Biology subjects

Jamshad, M.

Publications and source records attributed to Jamshad, M..

4 recordsLinked to original sources

bioRxiv2018

The C-terminal tail of the bacterial translocation ATPase SecA modulates its activity

In bacteria, the translocation of a subset of proteins across the cytoplasmic membrane by the Sec machinery requires SecA. Although SecA can recognise nascent polypeptides, the mechanism of cotranslational substrate protein recognition is not known. Here, we investigated the role of the C-terminal tail (CTT) of SecA, which consists of a flexible linker (FLD) and a small metal-binding domain (MBD), in its interaction with nascent polypeptides. Phylogenetic analysis and ribosome binding experiments indicated that the MBD interacts with 70S ribosomes. Disruption of the entire CTT or the MBD alone had opposing effects on ribosome binding, substrate-protein binding, ATPase activity and in vivo function. Autophotocrosslinking, mass spectrometry, x-ray crystallography and small-angle x-ray scattering experiments provided insight into the CTT-mediated conformational changes in SecA. Finally, photocrosslinking experiments indicated that binding of SecA to substrate protein affected its interaction with the ribosome. Taken together, our results suggest a mechanism for substrate protein recognition.\n\nImpact StatementSecA is an evolutionarily conserved ATPase that is required for the translocation of a subset of proteins across the cytoplasmic membrane in bacteria. We investigated how SecA recognises its substrate proteins at the ribosome as they are still being synthesised (i.e. cotranslationally).

biochemistry↗

bioRxiv2018

Evidence for phospholipid export from the gram-negative inner membrane: time to rethink the Mla pathway?

The Mla pathway is believed to be involved in maintaining the asymmetrical Gram-negative outer membrane via retrograde phospholipid transport. The pathway is composed of 3 components: the outer membrane MlaA-OmpC/F complex, a soluble periplasmic protein, MlaC, and the inner membrane ATPase, MlaFEDB complex. Here we solve the crystal structure of MlaC in its phospholipid free closed apo conformation, revealing a novel pivoting {beta}-sheet mechanism which functions to open and close the phospholipid-binding pocket. Using the apo form of MlaC we provide evidence that the Mla pathway functions in an anterograde rather than a retrograde direction by showing the inner membrane MlaFEDB machinery exports phospholipids and transfers them to MlaC in the periplasm. We confirm that the lipid export process occurs through the MlaD component of the MlaFEDB complex. This lipid export process is shown to be independent of ATP. Our data provides, for the first time, evidence of an apparatus for lipid export to the outer membrane.

biophysics↗

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↗

bioRxiv2017

MCE domain proteins: conserved inner membrane lipid-binding proteins required for outer membrane homeostasis

Bacterial proteins with MCE domains were first described as being important for Mammalian Cell Entry. More recent evidence suggests they are components of lipid ABC transporters. In Escherichia coli, the single-domain protein MlaD is known to be part of an inner membrane transporter that is important for maintenance of outer membrane lipid asymmetry. Here we describe two multi MCE domain-containing proteins in Escherichia coli, PqiB and YebT, the latter of which is an orthologue of MAM-7 that was previously reported to be an outer membrane protein. We show that all three MCE domain-containing proteins localise to the inner membrane. Bioinformatic analyses revealed that MCE domains are widely distributed across bacterial phyla but multi MCE domain-containing proteins evolved in Proteobacteria from single-domain proteins. Mutants defective in mlaD, pqiAB and yebST were shown to have distinct but partially overlapping phenotypes, but the primary functions of PqiB and YebT differ from MlaD. Complementing our previous findings that all three proteins bind phospholipids, results presented here indicate that multi-domain proteins evolved in Proteobacteria for specific functions in maintaining cell envelope homeostasis.

microbiology↗