Conformational States of a Bacterial α2-Macroglobulin Resemble Those of Human Complement C3

Abstract

α2 macroglobulins (α2Ms) are broad-spectrum protease inhibitors that play essential roles in the innate immune system of eukaryotic species. These large, multi-domain proteins are characterized by a broad-spectrum bait region and an internal thioester, which, upon cleavage, becomes covalently associated to the target protease, allowing its entrapment by a large conformational modification. Notably, α2Ms are part of a larger protein superfamily that includes proteins of the complement system, such as C3, a multi-domain macromolecule which is also characterized by an internal thioester-carrying domain and whose activation represents the pivotal step in the complement cascade. Recently, α2M/C3-like genes were identified in a large number of bacterial genomes, and the Escherichia coli α2M homolog (ECAM) was shown to be activated by proteases. In this work, we have structurally characterized ECAM by electron microscopy and small angle scattering (SAXS) techniques. ECAM is an elongated, flexible molecule with overall similarities to C3 in its inactive form; activation by methylamine, chymotrypsin, or elastase induces a conformational modification reminiscent of the one undergone by the transformation of C3 into its active form, C3b. In addition, the proposed C-terminus of ECAM displays high flexibility and different conformations, and could be the recognition site for partner macromolecules. This work sheds light on a potential bacterial defense mechanism that mimics structural rearrangements essential for activation of the complement cascade in eukaryotes, and represents a possible novel target for the development of antibacterials.

Structure of a bacterial α2-macroglobulin reveals mimicry of eukaryotic innate immunity

“…4). These observations are thus supportive of fluorescence, analytical ultracentrifugation and native polyacrylamide gel electrophoresis (PAGE) studies of E. coli A2M that suggested that thioester cleavage did not lead to major conformational differences 32 , but differ from SAXS results obtained on the latter molecule, which indicated some degree of conformational modification on methylamine treatment 33 . Despite the fact that a major shift in domain positions could not be detected for Sa-A2M, reaction with methylamine caused cleavage of the thioester bond (Fig.…”

“…1b). Previous studies with human and E. coli A2M have shown that reaction with methylamine inactivates the thioester and causes a major conformational change in the eukaryotic variant 5,19,32,33,[37][38][39] . To address the issue of a potential conformational change in a bacterial A2M on activation, we solved the crystal structure of methylamine-treated Sa-A2M.…”

“…In the event of a cell wall breach, the bacterial A2M would inhibit the antibacterial proteases, while PBP1c would repair the cell wall by catalysing the polymerization of glycan chains within the damaged peptidolgycan 28,31 . Biochemical characterization of the thioester-containing E. coli A2M (ECAM) showed that, unlike human A2M, it is a monomer, and is anchored to the inner membrane, thus being localized in the periplasmic space 32,33 . ECAM is capable of binding proteases and hindering their access to substrates, which supports the potentially defensive role of A2M-like proteins in bacteria.…”

Neutrophil serine proteases in antibacterial defense

“…Like αMG, proteolytic cleavage of a bait region in bMGs results in a conformational change that allows the inhibitor to covalently capture the protease. The E. coli protein ECAM has a similar secondary structure to human plasma α 2 -MG and likewise contains an internal thioester to form covalent complexes with NE, trypsin and chymotrypsin [45]. …”

Structural and functional insights into Escherichia coli α 2 -macroglobulin endopeptidase snap-trap inhibition

“…For successful encaging, at least two protomers are required to wrap around a standard-size endopeptidase (12), but the detailed molecular mechanism of tetrameric α 2 M inhibition is unknown, as only the molecular structure of induced hα 2 M is available (8). Little is also known about the physiology and function of bα 2 Ms, as only a YfaS-ortholog from Pseudomonas aeruginosa and ECAM have been partially studied to date (13)(14)(15)(16). The crystal structure of native α 2 M from Salmonella enterica (SEAM) is available (16), but its working mechanism is also unknown so far.…”