The membrane bound bacterial lipocalin Blc is a functional dimer with binding preference for lysophospholipids

Lipocalins, a widespread multifunctional family of small proteins (15-25kDa) have been first described in eukaryotes and more recently in Gram-negative bacteria. Bacterial lipocalins belonging to class I are outer membrane lipoproteins, among which Blc from E. coli is the better studied. Blc is expressed under conditions of starvation and high osmolarity, conditions known to exert stress on the cell envelope. The structure of Blc that we have previously solved (V. Campanacci, D. Nurizzo, S. Spinelli, C. Valencia, M. Tegoni, C. Cambillau, FEBS Lett. 562 (2004) 183-188.) suggested its possible role in binding fatty acids or phospholipids. Both physiological and structural data on Blc, therefore, point to a role in storage or transport of lipids necessary for membrane maintenance. In order to further document this hypothesis for Blc function, we have performed binding studies using fluorescence quenching experiments. Our results indicate that dimeric Blc binds fatty acids and phospholipids in a micromolar K(d) range. The crystal structure of Blc with vaccenic acid, an unsaturated C18 fatty acid, reveals that the binding site spans across the Blc dimer, opposite to its membrane anchored face. An exposed unfilled pocket seemingly suited to bind a polar group attached to the fatty acid prompted us to investigate lyso-phospholipids, which were found to bind in a nanomolar K(d) range. We discuss these findings in terms of a potential role for Blc in the metabolism of lysophospholipids generated in the bacterial outer membrane.     

The bacterial lipocalins

The lipocalins were once regarded as a eukaryotic protein family, but new members have been recently discovered in bacteria. The first bacterial lipocalin (Blc) was identified in Escherichia coli as an outer membrane lipoprotein expressed under conditions of environmental stress. Blc is distinguished from most lipocalins by the absence of intramolecular disulfide bonds, but the presence of a membrane anchor is shared with two of its closest homologues, apolipoprotein D and lazarillo. Several common features of the membrane-anchored lipocalins suggest that each may play an important role in membrane biogenesis and repair. Additionally, Blc proteins are implicated in the dissemination of antibiotic resistance genes and in the activation of immunity. Recent genome sequencing efforts reveal the existence of at least 20 bacterial lipocalins. The lipocalins appear to have originated in Gram-negative bacteria and were probably transferred horizontally to eukaryotes from the endosymbiotic alpha-proteobacterial ancestor of the mitochondrion. The genome sequences also reveal that some bacterial lipocalins exhibit disulfide bonds and alternative modes of subcellular localization, which include targeting to the periplasmic space, the cytoplasmic membrane, and the cytosol. The relationships between bacterial lipocalin structure and function further illuminate the common biochemistry of bacterial and eukaryotic cells.     

Structural studies of the Cpx pathway activator NlpE on the outer membrane of Escherichia coli

NlpE, an outer membrane lipoprotein, functions during envelope stress responses in Gram-negative bacteria. In Escherichia coli, adhesion to abiotic surfaces has been reported to activate the Cpx pathway in an NlpE-dependent manner. External copper ions are also thought to activate the Cpx pathway mediated by NlpE. We determined the crystal structure of NlpE from E. coli at 2.6 A resolution. The structure showed that NlpE consists of two beta barrel domains. The N-terminal domain resembles the bacterial lipocalin Blc, and the C-terminal domain has an oligonucleotide/oligosaccharide-binding (OB) fold. From both biochemical analyses and the crystal structure, it can be deduced that the cysteine residues in the CXXC motif may be chemically active. Furthermore, two monomers in the asymmetric unit form an unusual 3D domain-swapped dimer. These findings indicate that tertiary and/or quaternary structural instability may be responsible for Cpx pathway activation.     

NMR structure of lipoprotein YxeF from Bacillus subtilis reveals a calycin fold and distant homology with the lipocalin Blc from Escherichia coli

The soluble monomeric domain of lipoprotein YxeF from the Gram positive bacterium B. subtilis was selected by the Northeast Structural Genomics Consortium (NESG) as a target of a biomedical theme project focusing on the structure determination of the soluble domains of bacterial lipoproteins. The solution NMR structure of YxeF reveals a calycin fold and distant homology with the lipocalin Blc from the Gram-negative bacterium E.coli. In particular, the characteristic β-barrel, which is open to the solvent at one end, is extremely well conserved in YxeF with respect to Blc. The identification of YxeF as the first lipocalin homologue occurring in a Gram-positive bacterium suggests that lipocalins emerged before the evolutionary divergence of Gram positive and Gram negative bacteria. Since YxeF is devoid of the α-helix that packs in all lipocalins with known structure against the β-barrel to form a second hydrophobic core, we propose to introduce a new lipocalin sub-family named 'slim lipocalins', with YxeF and the other members of Pfam family PF11631 to which YxeF belongs constituting the first representatives. The results presented here exemplify the impact of structural genomics to enhance our understanding of biology and to generate new biological hypotheses.     

Structure and biochemical analysis of a secretin pilot protein

The ability to translocate virulence proteins into host cells through a type III secretion apparatus (TTSS) is a hallmark of several Gram-negative pathogens including Shigella, Salmonella, Yersinia, Pseudomonas, and enteropathogenic Escherichia coli. In common with other types of bacterial secretion apparatus, the assembly of the TTSS complex requires the preceding formation of its integral outer membrane secretin ring component. We have determined at 1.5 A the structure of MxiM28-142, the Shigella pilot protein that is essential for the assembly and membrane association of the Shigella secretin, MxiD. This represents the first atomic structure of a secretin pilot protein from the several bacterial secretion systems containing an orthologous secretin component. A deep hydrophobic cavity is observed in the novel 'cracked barrel' structure of MxiM, providing a specific binding domain for the acyl chains of bacterial lipids, a proposal that is supported by our various lipid/MxiM complex structures. Isothermal titration analysis shows that the C-terminal domain of the secretin, MxiD525-570, hinders lipid binding to MxiM.     

Membrane proteins: A tale of barrels and corks

Crystal structures have been solved for two bacterial outer membrane proteins, FhuA and FepA, which mediate active transport of chelated iron. Analysis of ligand-induced changes in the structure of FhuA has provided our first structural insights into an active transport mechanism for a complex solute.     

High level bacterial expression of uteroglobin, a dimeric eukaryotic protein with two interchain disulfide bridges, in its natural quaternary structure

Bacterial expression of eukaryotic proteins is a tool of ever-increasing importance in biochemistry and molecular biology. However, the majority of the recombinant eukaryotic proteins that have been expressed in bacteria are produced as fusion proteins and not in their native conformation. In particular, correct formation of quaternary structures by recombinant proteins in bacterial hosts has been reported very rarely. To our knowledge, correct intracellular formation of multimeric structures containing more than one interchain disulfide bridge has not been reported so far. We have constructed three plasmids which are able to direct expression of recombinant rabbit uteroglobin, a homodimeric protein with two interchain disulfide bridges, in Escherichia coli. Among these, the plasmid pLE103-1, in which the expression of recombinant uteroglobin is controlled by a bacteriophage T7 late promoter, is by far the most efficient. With pLE103-1, recombinant uteroglobin production reached about 10% of total bacterial soluble proteins. This protein accumulated in bacterial cells in dimeric form, as it is naturally found in the rabbit uterus. Recombinant uteroglobin was purified to near-homogeneity and its NH2-terminal amino acid sequence was confirmed to be identical to that of its natural counterpart, except for 2 Ala residues the codons for which were added during the plasmid construction. This protein was found to be as active a phospholipase A2 inhibitor as natural uteroglobin on a molar basis. To our knowledge, this is the first report of high level bacterial expression of a full length eukaryotic homodimeric protein with two interchain disulfide bridges in its natural, biologically active form. The plasmid pLE103-1 may be useful to explore structure-function relationships of rabbit uteroglobin. In addition, this plasmid may be useful in obtaining high level bacterial expression of other eukaryotic proteins with quaternary structure, as well as for other general applications requiring efficient bacterial expression of cDNAs.     

On the mode of integration of the thylakoid membrane protein cytochrome b(6) into cytoplasmic membrane of Escherichia coli

In the stroma compartment, several pathways are used for integration/translocation of chloroplast proteins into or across the thylakoid membrane. In this study we investigated the mode of incorporation of the chloroplast-encoded cytochrome b(6) into the bacterial membrane. Cytochrome b(6) naturally comprises of four transmembrane helices (A,B,C,D) and contains two b-type hemes. In the present study, mature cytochrome b(6) or constructed deletion mutants of cytochrome were expressed in E. coli cells. The membrane insertion of cytochrome b(6) in this bacterial model system requires an artificially added presequence that directs the protein to use an E. coli membrane-insertion pathway. This could be accomplished by fusion to maltose-binding protein (MBP) or to the bacterial Sec-dependent signal peptide (SSpelB). The integration of mature cytochrome b(6) into the bacterial cytoplasmic membrane by the Sec pathway has been reported previously by our group (Kroliczewski et al., 2005, Biochemistry, 44: 7570). The results presented here show that cytochrome b(6) devoid of the first helix A can be inserted into the membrane, as can the entire ABCD. On the other hand, the construct devoid of helices A and B is translocated through the membrane into the periplasm without any effective insertion. This suggests the importance of the membrane-anchoring sequences that are likely to be present in only the A and B part, and it is consistent with the results of computational prediction which did not identify any membrane-anchoring sequences for the C or D helices. We also show that the incorporation of hemes into the truncated form of cytochrome b(6) is possible, as long as the B and D helices bearing axial ligands to heme are present.     

Bacteria recovered without subculture from infected human urines expressed iron-regulated outer membrane proteins

Abstract Twelve enteric bacterial strains were recovered by differential centrifugation of urines which were collected from clinically diagnosed and microbiologically confirmed cases of urinary tract infection. The outer membrane protein (OMP) profiles of the clinical isolates were then analysed by sodiumdodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE). It was found that 5 of the 12 isolates (3 Escherichia coli strains, 1 Klebsiella pneumoniae and 1 Proteus mirabilis strain) expressed 2 or more high M _r proteins in the range of 66000 to 85000. These high M _r proteins were expressed by the same organisms during growth in vitro in iron-restricted conditions but not in iron-sufficient media.
In addition, it was found that the major outer membrane proteins expressed by the clinical isolates varied considerably and that, in many cases, fresh isolates expressed fewer porin proteins than the same bacterial strains after growth in vitro in trypticase soy broth. This is thus the first evidence the E. coli, K. pneumoniae and P. mirabilis grow under iron-restricted conditions in the urinary tract of humans and that the outer membrane protein profile of clinical isolates differ from in vitro grown bacteria.     

Forces involved in the assembly and stabilization of membrane proteins.

Hydrophobic organization: Determination of the structure of the bacterial photosynthetic reaction center, bacterial porins, and bacteriorhodopsin allows a comparison of the basic structural features of integral membrane proteins. Structure parameters of membrane- and water-soluble proteins are surprisingly similar, given the different dielectric environments, except for the polarity of residues on the protein surface. Hydrophobic and electrostatic forces: 1) Intramembrane helix-helix interactions that are sensitive to small structure changes can dictate assembly of membrane proteins, as indicated by reconstitution of bacteriorhodopsin from proteolytic fragments and specific dimer formation of the human erythrocyte sialoglycoprotein glycophorin A. 2) Electrostatic interactions have an important role in determining the trans-membrane orientation of integral membrane proteins of the bacterial inner membrane, as expressed by the "positive-inside" rule for the distribution of basic residues on the cis relative to the trans side of the membrane-spanning alpha-helices. The use of this charge asymmetry rule, in conjunction with a hydrophobicity algorithm for prediction of membrane-spanning domains, allows accurate prediction of the folding patterns of such polypeptides across the membrane. A role of electrostatic interactions in assembly and maintenance of the structure of oligomeric integral membrane protein complexes is also implied by the separation and extrusion from the membrane, at high pH, of the major hydrophobic subunits of the cytochrome b6f complex from the chloroplast thylakoid membrane. It is inferred that the hydrophobic helix-helix interactions between the subunits of this complex, whose function is electron transfer and proton translocation, are relatively weak compared to those in bacteriorhodopsin.     

Functional expression and characterization of a bacterial light-harvesting membrane protein in Escherichia coli and cell-free synthesis systems

Heterologous expression of a bacterial light-harvesting (LH) integral membrane protein was attempted using Escherichia coli cells and cell-free synthesis systems prepared from E. coli extracts. The alpha-apoprotein of LH1 complex from purple photosynthetic bacterium Rhodospirillum rubrum was overexpressed as a recombinant protein with a histidine (His6) tag added to the carboxyl terminus. Both of the expression systems produced alpha-apoprotein in a fully functional form as can judged by its ability to form a structural subunit with native beta-apoprotein and the pigment molecule bacteriochlorophyll a. The expression product in E. coli appears to be located in the inner cell membrane and can be almost completely extracted by 0.5% (w/v) Triton X-100. Circular dichroism measurement indicated that the expressed alpha-apoproteins from both systems had alpha-helical contents essentially identical with that of the native one. About two thirds of the alpha-apoprotein expressed in E. coli was found to have the amino terminal methionine residue modified by a formyl group. About one third of the alpha-apoprotein expressed in the cell-free system was found to be oxidized at the side chain of the amino terminal methionine residue. Functional expression of the alpha-apoprotein using the cell-free system provides an useful example for producing highly hydrophobic integral membrane proteins with relatively large quantities sufficient for biophysical and structural analysis.     

Practical aspects of overexpressing bacterial secondary membrane transporters for structural studies

Membrane transporter proteins play critical physiological roles in the cell and constitute 5-10% of prokaryotic and eukaryotic genomes. High-resolution structural information is essential for understanding the functional mechanism of these proteins. A prerequisite for structural study is to overexpress such proteins in large quantities. In the last few years, over 20 bacterial membrane transporters were overexpressed at a level of 1 mg/l of culture or higher, most often in Escherichia coli. In this review, we analyzed those factors that affect the quantity and quality of the protein produced, and summarized recent progress in overexpression of membrane transporters from bacterial inner membrane. Rapid progress in genome sequencing provides opportunities for expressing several homologues and orthologues of the target protein simultaneously, while the availability of various expression vectors allows flexible experimental design. Careful optimization of cell culture conditions can drastically improve the expression level and homogeneity of the target protein. New sample preparation techniques for mass spectrometry of membrane proteins have enabled one to identity the rigid protein core, which can be subsequently overexpressed. Size-exclusion chromatography on HPLC has proven to be an efficient method in screening detergent, pH an other conditions required for maintaining the stability and monodispersity of the protein. Such high-quality preparations of membrane transporter proteins will probably lead to successful crystallization and structure determination of these proteins in the next few years.     

Crystal structure of human thioesterase superfamily member 2

Hotdog-fold has been identified in more than 1000 proteins, yet many of which in eukaryotes are less studied. No structural or functional studies of human thioesterase superfamily member 2 (hTHEM2) have been reported before. Since hTHEM2 exhibits about 20% sequence identity to Escherichia coli PaaI protein, it was proposed to be a thioesterase with a hotdog-fold. Here, we report the crystallographic structure of recombinant hTHEM2, determined by the single-wavelength anomalous dispersion method at 2.3A resolution. This structure demonstrates that hTHEM2 indeed contains a hotdog-fold and forms a back-to-back tetramer as other hotdog proteins. Based on structural and sequence conservation, the thioesterase active site in hTHEM2 is predicted. The structure and substrate specificity are most similar to those of the bacterial phenylacetyl-CoA hydrolase. Asp65, located on the central alpha-helix of subunit B, was shown by site-directed mutagenesis to be essential to catalysis.     

Target selection for structural genomics: a single genome approach

We describe our strategy for selecting targets for protein structure determination in context of structural genomics of a single genome. In the course of target selection, we have studied two of the smallest microbial genomes, Mycoplasma genitalium and Mycoplasma pneumoniae. To our surprise, we found that only 71 Mycoplasma genes or their orthologues can be considered as easy targets for high-throughput structural studies--far fewer than expected. We discuss the methods and criteria used for target selection and the reasons explaining rarity of easy targets. First, despite the common opinion that protein folds can be predicted for only 30-50% of genes, the number of "truly unknown" structures is less than one-third. Second, due to the different codon usage, two thirds of Mycoplasma proteins cannot be directly expressed in E. coli in high-throughput manner and require substitution by their homologues from other organisms. Third, membrane or large multi-domain proteins are difficult targets because of solubility and size issues and often require identification and structure determination of protein domains. Finally, we propose different approaches to address the difficult targets.     

Architecture and selectivity in aquaporins: 2.5 a X-ray structure of aquaporin Z

Aquaporins are a family of water and small molecule channels found in organisms ranging from bacteria to animals. One of these channels, the E. coli protein aquaporin Z (AqpZ), has been shown to selectively conduct only water at high rates. We have expressed, purified, crystallized, and solved the X-ray structure of AqpZ. The 2.5 A resolution structure of AqpZ suggests aquaporin selectivity results both from a steric mechanism due to pore size and from specific amino acid substitutions that regulate the preference for a hydrophobic or hydrophilic substrate. This structure provides direct evidence on the molecular mechanisms of specificity between water and glycerol in this family of channels from a single species. It is to our knowledge the first atomic resolution structure of a recombinant aquaporin and so provides a platform for combined genetic, mutational, functional, and structural determinations of the mechanisms of aquaporins and, more generally, the assembly of multimeric membrane proteins.     

Crystallization of the membrane protein hVDAC1 produced in cell-free system

Structural studies of membrane proteins are in constant evolution with the development of new improvements for their expression, purification, stabilization and crystallization. However, none of these methods still provides a universal approach to solve the structure of membrane proteins. Here we describe the crystallization of the human voltage-dependent anion channel-1 produced by a bacterial cell-free expression system. While VDAC structures have been recently solved, we propose an alternative strategy for producing the recombinant protein, which can be applied to other membrane proteins reluctant to expression, purification and crystallization by classical approaches. Despite a lot of efforts to crystallize a cell-free expressed membrane protein, this study is to our knowledge one of the first reports of a successful crystallization. Focusing on expression in a soluble and functional state, in a detergent environment, is the key to get crystals. Although the diffraction of VDAC crystals is limited, the simplicity and the rapidity to set-up and optimize this technology are drastic advantages in comparison to other methods.     

β-lactamase as a probe of membrane protein assembly and protein export

The enzyme TEM beta-lactamase constitutes a versatile gene-fusion marker for studies on membrane proteins and protein export in bacteria. The mature form of this normally periplasmic enzyme displays readily detectable and distinctly different phenotypes when localized to the bacterial cytoplasm versus the periplasm, and thus provides a useful alternative to alkaline phosphatase for probing the topology of cytoplasmic membrane proteins. Cells producing translocated forms of beta-lactamase can be directly selected as ampicillin-resistant colonies, and consequently a beta-lactamase fusion approach can be used for positive selection for export signals, and for rapid assessment of whether any protein expressed in Escherichia coli inserts into the bacterial cytoplasmic membrane. The level of ampicillin resistance conferred on a cell by an extracytoplasmic beta-lactamase derivative depends on its level of expression, and therefore a beta-lactamase fusion approach can be used to directly select for increased yields of any periplasmic or membrane-bound gene products expressed in E. coli.     

Protein secretion controlled by a synthetic gene in Escherichia coli

The inability of Escherichia coli to secrete proteins in growth medium is one of the major drawbacks in its use in genetic engineering. A synthetic gene, homologous to the one coding for the kil peptide of pColE1, was made and cloned under the control of the lac promoter, in order to obtain the inducible secretion of homologous or heterologous proteins by E. coli. The efficiency of this synthetic gene to promote secretion was assayed by analysing the production and secretion of two proteins, the R-TEM1 beta-lactamase, and the alpha-amylase from Bacillus licheniformis. This latter protein was expressed in E. coli from its gene either on the same plasmid as the kil gene or on a different plasmid. The primary effect of the induction of the kil gene is the overproduction of the secreted proteins. When expressed at a high level, the kil gene promotes the overproduction of all periplasmic proteins and the total secretion in the culture medium of both the beta-lactamase or the alpha-amylase. This secretion is semi-selective for most periplasmic proteins are not secreted. The kil peptide induces the secretion of homologous or heterologous proteins in two steps, first acting on the cytoplasmic membrane, then permeabilizing the outer membrane. This system, which is now being assayed at the fermentor scale, is the first example of using a synthetic gene to engineer a new property into a bacterial strain.     

High resolution structure of Deinococcus bacteriophytochrome yields new insights into phytochrome architecture and evolution

Phytochromes are red/far red light photochromic photoreceptors that direct many photosensory behaviors in the bacterial, fungal, and plant kingdoms. They consist of an N-terminal domain that covalently binds a bilin chromophore and a C-terminal region that transmits the light signal, often through a histidine kinase relay. Using x-ray crystallography, we recently solved the first three-dimensional structure of a phytochrome, using the chromophore-binding domain of Deinococcus radiodurans bacterial phytochrome assembled with its chromophore, biliverdin IXalpha. Now, by engineering the crystallization interface, we have achieved a significantly higher resolution model. This 1.45A resolution structure helps identify an extensive buried surface between crystal symmetry mates that may promote dimerization in vivo. It also reveals that upon ligation of the C3(2) carbon of biliverdin to Cys(24), the chromophore A-ring assumes a chiral center at C2, thus becoming 2(R),3(E)-phytochromobilin, a chemistry more similar to that proposed for the attached chromophores of cyanobacterial and plant phytochromes than previously appreciated. The evolution of bacterial phytochromes to those found in cyanobacteria and higher plants must have involved greater fitness using more reduced bilins, such as phycocyanobilin, combined with a switch of the attachment site from a cysteine near the N terminus to one conserved within the cGMP phosphodiesterase/adenyl cyclase/FhlA domain. From analysis of site-directed mutants in the D. radiodurans phytochrome, we show that this bilin preference was partially driven by the change in binding site, which ultimately may have helped photosynthetic organisms optimize shade detection. Collectively, these three-dimensional structural results better clarify bilin/protein interactions and help explain how higher plant phytochromes evolved from prokaryotic progenitors.     

Structural resemblance between the families of bacterial signal-transduction proteins and of G proteins revealed by graph theoretical techniques

The first application of a novel technique for the identification of common folding motifs in proteins is presented. Using techniques derived from graph theory, developed in order to compare secondary structure motifs in proteins, we have established that there is a striking resemblance in the tertiary fold of the Salmonella typhimurium Che Y chemotaxis protein and that of the GDP-binding domain of Escherichia coli elongation factor Tu (EF Tu). These two protein structures are representatives of two major macromolecular classes: CheY is a signal-transduction protein with sequence homologies to a wide range of bacterial proteins involved in regulation of chemotaxis, membrane synthesis and sporulation; whilst EF Tu is one of a family of guanosine-nucleotide-binding proteins which include the ras oncogene proteins and signal-transducing G proteins. The similarity we have found extends far beyond the previously recognized resemblances of each protein's fold to that of a generic nucleotide-binding domain. The lack of significant sequence homology between the two classes of proteins may mean that the common fold of the two proteins constitutes a particularly stable folding motif. However, an alternative possibility is that the strong three-dimensional structural resemblance may be indicative of a remote shared common ancestry between the bacterial signal-transduction proteins and the GDP-binding proteins.