Bencsath Kalman P, Podgorski Michael S, Pagala Vishwajeeth R, Slaughter Clive A, Schulman Brenda A
Department of Structural Biology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, USA.
J Biol Chem. 2002 Dec 6;277(49):47938-45. doi: 10.1074/jbc.M207442200. Epub 2002 Sep 26.
Ubiquitin-like proteins (ub-lps) are conjugated by a conserved enzymatic pathway, involving ATP-dependent activation at the C terminus by an activating enzyme (E1) and formation of a thiolester intermediate with a conjugating enzyme (E2) prior to ligation to the target. Ubc9, the E2 for SUMO, synthesizes polymeric chains in the presence of its E1 and MgATP. To better understand conjugation of ub-lps, we have performed mutational analysis of Saccharomyces cerevisiae Ubc9p, which conjugates the SUMO family member Smt3p. We have identified Ubc9p surfaces involved in thiolester bond and Smt3p-Smt3p chain formation. The residues involved in thiolester bond formation map to a surface we show is the E1 binding site, and E2s for other ub-lps are likely to bind to their E1s at a homologous site. We also find that this same surface binds Smt3p. A mutation that impairs binding to E1 but not Smt3p impairs thiolester bond formation, suggesting that it is the E1 interaction at this site that is crucial. Interestingly, other E2s and their relatives also use this same surface for binding to ubiquitin, E3s, and other proteins, revealing this to be a multipurpose binding site and suggesting that the entire E1-E2-E3 pathway has coevolved for a given ub-lp.
类泛素蛋白（ub-lps）通过一条保守的酶促途径进行缀合，该途径包括在C末端由激活酶（E1）进行ATP依赖性激活，并在与靶标连接之前与缀合酶（E2）形成硫酯中间体。SUMO的E2——Ubc9，在其E1和MgATP存在的情况下合成聚合物链。为了更好地理解ub-lps的缀合过程，我们对酿酒酵母Ubc9p进行了突变分析，Ubc9p可与SUMO家族成员Smt3p缀合。我们确定了Ubc9p中参与硫酯键和Smt3p-Smt3p链形成的表面。参与硫酯键形成的残基定位到一个我们显示为E1结合位点的表面，其他ub-lps的E2可能在同源位点与其E1结合。我们还发现同一表面结合Smt3p。一个损害与E1结合但不影响与Smt3p结合的突变会损害硫酯键的形成，这表明正是该位点的E1相互作用至关重要。有趣的是，其他E2及其相关蛋白也利用同一表面结合泛素（ubiquitin）、E3和其他蛋白质，这表明该表面是一个多功能结合位点，也表明整个E1-E2-E3途径针对特定的ub-lp共同进化。
