Physiologically-Relevant Crowding Effects on the SH3-Son of Sevenless Interaction

Abstract

Nearly all biological processes, including tightly regulated protein-protein interactions implicated in cell signaling, occur inside living cells where the concentration of macromolecules can exceed 300 g/L. One such interaction that occurs along the cellular membrane, is between a globular 7-kDa SH3 domain and a 25-kDa intrinsically disordered region of Son of Sevenless (SOS). Despite its importance as a mediator in the mitogen-activated protein kinase signaling pathway present in all eukaryotic cells, most biophysical characterizations of this complex are performed in dilute buffered solutions where cosolute concentrations rarely exceed 10 g/L. We are investigating the effects of crowding, or high concentrations of physiologically-relevant cosolutes, on the equilibrium thermodynamics and kinetics of binding between SH3 and SOS. The SH3 domain is labeled with a fluorine atom on its sole tryptophan, allowing the interaction to be monitored with 19F NMR. Subsequent NMR lineshape analysis allows quantification of both rate constants, and hence the equilibrium constant. Two systems are used. The first system, a control, comprises the SH3 domain and a SOS-derived peptide. Experiments using a range of biologically-relevant molecules reveal that the kinetics are affected more than the stability of the complex. The origins of these effects are likely increased viscosity and electrostatic interactions with crowder molecules. The second system is that between SH3 and the full-length disordered region of SOS. Crowding effects are expected to be larger in magnitude than those observed for the peptide studies based on hypotheses from traditional crowding theory. These experiments are the first to analyze the effects of crowding on a protein-intrinsically disordered protein complex and have immediate implications for understanding the effects of the cellular environment on protein-protein interactions.