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Binding surface mapping of intra- and interdomain interactions among hHR23B, ubiquitin, and polyubiquitin binding site 2 of S5a.

hHR23B is the human homologue of the yeast protein RAD23 and is known to participate in DNA repair by stabilizing xeroderma pigmentosum group C protein. However, hHR23B and RAD23 also have many important functions related to general proteolysis. hHR23B consists of N-terminal ubiquitin-like (UbL), ubiquitin association 1 (UBA1), xeroderma pigmentosum group C binding, and UBA2 domains. The UBA domains interact with ubiquitin (Ub) and inhibit the assembly of polyubiquitin. On the other hand, the UbL domain interacts with the poly-Ub binding site 2 (PUbS2) domain of the S5a protein, which can carry polyubiquitinated substrates into the proteasome. We calculated the NMR structure of the UbL domain of hHR23B and determined binding surfaces of UbL and Ub to UBA1, UBA2, of hHR23B and PUbS2 of S5a by using chemical shift perturbation. Interestingly, the surfaces of UbL and Ub that bind to UBA1, UBA2, and PUbS2 are similar, consisting of five beta-strands and their connecting loops. This is the first report that an intramolecular interaction between UbL and UBA domains is possible, and this interaction could be important for the control of proteolysis by hHR23B. The binding specificities of UbL and Ub for PUbS1, PUbS2, and general ubiquitin-interacting motifs, which share the LALA motif, were evaluated. The UBA domains bind to the surface of Ub including Lys-48, which is required for multiubiquitin assembly, possibly explaining the observed inhibition of multiubiquitination by hHR23B. The UBA domains bind to UbL through electrostatic interactions supported by hydrophobic interactions and to Ub mainly through hydrophobic interactions supported by electrostatic interactions.

Pubmed ID: 12832454 RIS Download

Mesh terms: Amino Acid Motifs | Amino Acid Sequence | Binding Sites | Carrier Proteins | Cloning, Molecular | Cysteine Endopeptidases | DNA Repair | DNA Repair Enzymes | DNA-Binding Proteins | Humans | Kinetics | Lysine | Magnetic Resonance Spectroscopy | Models, Molecular | Molecular Sequence Data | Multienzyme Complexes | Proteasome Endopeptidase Complex | Protein Binding | Protein Conformation | Protein Structure, Tertiary | Saccharomyces cerevisiae Proteins | Sequence Homology, Amino Acid | Ubiquitin