Topological analysis of a bacterial DedA protein associated with alkaline tolerance and antimicrobial resistance

Abstract

Maintaining membrane integrity is of paramount importance to the survival of bacteria as the membrane is the site of multiple crucial cellular processes including energy generation, nutrient uptake and antimicrobial efflux. The DedA family of integral membrane proteins are widespread in bacteria and are associated with maintaining the integrity of the membrane. In addition, DedA proteins have been linked to resistance to multiple classes of antimicrobials in various microorganisms. Therefore, the DedA family are attractive targets for the development of new antibiotics. Despite DedA family members playing a key physiological role in many bacteria, their structure, function and physiological role remain unclear. To help illuminate the structure of the bacterial DedA proteins, we performed substituted cysteine accessibility method (SCAM) analysis on the most comprehensively characterized bacterial DedA protein, YqjA from Escherichia coli . By probing the accessibility of 15 cysteine residues across the length of YqjA using thiol reactive reagents, we mapped the topology of the protein. Using these data, we experimentally validated a structural model of YqjA generated using evolutionary covariance, which consists of an α-helical bundle with two re-entrant hairpin loops reminiscent of several secondary active transporters. In addition, our cysteine accessibility data suggest that YqjA forms an oligomer wherein the protomers are arranged in a parallel fashion. This experimentally verified model of YqjA lays the foundation for future work in understanding the function and mechanism of this interesting and important family.

Three bacterial DedA subfamilies with distinct functions and phylogenetic distribution

“…N- and C-terminal location and orientation were conserved within subfamilies (Fig. 2C-E), indicating that they are not caused solely by the mishandling of the C-terminal dimerization helices (2), but may reflect functional differences. The structural conservation of the DedA core domain and the variability of N- and C-terminal helices suggest that DedA subfamilies share a basic function (lipid scrambling/flipping) but vary in their substrate.…”

“…2A): a conserved (Fig. S1) structure similar to other transporter families (2). In such transporters, the residues at the tips of the two re-entrant helices are invariably involved in substrate interactions.…”

“…DedA-family proteins are broadly distributed and nearly ubiquitous in eukaryotes, archaea, and bacteria. The structure of the DedA domain has not been solved, but computational (1) and topological (2) approaches indicate similarity to other transporter families (2). Eukaryotic VMP1, TVP38, and TMEM41B DedA proteins are central players in autophagy, while bacterial DedA family members play roles in colistin resistance, cell division, and pH sensitivity (detailed below).…”

Regulation of ER‐derived membrane dynamics by the DedA domain‐containing proteins VMP1 and TMEM41B

“…In general, the reentrant loop is considered to be rather unstable in the lipid bilayer due to its low hydrophobicity (Yan & Luo, 2010). Self-binding of the DedA family protein YqjA may stabilize its reentrant loops by sequestering them inside (Keller et al, 2015;Scarsbrook et al, 2021). Therefore, it is possible that VMP1 and TMEM41B also interact with themselves or other proteins.…”

Microbial Musings – December 2021

“…One of those eight proteins was YqjA, which the paper in this month’s provides the first experimentally derived membrane topology of a DedA protein. The work, from the group of Chris Mulligan ( @Chris_mulligan ) at the University of Kent, UK, combined new methods of predicting protein structure by evolutionary covariance with the experimental testing of this using topological mapping [ 17 ]. The experimental method requires first removing all of the codons for cysteine from the gene, and then making versions with single cysteine codons added back in.…”