Rad6, an E2 ubiquitin-conjugating enzyme conserved from yeast to humans, functions in transcription, genome maintenance, and proteostasis. The contributions of many conserved secondary structures of Rad6 and its human homologs UBE2A and UBE2B to their biological functions are not understood. A mutant RAD6 allele with a missense substitution at alanine-126 (A126) of helix-3 that causes defects in telomeric gene silencing, DNA repair, and protein degradation was reported over 2 decades ago. Here, using a combination of genetics, biochemical, biophysical, and computational approaches, we discovered that helix-3 A126 mutations compromise the ability of Rad6 to ubiquitinate target proteins without disrupting interactions with partner E3 ubiquitin-ligases that are required for their various biological functions in vivo. Explaining the defective in vitro or in vivo ubiquitination activities, molecular dynamics simulations and NMR showed that helix-3 A126 mutations cause local disorder of the catalytic pocket of Rad6 in addition to disorganizing the global structure of the protein to decrease its stability in vivo. We also show that helix-3 A126 mutations deform the structures of UBE2A and UBE2B, the human Rad6 homologs, and compromise the in vitro ubiquitination activity and folding of UBE2B. Providing insights into their ubiquitination defects, we determined helix-3 A126 mutations impair the initial ubiquitin charging and the final discharging steps during substrate ubiquitination by Rad6. In summary, our studies reveal that the conserved helix-3 is a crucial structural constituent that controls the organization of catalytic pockets, enzymatic activities, and biological functions of the Rad6-family E2 ubiquitin-conjugating enzymes.

• PMID: 36162503
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Journal Article | Research Support, N.I.H., Extramural | Research Support, Non-U.S. Gov't

Authors

Shukla PK, Sinha D, Leng AM, Bissell JE, Thatipamula S, Ganguly R, Radmall KS, Skalicky JJ, Shrieve DC, Chandrasekharan MB

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