Department of Biochemistry, The University of Western Ontario, London, ON, Canada, N6A 5C1.
Biochem J. 2012 Jul 15;445(2):167-74. doi: 10.1042/BJ20120504.
E2 conjugating enzymes are the central enzymes in the ubiquitination pathway and are responsible for the transfer of ubiquitin and ubiquitin-like proteins on to target substrates. The secondary structural elements of the catalytic domain of these enzymes is highly conserved, including the sequence conservation of a three-residue HPN (His-Pro-Asn) motif located upstream of the active-site cysteine residue used for ubiquitin conjugation. Despite the vast structural knowledge of E2 enzymes, the catalytic mechanism of these enzymes remains poorly understood, in large part due to variation in the arrangements of the residues in the HPN motif in existing E2 structures. In the present study, we used the E2 enzyme HIP2 to probe the structures of the HPN motif in several other E2 enzymes. A combination of chemical-shift analysis, determination of the histidine protonation states and amide temperature coefficients were used to determine the orientation of the histidine ring and hydrogen-bonding arrangements within the HPN motif. Unlike many three-dimensional structures, we found that a conserved hydrogen bond between the histidine imidazole ring and the asparagine backbone amide proton, a common histidine protonation state, and a common histidine orientation exists for all E2 enzymes examined. These results indicate that the histidine within the HPN motif is orientated to structurally stabilize a tight turn motif in all E2 enzymes and is not orientated to interact with the asparagine side chain as proposed in some mechanisms. These results suggest that a common catalysis mechanism probably exists for all E2 conjugating enzymes to facilitate ubiquitin transfer.
E2 连接酶是泛素化途径中的核心酶，负责将泛素和泛素样蛋白转移到靶底物上。这些酶的催化结构域的二级结构元件高度保守，包括位于活性半胱氨酸残基上游的三残基 HPN（组氨酸-脯氨酸-天冬酰胺）基序的序列保守性，该基序用于泛素连接。尽管 E2 酶具有广泛的结构知识，但这些酶的催化机制仍知之甚少，部分原因是现有 E2 结构中 HPN 基序中残基的排列存在差异。在本研究中，我们使用 E2 酶 HIP2 来探测其他几种 E2 酶中 HPN 基序的结构。通过化学位移分析、组氨酸质子化状态的确定和酰胺温度系数的测定，确定了组氨酸环的取向和 HPN 基序中的氢键排列。与许多三维结构不同，我们发现，在所有检查的 E2 酶中，组氨酸咪唑环与天冬酰胺骨架酰胺质子之间存在保守的氢键、常见的组氨酸质子化状态和常见的组氨酸取向。这些结果表明，HPN 基序中的组氨酸定向构象稳定所有 E2 酶中的紧密转弯基序，而不是像一些机制中提出的那样定向与天冬酰胺侧链相互作用。这些结果表明，可能存在一种常见的催化机制，用于所有 E2 连接酶促进泛素转移。
