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

Abstract

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.Protein folding | stopped flow | coarse-grained simulation | protein-protein interactions | BCL-2

Conformational propensities of intrinsically disordered proteins influence the mechanism of binding and folding

“…The transition state for binding of c-Myb to KIX differs from those of other IDPs that have been studied to date in that Φ values and linear free energy relationships show that near-native secondary and tertiary structure has already formed at the top of the rate-limiting energy barrier (27). In contrast, the transition states for binding and folding of S peptide to S protein (33), the IDP PUMA to its MCL-1 target (34), and the activation domain of ACTR to the nuclear coactivator binding domain (NCBD) of CBP (35) are located early in the binding pathway; folding of the IDP and formation of native intermolecular interactions occur after the rate-limiting step for binding, i.e., by an induced-fit mechanism. Whereas native-like ACTR-NCBD interactions are mostly formed late in the reaction pathway, mutations that increase the propensity for spontaneous helix formation in the N-terminal region of ACTR accelerate binding by a conformational selection mechanism (36).…”

Intrinsically Disordered Proteins as Drug Targets

“…These structured elements can exist in the free protein, emerging upon conformational transitions or the combinations of both. [18][19][20] The order-disordered transitions suffered by IDPs can be produced by several factors such as exogenous perturbations (for instance pH, [21][22][23] Tª 22,[24][25][26] or macromolecular crowding) 23,[27][28][29][30] or binding (multiple) diverse molecular entities, such as proteins 18,31,32 or small molecules. 10,[33][34][35] As they are very promiscuous molecules, IDPs can bind multiple partners.…”

Untitled

“…These structured elements can exist in the free protein, emerging upon conformational transitions or the combinations of both [18][19][20]. The order-disordered transitions suffered by IDPs can be produced by several factors such as exogenous perturbations (for instance pH [21][22][23], Tª [22,[24][25][26] or macromolecular crowding [23,[27][28][29][30]. or binding (multiple) diverse molecular entities, such as proteins [18,31,32].…”