Abstract
In many cases protein assemblies are stabilized by covalent bonds, one example of which is the formation of intra- or intermolecular epsilon-(-glutamyl)lysil cross-links catalyzed by transglutaminases (TGases). Because of the potential for unwanted cross-linking reactions, the activities of many TGases have been shown to be tightly controlled. Bacterial endospores are highly resilient cells in part because they are surrounded by a complex protein coat. Proteins in the coat that surrounds Bacillus subtilis endospores are crosslinked by a TGase (Tgl). Unlike other TGases, however, Tgl is produced in an active form, and efficiently catalyzes amine incorporation and protein cross-linking in vitro with no known additional requirements. The absence of regulatory factors raises questions as to how the activity of Tgl is controlled during spore coat assembly. Here, we show that substrates assembled onto the spore coat prior to Tgl production govern the localization of Tgl to the surface of the developing spore. We also show that Tgl residues important for substrate recognition are crucial for its localization. We identified the glutamyl (Q) and lysil (K) substrate docking sites and we show that residues on the Q side of Tgl are more important for the assembly of Tgl than those on the K side. Thus, the first step in the reaction cycle, the interaction with Q-substrates and formation of an acyl-enzyme intermediate, is also the determinant step in the localization of Tgl. Consistent with the idea that Tg exerts a spotwelding activity, cross-linking pre-formed assemblies, we show that C30 is an oblong hexamer in solution that is cross-linked in vitro into high molecular weight forms. Moreover, during the reaction, Tgl becomes part of the cross-linked products. We suggest that the dependency of Tgl on its substrates is used to accurately control the time, location and extent of the enzymes activity, directed at the covalent fortification of pre-assembled complexes at the surface of the developing spore.
Author summary The orderly recruitment of proteins during the assembly of complex macromolecular structures poses challenges throughout cell biology. During endospore development in the bacterium Bacillus subtilis at least 80 proteins synthesized in the mother cell are assembled around the developing spore to form a protective coat. Regulation of coat gene expression has been described in detail but it is unknown how the information encoded by the structures of the proteins guide their assembly. We have examined the assembly of a transglutaminase, Tgl, which introduces epsilon-(-glutamyl)lysil cross-links in coat protein substrates. We describe with molecular detail a substrate-driven assembly model that directs the enzyme to the locations of its substrates where, as we suggest, it exerts a spotwelding activity to fortify pre-assembled complexes. The catalytic cysteine, located in a tunnel that spans the Tgl structure, first forms an acyl enzyme intermediate with a glutamine (Q) donor substrate. Then, it engages a lysine (K) donor substrate to form the cross-linked product. We have identified the Q and K acceptor ends of the Tgl tunnel, and we show that substitutions in substrate recognition residues at the Q side impair assembly more strongly than at the K side. Thus, assembly of Tgl parallels its catalytic cycle, directing the enzyme to the pre-formed complexes that are to be cross-linked.
