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pathways. in recent years, in vivo studies have shown that the protein Histidine triad nucleotide-binding protein 1 (HINT1) inhibits the ubiquitylation activity of various SCF ligases that regulate the NF-kB signaling pathway and cell cycle progression by promoting the degradation of substratesIkBa and p27 Kip1 , respectively. However, the mechanism of interaction between HINT1 and SCF ligases and how HINT1 causes their inhibition is not understood. Many questions remain unanswered such as the specific binding partner of HINT1 within the SCF complex and how HINT1 is able to inhibit the activity of unique SCF ligases. in total, the 173 kDa SCF ligase complex consists of a scaffolding protein, Cullin1 (90 kDa), that utilizes Rbx1 (12 kDa) to recruit E2 ubiquitin conjugating enzymes (e.g. UBE2D2) and substrates through the adaptor and substrate receptor proteins, Skp1 (17 kDa) and b-TrCP1 (54 kDa) respectively, to promote substrate ubiquitylation. Through co-elution in gel-filtration chromatography, I have assembled the SCF E3 ligase that ubiquitylates IkBa and have demonstrated that HINT1 (28 kDa) binds to this ligase. I have performed biochemical experiments to measure ubiquitylation combined with HDX-MS and other biophysical experiments to characterize the binding affinity and the specific binding interface of HINT1 on the SCF E3 ligase. The results shed light on the molecular mechanism of inhibition of SCF E3 ligases by HINT1.

The WWC family consists of three proteins‐ KIBRA/WWC1, WWC2, and WWC3. Each protein contains two WW domains which recognize proline‐rich sequences of the type PPxY (P is proline, Y is tyrosine, x is any amino acid). WW domains are named for two conserved tryptophans spaced 23 residues apart and adopt a three‐stranded antiparallel beta sheet fold. The WWC proteins are “atypical” in that, the second WW domain (WW2) contains an isoleucine rather than a conserved tryptophan. Recent studies of the KIBRA WW domains shows that this substitution leads to unfolding of the WW2 domains. However, the domain undergoes binding‐induced folding when bound to some partners but remains largely unfolded when bound to others. High sequence similarity of the WW domains in the WWC family suggests that unfolding and binding induced folding of the WW2 domain may be conserved in this family, but it is unclear why some partners induce folding of the WW2 domain while others do not. Our working hypothesis is that partners which contain PPxY motifs in close proximity will bind the WWC proteins with relatively high affinity and induce folding of the WW2 domain. In this study, we test this hypothesis with two binding partners, Large Tumor Suppressor 1 (LATS1), a kinase and tumor suppressor, and Synaptopodin (SYNPO), a synaptic and podocyte protein. We demonstrate by isothermal titration calorimetry that wildtype LATS1 and SYNPO which have two PPxY motifs each, separated by 194 and 15 residues respectively, bound KIBRA and WWC3 with modest micromolar binding affinities. However, mutant LATS1 (LATS1‐VA) and SYNPO (SYNPO_AA) constructs in which the PPxY motifs are separated by two amino acids, bound KIBRA and WWC3 with 20 to 180‐fold enhanced affinity. Using solution magnetic resonance spectroscopy, we demonstrated that the WW2 domain of KIBRA and WWC3 remain unfolded when bound to wildtype LATS1 and SYNPO, while the enhanced binding of the mutants LATS1_VA and SYNPO_AA is accompanied by folding of the WW2 domain. The structural heterogeneity of the WW2 domain may be an important for modulating binding to diverse PPxY motif partners. Support or Funding Information This work is supported by start‐up funds from Oregon State University (OSU), OSU College of Science Disease Mechanism and Prevention Fund, and a New Investigator Award from the Medical Research Foundation of Oregon. NMR experiments were collected at the Oregon State University NMR Facility funded in part by the National Institutes of Health, HEI Grant 1S10OD018518, and by the M. J. Murdock Charitable Trust grant # 2014162