Binding of polyubiquitin chains to ubiquitin-associated (UBA) domains of HHR23A.
Ubiquitin-associated (UBA) domains are small protein domains that occur in the context of larger proteins and are likely to function as inter- and intramolecular communication elements in ubiquitin/polyubiquitin signaling. Although monoubiquitin/UBA complexes are well characterized, much less is known about UBA/polyubiquitin complexes, even though polyubiquitin chains are believed to be biologically relevant ligands of many UBA domain proteins. Here, we report the results of a quantitative study of the interaction of K48-linked polyubiquitin chains with UBA domains of the DNA repair/proteolysis protein HHR23A, using surface plasmon resonance and other approaches. We present evidence that the UBL domain of HHR23A negatively regulates polyubiquitin/UBA interactions and identify leucine 8 of ubiquitin as an important determinant of chain recognition. A striking relationship between binding affinity and chain length suggests that maximum affinity is associated with a conformational feature that is fully formed in chains of n = 4-6 and can be recognized by a single UBA domain of HHR23A. Our findings provide new insights into polyubiquitin chain recognition and set the stage for future structural investigations of UBA/polyubiquitin complexes.
Raasi
Shahri
S
Department of Biochemistry and Molecular Biology/Bloomberg School of Public Health, Johns Hopkins University, 615 North Wolfe Street, Baltimore, MD 21205, USA.
Orlov
Irina
I
Fleming
Karen G
KG
Pickart
Cecile M
CM
D003432:Cross-Linking Reagents / Q000378:metabolism; D045643:DNA Repair Enzymes; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D046911:Macromolecular Substances; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D020349:Surface Plasmon Resonance; D025801:Ubiquitin / Q000737:chemistry / Q000378:metabolism*
D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D025801:Ubiquitin; C086985:RAD23A protein, human; D045643:DNA Repair Enzymes