Lim Lab | Papers

Structure of the N-WASP EVH1 Domain-WIP Complex: Insight into the Molecular Basis of Wiskott-Aldrich Syndrome

Brian F. Volkman, Kenneth E. Prehoda, Jessica A. Scott, Francis C. Peterson, and Wendell A. Lim
Cell 111, 565–576 (2002)

Supplemental Figure S1. Presence of Covalent Linker Does Not Effect WIP-EVH1 Complex Structure

(A) In addition to the covalently linked construct used for the bulk of these studies, we also generated a construct containing a thrombin cleavage site between the WIP peptide and the EVH1 domain. Samples were prepared and the second construct cleaved to completion as described in Experimental Procedures.

(B) 2D 15N-1H HSQC spectra of the covalently tethered complex (black) and the cleaved complex (red). Bottom image shows an overlay of the two spectra. In all images EVH1 domain residues at the binding surface are labeled in violet and WIP peptide residues are labeled in gold. Labeled EVH1 residues include those that that bind the N-terminal part of the peptide (Y46, W56, F104, T106; see Figure 5) and those that bind near the C terminus of the peptide (E90, Y92, G109, C112). Visible peptide residues range from 461 to 480. The spectra are virtually identical except for minor shifts (green) at residues near the cleavage termini. A few new resonances in the cleaved spectrum correspond to the new residues introduced by the thrombin site.

Conclusion: the structure of the EVH1-WIP complex remains the same even after cleavage of the covalent tether.