The patatin-like protein PlpD forms structurally dynamic homodimers in the Pseudomonas aeruginosa outer membrane.

Members of the Omp85 superfamily of outer membrane proteins (OMPs) found in Gram-negative bacteria, mitochondria and chloroplasts are characterized by a distinctive 16-stranded ß-barrel transmembrane domain and at least one periplasmic POTRA domain. All previously studied Omp85 proteins promote critical OMP assembly and/or protein translocation reactions. Pseudomonas aeruginosa PlpD is the prototype of an Omp85 protein family that contains an N-terminal patatin-like (PL) domain that is thought to be translocated across the OM by a C-terminal ß-barrel domain. Challenging the current dogma, we find that the PlpD PL-domain resides exclusively in the periplasm and, unlike previously studied Omp85 proteins, PlpD forms a homodimer. Remarkably, the PL-domain contains a segment that exhibits unprecedented dynamicity by undergoing transient strand-swapping with the neighboring ß-barrel domain. Our results show that the Omp85 superfamily is more structurally diverse than currently believed and suggest that the Omp85 scaffold was utilized during evolution to generate novel functions.

ß-barrel membrane proteins fold via hybrid-barrel intermediate states.

Gram-negative bacteria and eukaryotic organelles of bacterial origin contain outer membrane proteins that possess a transmembrane "ß-barrel" domain. The conserved ß-barrel assembly machine (BAM) and the sorting and assembly machine (SAM) are required for the folding and membrane insertion of ß-barrels in Gram-negative bacteria and mitochondria, respectively. Although the mechanisms by which ß-barrels are folded are incompletely understood, advances in cryo-electron microscopy (cryo-EM) have recently yielded unprecedented insights into their folding process. Here we highlight recent studies that show that both bacterial and mitochondrial ß-barrels fold via the formation of remarkable "hybrid-barrel" intermediate states during their interaction with the folding machinery. We discuss how these results align with a general model of ß-barrel folding.

Molecular Machines that Facilitate Bacterial Outer Membrane Protein Biogenesis.

Almost all outer membrane proteins (OMPs) in Gram-negative bacteria contain a ß-barrel domain that spans the outer membrane (OM). To reach the OM, OMPs must be translocated across the inner membrane by the Sec machinery, transported across the crowded periplasmic space through the assistance of molecular chaperones, and finally assembled (folded and inserted into the OM) by the ß-barrel assembly machine. In this review, we discuss how considerable new insights into the contributions of these factors to OMP biogenesis have emerged in recent years through the development of novel experimental, computational, and predictive methods. In addition, we describe recent evidence that molecular machines that were thought to function independently might interact to form dynamic intermembrane supercomplexes. Finally, we discuss new results that suggest that OMPs are inserted primarily near the middle of the cell and packed into supramolecular structures (OMP islands) that are distributed throughout the OM.

In Vivo Disulfide-Bond Crosslinking to Study ß-Barrel Membrane Protein Interactions, Dynamicity, and Folding Intermediates.

Membrane-embedded ß-barrels are the major building blocks of the Gram-negative outer membrane and are involved in antibiotic resistance, virulence, and the maintenance of bacterial cell physiology. The increased frequency of multidrug resistant Gram-negative infections warrants the sharing of accessible methods for the study of ß-barrels. One such method is "in vivo disulfide-bond crosslinking" which is a highly informative and cost-effective approach to study the structure, topology, dynamicity, and function of ß-barrels in situ. The approach can also be used to identify and finely map both stable or transient interactions between ß-barrels and other interacting proteins. In this chapter, I describe the conceptual basis of in vivo disulfide-bond crosslinking and the potential pitfalls in experimental design. I also provide a general protocol for high-efficiency in vivo disulfide-bond crosslinking and modified protocols as examples for how the method can be adapted to different scenarios.