Isomer-Specific Interactions of X-Pro Motifs: Investigating Fundamental Mechanisms of Signaling by Pro-Rich Sequences

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This project addresses a novel hypothesis regarding how a particular "on/off" switch inside a cell is regulated. The switch of interest involves a biological signaling interaction between two specific proteins that connect innate immunity signaling (a cell's first line of defense) to cell migration (the directed movement of a cell). This work builds on the experimental observation that IRAK1 (a key signaling protein in innate immunity) adopts two very different and slowly interconverting structures (cis and trans isomers of a specific Trp-Pro peptide bond), exactly in the region where it binds to VASP (a protein that regulates cell migration). VASP has two distinct domains (EVH1 and EVH2). Only the VASP-EVH1 domain directly binds to IRAK1, and this interaction is selective for only the trans isomer of IRAK1. Since IRAK1 has nearly equal populations of cis and trans structures before VASP enters the picture, the initial IRAK1:VASP binding utilizes only about half of the total IRAK1 that is present. This fast initial binding step is followed by a slow binding phase, the rate of which depends on the rate of flipping back and forth of the cis and trans isomers. This slow rate can be greatly accelerated by an enzyme (cyclophilin A, or CypA) that catalyzes the flipping process. Hence, CypA effectively catalyzes the IRAK1-VASP binding interaction by facilitating rapid conversion of cis to trans to replace the trans that has bound to VASP. The EVH2 domain of VASP self-associates as a cluster of four (a tetramer); consequently, full-length VASP is a tetramer that displays four EVH1 domains in close proximity. Interestingly, recent discoveries suggest that when innate immunity signaling is triggered, activated IRAK1 is also a tetramer. When placed into the context of a tetramer:tetramer interaction (the "on" state of the switch), there is far less IRAK1 tetramer in which all 4 IRAK1 components are in the VASP-binding competent trans state. In this case, enzyme-catalyzed acceleration of the cis/trans flipping (isomerization) rate is even more critical for achieving a binary "on/off" switch, in which the time constants for turning the switch on or off are short enough to be effective in the fast paced dynamics of the inner workings of a cell. This project will provide 1) a kinetic and thermodynamic description of the monomer:monomer IRAK1:VASP interaction, and 2) a quantitative analysis of the binding enhancement associated with the specific clustering of IRAK1 and the role of cis-trans isomerization in achieving this enhancement on a biologically relevant timescale. Together, these results will reveal how a key biological signaling interaction works to rapidly connect innate immunity signaling to cell migration. This work synergistically couples to cell biologists who study the participation of IRAK1 and VASP in biological processes such as innate immunity and cell migration, and to systems biologists who mathematically model signaling pathways and who require thermodynamic and kinetic data for critical steps in such pathways.The research in this project will be integrated in several ways into the teaching of a large (70-80 students) graduate level 3-credit course on Protein Structure, Function and Dynamics (BioMG 6310). As part of this class, a team-based project is assigned in which students write a proposal to "save the world" using proteins (with focus on energy and the environment), and projects are presented at the end of the semester at a public poster session. This activity has fostered connections with Cornell's Atkinson Center for a Sustainable Future (ACSF), including display of the posters at ACSF to a diverse audience of visitors, and the appointment of the PI as an ACSF Faculty Fellow. This NSF-sponsored research will be incorporated into BioMG 6310 lectures to illustrate fundamental concepts. Outreach to underrepresented groups includes giving lab tours for The Learning Web (a local youth development agency that serves a diversity of rural and city children), and bringing excitement for science to the 7th and 9th Wards of New Orleans during annual rebuilding trips with GEEKS (Graduates Employing Empathy, Knowledge and Service), a Cornell graduate student organization that the PI advises. The PI serves as research mentor for undergraduate (independent study and summer research) and graduate students in her lab. Students are trained in an interdisciplinary set of techniques that include NMR, biophysical methods, mathematical modeling, biochemistry, and cell biology.