Disordered proteins mitigate the temperature dependence of site-specific binding free energies

Abstract

Biophysical characterization of protein-protein interactions involving disordered proteins is challenging. A common simplification is to measure the thermodynamics and kinetics of disordered site binding using peptides containing only the minimum residues necessary. We should not assume, however, that these few residues tell the whole story. Son of sevenless, a multidomain signaling protein from Drosophila melanogaster, is critical to the mitogen-activated protein kinase pathway, passing an external signal to Ras, which leads to cellular responses. The disordered 55 kDa C-terminal domain of Son of sevenless is an autoinhibitor that blocks guanidine exchange factor activity. Activation requires another protein, Downstream of receptor kinase (Drk), which contains two Src homology 3 domains. Here, we utilized NMR spectroscopy and isothermal titration calorimetry to quantify the thermodynamics and kinetics of the N-terminal Src homology 3 domain binding to the strongest sites incorporated into the flanking disordered sequences. Comparing these results to those for isolated peptides provides information about how the larger domain affects binding. The affinities of sites on the disordered domain are like those of the peptides at low temperatures but less sensitive to temperature. Our results, combined with observations showing that intrinsically disordered proteins become more compact with increasing temperature, suggest a mechanism for this effect.

Keywords: Src homology 3 domain; biophysics; intrinsically disordered protein; protein–protein interaction; thermodynamics.

Figure 1

Sos (Son of sevenless)–Src homology 3 (SH3) binding.A, simplified model of Ras activation by Sos. B, representation of SH3-binding sites on Sos disordered C-terminal tail. Simulation of PepS2 binding to SH3 domain (70). Sequence alignment of binding motif with Sos-binding sites (21, 24). C, model of site 2, site 4, and knockout constructs with conserved amino acid positions highlighted at each site (as in B).

Figure 2

ITC analysis of Sos–SH3 binding at 4.2 °C.A, Sos site 2. B, Sos site 2-matched knockout. C, Sos site 4. D, Sos site 4-matched knockout. A and B were performed with 229 μM Sos and 4 mM SH3. C and D were performed with 250 μM Sos and 3.56 mM SH3. ITC, isothermal titration calorimetry; SH3, Src homology 3; Sos, Son of sevenless.

Figure 3

¹H–¹³C HMQC titration of Sos site 4 at 35 °C.A, spectra. B, simulated spectra from TITAN fits. C, van’t Hoff and Eyring analyses of ¹H–¹³C HMQC titrations. Measurements were made with a constant concentration of 51.5 μM Sos site 4, with titration points signified by crosspeaks of different colors. SH3 concentrations are 270, 122, 100, 75.0, 50.0, 37.5, 25.0, 12.5, 6.25, 3.13, 1.65, 0.781, 0.391, and 0 μM. HMQC, heteronuclear multiple quantum correlation; Sos, Son of sevenless.

Figure 4

Temperature dependence of SH3–Sos dissociation free energy. SH3, Src homology 3; Sos, Son of sevenless.