Temporal and spatial regulation of protein cross-linking by the pre-assembled substrates of a Bacillus subtilis spore coat transglutaminase

• Type of Material: Article
• Authors: Catarina G. Fernandes, Universidade Nova de Lisboa; Diogo Martins, Universidade Nova de Lisboa; Guillem Hernandez, Universidade Nova de Lisboa; Ana L. Sousa, Instituto Gulbenkian de Ciencias; Carolina Freitas, Universidade Nova de Lisboa; Erin M. Tranfield, Instituto Gulbenkian de Ciencias; Tiago N. Cordeiro, Universidade Nova de Lisboa; Monica Serrano, Universidade Nova de Lisboa; Charles Moran Jr., Emory University; Adriano O. Henriques, Universidade Nova de Lisboa
• Language: English
• Date: 2019-04-01
• Publisher: Public Library of Science
• Publication Version: Final Published Version
• Copyright Statement: © 2019 Fernandes et al
• License: Creative Commons Attribution 4.0 International
• Final Published Version (URL): http://dx.doi.org/10.1371/journal.pgen.1007912
• Title of Journal or Parent Work: PLoS Genetics
• ISSN: 1553-7390
• Volume: 15
• Issue: 4
• Start Page: e1007912
• End Page: e1007912
• Grant/Funding Information: This work was partially supported by PPBI - Portuguese Platform of BioImaging (PPBI-POCI-01-0145-FEDER-022122) co-funded by national funds from OE - "Orçamento de Estado"; and by european funds from FEDER - "Fundo Europeu de Desenvolvimento Regional"; By program IF of the FCT (IF/00268/2013/CP1173/CT0006) to MS.; C.F. was the recipient of a doctoral fellowship from the FCT (SFRH/BD/43200/2008).; This work was financially supported by Project LISBOA-01-0145-FEDER-007660 (“Microbiologia Molecular, Estrutural e Celular”) funded by FEDER funds through COMPETE2020 – “Programa Operacional Competitividade e Internacionalização” (POCI); by national funds through the FCT (“Fundação para a Ciência e a Tecnologia") grant POCI/BIA-BCM/60855/2004 to A.O.; By the National Institute of General and Medical Sciences Public Health Services Grant R01 GM110000-04 to CPM.
• Keywords: GERMINATION; BINDING; Life Sciences & Biomedicine; REVEALS; GENE; Science & Technology; LOCALIZATION; FAMILY; GROWTH; Genetics & Heredity; DIVISION; FACTOR-XIII; SPORULATION
• Research Categories: Biology, Genetics; Biology, Microbiology

Abstract

In many cases protein assemblies are stabilized by covalent bonds, one example of which is the formation of intra- or intermolecular ε-(γ-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 enzyme´s activity, directed at the covalent fortification of pre-assembled complexes at the surface of the developing spore.