Identification of molecular determinants required for interaction of ubiquitin-conjugating enzymes and RING finger proteins.
Recent results from several laboratories suggest that the interaction of E2 ubiquitin-conjugating enzymes with the RING finger domain has a central role in mediating the transfer of ubiquitin to proteins. Here we present a mutational analysis of the interaction between the E2 enzyme UbcM4/UbcH7 and three different RING finger proteins, termed UIPs, which, like Parkin, contain a RING1-IBR-RING2 motif. The results show that the E2 enzyme binds to the RING1 domain but not to the other cysteine/histidine-rich domains of the RING1-IBR-RING2 motif. Three regions within the UbcM4 molecule are involved in this interaction: the H1 alpha helix, loop L1, connecting the third and fourth strand of the beta sheet, and loop L2, located between the fourth beta strand and the second alpha helix. Loop L2 plays an important role in determining the specificity of interaction. The effects of L2 mutations on UbcM4/UIP interaction are different for each UIP, indicating that RING finger domains can vary considerably in their structural requirements for binding to E2 enzymes. The result that single amino-acid changes can regulate binding of E2 enzymes to different RING finger proteins suggests a novel approach to experimentally manipulate proteolytic pathways mediated by RING finger proteins.
Martinez-Noel
G
G
Heinrich-Pette-Institut für Experimentelle Virologie und Immunologie an der Universität Hamburg, Hamburg, Germany.
Müller
U
U
Harbers
K
K
D000595:Amino Acid Sequence; D000818:Animals; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008025:Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid; D025801:Ubiquitin / Q000378:metabolism*; D044763:Ubiquitin-Conjugating Enzymes; D016335:Zinc Fingers
D004268:DNA-Binding Proteins; D025801:Ubiquitin; D044763:Ubiquitin-Conjugating Enzymes; D008025:Ligases