Department of Biology, Washington University, Campus Box 1137, 1 Brookings Drive, St Louis, MO 63130, USA.
J Mol Biol. 2010 Aug 20;401(3):350-62. doi: 10.1016/j.jmb.2010.06.041. Epub 2010 Jun 25.
A superfamily of integral membrane proteins is characterized by a conserved tryptophan-rich region (called the WWD domain) in an external loop at the inner membrane surface. The three major members of this family (CcmC, CcmF, and CcsBA) are each involved in cytochrome c biosynthesis, yet the function of the WWD domain is unknown. It has been hypothesized that the WWD domain binds heme to present it to an acceptor protein (apoCcmE for CcmC or apocytochrome c for CcmF and CcsBA) such that the heme vinyl group(s) covalently attaches to the acceptors. Alternative proposals suggest that the WWD domain interacts directly with the acceptor protein (e.g., apoCcmE for CcmC). Here, it is shown that CcmC is only trapped with heme when its cognate acceptor protein CcmE is present. It is demonstrated that CcmE only interacts stably with CcmC when heme is present; thus, specific residues in each protein provide sites of interaction with heme to form this very stable complex. For the first time, evidence that the external WWD domain of CcmC interacts directly with heme is presented. Single and multiple substitutions of completely conserved residues in the WWD domain of CcmC alter the spectral properties of heme in the stable CcmC:heme:CcmE complexes. Moreover, some mutations reduce the binding of heme up to 100%. It is likely that endogenously synthesized heme enters the external WWD domain of CcmC either via a channel within this six-transmembrane-spanning protein or from the membrane. The data suggest that a specific heme channel (i.e., heme binding site within membrane spanning helices) is not present in CcmC, in contrast to the CcsBA protein. We discuss the likelihood that it is not important to protect the heme via trafficking in CcmC whereas it is critical in CcsBA.
一个由整合膜蛋白组成的超家族的特征是在内膜表面的外部环中有一个保守的富含色氨酸的区域（称为 WWD 结构域）。该家族的三个主要成员（CcmC、CcmF 和 CcsBA）都参与细胞色素 c 的生物合成，但 WWD 结构域的功能尚不清楚。有人假设 WWD 结构域结合血红素并将其呈现给受体蛋白（CcmC 的 apoCcmE 或 CcmF 和 CcsBA 的脱细胞色素 c），使血红素的乙烯基共价连接到受体上。替代方案表明，WWD 结构域与受体蛋白直接相互作用（例如，CcmC 的 apoCcmE）。在这里，当存在其同源受体蛋白 CcmE 时，仅发现 CcmC 被血红素捕获。结果表明，只有当存在血红素时，CcmE 才能与 CcmC 稳定相互作用；因此，每种蛋白质中的特定残基提供了与血红素相互作用的位点，形成了这种非常稳定的复合物。首次提出 CcmC 的外部 WWD 结构域与血红素直接相互作用的证据。CcmC 的 WWD 结构域中完全保守残基的单个和多个取代会改变稳定的 CcmC：血红素：CcmE 复合物中血红素的光谱特性。此外，一些突变会降低血红素的结合能力高达 100%。血红素很可能通过跨膜蛋白内的通道或从膜进入 CcmC 的外部 WWD 结构域。数据表明，CcmC 中不存在特定的血红素通道（即跨膜螺旋内的血红素结合位点），这与 CcsBA 蛋白相反。我们讨论了在 CcmC 中通过运输来保护血红素不重要的可能性，而在 CcsBA 中这是至关重要的。
