Staphylococcus aureus peptidoglycan stem packing by rotational-echo double resonance NMR spectroscopy

Abstract

Staphylococcus aureus grown in the presence of an alanine-racemase inhibitor was labeled with d-[1-(13)C]alanine and l-[(15)N]alanine to characterize some details of the peptidoglycan tertiary structure. Rotational-echo double-resonance NMR of intact whole cells was used to measure internuclear distances between (13)C and (15)N of labeled amino acids incorporated in the peptidoglycan, and from those labels to (19)F of a glycopeptide drug specifically bound to the peptidoglycan. The observed (13)C-(15)N average distance of 4.1-4.4 Å between d- and l-alanines in nearest-neighbor peptide stems is consistent with a local, tightly packed, parallel-stem architecture for a repeating structural motif within the peptidoglycan of S. aureus.

Figure 1

Chemical structure of S. aureus peptidoglycan (PG). The PG monomer is highlighted in gray. The monomer consists of disaccharide, stem, and bridge structures. A stem structure having the sequence, L-Ala-D-iso-Glu-L-Lys-D-Ala-D-Ala, is attached to N-acetylgluosamine (NAG) of the disaccharide unit. Cross-linking between monomers is formed between the N-terminus of the pentaglycyl bridge and the carbonyl carbon of D-Ala of the adjacent stem. Cross-linked stems terminate in a single D-Ala (stem on the left). The five-residue stem in the middle has no cross-link. A bridge-link is an amide bond between the ε nitrogen of L-Lys (3^rd position of stem) to the carboxyl terminus of the bridge. The incorporation of ¹⁵N (blue dots) and ¹³C (red dots) isotopic labels identify stems and bridges.

Figure 2

Schematic representations of disaccharide backbone arrangements proposed for the glycan chains of PG, adapted from reference (36). The repeating disaccharide is represented by the alternating dark gray (NAM) and light gray (NAG) boxes. PG stems are represented by arrows attached to NAM. The PG bridge structure is omitted for clarity. Side views of the proposed glycan conformations with periodicities of 20 Å (top), 40 Å (middle), and 30 Å (bottom) are shown on the left, and their end views on the right.

Figure 3

¹³C{¹⁵N} REDOR spectra of whole cells of S. aureus grown on media containing D-[1-¹³C]alanine and L-[¹⁵N]alanine with the alanine racemase inhibitor, alaphosphin (5 μg/ml), after dipolar evolution of 12.8 msec. The full-echo spectrum (S₀) is at the bottom of the figure, and the REDOR difference (ΔS) is at the top. The dephasing of natural-abundance ¹³C peaks at 75, 54, and 17 ppm are apparent in the ΔS spectrum. Magic-angle spinning was at 5000 Hz.

Figure 4

¹³C{¹⁵N} REDOR dephasing (ΔS/S₀) of whole cells of S. aureus grown on media containing D-[1-¹³C]alanine and L-[¹⁵N]alanine, with increasing concentrations of the alanine racemase inhibitor, alaphosphin, as a function of the dipolar evolution (open circles). The calculated dephasings for two single-distance components are shown as dashed lines. The total dephasing (solid lines) is a weighted sum of calculations for a single distance, and for a narrow Gaussian distribution of distances (see Table 1 for details). The error in the integrated REDOR difference is estimated as the diameter of the open-circle symbols.

Figure 5

¹³C{¹⁹F} REDOR dephasing (ΔS/S₀) of ¹⁹F-labeled LCTA-1110 complexed to whole cells of S. aureus grown on media containing D-[1-¹³C]alanine and L-[¹⁵N]alanine, with the alanine racemase inhibitor, alaphosphin (10 μg/ml), as a function of the dipolar evolution (open circles). The calculated dephasings for two single-distance components are shown as dashed lines, and the combined dephasing curve (24% shorter-distance component and 76% longer-distance component), as a solid line. The calculated dephasing for a single ¹³C-¹⁹F distance of 8.1 Å does not match experiment.

Figure 6

¹⁵N CPMAS NMR spectra of whole cells S. aureus grown on defined media containing L-[¹⁵N]alanine in the absence (left) and presence (right) of the alanine racemase inhibitor, alaphosphin (5 μg/ml). When alaphosphin is not present, the ¹⁵N label of L-alanine is scrambled and appears in the D-alanine of teichoic acid. The ¹⁵N chemical-shift scale is referenced to solid ammonium sulfate.

Figure 7

(Top) Peptidoglycan architecture for S. aureus with anti-parallel (left) and parallel (right) stems and a 4-fold glycan symmetry axis. Stems are highlighted in dark red to identify the 40-Å glycan periodicity. Bridges are in pink. Orientations of selected stems (red and green) relative to the glycan backbones (gray) are numbered for clarity. (Bottom) Tesserae and pores for the peptidoglycan architectures at the top of the figure. A tessera is a hypothetical PG cell-unit structure defined by two glycan chains connected by two pairs of cross-links. Tesserae for the architectures at the top of the figure are outlined in yellow. The tesserae at the bottom of the figure are two-dimensional projections, in which disaccharides are represented in alternating shades of gray with PG stems as arrows. The inter-bridge structures are omitted for clarity. A true tessera structure requires that the orientation of cross-linked PG stems is antiparallel. For the parallel-stem architecture, we define a pseudo-tessera (yellow box) defined by two pairs of parallel PG stems.

Figure 8

(Top right) A cross-section of the proposed PG-tertiary structure for S. aureus. The cross-section consists of nine glycan chains in a 3x3 matrix where the glycan backbones (represented by gray circles) are propagating perpendicular to the plane of the paper. The stems and bridges are represented by green and red rectangles, respectively. A cartoon of the glycopeptide LCTA-1110 (bottom right) is shown bound to a D-Ala-D-Ala uncross-linked peptide stem based on the lattice model of ref. . Details of the highlighted region of the top-right panel are shown in the light-blue insert (top left). REDOR distance measurements (blue numbers) connect labels by red arrows. (Bottom left) Chemical structure of LCTA-1110.

Figure 9

Schematic representation of the peptidoglycan of S. aureus as a multi-layered brick wall. Each brick is a structural motif (determined by REDOR NMR) of just a few glycan chains with tightly packed parallel stems (see Figure 7). One side of the structure is imagined as the cell surface and the other, the cell membrane. Proteins and teichoic acids are accommodated by portals, spacings, and gaps in the wall.