Backbone and side-chain (1)H, (13)C, and (15)N NMR assignments of the N-terminal domain of Escherichia coli LpoA.

The peptidoglycan is a major component of the bacterial cell wall and is essential to maintain cellular integrity and cell shape. Penicillin-Binding Proteins (PBPs) catalyze the final biosynthetic steps of peptidoglycan synthesis from lipid II precursor and are the main targets of ß-lactam antibiotics. The molecular details of peptidoglycan growth and its regulation are poorly understood. Presumably, PBPs are active in peptidoglycan synthesizing multi-enzyme complexes that are controlled from inside the cell by cytoskeletal elements. Recently, two outer-membrane lipoproteins, LpoA and LpoB, were shown to be required in Escherichia coli for the function of the main peptidoglycan synthases, PBP1A and PBP1B, by stimulating their transpeptidase activity. However, the mechanism of PBP-activation by Lpo proteins is not known, and the Lpo proteins await structural characterization at atomic resolution. Here we present the backbone and side-chain (1)H, (13)C, (15)N NMR assignments of the N-terminal domain of LpoA from E. coli for structural and functional studies.

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

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.