Crystal structure of LpxK, the 4′-kinase of lipid A biosynthesis and atypical P-loop kinase functioning at the membrane interface

Abstract

In Gram-negative bacteria, the hydrophobic anchor of the outer membrane lipopolysaccharide is lipid A, a saccharolipid that plays key roles in both viability and pathogenicity of these organisms. The tetraacyldisaccharide 4′-kinase (LpxK) of the diverse P-loopcontaining nucleoside triphosphate hydrolase superfamily catalyzes the sixth step in the biosynthetic pathway of lipid A, and is the only known P-loop kinase to act upon a lipid substrate at the membrane. Here, we report the crystal structures of apo-and ADP/ Mg 2+ -bound forms of Aquifex aeolicus LpxK to a resolution of 1.9 Å and 2.2 Å, respectively. LpxK consists of two α/β/α sandwich domains connected by a two-stranded β-sheet linker. The N-terminal domain, which has most structural homology to other family members, is responsible for catalysis at the P-loop and positioning of the disaccharide-1-phosphate substrate for phosphoryl transfer on the inner membrane. The smaller C-terminal domain, a substructure unique to LpxK, helps bind the nucleotide substrate and Mg 2+ cation using a 25°hinge motion about its base. Activity was severely reduced in alanine point mutants of conserved residues D138 and D139, which are not directly involved in ADP or Mg 2+ binding in our structures, indicating possible roles in phosphoryl acceptor positioning or catalysis. Combined structural and kinetic studies have led to an increased understanding of the enzymatic mechanism of LpxK and provided the framework for structurebased antimicrobial design.lipid metabolism | endotoxin | integral monotopic protein | Walker motif

Structural Basis of Lipid Binding for the Membrane-embedded Tetraacyldisaccharide-1-phosphate 4′-Kinase LpxK

“…Sequencing confirmed the correct construction of the ⌬12LpxK (pRPE72) and ⌬29LpxK (pRPE73) mutants. These plasmids were transformed into C41(DE3) cells, and membranes expressing these constructs were generated as with wild type LpxK (6,19). All constructs expressed to comparable levels as judged by SDS-PAGE.…”

“…Crystal Structure of LpxK Bound to Lipid IV A -Wild type A. aeolicus LpxK was generated by growth of C41(DE3) (19) cultures expressing the construct pRPE7 and purified as described previously (6). Crystals were grown in 24-well sitting drop vapor diffusion trays from Hampton (Aliso Viejo, CA) at 20°C.…”

“…The other assay components were 50 mM Tris, pH 8.5, 0.5% (w/v) Triton X-100 (Thermo Scientific, Rockford, IL), 1 mg/ml BSA (Sigma-Aldrich), 0.1 M NaCl, and enzyme. The TLC-based assay was performed as described previously (6,7), and the data were fit to the Michaelis-Menten equation using KaleidaGraph (Synergy Software, Reading, PA) in order to determine apparent K m and k cat with respect to DSMP. Enzyme concentration was varied to capture the linear range of activity for each mutant.…”

“…1A) (4,5). Crystal structures of Aquifex aeolicus LpxK in apo-, nucleotide-bound, and nucleotide analog-bound forms have been reported (6,7), revealing LpxK as a distinct member of the P-loop-containing nucleoside triphosphate hydrolase superfamily that acts upon a lipid substrate at the membrane surface (8 -10). The LpxK C-terminal domain, a substructure unique to this enzyme, connects to the N-terminal domain by a long two-stranded ␤-sheet linker and serves as the "lid" to trap the nucleotide substrate between the two domains, forming the "closed" conformation (Fig.…”

“…1B). In the apo-form, the C-terminal lid domain is rotated away from the active site by a ϳ25°angle, resulting in an "open" conformation of the ATP-binding pocket (6).…”