Structures of lipoprotein signal peptidase II from Staphylococcus aureus complexed with antibiotics globomycin and myxovirescin

Abstract

Antimicrobial resistance is a major global threat that calls for new antibiotics. Globomycin and myxovirescin are two natural antibiotics that target the lipoprotein-processing enzyme, LspA, thereby compromising the integrity of the bacterial cell envelope. As part of a project aimed at understanding their mechanism of action and for drug development, we provide high-resolution crystal structures of the enzyme from the human pathogen methicillinresistant Staphylococcus aureus (MRSA) complexed with globomycin and with myxovirescin. Our results reveal an instance of convergent evolution. The two antibiotics possess different molecular structures. Yet, they appear to inhibit identically as non-cleavable tetrahedral intermediate analogs. Remarkably, the two antibiotics superpose along nineteen contiguous atoms that interact similarly with LspA. This 19-atom motif recapitulates a part of the substrate lipoprotein in its proposed binding mode. Incorporating this motif into a scaffold with suitable pharmacokinetic properties should enable the development of effective antibiotics with built-in resistance hardiness.

Unstable Mechanisms of Resistance to Inhibitors of Escherichia coli Lipoprotein Signal Peptidase

“…While essential for growth of most Gram-negative bacteria, lspA is not essential for in vitro growth of Gram-positive bacteria but does lead to attenuation in virulence ( 16 – 18 ). LspA is the target of the natural product antibiotics globomycin (GBM) and myxovirescin (TA) synthesized by Streptomyces species and Myxococcus xanthus , respectively ( 19 , 20 ), which inhibit LspA function by targeting the catalytic dyad aspartic acid residues ( 21 ). Escherichia coli harbors >90 lipoproteins, many of which are localized to the inner leaflet of the outer membrane but can also be exposed on the bacterial cell surface ( 22 , 23 ).…”

Mode of action of lipoprotein modification enzymes—Novel antibacterial targets

“…Once diacylation of the lipobox cysteine by Lgt has occurred, Lsp cleaves the signal peptide liberating the α‐amino group of the prolipoprotein (Figure 2a). The X‐ray crystal structure of signal peptidase II (Lsp) from P. aeruginosa (Vogeley et al, 2016) and S. aureus (Olatunji et al, 2020) reveals two domains; a membrane domain consisting of the four transmembrane helices, with both the N and C terminus located in the cytoplasm (Munoa et al, 1991), and a periplasmic domain consisting of two sub domains—the β‐cradle, resting on the outer leaflet of the inner membrane, and α‐helix, with a single helical turn also resting on the membrane surface (Vogeley et al, 2016). Lsp belongs to the aspartate protease family (Tjalsma et al, 1999), where the catalytic aspartate residues reside at the membrane‐periplasm interface in TH1 and TH4.…”

“…The incoming prolipoprotein likely enters between the β‐cradle and the periplasmic helix, which form two arms extending away from the core of the enzyme (Vogeley et al, 2016). The scissile bond between the diacylglyceryl‐modified cysteine and the amino acids at position‐1 in the lipobox extends between the catalytic dyad (D124 and D143 in Lsp of P. aeruginosa ) (Figure 2b) and is clamped by the β‐cradle and the periplasmic helix (Olatunji et al, 2020; Vogeley et al, 2016). The catalytic site contains a water molecule in a deprotonated state.…”

“…The mechanism of host protection is not fully understood but has been hypothesized due to either (over‐)expression of lspA3 , which conferred highest resistance when expressed in E. coli or regulation in antibiotic levels by LspA4 (Xiao and Wall, 2014). In the S. aureus Lsp structures, globomycin and myxovirescin share a 19‐atom core structure bound in the central cavity of the enzyme, blocking the catalytic dyad (Olatunji et al, 2020), and is presumably where the signal peptide of prolipoprotein binds, whereas the macrocycles each occupy opposite sides of the catalytic site.…”

Novel inhibitors ofE. colilipoprotein diacylglyceryl transferase are insensitive to resistance caused bylppdeletion

“…After secretion through the IM, Lgt catalyzes the attachment of a diacylglyceryl moiety from phosphatidylglycerol to the thiol group of the conserved +1 position cysteine via a thioether bond (Sankaran & Wu, 1994). The second enzyme, prolipoprotein signal peptidase (LspA), is an aspartyl endopeptidase which cleaves off the signal peptide N-terminal of the conserved diacylated +1 cysteine (M. , and is the molecular target of the Gram-negative-specific natural-product antibiotics globomycin and myxovirescin (Dev, Harvey, & Ray, 1985;Gerth, Irschik, Reichenbach, & Trowitzsch, 1982;Olatunji et al, 2020;Xiao, Gerth, Müller, & Wall, 2012). In Gram-negative and high-GC Gram-positive bacteria, a third enzyme, lipoprotein N-acyl transferase (Lnt), catalyzes the addition of a third acyl chain to the amino group of the N-terminal cysteine via an amide linkage.…”