Abstract
Coupled folding and binding of intrinsically disordered proteins (IDPs) is prevalent in biology. As the first step toward understanding the mechanism of binding, it is important to know if a reaction is ‘diffusion-limited’ as, if this speed limit is reached, the association must proceed through an induced fit mechanism. Here, we use a model system where the ‘BH3 region’ of PUMA, an IDP, forms a single, contiguous α-helix upon binding the folded protein Mcl-1. Using stopped-flow techniques, we systematically compare the rate constant for association (k+) under a number of solvent conditions and temperatures. We show that our system is not ‘diffusion-limited’, despite having a k+ in the often-quoted ‘diffusion-limited’ regime (105–106 M–1 s–1 at high ionic strength) and displaying an inverse dependence on solvent viscosity. These standard tests, developed for folded protein–protein interactions, are not appropriate for reactions where one protein is disordered.
Highlights
It has long been assumed that the specific, folded, threedimensional structure of a protein was a prerequisite for its function in the cell
NMR structures have been solved for Mcl-1 in isolation[28] and bound to a 27 aa PUMA peptide[26] (Figure 1A and Figure S1)
The backbone atoms for Mcl-1 in these two structures overlay with an RMSD of 1.78 Å, consistent with only minor conformational changes in Mcl-1: a slight opening of the surface groove, upon binding the PUMA peptide (Figure S1)
Summary
It has long been assumed that the specific, folded, threedimensional structure of a protein was a prerequisite for its function in the cell. It has been recognized that many of Nature’s proteins have no fixed structure,[1] instead occupying an enormous number of rapidly interconverting conformations.[2] These intrinsically disordered proteins (IDPs) are widespread in biology[3] and, despite their lack of structure, perform many important functions in the cell.[4] One of the ways Nature has utilized this disorder, and maintained it in evolution, is in the form of ‘coupled folding and binding’ whereby an unstructured IDP gains structure only when bound to a target protein.[5] This mode of protein−protein interaction presents an alternative to that between the typically large, flat interfaces between two already folded proteins. There are many potential advantages for these protein−protein interactions[6] which could explain the high abundance of IDPs in signaling processes and their abundance in eukaryotic cells that rely more on complex signaling pathways.[3]
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