Comparative peptide binding studies of the PABC domains from the ubiquitin-protein isopeptide ligase HYD and poly(A)-binding protein. Implications for HYD function.
The PABC domain is a peptide-binding domain that is specifically found in poly(A)-binding protein (PABP) and a HECT ubiquitin-protein isopeptide ligase (E3) known as HYD (hyperplastic discs), EDD (E3 isolated by differential display), or Rat100. The PABC domain of PABP recruits various regulatory proteins and translation factors to poly(A) mRNAs through binding of a conserved 12-amino acid peptide motif, PAM2 (PABP-interacting motif 2). In contrast, little is known about the specificity or function of the domain from HYD. Here, we used isothermal calorimetry and surface plasmon resonance titrations to show that the PABC domain of HYD binds PAM2 peptides with micromolar affinity. NMR chemical shift perturbations were used to map the peptide-binding site in the PABC domain of HYD. The structural features of binding are very similar to those of the interactions with the domain of PABP, which explains the overlapping peptide specificity and binding affinity. We identified the anti-proliferative Tob proteins as potential binding partners of HYD. This was confirmed by glutathione S-transferase pulldown and immunoprecipitation experiments demonstrating the interaction with full-length Tob2. Altogether, our results point to a role of the PABC domain as a protein-protein interaction domain that brings together the processes of translation, ubiquitin-mediated protein degradation, and cell cycle control.
Lim
Nadia S
NS
Department of Biochemistry, McGill University, 3655 Promenade Sir William Osler, Montréal, Québec H3G 1Y6, Canada.
Kozlov
Guennadi
G
Chang
Tsung-Cheng
TC
Groover
Olivia
O
Siddiqui
Nadeem
N
Volpon
Laurent
L
De Crescenzo
Gregory
G
Shyu
Ann-Bin
AB
Gehring
Kalle
K
D000595:Amino Acid Sequence; D000818:Animals; D005982:Glutathione Transferase / Q000378:metabolism; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*; D039102:Poly(A)-Binding Proteins / Q000737:chemistry*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D017386:Sequence Homology, Amino Acid; D020349:Surface Plasmon Resonance; D025801:Ubiquitin / Q000737:chemistry*; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry*
D010455:Peptides; D039102:Poly(A)-Binding Proteins; D025801:Ubiquitin; C466763:Ubr5 protein, rat; D044767:Ubiquitin-Protein Ligases; D005982:Glutathione Transferase