POTRA: a conserved domain in the FtsQ family and a class of β-barrel outer membrane proteins

Bacterial cytokinesis requires the coordinated assembly of a complex of proteins, collectively known as the divisome, at the incipient division site. DivIB͞FtsQ is a conserved component of the divisome in bacteria with cell walls, suggesting that it plays a role in synthesis and͞or remodeling of septal peptidoglycan. We demonstrate that the extracytoplasmic region of DivIB comprises three discrete domains that we designate ␣, ␤, and ␥ from the N to C terminus. The ␣-domain is proximal to the cytoplasmic membrane and coincident with the polypeptide transport-associated domain that was proposed previously to function as a molecular chaperone. The ␤-domain has a unique 3D fold, with no eukaryotic counterpart, and we show that it interconverts between two discrete conformations via cis-trans isomerization of a Tyr-Pro peptide bond. We propose that this isomerization might modulate protein-protein interactions of the flanking ␣-and ␥-domains. The C-terminal ␥-domain is unstructured in the absence of other divisomal proteins, but we show that it is critical for DivIB function.bacterial cytokinesis ͉ divisome ͉ NMR ͉ protein structure B acterial cytokinesis requires spatiotemporal coordination of the assembly of a complex of cell division proteins, collectively known as the divisome, at the incipient division site (1, 2). Divisome assembly is initiated by the formation of a circumferential ring of FtsZ (the Z ring) around the inner surface of the cytoplasmic membrane (3, 4). FtsZ, the precursor of eukaryotic tubulin, is a GTPase that most likely provides at least part of the contractile force for septal invagination through dynamic remodeling of the Z ring (5-7). In addition, the Z ring promotes the switch from lateral to preseptal murein synthesis (8), and it provides a scaffold for recruitment of downstream divisomal proteins.In Escherichia coli the known divisomal proteins are thought to be recruited to the division site in the following hierarchical order: FtsZ4ZipA,FtsA4FtsEX4FtsK4FtsQ4(FtsL, FtsB)4FtsW4FtsI 4FtsN4AmiC, where the arrow indicates that recruitment of a particular protein requires prior assembly of all previously listed components, and parentheses enclose proteins whose recruitment to the divisome is interdependent (2, 9, 10). However, recent studies suggest that some subcomplexes, such as that formed among FtsQ, FtsL, and FtsB, might be preassembled before Z ring formation and trafficked to the developing division site as a preformed complex (2, 11). This latter situation is likely more akin to that in Bacillus subtilis, where the observed interdependent localization of most divisomal proteins argues for a more concerted recruitment to the division site (1,12).Whereas the earliest recruits to the division site are either wholly or mostly cytoplasmic (e.g., FtsZ, FtsA, and FtsK), the later recruits are typically bitopic membrane proteins with a short N-terminal cytoplasmic region, a single transmembrane segment, and a larger extracytoplasmic (ec) domain (i.e., FtsQ, FtsL, FtsB, FtsI, and FtsN). Rema...

Section: Discussionsupporting

confidence: 65%

Section: Resultsmentioning

confidence: 99%

Domain architecture and structure of the bacterial cell division protein DivIB

Robson

King²

2006

“…Insertion and assembly of these proteins is catalyzed by the highly conserved Omp85 protein family (outer membrane protein of 85 kDa) (1, 2) in a seemingly conserved process. Omp85's consist of a C-terminal 16-stranded pore-forming β-barrel and an N-terminal hydrophilic domain, with up to six polypeptide-transport-associated (POTRA) repeats (3)(4)(5)(6). The bacterial Omp85-also known as YaeT, D15, Oma87, or BamA (β-barrel assembly machinery protein A)-is part of a lipoprotein complex that facilitates the folding and insertion of the OMPs from the periplasm into the outer membrane (7,8).…”

mentioning

confidence: 99%

Chloroplast Omp85 proteins change orientation during evolution

Sommer

Daum

Groß

et al. 2011

The majority of outer membrane proteins (OMPs) from Gramnegative bacteria and many of mitochondria and chloroplasts are β-barrels. Insertion and assembly of these proteins are catalyzed by the Omp85 protein family in a seemingly conserved process. All members of this family exhibit a characteristic N-terminal polypeptide-transport-associated (POTRA) and a C-terminal 16-stranded β-barrel domain. In plants, two phylogenetically distinct and essential Omp85's exist in the chloroplast outer membrane, namely Toc75-III and Toc75-V. Whereas Toc75-V, similar to the mitochondrial Sam50, is thought to possess the original bacterial function, its homolog, Toc75-III, evolved to the pore-forming unit of the TOC translocon for preprotein import. In all current models of OMP biogenesis and preprotein translocation, a topology of Omp85 with the POTRA domain in the periplasm or intermembrane space is assumed. Using selfassembly GFP-based in vivo experiments and in situ topology studies by electron cryotomography, we show that the POTRA domains of both Toc75-III and Toc75-V are exposed to the cytoplasm. This unexpected finding explains many experimental observations and requires a reevaluation of current models of OMP biogenesis and TOC complex function.

“…O mp85-like proteins represent a large family defined by phylogenetic relatedness, secondary-structure predictions, and a role in protein translocation (1)(2)(3)(4)(5). Members of this family are present in diverse organisms across the evolutionary spectrum, including bacteria, fungi, plants, and animals (1,4).…”

mentioning

confidence: 99%

Evidence for conservation of architecture and physical properties of Omp85-like proteins throughout evolution

Surana

Grass

Hardy

et al. 2004

Omp85-like proteins represent a family of proteins involved in protein translocation, and they are present in all domains of life, except archaea. In eukaryotes, Omp85-like proteins have been demonstrated to form tetrameric pore-forming complexes that interact directly with their substrate proteins. Studies performed with bacterial Omp85-like proteins have demonstrated pore-forming activity but no evidence of multimerization. In this article, we characterize the Haemophilus influenzae HMW1B protein, an Omp85-like protein that has been demonstrated to be critical for secretion of the H. influenzae HMW1 adhesin. Analysis of purified protein by biochemical and electron microscopic techniques revealed that HMW1B forms a tetramer. Examination using liposomeswelling assays demonstrated that HMW1B has pore-forming activity, with a pore size of Ϸ2.7 nm. Far-Western blot analysis established that HMW1B interacts with the N terminus of HMW1. These results provide evidence that a bacterial Omp85-like protein forms a tetramer and interacts directly with a substrate protein, suggesting that the architecture and physical properties of Omp85-like proteins have been conserved throughout evolution. O mp85-like proteins represent a large family defined by phylogenetic relatedness, secondary-structure predictions, and a role in protein translocation (1-5). Members of this family are present in diverse organisms across the evolutionary spectrum, including bacteria, fungi, plants, and animals (1, 4). The widespread nature of Omp85-like proteins underscores their critical role in cellular processes, highlighted by the fact that many are required for cell viability (3,4,(6)(7)(8)(9). Although all Omp85-like proteins share sequence homology, they can be grouped into two classes based on the specific role that they play in protein translocation. One class is typified by Saccharomyces cerevisiae Sam50 and Neurospora crassa Tob55, which are chiefly involved in insertion of ␤-barrel proteins into the outer membrane (OM) of mitochondria (6, 9). The second class is epitomized by the chloroplast protein Toc75, which is involved in secretion of proteins across a membrane (10, 11). Interestingly, both classes of Omp85-like proteins are present in Gramnegative bacteria. Neisseria meningitidis Omp85, the namesake of this family of proteins, has been demonstrated recently to be critical for the insertion of proteins into the bacterial OM (7). Other Gram-negative bacterial Omp85-like proteins, including Serratia marcescens ShlB and Bordetella pertussis FhaC [and other members of the so-called two-partner secretion (TPS) pathway], have been demonstrated to function in protein export across the OM (12).The high level of amino acid homology among Omp85-like proteins from bacteria to humans raises the possibility that members of this family have similar structures. Although no crystal structures exist so far for Omp85-like proteins, several of these proteins have been subjected to biochemical studies of overall architecture. In eukaryotes, both classes...