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. 2017 Dec 19;113(12):2713-2722.
doi: 10.1016/j.bpj.2017.10.016.

pKID Binds to KIX via an Unstructured Transition State with Nonnative Interactions

Liza Dahal  1 , Tristan O C Kwan  1 , Sarah L Shammas  2 , Jane Clarke  3
Affiliations

pKID Binds to KIX via an Unstructured Transition State with Nonnative Interactions

Liza Dahal et al. Biophys J. .

Abstract

Understanding the detailed mechanism of interaction of intrinsically disordered proteins with their partners is crucial to comprehend their functions in signaling and transcription. Through its interaction with KIX, the disordered pKID region of CREB protein is central in the transcription of cAMP responsive genes, including those involved in long-term memory. Numerous simulation studies have investigated these interactions. Combined with experimental results, these can provide valuable and comprehensive understanding of the mechanisms involved. Here, we probe the transition state of this interaction experimentally through analyzing the kinetic effect of mutating both interface and solvent exposed residues in pKID. We show that very few specific interactions between pKID and KIX are required in the initial binding process. Only a small number of weak interactions are formed at the transition state, including nonnative interactions, and most of the folding occurs after the initial binding event. These properties are consistent with computational results and also the majority of experimental studies of intrinsically disordered protein coupled folding and binding in other protein systems, suggesting that these may be common features.

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Figures

Figure 1
Figure 1
Structure of pKID (cartoon) and KIX (gray spheres on top and gray cartoon on bottom) showing interface and solvent-exposed residues of pKID that were mutated in the study. The phosphorylated serine of pKID is shown as sticks (magenta). The residues of pKID that contact the KIX interface are shown as spheres (top) and sticks (bottom); S121 (pale pink), R124 (olive), R125 (green), L128 (purple), R131 (orange), Y134 (brown), I137 (cyan), L138 (yellow), D140 (red), and S142 (dark blue). The mutated solvent-exposed residues of pKID are shown in white.
Figure 2
Figure 2
Sample biophysical data. (A) Circular dichroism (CD), (B) equilibrium anisotropy, and (C) association and (D) dissociation kinetic plots for mutants of three different residues along the length of pKID. Wild-type data are shown in black/gray for comparison. The left panels show data for R125A (green) in αA of pKID. The middle panels show data for R131A (orange) in the interhelical kink region. The right panels show data for I137A (cyan) in αB of pKID.
Figure 3
Figure 3
Linear free-energy plot. kass,fast (●) and kdiss (◪) plotted against Kd for all mutants of pKID binding to KIX. Changes in Kd are mostly due to changes in kdiss.
Figure 4
Figure 4
Comparison between kinetic and equilibrium Kd and ΔΔG. (Top plot) KdKin plotted against KdEqub for all pKID mutants. (Bottom plot) ΔΔGKin plotted against ΔΔGEqub calculated for all pKID mutants. The straight line of y = x shown in black is presented to guide the eye for comparison between kinetic and equilibrium measurements. Wild-type parameters are shown in black and all other mutants are shown in red. Where affinities are lower this results in more uncertainty in the Kd measurements, and thus larger differences between kinetic and equilibrium data.
Figure 5
Figure 5
Φ-values for interface and solvent-exposed mutants. Top: ΦAverage, average of Φ-values calculated using both kinetic and equilibrium methods are plotted for mutants with ΔΔG > 0.34 kcal mol−1. The residues that were investigated but where Φ-values could not be calculated are underlined. Solvent-exposed (Ala-Gly) mutants are highlighted by an asterisk. Bottom: the pattern of Φ-values is mapped on the structure of pKID/KIX (1KDX). Φ-values are generally low, so negative Φ-values are shown in pale pink and positive Φ-values are shown in red.
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