From the Institute for Molecular Bioscience, Division of Chemistry and Structural Biology and.
J Biol Chem. 2014 Jan 31;289(5):2563-76. doi: 10.1074/jbc.M113.516898. Epub 2013 Dec 5.
The multidrug resistance-encoding IncA/C conjugative plasmids disseminate antibiotic resistance genes among clinically relevant enteric bacteria. A plasmid-encoded disulfide isomerase is associated with conjugation. Sequence analysis of several IncA/C plasmids and IncA/C-related integrative and conjugative elements (ICE) from commensal and pathogenic bacteria identified a conserved DsbC/DsbG homolog (DsbP). The crystal structure of DsbP reveals an N-terminal domain, a linker region, and a C-terminal catalytic domain. A DsbP homodimer is formed through domain swapping of two DsbP N-terminal domains. The catalytic domain incorporates a thioredoxin-fold with characteristic CXXC and cis-Pro motifs. Overall, the structure and redox properties of DsbP diverge from the Escherichia coli DsbC and DsbG disulfide isomerases. Specifically, the V-shaped dimer of DsbP is inverted compared with EcDsbC and EcDsbG. In addition, the redox potential of DsbP (-161 mV) is more reducing than EcDsbC (-130 mV) and EcDsbG (-126 mV). Other catalytic properties of DsbP more closely resemble those of EcDsbG than EcDsbC. These catalytic differences are in part a consequence of the unusual active site motif of DsbP (CAVC); substitution to the EcDsbC-like (CGYC) motif converts the catalytic properties to those of EcDsbC. Structural comparison of the 12 independent subunit structures of DsbP that we determined revealed that conformational changes in the linker region contribute to mobility of the catalytic domain, providing mechanistic insight into DsbP function. In summary, our data reveal that the conserved plasmid-encoded DsbP protein is a bona fide disulfide isomerase and suggest that a dedicated oxidative folding enzyme is important for conjugative plasmid transfer.
多药耐药基因编码的 IncA/C 可移动质粒在临床相关肠细菌中传播抗生素耐药基因。一种质粒编码的二硫键异构酶与接合有关。对来自共生菌和病原菌的几种 IncA/C 质粒和 IncA/C 相关整合和共轭元件（ICE）的序列分析鉴定了保守的 DsbC/DsbG 同源物（DsbP）。DsbP 的晶体结构揭示了一个 N 端结构域、一个连接区和一个 C 端催化结构域。DsbP 同源二聚体通过两个 DsbP N 端结构域的结构域交换形成。催化结构域包含一个具有特征 CXXC 和顺式-Pro 基序的硫氧还蛋白折叠。总体而言，DsbP 的结构和氧化还原特性与大肠杆菌 DsbC 和 DsbG 二硫键异构酶不同。具体而言，与 EcDsbC 和 EcDsbG 相比，DsbP 的 V 形二聚体是倒置的。此外，DsbP 的氧化还原电位（-161 mV）比 EcDsbC（-130 mV）和 EcDsbG（-126 mV）更还原。DsbP 的其他催化特性更类似于 EcDsbG，而不是 EcDsbC。这些催化差异部分是由于 DsbP 的不寻常活性位点基序（CAVC）所致；用 EcDsbC 样（CGYC）基序取代，将催化特性转化为 EcDsbC 的特性。我们确定的 12 个独立 DsbP 亚基结构的结构比较表明，连接区的构象变化有助于催化结构域的运动，为 DsbP 功能提供了机制见解。总之，我们的数据表明，保守的质粒编码 DsbP 蛋白是一种真正的二硫键异构酶，并表明专门的氧化折叠酶对于可移动质粒的转移很重要。
