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. 2017 Apr 7;429(7):987-998.
doi: 10.1016/j.jmb.2017.03.003. Epub 2017 Mar 8.

Controllable Activation of Nanoscale Dynamics in a Disordered Protein Alters Binding Kinetics

David J E Callaway  1 , Tsutomu Matsui  2 , Thomas Weiss  2 , Laura R Stingaciu  3 , Christopher B Stanley  4 , William T Heller  4 , Zimei Bu  5
Affiliations

Controllable Activation of Nanoscale Dynamics in a Disordered Protein Alters Binding Kinetics

David J E Callaway et al. J Mol Biol. .

Abstract

The phosphorylation of specific residues in a flexible disordered activation loop yields precise control of signal transduction. One paradigm is the phosphorylation of S339/S340 in the intrinsically disordered tail of the multi-domain scaffolding protein NHERF1, which affects the intracellular localization and trafficking of NHERF1 assembled signaling complexes. Using neutron spin echo spectroscopy (NSE), we show salt-concentration-dependent excitation of nanoscale motion at the tip of the C-terminal tail in the phosphomimic S339D/S340D mutant. The "tip of the whip" that is unleashed is near the S339/S340 phosphorylation site and flanks the hydrophobic Ezrin-binding motif. The kinetic association rate constant of the binding of the S339D/S340D mutant to the FERM domain of Ezrin is sensitive to buffer salt concentration, correlating with the excited nanoscale dynamics. The results suggest that electrostatics modulates the activation of nanoscale dynamics of an intrinsically disordered protein, controlling the binding kinetics of signaling partners. NSE can pinpoint the nanoscale dynamics changes in a highly specific manner.

Keywords: disordered protein; nanoscale protein motion; neutron spin echo spectroscopy; protein binding kinetics; protein dynamics.

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Figures

Figure 1
Figure 1
Domain organization of NHERF1. The full-length protein consists of PDZ1, PDZ2, disordered C-terminal tail (CT), EBD (magenta), and S339/S340 (red). Shown is the activation of “tip of whip” motion in the phosphomimic mutant revealed by NSE. The graphics were generated using the program UCSF Chimera [71].
Figure 2
Figure 2
SEC-SAXS results of NHERF1(wt) and NHERF1(DM) in 150 mM NaCl, 20 mM Tris-HCl, pH=7.5, 5 mM DTT, 0.5 mM EDTA. I(0) and Rg vs. image number (proportional to elution time) of (A) NHERF1(wt) and (B) NHERF1(S339D/S340D). (C) SAXS profile of NHERF1(wt) (filled black square) and NHERF1(S339D/S340D) (open circle) collected after the SEC peak. Lines are fit when computing P(r). (D) P(r) functions of NHERF1(wt) (black) and NHERF1(S339D/S340D) (red).
Figure 3
Figure 3
SANS results of deuterated dNHERF1(wt) and dNHERF1(S339D/S340D) in H2O buffer. (A) I(Q) vs. Q profile. Lines are fit when computing P(r). (B) P(r) function. The SANS data were collected in 20 mM Tris-HCl, 1 mM DTT, 0.5 mM EDTA at the 150 and 300 mM NaCl concentrations specified in the graph.
Figure 4
Figure 4
NSE spectra at different Q values for NHERF1(S339D/S340D) in (A) 150 mM NaCl, and (B) 150 mM NaCl, 20 mM dTris D2O (pD=7.5) buffer solution. The lines are a single exponential fit to the spectra. (C) Deff(Q) as a function of Q for NHERF1(S339D/S340D) in 300 mM NaCl (Black) and 150 mM NaCl (red) solution. Open black circles are the center-of-mass diffusion constant Do. The black lines are Deff(Q) calculated assuming rigid-body diffusion. Red lines are calculated Deff(Q) assuming motion at the “tip of whip” shown in Fig. 1.
Figure 5
Figure 5
Equilibrium dissociation constant Kd of NHERF1(wt) (black symbols) and NHERF1(S339D/S340D) (red symbols) binding to FERM at different ionic strength I from SPR. The different symbols are from different sets of SPR measurements of different ligand immobilization levels. Representative sensorgrams used to determine Kd are shown in Fig. S3.
Figure 6
Figure 6
(A) Kinetic association rate constant ka1 of NHERF1(wt) (black) and NHERF1(S339D/S340D) (red) binding to FERM from SPR. The different symbols are from different sets of SPR measurements of different ligand immobilization levels. Typical sensorgrams are shown in Fig. S4. (B) logka1 vs. logγ±, where logγ± is calculated from the Debye–Hückel equation (Eq. 4).
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