REDOR constraints on the peptidoglycan lattice architecture of Staphylococcus aureus and its FemA mutant

Abstract

The peptidoglycan of Gram-positive bacteria consists of glycan chains with attached short peptide stems cross-linked to one another by glycyl bridges. The bridge of Staphylococcus aureus has five glycyl units and that of its FemA mutant has one. These long- and short-bridge cross-links create totally different cell-wall architectures. S. aureus and its FemA mutant grown in the presence of an alanine-racemase inhibitor were labeled with d-[1-¹³C]alanine, l-[3-¹³C]alanine, [2-¹³C]glycine, and l-[5-¹⁹F]lysine to characterize some details of the peptidoglycan tertiary structure. Rotational-echo double-resonance (REDOR) NMR of isolated cell walls was used to measure internuclear distances between ¹³C-labeled alanines and ¹⁹F-labeled lysine incorporated in the peptidoglycan. The alanyl ¹³C labels in the parent strain were preselected for C{F} and C{P} REDOR measurement by their proximity to the glycine label using ¹³C¹³C spin diffusion. The observed ¹³C¹³C and ¹³C³¹P distances are consistent with a tightly packed architecture containing only parallel stems in a repeating structural motif within the peptidoglycan. Dante selection of d-alanine and l-alanine frequencies followed by ¹³C¹³C spin diffusion rules out scrambling of carbon labels. Cell walls of FemA were also labeled by a combination of d-[1-¹³C]alanine and l-[¹⁵N]alanine. Proximity of chains was measured by C{N} and N{C} REDOR distances and asymptotic plateaus, and both were consistent with a mixed-geometry model. Binding of an ¹⁹F-labeled eremomycin analog in the FemA cell wall matches that of binding to the parent-strain cell wall and reveals the proximity of parallel stems in the alternating parallel-perpendicular mixed-geometry model for the FemA peptidoglycan lattice.

Keywords: Alanine racemase; Bacterial cell-walls; Solid-state NMR.

Figure 1

Location of the labels (red, ¹³C; green, ¹⁹F) of FemA peptidoglycan, and (red, ¹³C; yellow, ³¹P) of the repeating unit of wall teichoic acid.

Figure 2

Dante-selected C{F} (left) and C{P} (right) REDOR of intact cell walls of the parent strain of S. aureus grown in media containing D-[1-¹³C]alanine, L-[3-¹³C]alanine, [2-¹³C]glycine, and L-[5-¹⁹F]lysine with the alanine racemase inhibitor, alaphosphin. Dante differencing with inversion of the glycyl-carbon peak (42 ppm) preceded REDOR dephasing. Four data blocks were collected resulting in spectra with and without Dante irradiation, each with and without ¹⁹F (or ³¹P) dephasing. The Dante differences (ΔS) are shown at the bottom of the figure and are the reference spectra for REDOR dephasing (ΔΔS) shown above. Spinning sidebands are designated by “ssb.”

Figure 3

Dante frequency selection for the FemA cell-wall sample. A train of 1-μs ¹³C radio-frequency pulses separated by 5 μs, with the carrier frequency centered at 178 ppm (top) or 175 ppm (bottom), was followed by z-axis storage for 200 ms. The resulting spectra are designated as “S.”

Figure 4

Dante difference spectra from the FemA cell-wall inversions of Figure 2 showing ¹³C-¹³C spin diffusion (for 200 ms) from the carbonyl-carbon label (175 ppm, left; 178 ppm, right) to the glycyl label (near 42 ppm) and the L-alanyl label (15 ppm). ΔS = S₀ – S. Spinning sidebands are designated by “ssb.”

Figure 5

Dante difference spectra from L-alanyl FemA cell-wall inversions showing ¹³C-¹³C spin diffusion (for 400 ms) from the methyl-carbon label (17.5 ppm, left; 21.2 ppm, right) to the glycyl label (near 42 ppm) and the D-alanyl label (near 175 ppm). Spinning sidebands are designated by “ssb.”

Figure 6

(Left) C{N} and N{C} REDOR dephasing (ΔS/S₀) of ¹⁹F-labeled LCTA-1110 complexed to cell walls of the FemA mutant 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 error in the integrated REDOR difference is estimated as the diameter of the open-circle symbols. (Right) C{F} and N{F} REDOR dephasing of the same sample (closed circles). The calculated dephasings for two single-distance components are shown in red and blue, and the combined dephasing curve (25% shorter-distance component and 75% longer-distance component), as a solid line.

Figure 7

(Top left) A cross-section of the proposed peptidoglycan-tertiary structure for the FemA mutant of S. aureus (ref 1). The cross-section consists of sixteen glycan chains in a 4×4 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-left panel are shown in the light-orange insert (bottom left). REDOR distance measurements (blue numbers) connect labels (red arrows). (Top right) Chemical structure of LCTA-1110.