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B-Barrel Proteins from Bacterial Outer Membranes: Structure, Function and Refolding Susan K Buchanan

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B-Barrel Proteins from Bacterial Outer Membranes: Structure, Function and Refolding Susan K Buchanan 455 b-Barrel proteins from bacterial outer membranes: structure, function and refolding Susan K Buchanan Recently solved outer membrane protein structures include the enzyme that hydrolyses endogenous phospholipids and is smallest and largest known β-barrel structures, with functions regulated by reversible dimerisation. Two active trans- distinct from the general and specific porins. Both protein porters of iron chelates, ferric enterobactin receptor expressed in outer membranes and protein deposited as (FepA) and ferrichrome receptor (FhuA), are the largest cytoplasmic aggregates have been used for the structure known β-barrel structures. They both contain a globular determinations. As most β-barrel proteins can be domain folded into the barrel lumen, which functions in overexpressed in an aggregated form (inclusion bodies) and ligand binding and transport. refolded to the native state, this provides an alternative to membrane-targeted expression strategies and yields sufficient Of the four new structures discussed in here, two were quantities of protein for future structural studies. solved using proteins isolated from Escherichia coli outer membranes (FepA and FhuA) and two were solved using Addresses proteins expressed in aggregated forms that were refolded Department of Crystallography, Birkbeck College, Malet Street, to the native state (OmpA and OMPLA). To show that London WC1E 7HX, UK; e-mail: [email protected] refolded protein is virtually identical to the native species, Current Opinion in Structural Biology 1999, 9:455–461 structures of the Rhodopseudomonas blastica porin were solved with both the native (membrane-inserted) protein http://biomednet.com/elecref/0959440X00900455 and the refolded material [7]. The root mean square devi- © Elsevier Science Ltd ISSN 0959-440X ation of the Cα atoms for the best superposition was within the limits of error for the structure determinations. Abbreviations cmc critical micelle concentration FepA ferric enterobactin receptor This review describes the structures and functions of FhuA ferrichrome receptor OmpA, OMPLA, FepA and FhuA. In addition, as more LPS lipopolysaccharide functional protein can be potentially obtained by refold- OmpA outer membrane protein A ing cytoplasmic aggregates than by solubilising OMPLA outer membrane phospholipase A membrane-inserted proteins (as a result of limitations on membrane-targeted expression), refolding methods that Introduction aim to provide large quantities of correctly folded protein Integral outer membrane proteins from Gram-negative for structural studies will be discussed. bacteria use amphipathic β-strands to traverse the mem- brane. In all known structures of this type, the β-sheet has Outer membrane protein A a simple meander topology. It is twisted to form a closed OmpA from E. coli is one of the most abundant proteins in barrel, in which the last β-strand is hydrogen bonded to the outer membrane and has been used to extensively the first. The versatility of transmembrane β-barrels is study membrane protein folding [8,9]. It is composed of illustrated by the variety of structures that has been 325 amino acids: residues 1–171 form the smallest mem- solved. The general porins, represented by porin from brane-spanning β-barrel so far observed and residues Rhodobacter capsulatus [1] and OmpF [2], use 16-stranded 172–325 form a periplasmic domain whose structure β-barrels that associate to form homotrimers. These chan- remains unknown. The periplasmic domain has been pro- nels carry out the passive diffusion of small (~600 Da) posed to bind the peptidoglycan layer, such that OmpA molecules. In comparison, the specific porins, such as mal- physically links the peptidoglycan layer to the outer mem- toporin (LamB) [3] and sucrose porin (ScrY) [4], are brane [10]. The extracellular loops of the barrel facilitate composed of 18 β-strands and also form homotrimers. F-mediated bacterial conjugation [11] and recognise spe- They recognise specific sugars and utilise facilitated diffu- cific colicins [12] and bacteriophages [13]. sion for uptake. Both classes of porins have been recently reviewed [5]. Yet another type of β-barrel is seen in Pautsch and Schulz [14••] have solved the structure of the α-hemolysin, a staphylococcal toxin that assembles as a membrane-spanning domain of OmpA (residues 1–171) at heptamer and inserts a 14-stranded β-barrel into the tar- 2.5 Å resolution (Figure 1a), starting from aggregated pro- geted cell membrane [6]. In the past year, several new tein that was subsequently refolded [15•]. The β-barrel types of β-barrel structures have been solved by X-ray consists of eight antiparallel strands with an average crystallography. These proteins differ both in architecture length of 13 residues. The strands are tilted approximate- and in function from the previous examples: outer mem- ly 45° with respect to the barrel axis. The shape of the brane protein A (OmpA) is the smallest known β-barrel barrel is almost circular, with an elliptical axis ratio of 5:4. structure and functions as a solid transmembrane anchor. The four extracellular loops consist of 7 to 17 residues and Outer membrane phospholipase A (OMPLA) is an are highly mobile. 456 Membrane proteins Figure 1 Ribbon diagrams of OmpA [14••], OMPLA [24] and FepA [30••], gradient-coloured from blue at the N termini, through green, to yellow at the C termini. (a) The eight-stranded transmembrane β-barrel of OmpA. The periplasmic domain, not present in the construct used for the crystal structure determination, would extend from the end of the yellow strand. (b) Monomeric OMPLA, a 12-stranded β-barrel. Residues S144, H142 and N156 comprising the catalytic triad are shown in red. (c) FepA, a 22-stranded β-barrel with a globular domain (blue) inserted into the barrel lumen. The TonB box is shown in red. This figure was drawn using SETOR [59]. Although ionophore activity for OmpA has been reported Outer membrane phospholipase A [16,17], the barrel interior contains no channel and has OMPLA is one of the few enzymes found in the outer been described by Pautsch and Schulz as a “solid inverse membrane. It hydrolyses endogenous phospholipids and is micelle” [14••]. The barrel interior contains an extensive activated upon cell rupture [18–20]. OMPLA is involved in hydrogen-bonding network that creates several large colicin secretion in E. coli and has been implicated in bac- aqueous cavities, but no passage that is large enough for terial virulence [21,22]. As this enzyme resides in its own the transport of water or small ions. Sequence alignment substrate, its activity must be tightly regulated to prevent with five bacterial homologues shows that the residues unwanted cell lysis. OMPLA is a 269-residue protein that that form the internal polar network are highly conserved; displays broad substrate specificity and shows no sequence the authors speculate that these residues may either be similarity to soluble phospholipases. It requires calcium for involved in protein folding or have some unknown func- activity and is regulated by reversible dimerisation [23]. tion. The structure of OmpA provides the first example of the β-barrel scaffold being used primarily as a solid trans- The structure of E. coli OMPLA in monomeric (2.2 Å res- membrane anchor for the separate functions of the olution) and dimeric (2.1 Å resolution) forms is presented extracellular loops and the periplasmic domain. in a soon-to-be published paper from Dijkstra’s group Figure 2 Conformational changes induced in the N-terminal domain of FhuA upon ferrichrome binding [28••]. The barrel is drawn in dark blue, portions of the N-terminal domain that do not move are in mid blue and portions of the N-terminal domain that show movement are drawn in cyan (no ligand) and red (ferrichrome-bound). Ferrichrome is drawn in green, with the iron atom represented by a red sphere. Residue R81 (indicated by the yellow arrow), located on the extracellular side of the N-terminal domain, shows a small shift upon ligand binding, while residue W22 (cyan and red arrows for the unbound and ligand-bound structures, respectively), located on the periplasmic side, undergoes a movement of 17 Å when ferrichrome is bound. This figure was kindly provided by K Locher and was made using the program DINO [60]. b-Barrel proteins from bacterial outer membranes Buchanan 457 Table 1 Refolding procedures for b-barrel membrane proteins. Protein Number of strands/ Expression Host IB Unfolding Refolding Scale Crystals References oligomeric system conditions conditions obtained state OmpF 16/3 Chromosomal E. coli No 6 M guanidine Dilution into mixed micelles 40–160 µg No [42] plus 95°C, then (asolectin plus C8-POE), from 200 µg exchange into dialysis unfolded 2% SDS protein OmpF 16/3 Chromosomal E. coli No 8 M urea Dilution into mixed micelles ND No [50] (DMPC–LM), dialysis OmpA 8/1 Chromosomal E. coli No 4 M urea Dilution into OG, reconstitution ND No [61] into DMPC vesicles OmpA 8/1 Chromosomal E. coli No 0.05% SDS, Addition of OG, then ND No [51] 100°C, 10 min reconstitution into DMPC vesicles PhoE 16/3 In vitro NA No Protein Addition of Triton X-100 ND No [55] synthesis obtained unfolded Rps. blastica 16/3 pET3b E. coli Yes 8 M urea Dilution into 10% LDAO, 25 mg/l Yes [7] porin BL21(DE3)– refolding on anion exchange pLysS column OMPLA 12/2 pJP29 E. coli Yes 8 M urea Dilution into 10 mM Triton 35 mg/l Yes [25] BL21(DE3) X-100, 0.87 M urea, 16 hr RT OmpA and 8/1 pET3b E. coli Yes 6 M guanidine Dilution into 5% C8-POE 150 mg/l Yes [15•] OmpX BL21(DE3) plus 0.6 M arginine H. influenzae 16?/3 pET11a E. coli Yes 8 M urea 1 M NaCl plus 10% SB3-14, 60 mg/l No [44] type-b porin BL21(DE3) gel filtration chromatography to reduce detergent concentration to 0.05%, storage at 4°C for 3 weeks H.
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