The structural and functional characterisation of the DedA family of integral membrane proteins

Bacterial membrane homeostasis requires the concerted efforts of a plethora of membrane proteins to regulate and to maintain numerous cellular events, such as membrane integrity, cell division, nutrient uptake as well as antimicrobial resistance. The DedA family of integral membrane proteins, have been shown to be significant in most aspects of bacterial cell membrane homeostasis. This importance is strengthened by their widespread inclusion in not just prokaryotes but also eukaryotes and archaea. Deletions of DedA genes have produced many detrimental phenotypes, such as cell division defects, altered membrane potential, temperature sensitivity, altered membrane lipid compositions, sensitivities to many antimicrobials, alongside some being essential for cell survival. The functions of DedA proteins have not been determined, however recent studies have suggested that some DedA proteins are able to transport lipids between membrane lipid leaflets. Lipid transport is essential for many aspects of bacterial membrane homeostasis, including importantly increasing resistance to antimicrobials. There is currently a lack of structural or functional studies on bacterial DedA proteins, with no determinate DedA structures. Gaining insight into the structural and functional aspects of DedA proteins will be important in identifying their roles in the cell and thus linking their function to phenotype. Here, we have sought to address this by producing a library of detergent purified DedA proteins, of high quality and yield that can then be used for biochemical and biophysical characterisations. We show here that five DedA proteins were able to be purified to this degree, including YqjA, YghB, YohD and YdjZ from E. coli and Pa4029 from P. aeruginosa. Using analytical ultracentrifugation, we determined that YqjA exists primarily in a dimeric state and with computational modelling and chemical crosslinking, we were able to model and map the dimer interface. Nanobodies were also generated against YqjA to aid in the determinations of a structure, but further optimisation was still required to acquire a YqjA-Nanobody complex that could be used for structural studies. Using a GFP-based thermal stability assay, we determined that YqjA is stabilised by lipids and that this stabilisation can be altered by the mutations of key evolutionarily conserved functional residues. We also performed liposome-based lipid transport assays to determine whether YqjA can transport lipids, but further optimisation was still required to identify any definitive lipid transport.