The Interaction Between The Evh1 Domain Of Vasp And Proline-Rich Sequences: Significance Of Isomerization, Phosphorylation, And A Secondary Binding Site
Ena/VASP proteins are cytoskeletal rearrangement proteins involved in processes such as phagocytosis, cell migration, and axon guidance in neurons. VASP contains an Nterminal EVH1 domain which localizes it to specific sites in the cell. The VASPEVH1 domain recognizes the proline-rich motif (F/W)Px[phi]P, which occurs in the immune cell signaling kinase IRAK1 and also as tandem repeats in the focal adhesion protein zyxin. Typically this motif is flanked by negatively charged amino acids that are necessary for a tight ( K D < 200 [MICRO SIGN]M) interaction. We have examined the interactions between VASP-EVH1 and the binding sites in IRAK1 and zyxin and describe three novel observations. First, we report that the W168-P169 peptide bond in the IRAK1 binding site partitions into nearly equal (43% cis, 57% trans) population of cis and trans isomers, and that EVH1 binding to this site is specific for the trans isomer. We use NMR and tryptophan fluorescence spectroscopy to show that the prolyl isomerase cyclophilin A (CypA) isomerizes this site and accelerates binding of VASP-EVH1 to peptides. Second, we report that phosphorylation of two serines flanking the WPPPP motif in IRAK1 increases the affinity for VASP-EVH1 by a factor of seven. This shows that phosphoserine is an effective substitute for glutamic or aspartic acid in VASP-binding sequences, suggesting that phosphorylation could be a novel mechanism to regulate VASP localization, especially in the case of IRAK1 and lamellipodin, which are known to be phosphorylated. Third, we show that VASPEVH1 contains a noncanonical second binding site on the face opposite its primary binding site. We show that this site shares some binding specificity with the canonical (F/W)Px[phi]P motif, and that this binding site is employed in the interaction with zyxin to recognize the third and fourth VASP-binding repeats of zyxin. We also show that this binding site is conserved in the homologous Ran-binding domain (RanBD), suggesting that it is widespread in EVH1 homologs. Separately, we report an NMR lineshape analysis-based protocol for dissecting the microscopic rate constants of a prolyl isomerase enzyme.
VASP; NMR; EVH1
Nicholson, Linda K
Wilson, David B; Oswald, Robert Edward
Ph. D., Biophysics
Doctor of Philosophy
dissertation or thesis