Expression, refolding, and initial structural characterization of the Y. pestis Ail outer membrane protein in lipids.
ABSTRACT: Ail is an outer membrane protein and virulence factor of Yersinia pestis, an extremely pathogenic, category A biothreat agent, responsible for precipitating massive human plague pandemics throughout history. Due to its key role in bacterial adhesion to host cells and bacterial resistance to host defense, Ail is a key target for anti-plague therapy. However, little information is available about the molecular aspects of its function and interactions with the human host, and the structure of Ail is not known. Here we describe the recombinant expression, purification, refolding, and sample preparation of Ail for solution and solid-state NMR structural studies in lipid micelles and lipid bilayers. The initial NMR and CD spectra show that Ail adopts a well-defined transmembrane ?-sheet conformation in lipids.
Project description:Ail is an outer membrane protein from Yersinia pestis that is highly expressed in a rodent model of bubonic plague, making it a good candidate for vaccine development. Ail is important for attaching to host cells and evading host immune responses, facilitating rapid progression of a plague infection. Binding to host cells is important for injection of cytotoxic Yersinia outer proteins. To learn more about how Ail mediates adhesion, we solved two high-resolution crystal structures of Ail, with no ligand bound and in complex with a heparin analog called sucrose octasulfate. We identified multiple adhesion targets, including laminin and heparin, and showed that a 40 kDa domain of laminin called LG4-5 specifically binds to Ail. We also evaluated the contribution of laminin to delivery of Yops to HEp-2 cells. This work constitutes a structural description of how a bacterial outer membrane protein uses a multivalent approach to bind host cells.
Project description:Yersinia pestis the causative agent of plague, is highly pathogenic and poses very high risk to public health. The outer membrane protein Ail (Adhesion invasion locus) is one of the most highly expressed proteins on the cell surface of Y. pestis, and a major target for the development of medical countermeasures. Ail is essential for microbial virulence and is critical for promoting the survival of Y. pestis in serum. Structures of Ail have been determined by X-ray diffraction and solution NMR spectroscopy, but the protein's activity is influenced by the detergents in these samples, underscoring the importance of the surrounding environment for structure-activity studies. Here we describe the backbone structure of Ail, determined in lipid bilayer nanodiscs, using solution NMR spectroscopy. We also present solid-state NMR data obtained for Ail in membranes containing lipopolysaccharide (LPS), a major component of the bacterial outer membranes. The protein in lipid bilayers, adopts the same eight-stranded ?-barrel fold observed in the crystalline and micellar states. The membrane composition, however, appears to have a marked effect on protein dynamics, with LPS enhancing conformational order and slowing down the 15N transverse relaxation rate. The results provide information about the way in which an outer membrane protein inserts and functions in the bacterial membrane.
Project description:Ail, a multifunctional outer membrane protein of Yersinia pestis, confers cell binding, Yop delivery and serum resistance activities. Resistance to complement proteins in serum is critical for the survival of Y. pestis during the septicemic stage of plague infections. Bacteria employ a variety of tactics to evade the complement system, including recruitment of complement regulatory factors, such as factor H, C4b-binding protein (C4BP) and vitronectin (Vn). Y. pestis Ail interacts with the regulatory factors Vn and C4BP, and Ail homologs from Y. enterocolitica and Y. pseudotuberculosis recruit factor H. Using co-sedimentation assays, we demonstrate that two surface-exposed amino acids, F80 and F130, are required for the interaction of Y. pestis Ail with Vn, factor H and C4BP. However, although Ail-F80A/F130A fails to interact with these complement regulatory proteins, it still confers 10,000-fold more serum resistance than a ?ail strain and prevents C9 polymerization, potentially by directly interfering with MAC assembly. Using site-directed mutagenesis, we further defined this additional mechanism of complement evasion conferred by Ail. Finally, we find that at Y. pestis concentrations reflective of early-stage septicemic plague, Ail weakly recruits Vn and fails to recruit factor H, suggesting that this alternative mechanism of serum resistance may be essential during plague infection.
Project description:The outer membrane protein Ail (Adhesion invasion locus) is one of the most abundant proteins on the cell surface of Yersinia pestis during human infection. Its functions are expressed through interactions with a variety of human host proteins, and are essential for microbial virulence. Structures of Ail have been determined by X-ray diffraction and solution NMR spectroscopy, but those samples contained detergents that interfere with functionality, thus, precluding analysis of the structural basis for Ail's biological activity. Here, we demonstrate that high-resolution solid-state NMR spectra can be obtained from samples of Ail in detergent-free phospholipid liposomes, prepared with a lipid to protein molar ratio of 100. The spectra, obtained with 13C or 1H detection, have very narrow line widths (0.40-0.60 ppm for 13C, 0.11-0.15 ppm for 1H, and 0.46-0.64 ppm for 15N) that are consistent with a high level of sample homogeneity. The spectra enable resonance assignments to be obtained for N, CO, CA and CB atomic sites from 75 out of 156 residues in the sequence of Ail, including 80% of the transmembrane region. The 1H-detected solid-state NMR 1H/15N correlation spectra obtained for Ail in liposomes compare very favorably with the solution NMR 1H/15N TROSY spectra obtained for Ail in nanodiscs prepared with a similar lipid to protein molar ratio. These results set the stage for studies of the molecular basis of the functional interactions of Ail with its protein partners from human host cells, as well as the development of drugs targeting Ail.
Project description:The outer membrane is a key virulence determinant of gram-negative bacteria. In Yersinia pestis, the deadly agent that causes plague, the protein Ail and lipopolysaccharide (LPS)6 enhance lethality by promoting resistance to human innate immunity and antibiotics, enabling bacteria to proliferate in the human host. Their functions are highly coordinated. Here we describe how they cooperate to promote pathogenesis. Using a multidisciplinary approach, we identify mutually constructive interactions between Ail and LPS that produce an extended conformation of Ail at the membrane surface, cause thickening and rigidification of the LPS membrane, and collectively promote Y. pestis survival in human serum, antibiotic resistance, and cell envelope integrity. The results highlight the importance of the Ail-LPS assembly as an organized whole, rather than its individual components, and provide a handle for targeting Y. pestis pathogenesis.
Project description:The surrounding environment has significant consequences for the structural and functional properties of membrane proteins. While native structure and function can be reconstituted in lipid bilayer membranes, the detergents used for protein solubilization are not always compatible with biological activity and, hence, not always appropriate for direct detection of ligand binding by NMR spectroscopy. Here we describe how the sample environment affects the activity of the outer membrane protein Ail (attachment invasion locus) from Yersinia pestis. Although Ail adopts the correct ?-barrel fold in micelles, the high detergent concentrations required for NMR structural studies are not compatible with the ligand binding functionality of the protein. We also describe preparations of Ail embedded in phospholipid bilayer nanodiscs, optimized for NMR studies and ligand binding activity assays. Ail in nanodiscs is capable of binding its human ligand fibronectin and also yields high quality NMR spectra that reflect the proper fold. Binding activity assays, developed to be performed directly with the NMR samples, show that ligand binding involves the extracellular loops of Ail. The data show that even when detergent micelles support the protein fold, detergents can interfere with activity in subtle ways.
Project description:The Yersinia pestis adhesin molecule Ail interacts with the extracellular matrix protein fibronectin (Fn) on host cells to facilitate efficient delivery of cytotoxic Yop proteins, a process essential for plague virulence. A number of bacterial pathogens are known to bind to the N-terminal region of Fn, comprising type I Fn (FNI) repeats. Using proteolytically generated Fn fragments and purified recombinant Fn fragments, we demonstrated that Ail binds the centrally located 120-kDa fragment containing type III Fn (FNIII) repeats. A panel of monoclonal antibodies (mAbs) that recognize specific epitopes within the 120-kDa fragment demonstrated that mAb binding to (9)FNIII blocks Ail-mediated bacterial binding to Fn. Epitopes of three mAbs that blocked Ail binding to Fn were mapped to a similar face of (9)FNIII. Antibodies directed against (9)FNIII also inhibited Ail-dependent cell binding activity, thus demonstrating the biological relevance of this Ail binding region on Fn. Bacteria expressing Ail on their surface could also bind a minimal fragment of Fn containing repeats (9-10)FNIII, and this binding was blocked by a mAb specific for (9)FNIII. These data demonstrate that Ail binds to (9)FNIII of Fn and presents Fn to host cells to facilitate cell binding and delivery of Yops (cytotoxins of Y. pestis), a novel interaction, distinct from other bacterial Fn-binding proteins.
Project description:The Yersinia pestis adhesin Ail mediates host cell binding and facilitates delivery of cytotoxic Yop proteins. Ail from Y. pestis and Y. pseudotuberculosis is identical except for one or two amino acids at positions 43 and 126 depending on the Y. pseudotuberculosis strain. Ail from Y. pseudotuberculosis strain YPIII has been reported to lack host cell binding ability, thus we sought to determine which amino acid difference(s) are responsible for the difference in cell adhesion. Y. pseudotuberculosis YPIII Ail expressed in Escherichia coli bound host cells, albeit at ~50% the capacity of Y. pestis Ail. Y. pestis Ail single mutants, Ail-E43D and Ail-F126V, both have decreased adhesion and invasion in E. coli when compared to wild-type Y. pestis Ail. Y. pseudotuberculosis YPIII Ail also had decreased binding to the Ail substrate fibronectin, relative to Y. pestis Ail in E. coli. When expressed in Y. pestis, there was a 30-50% decrease in adhesion and invasion depending on the substitution. Ail-mediated Yop delivery by both Y. pestis Ail and Y. pseudotuberculosis Ail were similar when expressed in Y. pestis, with only Ail-F126V giving a statistically significant reduction in Yop delivery of 25%. In contrast to results in E. coli and Y. pestis, expression of Ail in Y. pseudotuberculosis led to no measurable adhesion or invasion, suggesting the longer LPS of Y. pseudotuberculosis interferes with Ail cell-binding activity. Thus, host context affects the binding activities of Ail and both Y. pestis and Y. pseudotuberculosis Ail can mediate cell binding, cell invasion and facilitate Yop delivery.
Project description:The outer membrane protein Ail (attachment invasion locus) is a virulence factor of Yersinia pestis that mediates cell invasion, cell attachment and complement resistance. Here we describe its three-dimensional backbone structure determined in decyl-phosphocholine (DePC) micelles by NMR spectroscopy. The NMR structure was calculated using the membrane function of the implicit solvation potential, eefxPot, which we have developed to facilitate NMR structure calculations in a physically realistic environment. We show that the eefxPot force field guides the protein towards its native fold. The resulting structures provide information about the membrane-embedded global position of Ail, and have higher accuracy, higher precision and improved conformational properties, compared to the structures calculated with the standard repulsive potential.
Project description:Yersinia pestis, the causative agent of plague, binds host cells to deliver cytotoxic Yop proteins into the cytoplasm that prevent phagocytosis and generation of proinflammatory cytokines. Ail is an eight-stranded ?-barrel outer membrane protein with four extracellular loops that mediates cell binding and resistance to human serum. Following the deletion of each of the four extracellular loops that potentially interact with host cells, the Ail-?loop 2 and Ail-?loop 3 mutant proteins had no cell-binding activity while Ail-?loop 4 maintained cell binding (the Ail-?loop 1 protein was unstable). Using the codon mutagenesis scheme SWIM (selection without isolation of mutants), we identified individual residues in loops 1, 2, and 3 that contribute to host cell binding. While several residues contributed to the binding of host cells and purified fibronectin and laminin, as well as Yop delivery, three mutations, F80A (loop 2), S128A (loop 3), and F130A (loop 3), produced particularly severe defects in cell binding. Combining these mutations led to an even greater reduction in cell binding and severely impaired Yop delivery with only a slight defect in serum resistance. These findings demonstrate that Y. pestis Ail uses multiple extracellular loops to interact with substrates important for adhesion via polyvalent hydrophobic interactions.