Identification of a Unique Fe-S Cluster Binding Site in a Glycyl-Radical Type Microcompartment Shell Protein

Abstract

Recently, progress has been made toward understanding the functional diversity of bacterial microcompartment (MCP) systems, which serve as protein-based metabolic organelles in diverse microbes. New types of MCPs have been identified, including the glycyl-radical propanediol (Grp) MCP. Within these elaborate protein complexes, BMC-domain shell proteins assemble to form a polyhedral barrier that encapsulates the enzymatic contents of the MCP. Interestingly, the Grp MCP contains a number of shell proteins with unusual sequence features. GrpU is one such shell protein, whose amino acid sequence is particularly divergent from other members of the BMC-domain superfamily of proteins that effectively defines all MCPs. Expression, purification, and subsequent characterization of the protein showed, unexpectedly, that it binds an iron-sulfur cluster. We determined X-ray crystal structures of two GrpU orthologs, providing the first structural insight into the homohexameric BMC-domain shell proteins of the Grp system. The X-ray structures of GrpU, both obtained in the apo form, combined with spectroscopic analyses and computational modeling, show that the metal cluster resides in the central pore of the BMC shell protein at a position of broken 6-fold symmetry. The result is a structurally polymorphic iron-sulfur cluster binding site that appears to be unique among metalloproteins studied to date.

Bacterial microcompartments

“…Indications of the presence of Fe-S clusters [G] in BMC-H 8, 146 and BMC-T 120, 135, 147 proteins suggest that electron transfer across the shell occurs in some types of BMCs. In developing synthetic BMCs for bioengineering applications, the incorporation of electron relays through shell proteins would greatly expand the possible repertoire of potential core chemistries.…”

Bioinformatic Characterization of Glycyl Radical Enzyme-Associated Bacterial Microcompartments

“…The occurrence of shell proteins that are predicted to form assemblies with iron-sulfur clusters in the pores (PduT-like BMC-Ts or a GrpU-like BMC-H) offers one possibility (67,72). The majority of GRM1, GRM3, and GRM5 loci encode at least one shell protein homolog potentially coordinating an Fe-S cluster in its predicted oligomeric state (Table 2; see also Data Set S1 in the supplemental material).…”

Bacterial microcompartments: widespread prokaryotic organelles for isolation and optimization of metabolic pathways

“…This suggests a functional link between ethanolamine metabolism and the redox state of the bacterial cell, which might be related to the use of tetrathionate as an alternative terminal electron acceptor for growth on ethanolamine by certain gut bacteria (Thiennimitr et al ., ). A final hint about transport and the involvement of cofactors comes from a recent finding of a binding interaction between cobalamin (B 12 ) and the interior surface of EutL from Clostridium perfringens (Thompson et al ., ,), though the lack of sequence conservation for the ligating histidine leaves that observation in need of further investigation.…”

“…A site for Fe‐S cluster binding was first identified in PduT, a trimeric tandem‐domain BMC shell protein from the Pdu MCP (Crowley et al ., ; Parsons et al ., ; Pang et al ., ). Likewise, an Fe‐S cluster has been characterized in the pore of a hexameric (single‐domain) BMC shell protein, GrpU, from an MCP thought to use a glycyl‐radical enzyme for propanediol degradation (Thompson et al ., ,). In the latter case, six cysteines are present in the pore, but it appears that the natural sixfold symmetry breaks to allow for alternating cysteine conformations to bind a 4Fe‐4S cluster in a threefold symmetric fashion.…”