Structural insights into lipoprotein N-acylation by Escherichia coli apolipoprotein N-acyltransferase

Abstract

Gram-negative bacteria express a diverse array of lipoproteins that are essential for various aspects of cell growth and virulence, including nutrient uptake, signal transduction, adhesion, conjugation, sporulation, and outer membrane protein folding. Lipoprotein maturation requires the sequential activity of three enzymes that are embedded in the cytoplasmic membrane. First, phosphatidylglycerol: prolipoprotein diacylglyceryl transferase (Lgt) recognizes a conserved lipobox motif within the prolipoprotein signal sequence and catalyzes the addition of diacylglycerol to an invariant cysteine. The signal sequence is then cleaved by signal peptidase II (LspA) to give an N-terminal S-diacylglyceryl cysteine. Finally, apolipoprotein N-acyltransferase (Lnt) catalyzes the transfer of the sn-1-acyl chain of phosphatidylethanolamine to this N-terminal cysteine, generating a mature, triacylated lipoprotein. Although structural studies of Lgt and LspA have yielded significant mechanistic insights into this essential biosynthetic pathway, the structure of Lnt has remained elusive. Here, we present crystal structures of wild-type and an activesite mutant of Escherichia coli Lnt. The structures reveal a monomeric eight-transmembrane helix fold that supports a periplasmic carbonnitrogen hydrolase domain containing a Cys-Glu-Lys catalytic triad. Two lipids are bound at the active site in the structures, and we propose a putative phosphate recognition site where a chloride ion is coordinated near the active site. Based on these structures and complementary cell-based, biochemical, and molecular dynamics approaches, we propose a mechanism for substrate engagement and catalysis by E. coli Lnt.lipoprotein biosynthesis | apolipoprotein N-acyltransferase | crystal structure | enzyme mechanism T he essential outer membrane (OM) of Gram-negative bacteria has a distinct composition, containing a diverse set of phospholipids, lipopolysaccharides, OM proteins, and lipoproteins (1). Escherichia coli expresses at least 90 lipoproteins that play critical roles in multiple cellular processes ranging from OM biogenesis to cell division and virulence (2). A common feature of these proteins is an N-terminal N-acyl, S-diacylglyceryl cysteine, which ensures their proper localization (3, 4). Before triacylation, preprolipoproteins are translated in the cytoplasm and transferred across the inner bacterial membrane via the Sec or Tat pathway machineries (5). These nascent preprolipoproteins have an N-terminal hydrophobic signal peptide that contains a conserved lipobox motif with the consensus sequence [LVI] -3 [ASTVI][GAS]C +1 , which directs them to the lipoprotein biosynthetic pathway (6-8). The maturation of lipoproteins is then catalyzed by the consecutive action of three essential integral membrane proteins (Fig. 1A). In the first step of this pathway, phosphatidylglycerol:prolipoprotein diacylglyceryl transferase (Lgt) catalyzes the covalent attachment of the sn-1,2-diacylglyceryl group from phosphatidylglycerol to the sulfydryl group of the ...

Conformational changes in Apolipoprotein N-acyltransferase (Lnt)

“…We interpret this as the thioester acyl-intermediate. This is entirely consistent with mass spectrometry analysis indicating that a large percentage of the protein is covalently bound to palmitate (Noland et al, 2017) and previous findings that Lnt exists as a thioester acyl-enzyme intermediate (Buddelmeijer and Young, 2010). Despite seeing a significant percentage of covalently modified enzyme no electron density is seen in the structures presented by Noland et al, 2017 or in the other solved structures of Lnt (Lu et al, 2017;Wiktor et al, 2017).…”

“…In chain A by contrast, although residues 352-362 were not visible in the electron density, the beginning and end of the arm can be modelled extending parallel to the membrane ( Figure 3B) similar to the other reported structures (pdb ids 5n6h, 5n6l, 5n6m, 5vrg, and 5vrh). When in this conformation, the active site is accessible to the surrounding environment, which could explain why the thioester intermediate is not seen in chain A or any of the other crystal structures despite mass spectral evidence (Noland et al, 2017). However, this loop, when in the upward position could offer some protection to the active site and thioester intermediate from the outside environment.…”

“…All three enzymes involved in lipid attachment are essential for survival in bacteria making them attractive targets for new antimicrobial agents (Xia et al, 2018). Recently both the structures of Lgt (Mao et al, 2015) and LspA (Vogeley et al, 2016) have been solved, and very recently a number of structures of Lnt became available (Noland et al, 2017;Lu et al, 2017;Wiktor et al, 2017). Aside from the transmembrane domain, the structures of Lnt confirm that the overall fold of soluble nitrilases is conserved in Lnt.…”

“…It has also been suggested that Lnt exists at a slight angle with the binding pocket angling slightly downwards, towards the lipid interface (Lu et al, 2017), thus the membrane interface could be higher than predicted by the PPM server. Regardless of the exact position of the membrane interface in relation to the binding pocket, the fact that the acyl tail can exist above the membrane interface can be easily explained by the high hydrophobicity of the binding groove (Noland et al, 2017) that could easily accommodate hydrophobic molecules such as lipids.…”

Lipoprotein Transport: Greasing the Machines of Outer Membrane Biogenesis

“…The maturation enzymes are both broadly conserved and essential, making them excellent drug targets; Lgt and Lsp are also conserved among Gram‐positive bacteria, offering broad‐spectrum activity for inhibitors . Very recently, structural information for each of these enzymes has become available and more detailed understanding of lipoprotein maturation reactions will likely follow …”