Toward the Characterization of Peptidoglycan Structure and Protein-Peptidoglycan Interactions by Solid-State NMR Spectroscopy Thomas Kern, † Sabine Hediger,* ,‡ Patrick Mu ¨ ller, § Ce ´ cile Giustini, † Bernard Joris, ¶ Catherine Bougault, † Waldemar Vollmer, # and Jean-Pierre Simorre* ,† Institut de Biologie Structurale, UMR5075 (CEA/CNRS/UJF), 38027 Grenoble, France, Laboratoire de Chimie Inorganique et Biologique, UMR-E3 (CEA/UJF), INAC, CEA, 38054 Grenoble, France, Mikrobielle Genetik, UniVersita ¨t Tu ¨bingen, 72076 Tu ¨bingen, Germany, Centre d’inge ´nierie des prote ´ines, Institut de Chimie B6A, UniVersite ´ de Lie `ge, Sart-Tilman B4000, Belgium, and Institute for Cell and Molecular Biosciences, Newcastle UniVersity, NE2 4HH Newcastle Upon Tyne, U.K. Received December 10, 2007; E-mail: jean-pierre.simorre@ibs.fr; sabine.hediger@cea.fr The peptidoglycan (murein) sacculus is an essential component of the bacterial cell wall. Surrounding the cytoplasmic membrane, it plays a crucial role in allowing cells to withstand osmotic pres- sure 1 and in defining cellular shape. 2 Unique to the bacterial world, this large polymer network is also the principal target of many antibiotics 3 and one of the main microbial products recognized by the immune system. 4 Consequently, great efforts have been invested in the past decade to determine its architecture and biosynthesis. 5,6 While chemical composition and structure of fragments have been established, and several enzymes involved in peptidoglycan as- sembly have been isolated, the 3D organization of this biopolymer is still debated 7 (orientation of the glycan strands parallel 5,8 or perpendicular 9,10 to the cell membrane has been proposed), and its growth mechanism remains an area of active research. Peptidoglycan is a heteropolymer made of linear glycan strands of two alternating -1,4-linked carbohydrates, N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc), that are cross- linked by short peptides (Figure 1a). In the Gram-negative model bacterium Escherichia coli, the glycan strands vary in length from 1 to ∼80 disaccharide units, with an average length of 21-35. They are terminated with a 1,6-anhydro-MurNAc instead of a MurNAc residue. 11 Peptides are covalently linked to the lactyl group of the muramic acid and can vary between different bacteria in the nature of the amino acids involved (for example, m-DAP versus L-Lys in position 3), the number of cross-linked peptides (for example, in E. coli, from monomers to tetramers), and in the structure of the peptide cross-links (for example, in E. coli, D-Ala- D-m-DAP or L-m-DAP-D-m-DAP). 5 The molecular weight of a sacculus from E. coli is in the range of 3 × 10 6 kDa, making the nonfragmented molecule inaccessible to most analytical methods. In this work, we show for the first time that solid-state NMR spectroscopy can successfully be applied to intact hydrated and fully labeled E. coli sacculi. Almost complete NMR assignment of the repetitive polymer has been obtained, allowing the subsequent study of protein-peptidoglycan interactions. 13 C, 15 N-isotopically labeled E. coli BL21(DE3) cells were grown in M9 minimal medium and harvested by centrifugation. Sacculi were then purified according to previously published procedure. 11 After centrifugation, 25 mg of the resulting paste was finally loaded into a 4 mm solid-state NMR rotor. Different 13 C-correlation experiments were performed, and spectra of astonishing good resolution were obtained, considering the size and noncrystallinity of the sample. Figure 1c illustrates the high spectral quality obtained using a through-bond correlation experiment. The spectrum was acquired using the sequence in Figure S1, which is closely related to previously published experiments. 12,13 Interestingly, the signals observed for the glycan part present symmetric line shapes, without any distortion due to chemical shift inhomogeneity, as has been observed, for example, in cellulose. 14 This suggests a structural inhomogeneity which is small enough to be averaged out by the amplitude-restricted molecular dynamics present in the cross-linked polymer. † Institut de Biologie Structurale, UMR5075 (CEA/CNRS/UJF). ‡ Laboratoire de Chimie Inorganique et Biologique, UMR-E3 (CEA/UJF). § Universita ¨t Tu ¨bingen. ¶ Universite ´ de Lie `ge. # Newcastle University. Figure 1. (a) Chemical structure of E. coli peptidoglycan. The letter R stands for a hydrogen atom or the peptide stem of another peptidoglycan chain; X stands for a peptide stem. (b) Schematic drawing of the peptidoglycan sacculus. (c) Through-bond 13 C-correlation spectrum of 13 C-labeled peptidoglycan, measured on a 400 MHz ( 1 H frequency) spectrometer. The resonance assignment follows the color code defined in panel (a) for the different spin systems: shades of blue for glycan, black for lactic acid, orange for alanines, green for glutamate, and red for diaminopimelic acid. Published on Web 04/05/2008 10.1021/ja7108135 CCC: $40.75  2008 American Chemical Society 5618 9 J. AM. CHEM. SOC. 2008, 130, 5618–5619