The Alpha-Proteobacteria Wolbachia Pipientis Protein Disulfide Machinery Has A Regulatory Mechanism Absent In Gamma-Proteobacteria

The alpha-proteobacterium Wolbachia pipientis infects more than 65% of insect species worldwide and manipulates the host reproductive machinery to enable its own survival. It can live in mutualistic relationships with hosts that cause human disease, including mosquitoes that carry the Dengue virus. Like many other bacteria, Wolbachia contains disulfide bond forming (Dsb) proteins that introduce disulfide bonds into secreted effector proteins. The genome of the Wolbachia strain wMel encodes two DsbA-like proteins sharing just 21% sequence identity to each other, alpha-DsbA1 and alpha-DsbA2, and an integral membrane protein, alpha-DsbB. alpha-DsbA1 and alpha-DsbA2 both have a Cys-X-X-Cys active site that, by analogy with Escherichia coli DsbA, would need to be oxidized to the disulfide form to serve as a disulfide bond donor toward substrate proteins. Here we show that the integral membrane protein alpha-DsbB oxidizes alpha-DsbA1, but not alpha-DsbA2. The interaction between alpha-DsbA1 and alpha-DsbB is very specific, involving four essential cysteines located in the two periplasmic loops of alpha-DsbB. In the electron flow cascade, oxidation of alpha-DsbA1 by alpha-DsbB is initiated by an oxidizing quinone cofactor that interacts with the cysteine pair in the first periplasmic loop. Oxidizing power is transferred to the second cysteine pair, which directly interacts with alpha-DsbA1. This reaction is inhibited by a non-catalytic disulfide present in alpha-DsbA1, conserved in other alpha-proteobacterial DsbAs but not in gamma-proteobacterial DsbAs. This is the first characterization of the integral membrane protein alpha-DsbB from Wolbachia and reveals that the non-catalytic cysteines of alpha-DsbA1 regulate the redox relay system in cooperation with alpha-DsbB.