Simulating disorder-order transitions in molecular recognition of unstructured proteins: where folding meets binding

Abstract

A microscopic study of functional disorder-order folding transitions coupled to binding is performed for the p27 protein, which derives a kinetic advantage from the intrinsically disordered unbound form on binding with the phosphorylated cyclin A-cyclin-dependent kinase 2 (Cdk2) complex. Hierarchy of structural loss during p27 coupled unfolding and unbinding is simulated by using high-temperature Monte Carlo simulations initiated from the crystal structure of the tertiary complex. Subsequent determination of the transition-state ensemble and the proposed atomic picture of the folding mechanism coupled to binding provide a microscopic rationale that reconciles the initiation recruitment of p27 at the cyclin A docking site with the kinetic benefit for a disordered alpha-helix in the unbound form of p27. The emerging structural polarization in the ensemble of unfolding/unbinding trajectories and in the computationally determined transition-state ensemble is not determined by the intrinsic folding preferences of p27 but rather is attributed to the topological requirements of the native intermolecular interface to order beta-hairpin and beta-strand of p27 that could be critical for nucleating rapid folding transition coupled to binding. In agreement with the experimental data, the disorder-order folding transition for p27 is largely determined by the functional requirement to form a specific intermolecular interface that ultimately dictates the folding mechanism and overwhelms any local folding preferences for creating a stable alpha-helix in the p27 structure before overcoming the major free energy barrier.

Figure 1

The crystal structure of the tertiary complex with the bound 69-aa p27 protein (residues 25–93) comprising sequentially the coil (residues from 25 to 34), α-helix (residues from 35 to 60), β-hairpin (residues from 61 to 71), β-strand (residues 75 to 81), and 3₁₀-helix (residues from 85 to 90). Schematic drawing shows p27 in blue, cyclin A in green, and Cdk2 in light blue.

Figure 2

Time-dependent history of the rmsd from the crystal structure for the p27 protein (a) and the total number of the intermolecular hydrogen bonds formed by p27 (b) during simulations at T = 300 K (black), T = 400 K (orange), T = 500 K (red), and T = 600 K (blue).

Figure 3

Occupancy histograms monitor the hierarchy of structural loss for p27 (a), coil (b), α-helix (c), β-hairpin (d), β-strand (e), and 3₁₀-helix (f). The histograms are generated by averaging the results of 20 independent unfolding/unbinding simulations for p27 at T = 600 K and are represented as a function of the total number of the intermolecular hydrogen bonds (NHB) formed by p27 in the complex versus the rmsd from the crystal structure for p27 and for each of the p27 elements.

Figure 4

(a) The distribution of the mean rmsd values from the native-bound p27 conformation for clusters representing the ensemble unstructured and weakly bound states (red filled bars), the ensemble of partially unfolded/unbound states (blue filled bars), and the ensemble of native-like bound states (green filled bars) with the rmsd values of β-strand (black filled bars) and with the rmsd values of β-hairpin (black filled bars) (c). (b) The distribution of the mean number of the intermolecular hydrogen bonds formed by p27 in clusters representing these conformations ensembles (red, blue, and green filled bars, respectively) with the distribution for the β-strand (black filled bars) and for the β-hairpin (black filled bars) (d). The mean values are obtained by averaging over all conformations in a given cluster. Conformational ensembles are ordered according to the decreasing number of conformations in a given cluster.

Figure 5

The outcome of the 20 short kinetic runs for a representative conformation in the TSE (a and b) and in the ensemble of postcritical conformations (c and d). (a and c) The distribution of the rmsd values from the native bound conformation of p27 in the tertiary complex (empty bars), and the rmsd values of the β-strand (filled bars). (b and d) The distribution of the number of the intermolecular hydrogen bonds formed with the complex by p27 (empty bars) and by the β-strand (filled bars).

Figure 6

A schematic representation of the proposed p27 binding mechanism: (a) the ensemble of nonspecific, largely unbound conformations; (b) initiation recruitment of p27 at the cyclin A docking site; (c) the TSE; and (d) the ensemble of postcritical states. The major conformational ensembles (red) are superimposed onto the crystal structure of the bound p27 (blue), cyclin A (green), and Cdk2 (light blue).