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Chemical Probing Discovers New Insights into the Native Assembly State of a Bacterial Microcompartment

ABSTRACT: Microcompartments (BMCs) are widespread in bacteria and are used for a variety of metabolic purposes, including catabolism of host metabolites. A suite of proteins self-assembles into the shell and cargo layers of BMCs. However, the native assembly state of these large complexes remains to be elucidated. Herein, chemical probes were used to discover structural features of a native BMC. While the exterior could be demarcated with fluorophores, the interior was unexpectedly permeable, suggesting the shell layer may be more dynamic than thought. This allowed access to cross-linking chemical probes, which were analyzed for discovery of the protein interactome. These cross-links revealed a complex multivalent network among cargo proteins that contained encapsulation peptides and demonstrated that the shell layer follows discreet rules in its assembly. These results are consistent overall with a model where biomolecular condensation drives interactions of cargo proteins prior to envelopment by shell layer proteins.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Salmonella Typhimurium (strain Lt2 / Sgsc1412 / Atcc 700720)

SUBMITTER: Conor Jenkins

LAB HEAD: Wade C. Winkler

PROVIDER: PXD029675 | Pride | 2022-08-19

REPOSITORIES: Pride

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Chemical probing provides insight into the native assembly state of a bacterial microcompartment.

Trettel Daniel S DS Resager William W Ueberheide Beatrix M BM Jenkins Conor C CC Winkler Wade C WC

Structure (London, England : 1993) 20220224 4

Bacterial microcompartments (BMCs) are widespread in bacteria and are used for a variety of metabolic purposes, including catabolism of host metabolites. A suite of proteins self-assembles into the shell and cargo layers of BMCs. However, the native assembly state of these large complexes remains to be elucidated. Herein, chemical probes were used to observe structural features of a native BMC. While the exterior could be demarcated with fluorophores, the interior was unexpectedly permeable, sug ...[more]

PMID: 35216657

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Project description:Background: The shells of various Haliotis species have served as models of invertebrate biomineralization and physical shell properties for more than 20 years. A focus of this research has been the nacreous inner layer of the shell with its conspicuous arrangement of aragonite platelets, resembling in cross-section a brick-and-mortar wall. In comparison, the outer, less stable, calcitic prismatic layer has received much less attention. One of the first molluscan shell proteins to be characterized at the molecular level was Lustrin A, a component of the nacreous organic matrix of Haliotis rufescens. This was soon followed by the C-type lectin perlucin and the growth factor-binding perlustrin, both isolated from H. laevigata nacre, and the crystal growth-modulating AP7 and AP24, isolated from H. rufescens nacre. Mass spectrometry-based proteomics was subsequently applied to to Haliotis biomineralization research with the analysis of the H. asinina shell matrix and yielded 14 different shell-associated proteins. That study was the most comprehensive for a Haliotis species to date. Methods: The shell proteomes of nacre and prismatic layer of the marine gastropod Haliotis laevigata were analyzed combining mass spectrometry-based proteomics and next generation sequencing. Results: We identified 297 proteins from the nacreous shell layer and 350 proteins from the prismatic shell layer from the green lip abalone H. laevigata. Considering the overlap between the two sets we identified a total of 448 proteins. Fifty-one nacre proteins and 43 prismatic layer proteins were defined as major proteins based on their abundance at more than 0.2% of the total. The remaining proteins occurred at low abundance and may not play any significant role in shell fabrication. The overlap of major proteins between the two shell layers was 17, amounting to a total of 77 major proteins. Conclusions: The H. laevigata shell proteome shares moderate sequence similarity at the protein level with other gastropod, bivalve and more distantly related invertebrate biomineralising proteomes. Features conserved in H. laevigata and other molluscan shell proteomes include short repetitive sequences of low complexity predicted to lack intrinsic three-dimensional structure, and domains such as tyrosinase, chitin-binding, and carbonic anhydrase. This catalogue of H. laevigata shell proteins represents the most comprehensive for a haliotid and should support future efforts to elucidate the molecular mechanisms of shell assembly.

			Action	DRS
	4_DSSO_XL_ETD.msf	Msf
	4_DSSO_XL_ETD.pdResult	Other
	4_DSSO_XL_ETD.raw	Raw
	4_DSSO_XL_MS2.msf	Msf
	4_DSSO_XL_MS2.pdResult	Other