Abstract
Alpha-2-macroglobulins (A2Ms) are large spectrum protease inhibitors that are major components of the eukaryotic immune system. Pathogenic and colonizing bacteria, such as the opportunistic pathogen Pseudomonas aeruginosa, also carry structural homologs of eukaryotic A2Ms. Two types of bacterial A2Ms have been identified: Type I, much like the eukaryotic form, displays a conserved thioester that is essential for protease targeting, and Type II, which lacks the thioester and to date has been poorly studied despite its ubiquitous presence in Gram-negatives. Here we show that MagD, the Type II A2M from P. aeruginosa that is expressed within the six-gene mag operon, specifically traps a target protease despite the absence of the thioester motif, comforting its role in protease inhibition. In addition, analytical ultracentrifugation and small angle scattering show that MagD forms higher order complexes with proteins expressed in the same operon (MagA, MagB, and MagF), with MagB playing the key stabilization role. A P. aeruginosa strain lacking magB cannot stably maintain MagD in the bacterial periplasm, engendering complex disruption. This suggests a regulated mechanism of Mag complex formation and stabilization that is potentially common to numerous Gram-negative organisms, and that plays a role in periplasm protection from proteases during infection or colonization.
Highlights
Alpha-2-macroglobulins (A2Ms) are broad-spectrum protease inhibitors present in all metazoans ranging from insects to humans, and play key roles in host defense[1]
We show that MagD can be actively recognized and cleaved by a specific protease within its bait region, an event which traps the protease in a stable complex, attesting to the ability of Type II A2Ms to display macroglobulin-like activity even in the absence of the classical thioester motif
Thioester motif involved in the covalent association to target proteases (Fig. 1)[11,13,14], we reasoned that if its biological role is linked to protection of the cell from protease attack, it should be targeted by different proteases as shown for Type I A2Ms19
Summary
Alpha-2-macroglobulins (A2Ms) are broad-spectrum protease inhibitors present in all metazoans ranging from insects to humans, and play key roles in host defense[1]. Upon recognition and cleavage of the bait region by an attacking protease, the thioester bond becomes exposed and subsequently cross-linked to the enzyme, causing the A2M to trap the attacking protease in a cage-like structure[4,5] This mechanism ensures that proteases secreted by infecting microorganisms are cleared effectively, and is thought to be part of an innate immune system that predates the immunoglobulin-based system[3]. In spite of the fact that molecules of the A2M/complement superfamily were believed to be limited to metazoans, genomic analyses revealed that genes for A2M-like proteins exist in several bacterial species, many of which are pathogenic or are common colonizers of higher eukaryotes[7]. The atomic details that describe a complex between a bacterial A2M and a protease are still an open question
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