The second WW domain (WW2) of the kidney and brain scaffolding protein, KIBRA, has an isoleucine (Ile-81) rather than a second conserved tryptophan and is primarily unstructured. However, it adopts the canonical triple-stranded antiparallel β-sheet structure of WW domains when bound to a two-PPXY motif peptide of the synaptic protein Dendrin. Here, using a series of biophysical experiments, we demonstrate that the WW2 domain remains largely disordered when bound to a 69-residue two-PPXY motif polypeptide of the synaptic and podocyte protein synaptopodin (SYNPO). Isothermal titration calorimetry and CD experiments revealed that the interactions of the disordered WW2 domain with SYNPO are significantly weaker than SYNPO's interactions with the well-folded WW1 domain and that an I81W substitution in the WW2 domain neither enhances binding affinity nor induces substantial WW2 domain folding. In the tandem polypeptide, the two WW domains synergized, enhancing the overall binding affinity with the I81W variant tandem polypeptide 2-fold compared with the WT polypeptide. Solution NMR results showed that SYNPO binding induces small but definite chemical shift perturbations in the WW2 domain, confirming the disordered state of the WW2 domain in this complex. These analyses also disclosed that SYNPO binds the tandem WW domain polypeptide in an antiparallel manner, that is, the WW1 domain binds the second PPXY motif of SYNPO. We propose a binding model consisting of a bipartite interaction mode in which the largely disordered WW2 forms a “fuzzy” complex with SYNPO. This binding mode may be important for specific cellular functions.
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.
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