SUMMARY Bacterial microcompartments (MCPs) are sophisticated protein-based organelles used to optimize metabolic pathways. They consist of metabolic enzymes encapsulated within a protein shell, which creates an ideal environment for catalysis and facilitates the channeling of toxic/volatile intermediates to downstream enzymes. The metabolic processes that require MCPs are diverse and widely distributed and play important roles in global carbon fixation and bacterial pathogenesis. The protein shells of MCPs are thought to selectively control the movement of enzyme cofactors, substrates, and products (including toxic or volatile intermediates) between the MCP interior and the cytoplasm of the cell using both passive electrostatic/steric and dynamic gated mechanisms. Evidence suggests that specialized shell proteins conduct electrons between the cytoplasm and the lumen of the MCP and/or help rebuild damaged iron-sulfur centers in the encapsulated enzymes. The MCP shell is elaborated through a family of small proteins whose structural core is known as a bacterial microcompartment (BMC) domain. BMC domain proteins oligomerize into flat, hexagonally shaped tiles, which assemble into extended protein sheets that form the facets of the shell. Shape complementarity along the edges allows different types of BMC domain proteins to form mixed sheets, while sequence variation provides functional diversification. Recent studies have also revealed targeting sequences that mediate protein encapsulation within MCPs, scaffolding proteins that organize lumen enzymes and the use of private cofactor pools (NAD/H and coenzyme A [HS-CoA]) to facilitate cofactor homeostasis. Although much remains to be learned, our growing understanding of MCPs is providing a basis for bioengineering of protein-based containers for the production of chemicals/pharmaceuticals and for use as molecular delivery vehicles.

Bacterial microcompartments are widespread prokaryotic organelles that have important and diverse roles ranging from carbon fixation to enteric pathogenesis. Current models for microcompartment function propose that their outer protein shell is selectively permeable to small molecules, but whether a protein shell can mediate selective permeability and how this occurs are unresolved questions. Here, biochemical and physiological studies of structureguided mutants are used to show that the hexameric PduA shell protein of the 1,2-propanediol utilization (Pdu) microcompartment forms a selectively permeable pore tailored for the influx of 1,2-propanediol (the substrate of the Pdu microcompartment) while restricting the efflux of propionaldehyde, a toxic intermediate of 1,2-propanediol catabolism. Crystal structures of various PduA mutants provide a foundation for interpreting the observed biochemical and phenotypic data in terms of molecular diffusion across the shell. Overall, these studies provide a basis for understanding a class of selectively permeable channels formed by nonmembrane proteins.

Summary Bacterial microcompartments (MCPs) are complex organelles that consist of metabolic enzymes encapsulated within a protein shell. In this study, we investigate the function of the PduJ MCP shell protein. PduJ is 80% identical in amino acid sequence to PduA and both are major shell proteins of the 1,2-propanediol (1,2-PD) utilization (Pdu) MCP of Salmonella. Prior studies showed that PduA mediates the transport of 1,2-PD (the substrate) into the Pdu MCP. Surprisingly, however, results presented here establish that PduJ has no role 1,2-PD transport. The crystal structure revealed that PduJ was nearly identical to that of PduA and, hence, offered no explanation for their differential functions. Interestingly, however, when a pduJ gene was placed at the pduA chromosomal locus, the PduJ protein acquired a new function, the ability to mediate 1,2-PD transport into the Pdu MCP. To our knowledge, these are the first studies to show that that gene location can determine the function of a MCP shell protein. We propose that gene location dictates protein-protein interactions essential to the function of the MCP shell.

Bacterial microcompartments are giant protein-based organelles that encapsulate special metabolic pathways in diverse bacteria. Structural and genetic studies indicate that metabolic substrates enter these microcompartments by passing through the central pores in hexameric assemblies of shell proteins. Limiting the escape of toxic metabolic intermediates created inside the microcompartments would confer a selective advantage for the host organism. Here, we report the first molecular dynamics (MD) simulation studies to analyze small-molecule transport across a microcompartment shell. PduA is a major shell protein in a bacterial microcompartment that metabolizes 1,2-propanediol via a toxic aldehyde intermediate, propionaldehyde. Using both metadynamics and replica-exchange umbrella sampling, we find that the pore of the PduA hexamer has a lower energy barrier for passage of the propanediol substrate compared to the toxic propionaldehyde generated within the microcompartment. The energetic effect is consistent with a lower capacity of a serine side chain, which protrudes into the pore at a point of constriction, to form hydrogen bonds with propionaldehyde relative to the more freely permeable propanediol. The results highlight the importance of molecular diffusion and transport in a new biological context.

Proteins' structures determine functions and the functions determine the structures. Yet, proteins and other biological macromolecules are dynamic systems under continuous conformational motion and the motions are keys to many of the biological processes in which they are involved. We argue

Efficient expression of human T-cell leukemia virus (HTLV) and human immunodeficiency virus structural proteins requires Rx and Rev proteins, respectively. Decreased expression of Gag and Env appears to be due, in part, to intragenic RNA sequences, termed cis-acting repressive sequences (CRS), and may be mediated by binding of specific cellular factors. We demonstrated previously that two cellular proteins, p60CRS and p40CRS, interact with HTLV type 2.5' long terminal repeat CRS RNA and that the interaction of both proteins with CRS RNA correlates with function (A. C. Black, C. T. Ruland, J. Luo, A. Bakker, J. K. Fraser, and J. D. Rosenblatt, Virology 200:29-41, 1994). By radioimmunoprecipitation of HeLa nuclear proteins UV cross-linked to CRS RNAs with murine monoclonal antibodies, we now show that p40CRS is heterogeneous nuclear ribonucleoprotein (hnRNP) A1 and p60CRS is polypyrimidine tract-binding protein or hnRNP I. These immunoprecipitation results were confirmed by an immunobinding assay with hnRNP I and hnRNP AI antibodies and by cross-competition electrophoretic mobility shift experiments. In addition, we mapped a putative hnRNP A1 binding site in U5 RNA and demonstrated that p40CRS (hnRNP A1) binding to that site correlates with CRS function. Since both hnRNP I and hnRNP A1 have been shown to influence splicing and potentially other steps in RNA processing, the binding of both hnRNP I and hnRNP A1 to HTLV RNA regulatory elements may alter retrovirus RNA processing and may be involved in regulation by Rex.

The transport of biomolecules and small particles such as viruses through constrained geometries is critical to understand for efficient molecular separations, detection and selective binding for biomedical and biodefense applications. To develop improved capabilities for isolating and controlling both particles and molecules, there is a need for models developed by computational techniques and matched by well-integrated experiments. Here, we use molecular dynamics simulations to study the flow of colloidal silica particles (diam-eter~50-100 nm) through cylindrical nanopores with diameter 200nm, and lengths 50-100 nm. Particle translocation is investigated in the presence of an applied electric field. We demonstrate the molecular origin of the pore resistance change that occurs once the colloid is inside the pore. The rate at which particles transit the nanopore is highly dependent on the dispersion forces and the electric field, which is in turn a function of both the applied voltage and the pore geometry. Models are supported by experimental findings obtained with resistive pulse sensing.

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