Structures of two bacterial resistance factors mediating tRNA-dependent aminoacylation of phosphatidylglycerol with lysine or alanine

Abstract

The cytoplasmic membrane is probably the most important physical barrier between microbes and the surrounding habitat. Aminoacylation of the polar head group of the phospholipid phosphatidylglycerol (PG) catalyzed by Ala-tRNA Ala -dependent alanyl-phosphatidylglycerol synthase (A-PGS) or by Lys-tRNA Lys -dependent lysyl-phosphatidylglycerol synthase (L-PGS) enables bacteria to cope with cationic peptides that are harmful to the integrity of the cell membrane. Accordingly, these synthases also have been designated as multiple peptide resistance factors (MprF). They consist of a separable C-terminal catalytic domain and an N-terminal transmembrane flippase domain. Here we present the X-ray crystallographic structure of the catalytic domain of A-PGS from the opportunistic human pathogen Pseudomonas aeruginosa. In parallel, the structure of the related lysyl-phosphatidylglycerol-specific L-PGS domain from Bacillus licheniformis in complex with the substrate analog L-lysine amide is presented. Both proteins reveal a continuous tunnel that allows the hydrophobic lipid substrate PG and the polar aminoacyl-tRNA substrate to access the catalytic site from opposite directions. Substrate recognition of A-PGS versus L-PGS was investigated using misacylated tRNA variants. The structural work presented here in combination with biochemical experiments using artificial tRNA or artificial lipid substrates reveals the tRNA acceptor stem, the aminoacyl moiety, and the polar head group of PG as the main determinants for substrate recognition. A mutagenesis approach yielded the complementary amino acid determinants of tRNA interaction. These results have broad implications for the design of L-PGS and A-PGS inhibitors that could render microbial pathogens more susceptible to antimicrobial compounds.A-PGS | L-PGS | MprF | structure | tRNA

Deciphering the tRNA-dependent lipid aminoacylation systems in bacteria: Novel components and structural advances

“…38 Several studies have tried to determine the phenotypical changes correlated with synthesis of Lys-PG compared with Ala-PG in S. aureus and P. aeruginosa . 24,30 These investigations showed that substitution of Lys-PG (which confers a net charge of +1) with Ala-PG (which bears a neutral net charge) did not affect bacterial susceptibility to CAMPs such as nisin and gallidermin, or to the CAMP-like antibiotic daptomycin. These findings suggest that the α amino group of the aa moiety of aa-PG alone is able to enhance antimicrobial resistance in bacteria.…”

“…Several enzymes with altered aa-tRNA and lipid substrate specificities have been identified, and the recently solved crystal structures of two aaPGSs have shed light on the molecular mechanism of tRNA-dependent systems for lipid aminoacylation. 30 …”

“…These observations were recently confirmed by the crystal structures of aaPGSs from Bacillus licheniformis and Pseudomonas aeruginosa . 30 In the Protein Family database (Pfam 41 ), the GNAT fold is found in 39 families of transferases which altogether constitute the N-acyltransferases clan (CL0257). This clan includes many acyl-CoA and aa-tRNA transferases.…”

Profiling and tandem mass spectrometry analysis of aminoacylated phospholipids in Bacillus subtilis

“…The pivotal protein for aminoacyl-PG biosynthesis from L-aminoacyl-tRNA and PG is the lysyl-PG synthase MprF (multiple peptide resistance factor) 8 which appears to have a broad range of specificity for L-aminoacyl-tRNAs 9, 10 . The crystal structures of the cytoplasmic catalytic domains of two MprFs, with one specific for lysyl- the other for alanyl-PG biosynthesis, have recently been elucidated 11 . The catalytic domains of the two MprF enzymes have a long tunnel for accommodating PG with the catalytic site located at the narrowest part of the tunnel 11 .…”

“…The crystal structures of the cytoplasmic catalytic domains of two MprFs, with one specific for lysyl- the other for alanyl-PG biosynthesis, have recently been elucidated 11 . The catalytic domains of the two MprF enzymes have a long tunnel for accommodating PG with the catalytic site located at the narrowest part of the tunnel 11 . The primary and tertiary structures of MprF resemble that of FemX which catalyzes L-alanyl transfer from tRNA to a peptidoglycan precursor 12 .…”