We are a Chemical Biology lab that combines chemistry, biochemistry, genetics and functional genomics to address problems in two main areas: the bacterial cell envelope and O-GlcNAc transferase (OGT). Our focus on these disparate areas stems from an early interest in the structure, function, and inhibition of glycosyltransferases.
The bacterial cell envelope is a complex system that serves as the interface between the organism and its environment. It includes: 1) the cell membrane with all its phospho- and glycolipids, 2) the proteins that are embedded in or associated with the membrane, and 3) the surrounding peptidoglycan layers, which in Gram-positive organisms are densely functionalized with other glycopolymers and contain both covalently and non-covalently bound proteins. The proteins of the cell envelope include a variety of two-component signaling systems as well as numerous enzymes that assemble or modify membrane lipids and cell wall polymers. Enzymes important for virulence are also found in the cell envelope. The activities of all enzymes in the cell envelope must be coordinated with intracellular processes for bacteria to grow and divide properly, but how intra- and extracellular processes are coordinated is not well understood. We are interested in developing a comprehensive understanding of bacterial cell envelope assembly and regulation, and in identifying and exploiting key vulnerabilities to disrupt bacterial growth. In line with our interests in developing approaches to kill bacteria, we primarily focus on pathogens, including Staphylococcus aureus and Streptococcus pneumoniae.
Over the years, we have developed a wide range of tools and methods to enable study of the bacterial cell wall. We developed the first synthetic routes to Lipid I and Lipid II, key building blocks of bacterial peptidoglycan, and in a fruitful collaboration with the Kahne laboratory at Harvard, we developed facile methods to accumulate, isolate, and label Lipid II from numerous different bacteria. Rapid access to this key substrate has made it possible to address problems that were formerly out of reach. For example, we were recently able to define the order of assembly of the Gram-positive cell wall by defining the substrate preferences of cell wall ligases that couple two polymers. The ability to obtain Lipid II easily also enables the study (and rapid discovery) of Lipid II-binding antibiotics.
For recent work on bacterial cell wall biosynthesis and its inhibition see:
Schaefer K, Owens TW, Kahne D, Walker S. "Substrate Preferences Establish the Order of Cell Wall Assembly in Staphylococcus aureus." J. Am. Chem. Soc. 2018; 140:2442-5.
Santiago M, Lee W, Fayad AA, Coe KA, Rajagopal M, Do T, Hennessen F, Srisuknimit V, Müller R, Meredith TC, Walker S. "Genome-wide mutant profiling predicts the mechanism of a Lipid II binding antibiotic." Nat. Chem. Biol. 2018; 14:601-8.
Schaefer K, Matano LM, Qiao Y, Kahne D, Walker S. "In vitro reconstitution demonstrates the cell wall ligase activity of LCP proteins." Nat. Chem. Biol. 2017; 13:396-401.
Welsh MA, Taguchi A, Schaefer K, Van Tyne D, Lebreton F, Gilmore MS, Kahne D, Walker S. "Identification of a Functionally Unique Family of Penicillin-Binding Proteins." J. Am. Chem. Soc. 2017; 139:17727-30.
Qiao Y, Srisuknimit V, Rubino F, Schaefer K, Ruiz N, Walker S, Kahne D. "Lipid II overproduction allows direct assay of transpeptidase inhibition by beta-lactams." Nat. Chem. Biol. 2017; 13:793-8.
Lee W, Schaefer K, Qiao Y, Srisuknimit V, Steinmetz H, Müller R, Kahne D, Walker S. "The mechanism of action of lysobactin." J. Am. Chem. Soc. 2016; 138:100-3.
Qiao Y, Lebar MD, Schirner K, Schaefer K, Tsukamoto H, Kahne D, Walker S. "Detection of Lipid-Linked Peptidoglycan Precursors by Exploiting an Unexpected Transpeptidase Reaction." J. Am. Chem. Soc. 2014; 136:14678-81.
For selected work on OGT, see:
Levine ZG, Fan C, Melicher MS, Orman M, Benjamin T, Walker S. "O-GlcNAc Transferase Recognizes Protein Substrates Using an Asparagine Ladder in the Tetratricopeptide Repeat (TPR) Superhelix." J. Am. Chem. Soc. 2018; 140:3510-3.
Janetzko J, Walker S. "Aspartate glycosylation triggers isomerization to isoaspartate." J. Am. Chem. Soc. 2017; 139:3332-5.
Janetzko J, Trauger SA, Lazarus MB, Walker S. "How the glycosyltransferase OGT catalyzes amide bond cleavage." Nat. Chem. Biol. 2016; 12:899-901.
Lazarus MB, Jiang J, Kapuria V, Bhuiyan T, Janetzko J, Zandberg WF, Vocadlo DJ, Herr W, Walker S. "HCF-1 is cleaved in the active site of O-GlcNAc transferase." Science 2013; 342:1235-9.
Lazarus MB, Jiang J, Gloster TM, Zandberg WF, Whitworth GE, Vocadlo DJ, Walker S. "Structural snapshots of the reaction coordinate for O-GlcNAc transferase." Nat. Chem. Biol. 2012; 8:966-8.
Lazarus MB, Nam Y, Jiang J, Sliz P, Walker S. "Structure of human O-GlcNAc transferase and its complex with a peptide substrate." Nature 2011; 469: 564-7.
Gross BJ, Kraybill BC, and Walker S. "Discovery of O-GlcNAc Transferase Inhibitors." J. Am. Chem. Soc. 2005; 127:14588-9.