A single shell protein plays a major role in choline transport across the shell of the choline utilization microcompartment of Escherichia coli 536.

Bacterial microcompartments (MCPs) are widespread protein-based organelles that play important roles in the global carbon cycle and in the physiology of diverse bacteria, including a number of pathogens. MCPs consist of metabolic enzymes encapsulated within a protein shell. The main roles of MCPs are to concentrate enzymes together with their substrates (to increase reaction rates) and to sequester harmful metabolic intermediates. Prior studies indicate that MCPs have a selectively permeable protein shell, but the mechanisms that allow selective transport across the shell are not fully understood. Here we examine transport across the shell of the choline utilization (Cut) MCP of Escherichia coli 536, which has not been studied before. The shell of the Cut MCP is unusual in consisting of one pentameric and four hexameric bacterial microcompartment (BMC) domain proteins. It lacks trimeric shell proteins, which are thought to be required for the transport of larger substrates and enzymatic cofactors. In addition, its four hexameric BMC domain proteins are very similar in amino acid sequence. This raises questions about how the Cut MCP mediates the selective transport of the substrate, products and cofactors of choline metabolism. In this report, site-directed mutagenesis is used to modify the central pores (the main transport channels) of all four Cut BMC hexamers to assess their transport roles. Our findings indicate that a single shell protein, CmcB, plays the major role in choline transport across the shell of the Cut MCP and that the electrostatic properties of the CmcB pore also impact choline transport. The implications of these findings with regard to the higher-order structure of MCPs are discussed.

Structural characterization of hexameric shell proteins from two types of choline-utilization bacterial microcompartments.

Bacterial microcompartments are large supramolecular structures comprising an outer proteinaceous shell that encapsulates various enzymes in order to optimize metabolic processes. The outer shells of bacterial microcompartments are made of several thousand protein subunits, generally forming hexameric building blocks based on the canonical bacterial microcompartment (BMC) domain. Among the diverse metabolic types of bacterial microcompartments, the structures of those that use glycyl radical enzymes to metabolize choline have not been adequately characterized. Here, six structures of hexameric shell proteins from type I and type II choline-utilization microcompartments are reported. Sequence and structure analysis reveals electrostatic surface properties that are shared between the four types of shell proteins described here.

Recent structural insights into bacterial microcompartment shells.

Bacterial microcompartments are organelle-like structures that enhance a variety of metabolic functions in diverse bacteria. Composed entirely of proteins, thousands of homologous hexameric shell proteins tesselate to form facets while pentameric proteins form the vertices of a polyhedral shell that encapsulates various enzymes, substrates and cofactors. Recent structural data have highlighted nuanced variations in the sequence and topology of microcompartment shell proteins, emphasizing how variation and specialization enable the construction of complex molecular machines. Recent studies engineering synthetic miniaturized microcompartment shells provide additional frameworks for dissecting principles of microcompartment structure and assembly. This review updates our current understanding of bacterial microcompartment shell proteins, providing new insights and highlighting outstanding questions.

MCPdb: The bacterial microcompartment database.

Bacterial microcompartments are organelle-like structures composed entirely of proteins. They have evolved to carry out several distinct and specialized metabolic functions in a wide variety of bacteria. Their outer shell is constructed from thousands of tessellating protein subunits, encapsulating enzymes that carry out the internal metabolic reactions. The shell proteins are varied, with single, tandem and permuted versions of the PF00936 protein family domain comprising the primary structural component of their polyhedral architecture, which is reminiscent of a viral capsid. While considerable amounts of structural and biophysical data have been generated in the last 15 years, the existing functionalities of current resources have limited our ability to rapidly understand the functional and structural properties of microcompartments (MCPs) and their diversity. In order to make the remarkable structural features of bacterial microcompartments accessible to a broad community of scientists and non-specialists, we developed MCPdb: The Bacterial Microcompartment Database (https://mcpdb.mbi.ucla.edu/). MCPdb is a comprehensive resource that categorizes and organizes known microcompartment protein structures and their larger assemblies. To emphasize the critical roles symmetric assembly and architecture play in microcompartment function, each structure in the MCPdb is validated and annotated with respect to: (1) its predicted natural assembly state (2) tertiary structure and topology and (3) the metabolic compartment type from which it derives. The current database includes 163 structures and is available to the public with the anticipation that it will serve as a growing resource for scientists interested in understanding protein-based metabolic organelles in bacteria.

Symmetry breaking and structural polymorphism in a bacterial microcompartment shell protein for choline utilization.

Bacterial microcompartments are protein-based organelles that carry out specialized metabolic functions in diverse bacteria. Their outer shells are built from several thousand protein subunits. Some of the architectural principles of bacterial microcompartments have been articulated, with lateral packing of flat hexameric BMC proteins providing the basic foundation for assembly. Nonetheless, a complete understanding has been elusive, partly owing to polymorphic mechanisms of assembly exhibited by most microcompartment types. An earlier study of one homologous BMC shell protein subfamily, EutS/PduU, revealed a profoundly bent, rather than flat, hexameric structure. The possibility of a specialized architectural role was hypothesized, but artifactual effects of crystallization could not be ruled out. Here we report a series of crystal structures of an orthologous protein, CutR, from a glycyl-radical type choline-utilizing microcompartment from the bacterium Streptococcus intermedius. Depending on crystal form, expression construct, and minor mutations, a range of novel quaternary architectures was observed, including two spiral hexagonal assemblies. A new graphical approach helps illuminate the variations in BMC hexameric structure, with results substantiating the idea that the EutS/PduU/CutR subfamily of BMC proteins may endow microcompartment shells with flexible modes of assembly.

High-symmetry protein assemblies: patterns and emerging applications.

The accelerated elucidation of three-dimensional structures of protein complexes, both natural and designed, is providing new examples of large supramolecular assemblies with intriguing shapes. Those with high symmetry - based on the geometries of the Platonic solids - are particularly notable as their innately closed forms create interior spaces with varying degrees of enclosure. We survey known protein assemblies of this type and discuss their geometric features. The results bear on issues of protein function and evolution, while also guiding novel bioengineering applications. Recent successes using high-symmetry protein assemblies for applications in interior encapsulation and exterior display are highlighted.