Research Interests Targeted protein degradation; Protein quality control; NMR spectroscopy

Ubiquitin-mediated protein degradation is vital, as it regulates major cellular events including cell cycle progression, transcription, and apoptosis. Research in the Walters laboratory focuses on how ubiquitinated substrates are recognized and processed by the proteasome and on how aberrant cytosolic proteins are trafficked to degradation machineries. NMR spectroscopy is used to provide atomic level information on ubiquitin receptors that enable the proteasome to recognize its substrates. Our recent work has focused on proteasome components S5a and Rpn13. By using NMR spectroscopy, we defined how these two proteins bind polyubiquitin (Figs below) and helped establish Rpn13 as a ubiquitin receptor. We also found that the two proteasome receptors can bind simultaneously to ubiquitinated substrates. We continue to study proteasome components and their interactions with each other and ubiquitinated substrates.

S5a (196-306) is shown in gray bound to ubiquitin subunits within K48-linked diubiquitin. S5a has a preference for its C-terminal ubiquitin interacting motif (UIM2) binding to the proximal (blue) subunit while its N-terminal UIM (UIM1) binds to the distal subunit (green). K48 of the proximal subunit is linked to the C-terminal glycine of the distal one and it is the proximal subunit that would be attached to a protein substrate. The two binding modes were established by intermolecular NOEs of half-filtered NOESY experiments.

By selectively labeling either the proximal of distal subunits of K48-linked diubiquitin, we found that Rpn13’s ubiquitin binding domain (displayed in yellow) binds preferentially to the proximal subunit.  Rpn13 binds ubiquitin with three loops, shown in blue. This binding mode would leave the distal subunit free to interact with other ubiquitin processing proteins of the proteasome.

Schreiner, P., Chen, X., Husnjak, K., Randles, L., Zhang, N., Elsasser, S., Finley, D., Dikic, I.*, Walters, K. J.*, Groll, M.*: “Ubiquitin docking at the proteasome via a novel PH domain interaction,” Nature 453, 548-552 (2008).
 
Husnjak, K., Elsasser, S., Zhang, N., Chen, X., Randles, L., Hofmann, K., Walters, K. J.*, Finley, D.*, Dikic, I.*: “Proteasome subunit Rpn13 is a novel ubiquitin receptor,” Nature 453, 481-488 (2008).
 
Zhang, N., Wang, Q., Ehlinger, A., Randles, L., Lary, J. W., Kang, Y., Haririnia, A., Cole, J. L., Fushman, D., Walters, K. J.: “Structure of the S5a:K48 linked diubiquitin complex and its interactions with Rpn13” Molecular Cell 35, 280-290 (2009).
 
Zhang, N., Walters, K. J.*: “Insights into how protein dynamics affects arylamine N-acetyltransferase catalysis” Biochem and Biophys Res Comm 385, 395-401 (2009).
 
Chen, X., Solomon, W. C., Kang, Y., Cerda-Maira, F., Darwin, K. H., Walters, K. J.: “Prokaryotic ubiquitin-like protein Pup is intrinsically disordered” J. of Mol. Biol. 392, 208-217 (2009).

Dikic, I., Wakatsuki, S. Walters, K. J.: “Ubiquitin binding domains-from structures to functions” Nature Reviews Molecular Cell Biology 10, 659-671 (2009).

Walters Lab Photo