Cranford Smith, T. | Search bioRxiv

bioRxiv2019

Iron Is a Physiological Ligand of SecA-like Metal Binding Domains

The ATPase SecA is an essential component of the bacterial Sec machinery, which transports proteins across the cytoplasmic membrane. Most SecA proteins contain a long C-terminal tail (CTT). In Escherichia coli, the CTT contains a structurally flexible linker domain and a small metal-binding domain (MBD). The MBD coordinates zinc via a conserved cysteine-containing motif and binds to SecB and ribosomes. In this study, we screened a high-density transposon library for mutants that affect the susceptibility of E. coli to sodium azide, which inhibits SecA-mediated translocation. Results from sequencing this library suggested that mutations removing the CTT make E. coli less susceptible to sodium azide at subinhibitory concentrations. Copurification experiments suggested that the MBD binds to iron and that azide disrupts iron binding. Azide also disrupted binding of SecA to membranes. Two other E. coli proteins that contain SecA-like MBDs, YecA and YchJ, also copurified with iron, and NMR spectroscopy experiments indicated that YecA binds iron via its MBD. Competition experiments and equilibrium binding measurements indicated that the SecA MBD binds preferentially to iron and that a conserved serine is required for this specificity. Finally, structural modelling suggested a plausible model for the octahedral coordination of iron. Taken together, our results suggest that SecA-like MBDs likely bind to iron in vivo.

biochemistry↗

bioRxiv2017

A genetic screen suggests an alternative mechanism for inhibition of SecA by azide

Sodium azide prevents bacterial growth by inhibiting the activity of SecA, which is required for translocation of proteins across the cytoplasmic membrane. Azide inhibits ATP turnover in vitro, but its mechanism of action in vivo is unclear. To investigate how azide inhibits SecA in cells, we used transposon directed insertion-site sequencing (TraDIS) to screen a library of transposon insertion mutants for mutations that affect the susceptibility of E. coli to azide. Insertions disrupting components of the Sec machinery generally increased susceptibility to azide, but insertions truncating the C-terminal tail (CTT) of SecA decreased susceptibility of E. coli to azide. Treatment of cells with azide caused increased aggregation of the CTT, suggesting that azide disrupts its structure. Analysis of the metal-ion content of the CTT indicated that SecA binds to iron and the azide disrupts the interaction of the CTT with iron. Azide also disrupted binding of SecA to membrane phospholipids, as did alanine substitutions in the metal-coordinating amino acids. Furthermore, treating purified phospholipid-bound SecA with azide in the absence of added nucleotide disrupted binding of SecA to phospholipids. Our results suggest that azide does not inhibit SecA by inhibiting the rate of ATP turnover in vivo. Rather, azide inhibits SecA by causing it to \"backtrack\" from the ADP-bound to the ATP-bound conformation, which disrupts the interaction of SecA with the cytoplasmic membrane.\n\nSignificance statementSecA is a bacterial ATPase that is required for the translocation of a subset of secreted proteins across the cytoplasmic membrane. Sodium azide is a well-known inhibitor of SecA, but its mechanism of action in vivo is poorly understood. To investigate this mechanism, we examined the effect of azide on the growth of a library of [~]1 million transposon insertion mutations. Our results suggest that azide causes SecA to backtrack in its ATPase cycle, which disrupts binding of SecA to the membrane and to its metal cofactor, which is iron. Our results provide insight into the molecular mechanism by which SecA drives protein translocation and how this essential biological process can be disrupted.

microbiology↗