Elongated structure of the outer-membrane activator of peptidoglycan synthesis LpoA: implications for PBP1A stimulation - PubMed (original) (raw)

Elongated structure of the outer-membrane activator of peptidoglycan synthesis LpoA: implications for PBP1A stimulation

Nicolas L Jean et al. Structure. 2014.

Abstract

The bacterial cell envelope contains the stress-bearing peptidoglycan layer, which is enlarged during cell growth and division by membrane-anchored synthases guided by cytoskeletal elements. In Escherichia coli, the major peptidoglycan synthase PBP1A requires stimulation by the outer-membrane-anchored lipoprotein LpoA. Whereas the C-terminal domain of LpoA interacts with PBP1A to stimulate its peptide crosslinking activity, little is known about the role of the N-terminal domain. Herein we report its NMR structure, which adopts an all-α-helical fold comprising a series of helix-turn-helix tetratricopeptide-repeat (TPR)-like motifs. NMR spectroscopy of full-length LpoA revealed two extended flexible regions in the C-terminal domain and limited, if any, flexibility between the N- and C-terminal domains. Analytical ultracentrifugation and small-angle X-ray scattering results are consistent with LpoA adopting an elongated shape, with dimensions sufficient to span from the outer membrane through the periplasm to interact with the peptidoglycan synthase PBP1A.

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Graphical abstract

Figure 1

Figure 1

LpoAN Has a TPR Domain-like Structure (A) Amino acid sequence of the LpoAN construct used for structure determination and secondary structure elements (α helices). The residues of the oligohistidine tag are shown in red. (B) Cartoon representation of the 20 lowest-energy structures of LpoAN, as determined by NMR spectroscopy, emphasizing the spatial arrangement of the 12 α-helices, which are numbered from the N to C terminus. (C) LpoAN is stabilized by numerous interhelical hydrophobic contacts. The ten α helices with more than four residues are shown as circles and labeled according to the description in (B). Interhelical hydrophobic contacts are represented with dashed lines, and short plain lines connect each of the residues to its corresponding helix. See also Figure S1.

Figure 2

Figure 2

Comparison of LpoAC from E. coli and H. influenzae (A) Superimposition of the X-ray structure of H. influenzae LpoAC (green, PDB code

3CKM

) and the structure of E. coli LpoAC predicted by PHYRE (red). Regions 1 (residues N285–P351) and 2 (residues S493–N531) are present in LpoAC from E. coli, but not from H. influenzae. Flexible residues for which backbone resonances have been assigned by NMR spectroscopy are sketched as blue sticks. (B) Disorder in E. coli LpoAC predicted by IUPred. The two main regions absent from the H. influenzae LpoA sequence (in gray) are predicted as being mostly unstructured by IUPred (score > 0.5). (C) Sequence alignment of H. influenzae and E. coli LpoAC, where the two E. coli inserts are highlighted in gray. NMR-assigned residues are shown in blue. (D) The linker between LpoAN and LpoAC (in gray and white hashes) starts at K249 and ends at K258. The criteria used to define this linker included the structuring of the N-terminal domain as quantified by the {1H}15N-NOE measured in LpoAN (black) and the definition of the first secondary-structure element in the C-terminal domain (red) of LpoA as modeled by PHYRE. See also Figure S2.

Figure 3

Figure 3

Full-Length LpoA Has an Extended Structure (A) SAXS curves of LpoA at concentrations of 1, 2, and 5 mg/ml. (B) Experimental distance distribution function, P(r), calculated from SAXS data collected on a 5 mg/ml 15N-LpoA sample (black) and theoretical P(r) function calculated for molecular models with three different, arbitrarily chosen orientations of the N- and C-terminal domains (....., globular model; ----, L-shaped model; – – –, extended model; see also Figure S3C). The theoretical Rg values extracted from these curves are 3.11 nm for the globular model, 3.58 nm for the L-shaped model, and 4.44 nm for the extended model. The experimental Rg value, 4.22 ± 0.01 nm, fits best to the extended model.

Figure 4

Figure 4

Schematic Representation of PBP1A Activation by LpoA The N-terminal domain of LpoA (blue) anchors to the outer membrane, whereas the C-terminal domain (orange) interacts with the outer-membrane PBP1A docking domain (Typas et al., 2010). LpoA has an estimated total width of ∼30 Å and length of ∼145 Å. These dimensions should enable the protein to reach PBP1A through the periplasm and cross the ∼60-Å-thick PG layer, which has ∼40- to 60-Å-wide pores (Demchick and Koch, 1996). TP, transpeptidase domain; GT, glycosyltransferase domain; IM, inner membrane; OM, outer membrane.

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