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 β-Tanford values, Leffler α, Φ-value analysis, and coarse-grained simulations, and propose a self-consistent mechanism for binding. Folding of the disordered protein before binding does not appear to be required and few, if any, specific interactions are required to commit to association. The majority of PUMA folding occurs after the transition state, in the presence of MCL-1. We also examine the role of the side chains of folded MCL-1 that make up the binding groove and find that many favor equilibrium binding but, surprisingly, inhibit the association process.For many proteins, correct folding to a specific 3D structure is essential for their function inside the cell; once folded, some of these have the appropriate shape and accessible chemical groups to interact specifically with, and bind to, another protein (1). However, for a number of protein–protein interactions, folding and binding do not appear to be separate, sequential events (2, 3). Many intrinsically disordered proteins (IDPs) will appear largely unfolded in isolation, only forming a specific structure when bound to an appropriate partner protein and undergoing coupled folding and binding (4–6). Such reactions are abundant in signaling and regulatory processes (7, 8). Protein folding does not simply provide correctly shaped building blocks for the cell; it can play an intimate role in molecular recognition.Over the past decade, bioinformatics studies have revealed that protein disorder (7, 9), and coupled folding and binding (10), are widespread in biology. Many structures of bound, folded IDPs have been solved and have shown the wide range of topologies that can be formed (11). Biophysical techniques (12), NMR in particular (13), can characterize isolated IDPs in detail. Despite this progress, the number of studies examining kinetics and the mechanisms of binding remains relatively small (14–21) given that the most commonly observed function of IDPs is in coupled folding and binding reactions (22).To describe coupled folding and binding, two extreme mechanisms are often discussed, focusing on whether an IDP needs to fold before interacting productively with its binding partner. In isolation an IDP could, perhaps only transiently, occupy a conformation that resembles the bound state. In the pure conformational selection mechanism, the IDP must be in this conformation at the start of the eventually successful encounter with the partner protein (23, 24) (Fig. 1A). Arguments in support of this mechanism largely come from NMR studies that have successfully detected these lowly populated, folded states in unbound IDPs (25–27). In the contrasting induced-fit mechanism, there is no requirement for the IDP to fold in isolation (28). Instead, the potentially transient interactions with the partner protein lead to the folding of the IDP (Fig. 1A). Complex mixtures of these two extreme mechanisms can also be imagined: e.g., perhaps only a proportion of the IDP needs to fold before the encounter, i.e., conformational selection followed by induced fit of the remaining peptide chain (29). To add to the potential complexity, flux through different pathways could occur simultaneously, and may depend on the concentrations of protein involved (23, 30). Further, confirming the degree of induced fit and conformational selection is only one aspect of the binding mechanism. There remain a large number of mechanistic possibilities beyond the state of the IDP prior to successful encounters.Open in a separate windowFig. 1.PUMA–MCL-1 binding. (A) Cartoon of binding mechanisms. IDP PUMA (blue) can undergo coupled folding and binding with structured MCL-1 (white) to form a single, contiguous α-helix. Structures based on PDB 2ROC (39) and 1WSX (58). Unbound PUMA and encounter complex built using Chimera (University of California, San Francisco). Figure prepared using PyMol. (B) Representative fluorescence stopped-flow traces for binding. Increasing the concentration of urea from 0 to 3.5 M (in 0.5-M increments) slows association. (C) The urea dependence of the natural log of the association rate constant (k+) for the wild-type PUMA peptide used in this study. (D) The urea dependence of the dissociation rate constant (k−). k− was determined by preforming the PUMA–MCL-1 complex at micromolar concentrations and manually diluting to nanomolar concentrations to induce dissociation. The resulting kinetic trace was fit to a reversible model, fixing k+ from the association experiments (41). Gradient of the linear fits corresponds to the m values discussed in the main text. A, B, and C adapted from ref. 37.It is largely agreed that most protein folding (and unfolding) reactions are limited by the requirement to populate a high-energy transition state (31). Kinetic, time-resolved experiments, in combination with site-directed mutagenesis and Φ-value analysis (32), have been applied successfully to describe these transition states (33, 34). With carefully chosen mutations, the distribution of Φ values (classically between 0 and 1) offers an average picture of the interactions formed at this critical stage of the folding reaction, at residue-level resolution. This picture, in conjunction with other evidence, can offer invaluable insights into the mechanisms of folding (35, 36).We have previously reported the kinetics of a model coupled folding and binding reaction (37, 38); the BH3 motif of PUMA (an IDP) can associate with the structured protein MCL-1 and fold to a single contiguous α-helix (39). The solvent and temperature dependence of the association reaction suggested that this reaction is limited by a free energy barrier, or transition state (TS) (37). Here we systematically make structurally conservative mutations to the IDP and the partner protein, apply Φ-value analysis, and describe the transition state for binding. Molecular dynamics simulations using a coarse-grained, topology-based model of the binding process are consistent with our experimental results. We bring together all available evidence to propose a mechanism of binding.