Structural Basis for the Utilization of Host Fatty Acids by Bacterial Fatty Acid Binding Proteins

Abstract

The Fatty acid kinase (Fak) system is a two‐protein system allowing Gram‐positive pathogens to incorporate exogenous fatty acids (FA) for membrane phospholipid synthesis. Exogenous FA bind to a FA binding protein, FakB, are phosphorylated by a kinase, FakA, and used to construct the membrane. Phosphorylated FA can also be interconverted to acyl‐acyl carrier protein, via the phosphate acyltransferase PlsX, and be elongated by the fatty acid synthase type II (FASII) system. We used a combination of cellular labeling experiments, genetics, and biochemistry to characterize two FakBs of Staphylococcus aureus and three FakBs of Streptococcus pneumoniae. FakBs have structure‐specific FA binding selectivity unlike mammalian FA binding proteins. Both bacteria use one FakB (FakB1) to exclusively bind saturates and use a second FakB (FakB2) to bind monounsaturates. The additional FakB in S. pneumoniae (FakB3) binds polyunsaturates, specifically linoleate which is an abundant human FA. High‐resolution crystal structures of five FakBs show nearly identical tertiary structures allowing all FakBs to interact with other proteins involved in lipid metabolism. FakBs differ in the shape and volume of their interior FA binding pockets resulting in FA selectivity. FakB1s have a highly tailored pocket and exclude unsaturates. The FA binding pockets of FakB2s are tailored to bind monounsaturates and have a kinked tunnel to match the conformation of the cis double bond. The S. pneumoniae‐specific FakB3 has an expanded acyl chain pocket allowing polyunsaturates to bind. FakB2s and FakB3 cannot completely exclude the binding of saturates because flexible saturated acyl chains fit into both kinked and expanded binding pockets. A lipidomic workflow was used to determine the impact of the two FakBs on the structure of the S. aureus membrane phosphatidylglycerol in a thigh infection model. Wild type S. aureus incorporated saturated and unsaturated host FA into the 1‐position of phosphatidylglycerol, whereas the fakB1 mutant was deficient in saturated FA utilization and the fakB2 mutant did not incorporate unsaturated FA. Mutagenesis, X‐ray crystallography, and NMR show that FakBs bind to anionic membranes using surface lysine residues. The membrane‐bound FA exchanges into FakB after two helices near the FA binding pocket shift outward. This mobile “flap” exposes the FA binding pocket and allows the carboxyl end of the FA to hydrogen bond to polar residues in the pocket. This work reveals the structural basis for the physiological functions of bacterial FA binding proteins in the acquisition of specific host fatty acids at the infection site.Support or Funding InformationThis work was supported by National Institutes of Health grant GM034496, Cancer Center Support Grant CA21765 and the American Lebanese Syrian Associated Charities. Use of the Advanced Photon Source was supported by the U. S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. W‐31‐109‐Eng‐38.Model for the function of FakB proteins in S. pneumoniae. Host fatty acids (FA) are incorporated into bacterial phospholipid membranes by binding to FakB. Distinct FakBs bind either saturated FA (SFA), monounsaturated FA (MUFA), or polyunsaturated FA (PUFA). FA are then phosphorylated by a kinase, FakA, and work in concert with the S. pneumoniae FASII machinery to produce substrates for acyltransferases (PlsX, PlsY) which acylate a G3P backbone and form phospholipids.Figure 1