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In vivo protein complex topologies: sights through a cross-linking lens.

ABSTRACT: Proteins are a remarkable class of molecules that exhibit wide diversity of shapes or topological features that underpin protein interactions and give rise to biological function. In addition to quantitation of abundance levels of proteins in biological systems under a variety of conditions, the field of proteome research has as a primary mission the assignment of function for proteins and if possible, illumination of factors that enable function. For many years, chemical cross-linking methods have been used to provide structural data on single purified proteins and purified protein complexes. However, these methods also offer the alluring possibility to extend capabilities to complex biological samples such as cell lysates or intact living cells where proteins may exhibit native topological features that do not exist in purified form. Recent efforts are beginning to provide glimpses of protein complexes and topologies in cells that suggest continued development will yield novel capabilities to view functional topological features of many proteins and complexes as they exist in cells, tissues, or other complex samples. This review will describe rationale, challenges, and a few success stories along the path of development of cross-linking technologies for measurement of in vivo protein interaction topologies.

SUBMITTER: Bruce JE

PROVIDER: S-EPMC3654877 | biostudies-literature | 2012 May

REPOSITORIES: biostudies-literature

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In vivo protein complex topologies: sights through a cross-linking lens.

Bruce James E JE

Proteomics 20120501 10

Proteins are a remarkable class of molecules that exhibit wide diversity of shapes or topological features that underpin protein interactions and give rise to biological function. In addition to quantitation of abundance levels of proteins in biological systems under a variety of conditions, the field of proteome research has as a primary mission the assignment of function for proteins and if possible, illumination of factors that enable function. For many years, chemical cross-linking methods h ...[more]

PMID: 22610688

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Project description:The coat of Bacillus subtilis spores is a multiprotein protective structure that also arbitrates many of the environmental interactions of the spore. The coat assembles around the cortex peptidoglycan layer and is differentiated into an inner and an outer layer and a crust. SafA governs assembly of the inner coat, whereas CotE drives outer coat assembly. SafA localizes to the cortex-coat interface. Both SafA and its short form C30 are substrates for Tgl, a coat-associated transglutaminase that cross-links proteins through ε-(γ-glutamyl)lysyl isopeptide bonds. We show that SafA and C30 are distributed between the coat and cortex layers. The deletion of tgl increases the extractability of SafA, mainly from the cortex. Tgl itself is mostly located in the inner coat and cortex. The localization of Tgl-cyan fluorescent protein (Tgl-CFP) is strongly, but not exclusively, dependent on safA However, the association of Tgl with the cortex requires safA Together, our results suggest an assembly pathway in which Tgl is first recruited to the forming spore in a manner that is only partially dependent on SafA and then is drafted to the cortex by SafA. Tgl, in turn, promotes the conversion of coat- and cortex-associated SafA into forms that resist extraction, possibly by catalyzing the cross-linking of SafA to other coat proteins, to the cortex, and/or to cortex-associated proteins. Therefore, the final assembly state of SafA relies on an autoregulatory pathway that requires the subcellular localization of a protein cross-linking enzyme. Tgl most likely exerts a "spotwelding" activity, cross-linking preformed complexes in the cortex and inner coat layers of spores.IMPORTANCE In this work, we show how two proteins work together to determine their subcellular location within the coat of bacterial endospores. Bacillus subtilis endospores are surrounded by a multilayer protein coat composed of over 80 proteins, which surrounds an underlying peptidoglycan layer (the spore cortex) protecting it from lytic enzymes. How specific coat proteins are targeted to specific layers of the coat is not well understood. We found that the protein SafA recruits a protein-cross-linking enzyme (a transglutaminase) to the cortex and inner layers of the coat, where both are cemented, by cross-linking, into macromolecular complexes.

Dataset Information

In vivo protein complex topologies: sights through a cross-linking lens.

Publications

In vivo protein complex topologies: sights through a cross-linking lens.

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