscape.

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

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 ß-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.

Allostery within a transcription coactivator is predominantly mediated through dissociation rate constants.

The kinase-inducible domain interacting (KIX) domain of CREB binding protein binds to multiple intrinsically disordered transcription factors in vivo at two distinct sites on its surface. Several reports have been made of allosteric communication between these two sites in this well-characterized model system. In this work, we have performed fluorescence stopped-flow measurements to investigate the kinetics of binding of five KIX binding proteins. We find that they all have similar association and dissociation rate constants for complex formation, despite their wide range of intrinsic helical propensities. Furthermore, by careful arrangement of pseudofirst-order conditions, we have been able to show that both association and dissociation rate constants are decreased when a partner is bound at the alternative site. These decreases suggest that positive allosteric effects are not mediated by structural changes in binding sites but rather, through a more general mechanism, largely mediated through dissociation, which we propose is largely related to changes in the flexibility of the KIX domain itself.

Mechanism of assembly of the non-covalent spectrin tetramerization domain from intrinsically disordered partners.

Interdomain interactions of spectrin are critical for maintenance of the erythrocyte cytoskeleton. In particular, "head-to-head" dimerization occurs when the intrinsically disordered C-terminal tail of ß-spectrin binds the N-terminal tail of α-spectrin, folding to form the "spectrin tetramer domain". This non-covalent three-helix bundle domain is homologous in structure and sequence to previously studied spectrin domains. We find that this tetramer domain is surprisingly kinetically stable. Using a protein engineering Φ-value analysis to probe the mechanism of formation of this tetramer domain, we infer that the domain folds by the docking of the intrinsically disordered ß-spectrin tail onto the more structured α-spectrin tail.

Remarkably fast coupled folding and binding of the intrinsically disordered transactivation domain of cMyb to CBP KIX.

Association rates for interactions between folded proteins have been investigated extensively, allowing the development of computational and theoretical prediction methods. Less is known about association rates for complexes where one or more partner is initially disordered, despite much speculation about how they may compare to those for folded proteins. We have attached a fluorophore to the N-terminus of the 25 amino acid cMyb peptide used previously in NMR and equilibrium studies (termed FITC-cMyb), and used this to monitor the kinetics of its interaction with the KIX protein. We have investigated the ionic strength and temperature dependence of the kinetics, and conclude that the association process is extremely fast, apparently exceeding the rates predicted by formulations applicable to interactions between pairs of folded proteins. This is despite the fact that not all collisions result in complex formation (there is an observable activation energy for the association process). We propose that this is partially a result of the disordered nature of the FITC-cMyb peptide itself.

Protein folding: adding a nucleus to guide helix docking reduces landscape roughness.

The elongated three-helix-bundle spectrin domains R16 and R17 fold and unfold unusually slowly over a rough energy landscape, in contrast to the homologue R15, which folds fast over a much smoother, more typical landscape. R15 folds via a nucleation-condensation mechanism that guides the docking of the A and C-helices. However, in R16 and R17, the secondary structure forms first and the two helices must then dock in the correct register. Here, we use variants of R16 and R17 to demonstrate that substitution of just five key residues is sufficient to alter the folding mechanism and reduce the landscape roughness. We suggest that, by providing access to an alternative, faster, folding route over their landscape, R16 and R17 can circumvent their slow, frustrated wild-type folding mechanism.