B-Barrel proteins from bacterial outer membranes: structure, function and refoldingIntroductionOuter membrane protein AOuter membrane phospholipase A Ferric enterobactin and ferrichrome receptorsIn vivo folding of outer membrane proteinsRefolding of outer membrane proteinsConclusionsAcknowledgementsReferences and recommended readingFigures and TableFigure 1Figure 2Table 1455Recently solved outer membrane protein structures include thesmallest and largest known β-barrel structures, with functionsdistinct from the general and specific porins. Both proteinexpressed in outer membranes and protein deposited ascytoplasmic aggregates have been used for the structuredeterminations. As most β-barrel proteins can beoverexpressed in an aggregated form (inclusion bodies) andrefolded to the native state, this provides an alternative tomembrane-targeted expression strategies and yields sufficientquantities of protein for future structural studies.AddressesDepartment of Crystallography, Birkbeck College, Malet Street,London WC1E 7HX, UK; e-mail: [email protected] Opinion in Structural Biology 1999, 9:455–461http://biomednet.com/elecref/0959440X00900455© Elsevier Science Ltd ISSN 0959-440XAbbreviationscmc critical micelle concentrationFepA ferric enterobactin receptorFhuA ferrichrome receptorLPS lipopolysaccharideOmpA outer membrane protein AOMPLA outer membrane phospholipase AIntroductionIntegral outer membrane proteins from Gram-negativebacteria use amphipathic β-strands to traverse the mem-brane. In all known structures of this type, the β-sheet hasa simple meander topology. It is twisted to form a closedbarrel, in which the last β-strand is hydrogen bonded tothe first. The versatility of transmembrane β-barrels isillustrated by the variety of structures that has beensolved. The general porins, represented by porin fromRhodobacter capsulatus [1] and OmpF [2], use 16-strandedβ-barrels that associate to form homotrimers. These chan-nels carry out the passive diffusion of small (~600 Da)molecules. In comparison, the specific porins, such as mal-toporin (LamB) [3] and sucrose porin (ScrY) [4], arecomposed of 18 β-strands and also form homotrimers.They recognise specific sugars and utilise facilitated diffu-sion for uptake. Both classes of porins have been recentlyreviewed [5]. Yet another type of β-barrel is seen inα-hemolysin, a staphylococcal toxin that assembles as aheptamer and inserts a 14-stranded β-barrel into the tar-geted cell membrane [6]. In the past year, several newtypes of β-barrel structures have been solved by X-raycrystallography. These proteins differ both in architectureand in function from the previous examples: outer mem-brane protein A (OmpA) is the smallest known β-barrelstructure and functions as a solid transmembrane anchor.Outer membrane phospholipase A (OMPLA) is anenzyme that hydrolyses endogenous phospholipids and isregulated by reversible dimerisation. Two active trans-porters of iron chelates, ferric enterobactin receptor(FepA) and ferrichrome receptor (FhuA), are the largestknown β-barrel structures. They both contain a globulardomain folded into the barrel lumen, which functions inligand binding and transport. Of the four new structures discussed in here, two weresolved using proteins isolated from Escherichia coli outermembranes (FepA and FhuA) and two were solved usingproteins expressed in aggregated forms that were refoldedto the native state (OmpA and OMPLA). To show thatrefolded protein is virtually identical to the native species,structures of the Rhodopseudomonas blastica porin weresolved with both the native (membrane-inserted) proteinand the refolded material [7]. The root mean square devi-ation of the Cα atoms for the best superposition waswithin the limits of error for the structure determinations.This review describes the structures and functions ofOmpA, OMPLA, FepA and FhuA. In addition, as morefunctional protein can be potentially obtained by refold-ing cytoplasmic aggregates than by solubilisingmembrane-inserted proteins (as a result of limitations onmembrane-targeted expression), refolding methods thataim to provide large quantities of correctly folded proteinfor structural studies will be discussed.Outer membrane protein AOmpA from E. coli is one of the most abundant proteins inthe outer membrane and has been used to extensivelystudy membrane protein folding [8,9]. It is composed of325 amino acids: residues 1–171 form the smallest mem-brane-spanning β-barrel so far observed and residues172–325 form a periplasmic domain whose structureremains unknown. The periplasmic domain has been pro-posed to bind the peptidoglycan layer, such that OmpAphysically links the peptidoglycan layer to the outer mem-brane [10]. The extracellular loops of the barrel facilitateF-mediated bacterial conjugation [11] and recognise spe-cific colicins [12] and bacteriophages [13].Pautsch and Schulz [14••] have solved the structure of themembrane-spanning domain of OmpA (residues 1–171) at2.5 Å resolution (Figure 1a), starting from aggregated pro-tein that was subsequently refolded [15•]. The β-barrelconsists of eight antiparallel strands with an averagelength of 13 residues. The strands are tilted approximate-ly 45° with respect to the barrel axis. The shape of thebarrel is almost circular, with an elliptical axis ratio of 5:4.The four extracellular loops consist of 7 to 17 residues andare highly mobile.b-Barrel proteins from bacterial outer membranes: structure,function and refoldingSusan K BuchananAlthough ionophore activity for OmpA has been reported[16,17], the barrel interior contains no channel and hasbeen described by Pautsch and Schulz as a “solid inversemicelle” [14••]. The barrel interior contains an extensivehydrogen-bonding network that creates several largeaqueous cavities, but no passage that is large enough forthe transport of water or small ions. Sequence alignmentwith five bacterial homologues shows that the residuesthat form the internal polar network are highly conserved;the authors speculate that these residues may either beinvolved in protein folding or have some unknown func-tion. The structure of OmpA provides the first example ofthe β-barrel scaffold being used primarily as a solid trans-membrane anchor for the separate functions of theextracellular loops and the periplasmic domain.Outer membrane phospholipase A OMPLA is one of the few enzymes found in the outermembrane. It hydrolyses