Structure snapshots reveal the mechanism of a bacterial membrane lipoprotein N-acyltransferase

Bacterial lipoproteins (BLPs) decorate the surface of membranes in the cell envelope. They function in membrane assembly and stability, as enzymes, and in transport. The final enzyme in the BLP synthesis pathway is the apolipoprotein N-acyltransferase, Lnt, which is proposed to act by a ping-pong mechanism. Here, we use x-ray crystallography and cryo–electron microscopy to chart the structural changes undergone during the progress of the enzyme through the reaction. We identify a single active site that has evolved to bind, individually and sequentially, substrates that satisfy structural and chemical criteria to position reactive parts next to the catalytic triad for reaction. This study validates the ping-pong mechanism, explains the molecular bases for Lnt’s substrate promiscuity, and should facilitate the design of antibiotics with minimal off-target effects.

[1]  W. Sandoval,et al.  Deletion of a previously uncharacterized lipoprotein lirL confers resistance to an inhibitor of type II signal peptidase in Acinetobacter baumannii , 2022, Proceedings of the National Academy of Sciences of the United States of America.

[2]  M. Caffrey,et al.  Bacterial Lipoprotein Posttranslational Modifications. New Insights and Opportunities for Antibiotic and Vaccine Development , 2021, Frontiers in Microbiology.

[3]  M. Caffrey,et al.  Structural basis of the membrane intramolecular transacylase reaction responsible for lyso-form lipoprotein synthesis , 2021, Nature Communications.

[4]  Qiangde Duan,et al.  Research progress on Toll-like receptor signal transduction and its roles in antimicrobial immune responses , 2021, Applied Microbiology and Biotechnology.

[5]  Christian N. Cunningham,et al.  Inhibition of Escherichia coli Lipoprotein Diacylglyceryl Transferase Is Insensitive to Resistance Caused by Deletion of Braun’s Lipoprotein , 2021, Journal of bacteriology.

[6]  M. Liao,et al.  Mechanism of LolCDE as a molecular extruder of bacterial triacylated lipoproteins , 2021, Nature Communications.

[7]  M. Caffrey,et al.  9.8 MAG: A New Host Lipid for In Meso (Lipid Cubic Phase) Crystallization of Integral Membrane Proteins , 2020 .

[8]  Z. Modrušan,et al.  Unstable Mechanisms of Resistance to Inhibitors of Escherichia coli Lipoprotein Signal Peptidase , 2020, mBio.

[9]  Conrad C. Huang,et al.  UCSF ChimeraX: Structure visualization for researchers, educators, and developers , 2020, Protein science : a publication of the Protein Society.

[10]  S. Gloor,et al.  Optimization of globomycin analogs as novel gram-negative antibiotics. , 2020, Bioorganic & medicinal chemistry letters.

[11]  W. Jonckheere,et al.  A dual constriction biological nanopore resolves homonucleotide sequences with high fidelity , 2020, Nature Biotechnology.

[12]  M. Caffrey,et al.  Structures of lipoprotein signal peptidase II from Staphylococcus aureus complexed with antibiotics globomycin and myxovirescin , 2020, Nature Communications.

[13]  M. Högbom,et al.  Conformational changes in Apolipoprotein N-acyltransferase (Lnt) , 2018, Scientific Reports.

[14]  Erik Lindahl,et al.  New tools for automated high-resolution cryo-EM structure determination in RELION-3 , 2018, eLife.

[15]  D. Wolan,et al.  Probing substrate recognition of bacterial lipoprotein signal peptidase using FRET reporters , 2018, FEBS letters.

[16]  D. Wolan,et al.  Lipoprotein Signal Peptidase Inhibitors with Antibiotic Properties Identified through Design of a Robust In Vitro HT Platform. , 2018, Cell chemical biology.

[17]  Randy J Read,et al.  Real-space refinement in PHENIX for cryo-EM and crystallography , 2018, bioRxiv.

[18]  Justyna Aleksandra Wojdyla,et al.  DA+ data acquisition and analysis software at the Swiss Light Source macromolecular crystallography beamlines , 2018, Journal of synchrotron radiation.

[19]  Kazutaka Katoh,et al.  MAFFT online service: multiple sequence alignment, interactive sequence choice and visualization , 2017, Briefings Bioinform..

[20]  Michele D. Kattke,et al.  Structural insights into lipoprotein N-acylation by Escherichia coli apolipoprotein N-acyltransferase , 2017, Proceedings of the National Academy of Sciences.

[21]  Y. Xiong,et al.  Crystal structure of E. coli apolipoprotein N-acyl transferase , 2017, Nature Communications.

[22]  M. Caffrey,et al.  Structural insights into the mechanism of the membrane integral N-acyltransferase step in bacterial lipoprotein synthesis , 2017, Nature Communications.

[23]  A. Gurtman,et al.  SA4Ag, a 4-antigen Staphylococcus aureus vaccine, rapidly induces high levels of bacteria-killing antibodies. , 2017, Vaccine.

[24]  David J. Fleet,et al.  cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination , 2017, Nature Methods.

[25]  Jason C. Rouse,et al.  The Dual Role of Lipids of the Lipoproteins in Trumenba, a Self-Adjuvanting Vaccine Against Meningococcal Meningitis B Disease , 2016, The AAPS Journal.

[26]  James Clarke,et al.  Nanopore development at Oxford Nanopore , 2016, Nature Biotechnology.

[27]  P. Balmer,et al.  Characteristics of a new meningococcal serogroup B vaccine, bivalent rLP2086 (MenB-FHbp; Trumenba®) , 2016, Postgraduate medicine.

[28]  O. Bunk,et al.  Fast two-dimensional grid and transmission X-ray microscopy scanning methods for visualizing and characterizing protein crystals , 2016, Journal of applied crystallography.

[29]  Itay Mayrose,et al.  ConSurf 2016: an improved methodology to estimate and visualize evolutionary conservation in macromolecules , 2016, Nucleic Acids Res..

[30]  M. Caffrey,et al.  Structural basis of lipoprotein signal peptidase II action and inhibition by the antibiotic globomycin , 2016, Science.

[31]  K. Diederichs,et al.  In meso in situ serial X-ray crystallography of soluble and membrane proteins at cryogenic temperatures , 2016, Acta crystallographica. Section D, Structural biology.

[32]  Robert D. Finn,et al.  HMMER web server: 2015 update , 2015, Nucleic Acids Res..

[33]  Erik G Marklund,et al.  Bayesian deconvolution of mass and ion mobility spectra: from binary interactions to polydisperse ensembles. , 2015, Analytical chemistry.

[34]  R. Read,et al.  Neisseria meningitidis serogroup B bivalent factor H binding protein vaccine , 2015, Expert review of vaccines.

[35]  N. Buddelmeijer.  The molecular mechanism of bacterial lipoprotein modification--how, when and why? , 2015, FEMS microbiology reviews.

[36]  S. Lory,et al.  Residues located on membrane‐embedded flexible loops are essential for the second step of the apolipoprotein N‐acyltransferase reaction , 2015, Molecular microbiology.

[37]  Peter B. McGarvey,et al.  UniRef clusters: a comprehensive and scalable alternative for improving sequence similarity searches , 2014, Bioinform..

[38]  M. Caffrey,et al.  Renaturing Membrane Proteins in the Lipid Cubic Phase, a Nanoporous Membrane Mimetic , 2014, Scientific Reports.

[39]  M. Resh.  Covalent lipid modifications of proteins , 2013, Current Biology.

[40]  Sjors H.W. Scheres,et al.  RELION: Implementation of a Bayesian approach to cryo-EM structure determination , 2012, Journal of structural biology.

[41]  Martin Caffrey,et al.  Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases , 2012, Journal of visualized experiments : JoVE.

[42]  Kevin W Eliceiri,et al.  NIH Image to ImageJ: 25 years of image analysis , 2012, Nature Methods.

[43]  F. Hillmann,et al.  Kinetics and Phospholipid Specificity of Apolipoprotein N-Acyltransferase , 2011, The Journal of Biological Chemistry.

[44]  Robert D. Finn,et al.  HMMER web server: interactive sequence similarity searching , 2011, Nucleic Acids Res..

[45]  M. Caffrey,et al.  Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases , 2010, Journal of visualized experiments : JoVE.

[46]  R. Titball,et al.  Lipoproteins of Bacterial Pathogens , 2010, Infection and Immunity.

[47]  Subbalakshmi S. Malladi,et al.  Structure-activity relationships in toll-like receptor-2 agonistic diacylthioglycerol lipopeptides. , 2010, Journal of medicinal chemistry.

[48]  N. Buddelmeijer,et al.  The essential Escherichia coli apolipoprotein N-acyltransferase (Lnt) exists as an extracytoplasmic thioester acyl-enzyme intermediate. , 2010, Biochemistry.

[49]  Vincent B. Chen,et al.  MolProbity: all-atom structure validation for macromolecular crystallography , 2009, Acta crystallographica. Section D, Biological crystallography.

[50]  S. Paik,et al.  Recognition of lipopeptide patterns by Toll-like receptor 2-Toll-like receptor 6 heterodimer. , 2009, Immunity.

[51]  S. Paik,et al.  Crystal Structure of the TLR1-TLR2 Heterodimer Induced by Binding of a Tri-Acylated Lipopeptide , 2007, Cell.

[52]  Randy J. Read,et al.  Phaser crystallographic software , 2007, Journal of applied crystallography.

[53]  M. Madan Babu,et al.  A Database of Bacterial Lipoproteins (DOLOP) with Functional Assignments to Predicted Lipoproteins , 2006, Journal of bacteriology.

[54]  Conrad C. Huang,et al.  UCSF Chimera—A visualization system for exploratory research and analysis , 2004, J. Comput. Chem..

[55]  H. Kogen,et al.  Structure-activity relationships of globomycin analogues as antibiotics. , 2004, Bioorganic & medicinal chemistry.

[56]  H. Kogen,et al.  Synthesis and antimicrobial activity of novel globomycin analogues. , 2003, Bioorganic & medicinal chemistry letters.

[57]  S. Akira,et al.  Cutting Edge: Role of Toll-Like Receptor 1 in Mediating Immune Response to Microbial Lipoproteins1 , 2002, The Journal of Immunology.

[58]  S. Akira,et al.  Discrimination of bacterial lipoproteins by Toll-like receptor 6. , 2001, International immunology.

[59]  M. Caffrey,et al.  A simple mechanical mixer for small viscous lipid-containing samples. , 1998, Chemistry and physics of lipids.

[60]  C. Schmid,et al.  Vaccination against Lyme disease with recombinant Borrelia burgdorferi outer-surface lipoprotein A with adjuvant. Lyme Disease Vaccine Study Group. , 1998, The New England journal of medicine.

[61]  Sita D Gupta,et al.  Phosphatidylethanolamine is not essential for the N-acylation of apolipoprotein in Escherichia coli. , 1991, The Journal of biological chemistry.

[62]  Sita D Gupta,et al.  Identification and subcellular localization of apolipoprotein N-acyltransferase in Escherichia coli. , 1991, FEMS microbiology letters.

[63]  C. Rock,et al.  Transfer of fatty acids from the 1-position of phosphatidylethanolamine to the major outer membrane lipoprotein of Escherichia coli. , 1986, The Journal of biological chemistry.

[64]  W. Bessler,et al.  Synthetic lipopeptide analogs of bacterial lipoprotein are potent polyclonal activators for murine B lymphocytes. , 1985, Journal of immunology.

[65]  A. Sharff,et al.  Data processing and analysis with the autoPROC toolbox , 2011, Acta crystallographica. Section D, Biological crystallography.

[66]  V. Cherezov,et al.  Crystallizing membrane proteins using lipidic mesophases , 2009, Nature Protocols.

[67]  Ennis,et al.  Vaccination against Lyme Disease with Recombinant Borrelia burgdorferi Outer-Surface Lipoprotein A with Adjuvant , 2000 .