The Escherichia coli disulfide bond isomerase DsbC rearranges incorrect disulfide bonds during oxidative protein folding. It is specifically activated by the periplasmic N‐terminal domain (DsbDα) of the transmembrane electron transporter DsbD. An intermediate of the electron transport reaction was trapped, yielding a covalent DsbC–DsbDα complex. The 2.3 Å crystal structure of the complex shows for the first time the specific interactions between two thiol oxidoreductases. DsbDα is a novel thiol oxidoreductase with the active site cysteines embedded in an immunoglobulin fold. It binds into the central cleft of the V‐shaped DsbC dimer, which assumes a closed conformation on complex formation. Comparison of the complex with oxidized DsbDα reveals major conformational changes in a cap structure that regulates the accessibility of the DsbDα active site. Our results explain how DsbC is selectively activated by DsbD using electrons derived from the cytoplasm.

SAMHD1 restricts HIV-1 infection of myeloid-lineage and resting CD4⁺ T-cells. Most likely this occurs through deoxynucleoside triphosphate triphosphohydrolase activity that reduces cellular dNTP to a level where reverse transcriptase cannot function, although alternative mechanisms have been proposed recently. Here, we present combined structural and virological data demonstrating that in addition to allosteric activation and triphosphohydrolase activity, restriction correlates with the capacity of SAMHD1 to form “long-lived” enzymatically competent tetramers. Tetramer disruption invariably abolishes restriction but has varied effects on in vitro triphosphohydrolase activity. SAMHD1 phosphorylation also ablates restriction and tetramer formation but without affecting triphosphohydrolase steady-state kinetics. However phospho-SAMHD1 is unable to catalyse dNTP turnover under conditions of nucleotide depletion. Based on our findings we propose a model for phosphorylation-dependent regulation of SAMHD1 activity where dephosphorylation switches housekeeping SAMHD1 found in cycling cells to a high-activity stable tetrameric form that depletes and maintains low levels of dNTPs in differentiated cells. SAMHD1 is a restriction factor that blocks infection of certain immune cells by HIV-1. It was discovered to be an enzyme that catalyses the breakdown of dNTPs, suggesting that it inhibits HIV-1 replication by reducing cellular dNTP pools to such low levels that reverse transcriptase cannot function. However, recently, alternative mechanisms have been proposed. SAMHD1 is also regulated by phosphorylation, although the effects of phosphorylation on protein function are unclear. In order to address these issues, we carried out combined structural and virological studies and have demonstrated that in addition to allosteric activation and triphosphohydrolase activity, restriction correlates with the capacity of SAMHD1 to form “long-lived” enzymatically competent tetramers. Disrupting the tetramer in various ways always abolished restriction but had differing effects on enzyme activity in vitro. SAMHD1 phosphorylation also prevented restriction and tetramer formation but without affecting enzyme catalysis under steady-state dNTP conditions. However phosphorylated SAMHD1 was unable to catalyse dNTP turnover at very low nucleotide levels that more accurately represent conditions in the cells in which restriction takes place. Based on our findings we propose a model for phosphorylation-dependent regulation of SAMHD1 activity and substantiate that degradation of dNTPs by SAMHD1 is sufficient to restrict HIV-1.

Significance: Retroviral infection of cells can be blocked by the action of the postentry restriction factors. The Trim5α and Fv1 factors do so by targeting the capsid that surrounds the viral core. The nature of the interaction of these factors with the viral assembly is unclear. We show that these factors form antiparallel dimers that display specificity domains spaced to target motifs on the capsid lattice surface. In doing so Fv1 and Trim5α take advantage of the regularly spaced array of binding sites on the capsid surface, generating avidity to aid recognition of retroviral pathogens.

There are two distinct pathways for disulfide formation in prokaryotes. The DsbA‐DsbB pathway introduces disulfide bonds de novo , while the DsbC‐DsbD pathway functions to isomerize disulfides. One of the key questions in disulfide biology is how the isomerase pathway is kept separate from the oxidase pathway in vivo . Cross‐talk between these two systems would be mutually destructive To force communication between these two systems we have selected dsbC mutants that complement a dsbA null mutation. In these mutants, DsbC is present as a monomer as compared with dimeric wild‐type DsbC. Based on these findings we rationally designed DsbC mutants in the dimerization domain. All of these mutants are able to rescue the dsbA null phenotype. Rescue depends on the presence of DsbB, the native re‐oxidant of DsbA, both in vivo and in vitro . Our results suggest that dimerization acts to protect DsbC's active sites from DsbB‐mediated oxidation. These results explain how oxidative and reductive pathways can co‐exist in the periplasm of Escherichia coli .