Identification of rabbit reticulocyte E2(17K) as a UBC7 homologue and functional characterization of its core domain loop

The structural basis by which ubiquitin (Ub)-conjugating enzymes (E2s) dete rmine substrate specificity remains unclear. We cloned rabbit reticulocyte E2(17K) because unlike the similarly sized class I E2s, E2(14K) and UBC4, i t is unable to support ubiquitin-protein ligase (E3)-dependent conjugation to endogenous proteins. RNA analysis revealed that this E2 was expressed in all tissues tested, with higher levels in the testis, Analysis of testis R NA from rats of different ages showed that E2(17K) mRNA was induced from da ys 15 to 30, The predicted amino acid sequence indicates that E2(17K) is a 19.5-kDa class I E2 but differs from other class I enzymes in possessing an insertion of 13 amino acids distal to the active site cysteine, E2(17K) sh ows 74% amino acid identity with Saccharomyces cerevisiae UBC7, and therefo re, we rename it mammalian UBC7, Yeast UBC7 crystal structure indicates tha t this insertion forms a loop out of the otherwise conserved folding struct ure. Sequence analysis of E2s had previously suggested that this loop is a hypervariable region and may play a role in substrate specificity. We creat ed mutant UBC7 lacking the loop (ubc7 Delta loop) and a mutant E2(17K) with an inserted loop (E2(14k)+loop) and characterized their biochemical functi ons. Ubc7 Delta loop had higher affinity for the E1-Ub thiol ester than nat ive UBC7 and permitted conjugation of Ub to selected proteins in the testis but did not permit the broad spectrum E3-dependent conjugation to endogeno us reticulocyte proteins. Surprisingly, E2(14k)+loop was unable to accept U b from ubiquitin-activating enzyme (E1) but was able to accept NEDDS from E 1. E2(14k)+loop was able to support conjugation of NEDD8 to endogenous reti culocyte proteins but with much lower efficiency than E2(14k). Thus, the lo op can influence interactions of the E2 with charged El as well as with E3s or substrates, but the exact nature of these interactions depends on diver gent sequences in the remaining conserved core domain.