The periplasmic domain of Escherichia coli outer membrane protein A can undergo a localized temperature dependent structural transition

Abstract

Gram-negative bacteria such as Escherichia coli are surrounded by two membranes with a thin peptidoglycan (PG)-layer located in between them in the periplasmic space. The outer membrane protein A (OmpA) is a 325-residue protein and it is the major protein component of the outer membrane of E. coli. Previous structure determinations have focused on the N-terminal fragment (residues 1-171) of OmpA, which forms an eight stranded transmembrane β-barrel in the outer membrane. Consequently it was suggested that OmpA is composed of two independently folded domains in which the N-terminal β-barrel traverses the outer membrane and the C-terminal domain (residues 180-325) adopts a folded structure in the periplasmic space. However, some reports have proposed that full-length OmpA can instead refold in a temperature dependent manner into a single domain forming a larger transmembrane pore. Here, we have determined the NMR solution structure of the C-terminal periplasmic domain of E. coli OmpA (OmpA(180-325)). Our structure reveals that the C-terminal domain folds independently into a stable globular structure that is homologous to the previously reported PG-associated domain of Neisseria meningitides RmpM. Our results lend credence to the two domain structure model and a PG-binding function for OmpA, and we could indeed localize the PG-binding site on the protein through NMR chemical shift perturbation experiments. On the other hand, we found no evidence for binding of OmpA(180-325) with the TonB protein. In addition, we have also expressed and purified full-length OmpA (OmpA(1-325)) to study the structure of the full-length protein in micelles and nanodiscs by NMR spectroscopy. In both membrane mimetic environments, the recombinant OmpA maintains its two domain structure that is connected through a flexible linker. A series of temperature-dependent HSQC experiments and relaxation dispersion NMR experiments detected structural destabilization in the bulge region of the periplasmic domain of OmpA above physiological temperatures, which may induce dimerization and play a role in triggering the previously reported larger pore formation.

Braun’s Lipoprotein Facilitates OmpA Interaction with the Escherichia coli Cell Wall

“…in OmpA from certain bacteria, which comprises the least stable region of the protein as shown by a NMR study (24) and previous computational simulations (29). The lysine and arginine residues are conserved in homologs from Salmonella enterica and Neisseria meningitides and, based on their crystal structures, are positioned similarly compared to the ones in E. coli (Fig.…”

“…The E. coli outer membrane porin OmpA is a multidomain protein whose N-terminal domain (NTD) is made of a b-barrel and C-terminal domain (CTD) is a globular periplasmic unit that binds to PGN, connected by an unstructured 20-residue linker region (18). The NTD has been subject to numerous functional and structural studies (19)(20)(21)(22)(23), whereas the structure of the CTD has recently emerged from a NMR study (24). Experimental evidence suggests that the full-length OmpA can form a homodimer (25,26), the model of which has been proposed and validated by mass spectrometry and MD simulations (27)(28)(29).…”

Probing the Protein Interaction Network of Pseudomonas aeruginosa Cells by Chemical Cross-Linking Mass Spectrometry

“…S4c). The predicted structure of the OprF C-terminal domain was superimposed onto the corresponding region of OmpA crystal structure (PDB identifier: 2MQE) (Ishida et al, 2014) to identify regions of variability and the two proteins showed high structural similarity, also reflected in the high TM-score of the predicted model. Importantly, the region of OmpA dimerization interface and the corresponding superimposed region of OprF monomer containing cross-link sites showed identical secondary structures.…”

The Shigella flexneri OmpA amino acid residues 188 EVQ 190 are essential for the interaction with the virulence factor PhoN2

“…2 D). The N- and the C- terminal domains of OmpA are connected by a short region (residues 173–190) with unresolved structure that forms a flexible linker [28] . Basing on our tridimensional model, a set of twelve ompA fragments were PCR-generated ( Fig.…”