RUI: Biophysical investigation of SH3 domain binding partners: How the binding motif and surrounding disordered sequence affect the finding pathway

Skidmore College, Saratoga Springs NY

Investigators

Abstract

The communication within cells that allows cellular processes to occur is mediated by interactions between proteins. The goal of this research is to understand how these fundamental interactions are controlled at the local site of interaction, as well as the surrounding regions. Understanding the details of these interactions would allow researchers to predict and modify cell behavior. Results of this project will provide deeper insights into how protein binding interactions function in different contexts, including in the presence of multiple sites that can bind the same protein partner, and help explain how a common interaction can specialize to perform many different cellular functions. Undergraduate students working on this project will have the opportunity to learn both computational and experimental biophysics skills. To allow a larger number of students to engage in undergraduate research, the investigator will offer a research-based lab course that will expose students to techniques in computational biophysics and molecular dynamics simulations. In this course, students will develop and carryout a research project contributing to the larger project goals. Cellular signaling interactions often involve binding of intrinsically disordered protein regions to small domains. The affinity and specificity of these interactions depend on the binding motif within the disordered region, but are also affected by the flanking regions and surrounding context. Additionally, the binding pathway, rather than just the structure of the bound state, can be important for understanding the functional adaptation of these interactions. Focusing on SH3 domain binding as a model system, the PI have previously shown that the disordered proline-rich peptide ArkA binds to the AbpSH3 domain in a multi-step process using molecular dynamics (MD) simulations. In this project, the PI will now examine how this binding pathway varies in different biologically relevant contexts by systematically studying this pathway at increasing levels of complexity using MD simulations, NMR spectroscopy, and ITC. The first aim will focus on the effects of altering the flexibility and electrostatic interactions of the binding motif itself by simulating the binding with ArkA prolines in the cis conformation, with salt screening electrostatic interactions, and with mutations to charged AbpSH3 residues involved in binding. In the second aim, different physiologically relevant IDR sequences will be compared to explore the effect of the motif-flanking regions on binding. In the third aim, the binding process in the context of multiple binding motifs will be examined. The completion of this project will add a new level of complexity to the understanding of how disordered protein regions bind to SH3 domains, as how both the binding motif and the surrounding sequences influence the binding pathway, and ultimately biological function will be revealed. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.