In a landmark 15^{15}N relaxation-dispersion study, Sugase, Dyson and Wright (2007) showed that the intrinsically disordered pKID domain of CREB binds the KIX domain of the coactivator CBP through a sequential pathway: a hydrophobic-dominated ensemble of transient encounter complexes that evolves on the KIX surface, without dissociation, through a partially folded intermediate to the fully bound state. The paper established this pathway for pKID–KIX while noting that the mechanism by which folding is coupled to binding is poorly understood; the same authors' follow-up review framed the resulting open question as whether this free→encounter→intermediate→bound (F→E→I→B) induced-folding mechanism is general across intrinsically disordered protein (IDP) systems, or whether other IDPs utilize different mechanisms such as conformational selection (Wright & Dyson 2009). Nearly two decades of follow-up work has established that mechanisms are diverse and system-dependent: c-Myb–KIX mixes conformational selection with induced fit, Sendai virus NT–PX stabilizes a pre-existing helical substate inside a dynamic encounter complex, PUMA–Mcl-1 requires no particular residual structure, the histone H1–prothymosin-α complex remains fully disordered, and YAP–TEAD proceeds largely by conformational selection. What still does not exist is a general, quantitative account of the mechanism space: a mapping from IDP and partner properties (residual conformational propensities, charge patterning and electrostatics versus hydrophobicity, post-translational modifications) to the binding pathway and its kinetics, and a measure of how ubiquitous the sequential F→E→I→B pathway is across IDP–folded-domain associations. The problem — still open — is to produce that account.

Background

Many proteins that regulate cellular signalling are intrinsically disordered proteins (IDPs): instead of folding into one stable three-dimensional structure in isolation, they populate a heterogeneous ensemble of rapidly interconverting conformations under physiological conditions. Disordered regions are abundant in eukaryotic regulatory proteins and frequently fold into a defined structure only upon binding a physiological partner — a process called coupled folding and binding.

Two limiting mechanisms have been proposed for such binding. In conformational selection, the free IDP transiently populates a binding-competent (partly folded) conformer that the partner recognizes directly. In induced fit (folding-after-binding), the partner first binds a disordered conformer and folding occurs afterwards, on the partner surface. Theoretical proposals such as fly-casting suggested that disorder might even accelerate association: a disordered chain binds weakly and non-specifically at a distance and folds as it approaches the cognate site. Distinguishing these mechanisms experimentally requires observing the transient, weakly populated species along the binding pathway, not only the end states.

A key experimental probe is NMR relaxation dispersion: measuring the transverse relaxation rate R2R_{2} of backbone 15^{15}N nuclei (the nitrogen isotope used to label the protein backbone for NMR) as a function of magnetic field strength and ligand concentration. Exchange between a major and a minor state on the microsecond-to-millisecond timescale increases R2R_{2} in a field-dependent way, and global fitting to a kinetic exchange model yields the populations, interconversion rates, and residue-specific chemical-shift differences of otherwise invisible transient states — including how far each residue is folded in them.

The paradigmatic system is the interaction between the phosphorylated kinase-inducible activation domain of the CREB transcription factor (pKID, disordered in isolation) and the KIX domain of the coactivator CBP (CREB-binding protein). Sugase, Dyson and Wright (2007) applied 15^{15}N R2R_{2} relaxation dispersion at two fields and four pKID:KIX concentration ratios and found that pKID forms a heterogeneous ensemble of encounter complexes — weak, transient, non-specific complexes — on the KIX surface, stabilized primarily by short-range hydrophobic contacts rather than the electrostatic steering typical of folded-protein association. These encounter complexes evolve, without leaving the KIX surface, through a folding intermediate in which the N-terminal helix of pKID is nearly fully formed while the C-terminal helix is only partially folded, into the fully bound state: a sequential free→encounter→intermediate→bound (F→E→I→B) pathway in which folding happens on the partner. The fastest step, free⇄encounter, lies in the very-fast-exchange regime and contributes negligibly to the observed dispersion, so it could be represented only by effective rate constants that merge the free and encounter states into one kinetic step.

This raised the question that defines the present problem: is the pKID–KIX pathway the general mechanism by which disordered proteins recognize their partners, or do other mechanisms — notably conformational selection — predominate in other systems? Subsequent work on other systems (reviewed below) has revealed a genuinely diverse landscape rather than a single answer, which makes the systematizing question sharper, not obsolete.

Problem Statement

For associations between intrinsically disordered proteins (or regions) and their binding partners, establish a general, quantitative account of the mechanism of coupled folding and binding. This comprises two linked deliverables serving one objective. First, determine — at residue-resolved kinetic and structural resolution, using evidence such as the multi-field, multi-concentration relaxation-dispersion analysis pioneered for pKID–KIX, together with independent kinetic, single-molecule, or simulation approaches — which binding pathways actually operate across the diversity of IDP–partner systems: the sequential scheme observed for pKID–KIX (free state → hydrophobic-dominated encounter ensemble → partially folded bound intermediate → fully bound state), conformational selection of pre-structured conformers from the free-state ensemble, segment-wise mixtures of the two, or qualitatively different schemes such as fully disordered complexes held together by distributed opposite-charge interactions (polyelectrostatic complexes). Second, identify the determinants that govern which mechanism, and which kinetic regime, a given IDP–partner pair follows — including intrinsic conformational and secondary-structure propensities of the disordered chain, charge patterning and long-range electrostatics versus short-range hydrophobicity, partner surface properties, post-translational modifications, and solution conditions — supporting each proposed determinant with the per-system kinetic or structural evidence that motivates it, and thereby quantifying how general the sequential F→E→I→B pathway is across intrinsically disordered–folded-domain associations. Resolving the very-fast free⇄encounter step and residue-local fit anomalies (e.g. Asp140) are methodological sub-goals in service of this objective, not separate problems. An account resolves this objective once it assigns every IDP–partner association it treats to one of these pathway classes on residue-resolved evidence, states explicitly which systems it covers — a set that must extend beyond the systems already named in this statement and be supported by an explicit argument that it is representative of the diversity of IDP–partner systems — identifies, for each system it covers, the determinants that govern its pathway class and kinetic regime, and reports the ubiquity of the sequential F→E→I→B pathway as a proportion over that stated set rather than as an unrestricted claim.

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