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Plekha7 (Pleckstrin homology [PH] domain containing, family A member 7) regulates the assembly of proteins of the cytoplasmic apical zonula adherens junction (AJ), thus ensuring cell-cell adhesion and tight-junction barrier integrity. Little is known of Plekha7 function in cancer. In colorectal cancer (CRC) Plekha7 expression is elevated compared to adjacent normal tissue levels, increasing with clinical stage. Plekha7 was present at plasma membrane AJ with wild-type KRas (wt-KRas) but was dispersed in cells expressing mutant KRas (mut-KRas). Fluorescence lifetime imaging microscopy (FLIM) indicated a direct Plekha7 interaction with wt-KRas but scantily with mut-KRas. Inhibiting Plekha7 specifically decreased mut-KRas cell signaling, proliferation, attachment, migration, and retarded mut-KRAS CRC tumor growth. Binding of diC8-phosphoinositides (PI) to the PH domain of Plekha7 was relatively low affinity. This may be because a D175 amino acid residue plays a “sentry” role preventing PI(3,4)P(2) and PI(3,4,5)P(3) binding. Molecular or pharmacological inhibition of the Plekha7 PH domain prevented the growth of mut-KRas but not wt-KRas cells. Taken together the studies suggest that Plekha7, in addition to maintaining AJ structure plays a role in mut-KRas signaling and phenotype through interaction of its PH domain with membrane mut-KRas, but not wt-KRas, to increase the efficiency of mut-KRas downstream signaling.

The human blood protein vitronectin (Vn) is a major component of the abnormal deposits associated with age-related macular degeneration, Alzheimer's disease, and many other age-related disorders. Its accumulation with lipids and hydroxyapatite (HAP) has been demonstrated, but the precise mechanism for deposit formation remains unknown. Using a combination of solution and solid-state NMR experiments, cosedimentation assays, differential scanning fluorimetry (DSF), and binding energy calculations, we demonstrate that Vn is capable of binding both soluble ionic calcium and crystalline HAP, with high affinity and chemical specificity. Calcium ions bind preferentially at an external site, at the top of the hemopexin-like (HX) domain, with a group of four Asp carboxylate groups. The same external site is also implicated in HAP binding. Moreover, Vn acquires thermal stability upon association with either calcium ions or crystalline HAP. The data point to a mechanism whereby Vn plays an active role in orchestrating calcified deposit formation. They provide a platform for understanding the pathogenesis of macular degeneration and other related degenerative disorders, and the normal functions of Vn, especially those related to bone resorption.

Bcl-xL is a major inhibitor of apoptosis, a fundamental homeostatic process of programmed cell death that is highly conserved across evolution. Because it plays prominent roles in cancer, Bcl-xL is a major target for anti-cancer therapy and for studies aimed at understanding its structure and activity. While Bcl-xL is active primarily at intracellular membranes, most studies have focused on soluble forms of the protein lacking both the membrane-anchoring C-terminal tail and the intrinsically disordered loop, and this has resulted in a fragmented view of the protein’s biological activity. Here we describe how these segments affect the protein’s conformation and ligand binding activity in both its soluble and membrane-anchored states. The combined data from nuclear magnetic resonance (NMR) spectroscopy, molecular dynamics (MD) simulations, and isothermal titration calorimetry (ITC) provide information about the molecular basis for the protein’s functionality and a view of its complex molecular mechanisms.SIGNIFICANCEThe human protein Bcl-xL is a key regulator of programmed cell death in health and disease. Structural studies, important for understating the molecular basis for its functions, have advanced primarily by deleting both the long disordered loop that regulates its activity and the C-terminal tail that anchors the protein to intracellular membranes Here we describe the preparation and conformations of full-length Bcl-xL in both its water-soluble and membrane-anchored states. The study provides new biophysical insights about Bcl-xL and its greater Bcl-2 protein family.

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.

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 β-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.

Vitronectin (Vn) is a major component of blood that controls many processes central to human biology. It is a drug target and a key factor in cell and tissue engineering applications, but despite long-standing efforts, little is known about the molecular basis for its functions. Here, we define the domain organization of Vn, report the crystal structure of its carboxyl-terminal domain, and show that it harbors the binding site for the Yersinia pestis outer membrane protein Ail, which recruits Vn to the bacterial cell surface to evade human host defenses. Vn forms a single four-bladed β/α-propeller that serves as a hub for multiple functions. The structure explains key features of native Vn and provides a blueprint for understanding and targeting this essential human protein.

B cell lymphoma gene 2 (Bcl-2) family proteins are key regulators of programmed cell death and important targets for drug discovery. Pro-apoptotic and anti-apoptotic Bcl-2 family proteins reciprocally modulate their activities in large part through protein interactions involving a motif known as BH3 (Bcl-2 homology 3). Nur77 is an orphan member of the nuclear receptor family that lacks a BH3 domain but nevertheless binds certain anti-apoptotic Bcl-2 family proteins (Bcl-2, Bfl-1, and Bcl-B), modulating their effects on apoptosis and autophagy. We used a combination of NMR spectroscopy-based methods, mutagenesis, and functional studies to define the interaction site of a Nur77 peptide on anti-apoptotic Bcl-2 family proteins and reveal a novel interaction surface. Nur77 binds adjacent to the BH3 peptide-binding crevice, suggesting the possibility of cross-talk between these discrete binding sites. Mutagenesis of residues lining the identified interaction site on Bcl-B negated the interaction with Nur77 protein in cells and prevented Nur77-mediated modulation of apoptosis and autophagy. The findings establish a new protein interaction site with the potential to modulate the apoptosis and autophagy mechanisms governed by Bcl-2 family proteins.

Yersinia pestis, the agent of plague, requires the Ail (attachment invasion locus) outer membrane protein to survive in the blood and tissues of its mammalian hosts. Ail is important for both attachment to host cells and for resistance to complement-dependent bacteriolysis. Previous studies have shown that Ail interacts with components of the extracellular matrix, including fibronectin, laminin and heparan sulfate proteoglycans, and with the complement inhibitor C4b-binding protein. Here, we demonstrate that Ail-expressing Y. pestis strains bind vitronectin - a host protein with functions in cell attachment, fibrinolysis and inhibition of the complement system. The Ail-dependent recruitment of vitronectin resulted in efficient cleavage of vitronectin by the outer membrane Pla (plasminogen activator protease). Escherichia coli DH5α expressing Y. pestis Ail bound vitronectin, but not heat-treated vitronectin. The ability of Ail to directly bind vitronectin was demonstrated by ELISA using purified refolded Ail in nanodiscs.

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 β-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.

BCL-XL is a major suppressor of apoptosis and a prominent member of the BCL-2 family. It is expressed in a variety of tissues and cell types and overexpressed in many tumors where it acts to promote tumor cell survival, tumor formation and tumor resistance to chemotherapy. Structure and activity studies have focused on truncated forms of the protein without the hydrophobic C-terminus that is essential for mediating the protein's association with intracellular membranes and cytoprotective activity. Hence, while the cytosolic state of the protein is relatively well understood, molecular information about the membrane-bound state is limited. We have used solution and solid-state NMR, together with isothermal titration calorimetry, to examine the structure and ligand binding activity of membrane-integrated BCL-XL, including its complete hydrophobic C-terminus. The fraction of BCL-XL expressed in E. coli as insoluble inclusion bodies can be isolated, purified and refolded into phospholipid bilayers. Here we describe the conformation of this membrane-bound state as a soluble globular domain anchored by a transmembrane C-terminal [[Unsupported Character - Symbol Font α]] helix. Membrane-bound BCL-XL binds a BID BH3 peptide with affinity similar to or slightly greater than the C-terminal truncated form. Implications for apoptosis from the conformation the membrane-integrated BCL-XL are discussed.Acknowledgements. This research was supported by grants from the National Institutes of Health (CA 179087; EB 002031).

CXCR1 is one of two high-affinity receptors for the CXC chemokine interleukin-8 (IL-8), a major mediator of immune and inflammatory responses implicated in many disorders, including tumour growth. IL-8, released in response to inflammatory stimuli, binds to the extracellular side of CXCR1. The ligand-activated intracellular signalling pathways result in neutrophil migration to the site of inflammation. CXCR1 is a class A, rhodopsin-like G-protein-coupled receptor (GPCR), the largest class of integral membrane proteins responsible for cellular signal transduction and targeted as drug receptors. Despite its importance, the molecular mechanism of CXCR1 signal transduction is poorly understood owing to the limited structural information available. Recent structural determination of GPCRs has advanced by modifying the receptors with stabilizing mutations, insertion of the protein T4 lysozyme and truncations of their amino acid sequences, as well as addition of stabilizing antibodies and small molecules that facilitate crystallization in cubic phase monoolein mixtures. The intracellular loops of GPCRs are crucial for G-protein interactions, and activation of CXCR1 involves both amino-terminal residues and extracellular loops. Our previous nuclear magnetic resonance studies indicate that IL-8 binding to the N-terminal residues is mediated by the membrane, underscoring the importance of the phospholipid bilayer for physiological activity. Here we report the three-dimensional structure of human CXCR1 determined by NMR spectroscopy. The receptor is in liquid crystalline phospholipid bilayers, without modification of its amino acid sequence and under physiological conditions. Features important for intracellular G-protein activation and signal transduction are revealed. The structure of human CXCR1 in a lipid bilayer should help to facilitate the discovery of new compounds that interact with GPCRs and combat diseases such as breast cancer.

BCL-XL is a dominant inhibitor of apoptosis and a significant anti-cancer drug target. Endogenous BCL-XL is integral to the mitochondrial outer membrane (MOM). Indeed, BCL-XL reconstituted in detergent-free lipid bilayer nanodiscs is anchored to the nanodisc lipid bilayer membrane by tight association of its C-terminal tail, while the N-terminal head retains the canonical structure determined for water-soluble, tail-truncated BCL-XL, with the surface groove solvent-exposed and available for BH3 ligand binding. To better understand the conformation and dynamics of this key region of BCL-XL we have developed methods for isolating the membrane-embedded C-terminal tail from its N-terminal head and for preparing protein suitable for structural and biochemical studies. Here, we outline the methods for sample preparation and characterization and describe previously unreported structural and dynamics features. We show that the C-terminal tail of BCL-XL forms a transmembrane α-helix that retains a significant degree of conformational dynamics. We also show that the presence of the intact C-terminus destabilizes the soluble state of the protein, and that the small fraction of soluble recombinant protein produced in E. coli is susceptible to proteolytic degradation of C-terminal residues beyond M218. This finding impacts the numerous previous studies where recombinant soluble BCL-XL was presumed to be full-length. Notably, however, the majority of recombinant BCL-XL produced in E. coli is insoluble and protected from proteolysis. This protein retains the complete C-terminal tail and can be reconstituted in lipid bilayers in a folded and active state.

The seven members of the FXYD protein family associate with the Na(+)-K(+) pump and modulate its activity. We investigated whether conserved cysteines in FXYD proteins are susceptible to glutathionylation and whether such reactivity affects Na(+)-K(+) pump function in cardiac myocytes and Xenopus oocytes. Glutathionylation was detected by immunoblotting streptavidin precipitate from biotin-GSH loaded cells or by a GSH antibody. Incubation of myocytes with recombinant FXYD proteins resulted in competitive displacement of native FXYD1. Myocyte and Xenopus oocyte pump currents were measured with whole-cell and two-electrode voltage clamp techniques, respectively. Native FXYD1 in myocytes and FXYD1 expressed in oocytes were susceptible to glutathionylation. Mutagenesis identified the specific cysteine in the cytoplasmic terminal that was reactive. Its reactivity was dependent on flanking basic amino acids. We have reported that Na(+)-K(+) pump β(1) subunit glutathionylation induced by oxidative signals causes pump inhibition in a previous study. In the present study, we found that β(1) subunit glutathionylation and pump inhibition could be reversed by exposing myocytes to exogenous wild-type FXYD3. A cysteine-free FXYD3 derivative had no effect. Similar results were obtained with wild-type and mutant FXYD proteins expressed in oocytes. Glutathionylation of the β(1) subunit was increased in myocardium from FXYD1(-/-) mice. In conclusion, there is a dependence of Na(+)-K(+) pump regulation on reactivity of two specifically identified cysteines on separate components of the multimeric Na(+)-K(+) pump complex. By facilitating deglutathionylation of the β(1) subunit, FXYD proteins reverse oxidative inhibition of the Na(+)-K(+) pump and play a dynamic role in its regulation.

The three-dimensional structure of the membrane-bound form of the major coat protein of Pf1 bacteriophage was determined in phospholipid bilayers using orientation restraints derived from both solid-state and solution NMR experiments. In contrast to previous structures determined solely in detergent micelles, the structure in bilayers contains information about the spatial arrangement of the protein within the membrane, and thus provides insights to the bacteriophage assembly process from membrane-inserted to bacteriophage-associated protein. Comparisons between the membrane-bound form of the coat protein and the previously determined structural form found in filamentous bacteriophage particles demonstrate that it undergoes a significant structural rearrangement during the membrane-mediated virus assembly process. The rotation of the transmembrane helix (Q16-A46) around its long axis changes dramatically (by 160 degrees) to obtain the proper alignment for packing in the virus particles. Furthermore, the N-terminal amphipathic helix (V2-G17) tilts away from the membrane surface and becomes parallel with the transmembrane helix to form one nearly continuous long helix. The spectra obtained in glass-aligned planar lipid bilayers, magnetically aligned lipid bilayers (bicelles), and isotropic lipid bicelles reflect the effects of backbone motions and enable the backbone dynamics of the N-terminal helix to be characterized. Only resonances from the mobile N-terminal helix and the C-terminus (A46) are observed in the solution NMR spectra of the protein in isotropic q1 bicelles, whereas only resonances from the immobile transmembrane helix are observed in the solid-state (1)H/(15)N-separated local field spectra in magnetically aligned bicelles. The N-terminal helix and the hinge that connects it to the transmembrane helix are significantly more dynamic than the rest of the protein, thus facilitating structural rearrangement during bacteriophage assembly.

Asn-Gly-Arg peptides have been designed as vehicles for the delivery of chemotherapeutics, magnetic resonance imaging contrast agents, and fluorescence labels to tumor cells, and cardiac angiogenic tissue. Specificity is derived via an interaction with aminopeptidase N, also known as CD13, a cell surface receptor that is highly expressed in angiogenic tissue. Peptides containing the CNGRC homing sequence tethered to a pro-apoptotic peptide sequence have the ability to specifically induce apoptosis in tumor cells. We have now identified a modification to the Asn-Gly-Arg homing sequence motif that improves overall binding affinity to aminopeptidase N. Through the addition of a proline residue, the new peptide with sequence, CPNGRC, inhibits aminopeptidase N proteolytic activity with an IC(50) of 10 microM, a value that is 30-fold lower than that for CNGRC. Both peptides are cyclized via a disulfide bridge between cysteines. Steady-state kinetic experiments suggest that efficient aminopeptidase N inhibition is achieved through the highly cooperative binding of two molecules of CPNGRC. We have used NMR-derived structural constraints for the elucidation of the solution structures CNGRC and CPNGRC. Resulting structures of CNGRC and CPNGRC have significant differences in the backbone torsion angles, which may contribute to the enhanced binding affinity and demonstrated enzyme inhibition by CPNGRC.

Tuberculosis (TB) is the seventh most common cause of death globally (1). Mycobacterium tuberculosis, its causative agent, has achieved a spread in the human population unmatched by any other bacterial pathogen (2), despite the availability of effective short-course chemotherapy and the Bacille Calmette-Guerin vaccine. Important factors contributing to the high pathogenicity of M. tuberculosis are its highly impermeable cell wall, which prevents the uptake of most hydrophilic antibiotic drugs, and its high adaptability to environmental changes during the course of infection, including: nutrient deprivation, hypoxia, various exogenous stress conditions and the phagosomal environment (3). The complete genome sequence of M. tuberculosis H37Rv, the best-characterized strain of the bacterium, has identified several stress response genes which contribute to pathogenicity (4). Among these, the membrane protein Rv0899, encoded by the rv0899 gene, is thought to confer adaptation of M. tuberculosis to the acidic environment of the phagosome (5). Sequence analysis of the mycobacterial genomes shows that the gene is restricted to pathogenic mycobacteria associated with TB (M. tuberculosis, M. bovis) and other TB-related diseases (M. marinum, M. ulcerans, M. kansaii) and, thus, is an attractive candidate for the development of anti-TB chemotherapeutic agents. Notably, two M. tuberculosis H37Rv genes (Rv0900 and Rv0901) adjacent to Rv0899 also encode putative membrane proteins, and are found exclusively in association with Rv0899 in the same pathogenic mycobacteria, suggesting that the three may constitute an operon dedicated to a common function. Amino acid sequence analysis reveals that the 326-residue Rv0899 contains three domains (Figure 1). The N-terminal domain (M; residues 1-72) includes a sequence of 20 hydrophobic amino acids (residues 28-50) that is required for membrane translocation but is not cleaved (6), thus constituting a membrane anchor. The central domain (B; residues 73-200) shares homology with the BON (Bacterial OsmY and Nodulation) superfamily (pfam04972), a conserved, putative lipid binding sequence that is found in some osmotic shock protection proteins, secretins, haemolysins, nodulation specificity proteins, and channels (7). Finally, the C-terminal domain (C; residues 201-326) has homology to the OmpA-C-like superfamily (pfam00691), a periplasmic peptidoglycan-binding domain that is found in several types of bacterial membrane proteins, including in the C-terminus of the outer membrane protein OmpA from E. coli. Owing to its homology with E. coli OmpA, the rv0899 gene was originally denoted as ompA in the published genome sequence of M. tuberculosis (4), and an outer membrane porin activity was proposed for its protein product, which was subsequently named OmpATb (8). Figure 1 Amino acid sequence (A), domain organization (B), and conserved domain homology (C) of M. tuberculosis Rv0899. The M domain contains 20 hydrophobic amino acids (residues 28-50) with transmembrane (TM) sequence similarity. The B domain contains a sequence ... Indeed, Rv0899 was isolated with the mycobacterial cell wall fraction (6, 9), and a pore-forming activity was measured in liposomes and in planar lipid bilayers for purified, recombinant Rv0899 produced in E. coli (6, 8, 10). The N-terminal hydrophobic sequence was found to be required for ion channel activity (6, 8) and proteolytic studies indicated that a trypsin-resistant polypeptide, spanning residues 73 to 326, was responsible for the pore-forming properties of Rv0899. Furthermore, a polypeptide spanning residues 73 to 220 was found to be sufficient for inducing the channel characteristics of the native protein in lipid bilayers, when it was expressed as a fusion with the E. coli OmpA signal sequence to direct it to its presumed outer membrane location (10). Transport experiments further led to the hypothesis that Rv0899 could function as a porin (5). The rv0899 gene was also shown to be significantly up-regulated in wild-type M. tuberculosis bacteria exposed to acidic conditions, whereas a mutant, lacking rv0899, displayed a substantially impaired ability to grow at acidic pH in macrophages and in mice (5), suggesting that rv0899 could facilitate the growth of M. tuberculosis in a mammalian host by helping it overcome host defense mechanisms. Indeed, pH adaptation is a common trait of pathogenic bacteria (11) and it has been shown that mycobacteria, including M. tuberculosis, maintain a neutral internal pH in an acidic environment (12-16). However, the mechanisms utilized by M. tuberculosis to adapt to acidic environments are poorly understood. The proposed porin activity and the pH adaption activity of Rv0899 seem to be at odds with each other, since a general response by Gram-negative bacteria to acid stress is to reduce porin expression (17-19). To gain further insights to the function of Rv0899 we have characterized the structure and dynamics of residues 73 to 326, spanning the B and C domains, and have determined the three-dimensional structure of the central B domain, including the BON homology sequence. Rv0899 does not form a transmembrane β-barrel. Residues 73 to 326 form a globular structure, which encompasses two independently folded modules, corresponding to the B and C domains connected by a flexible linker. Both domains adopt mixed α/β secondary structures. The B domain adopts a fold that had not been previously documented in the Protein Data Bank (PDB), with three parallel/antiparallel α-helices on one face of the protein, packed against six parallel/antiparallel β-strands which form a β-sheet on the opposite face. The structure reveals the fold of a BON homology domain for the first time. The C domain adopts the typical α/β structure of peptidoglycan-binding domains in the OmpA-C-like superfamily. The overall architecture of the protein and the unexpected structure of the B domain make it difficult to reconcile a porin activity with its central domain, but suggest alternative modes of membrane association. Throughout this article, we refer to the protein as Rv0899, rather than OmpATb, because its homology to E. coli OmpA is limited to the C domain, and because our studies show that it is not a β-barrel.

FXYD1 (phospholemman) is a member of an evolutionarily conserved family of membrane proteins that regulate the function of the Na,K-ATPase enzyme complex in specific tissues and specific physiological states. In heart and skeletal muscle sarcolemma, FXYD1 is also the principal substrate of hormone-regulated phosphorylation by c-AMP dependent protein kinase A and by protein kinase C, which phosphorylate the protein at conserved Ser residues in its cytoplasmic domain, altering its Na,K-ATPase regulatory activity. FXYD1 adopts an L-shaped alpha-helical structure with the transmembrane helix loosely connected to a cytoplasmic amphipathic helix that rests on the membrane surface. In this paper we describe NMR experiments showing that neither PKA phosphorylation at Ser68 nor the physiologically relevant phosphorylation mimicking mutation Ser68Asp induces major changes in the protein conformation. The results, viewed in light of a model of FXYD1 associated with the Na,K-ATPase alpha and beta subunits, indicate that the effects of phosphorylation on the Na,K-ATPase regulatory activity of FXYD1 could be due primarily to changes in electrostatic potential near the membrane surface and near the Na(+)/K(+) ion binding site of the Na,K-ATPase alpha subunit.

The BH3-only BCL-2 family protein BID is activated by caspase-8 cleavage upon engagement of cell surface death receptors. The resulting 15 kDa C-terminal fragment, tBID, translocates to mitochondria, triggering the release of cytotoxic molecules and cell death. The pro-apoptotic activity of tBID is regulated by its interactions with pro-survival BCL-XL and pro-death BAX, both in the cytosol and at the mitochondrial membrane. In this study, we characterize the molecular interactions between full-length tBID and BCL-XL using NMR spectroscopy and isothermal titration calorimetry (ITC). In aqueous solution, tBID adopts an alpha-helical but dynamically disordered conformation; however, the three-dimensional conformation is stabilized when tBID engages its BH3 domain in the BH3-binding hydrophobic groove of BCL-XL to form a stable heterodimeric complex. Characterization of the binding thermodynamics by ITC reveals that the interaction between tBID and BCL-XL is driven by enthalpy but disfavored by the entropy associated with the conformational order induced in tBID upon binding BCL-XL.

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.

Background: Phospholemman (PLM) is an abundant phosphoprotein in the plasma membrane of cardiac, skeletal and smooth muscle. It is a member of the FXYD family of proteins that bind to and regulate the Na,K-ATPase. Protein kinase A (PKA) is known to phosphorylate PLM on serine 68 (S68), although the functional effect of S68 PLM phosphorylation is unclear. We therefore evaluated S68 PLM phosphorylation in swine carotid arteries. Methods: Two anti-PLM antibodies, one to S68 phosphorylated PLM and one to unphosphorylated PLM, were made to PLM peptides in rabbits and tested with purified PLM and PKA-treated PLM. Swine carotid arteries were mounted isometrically, contracted, relaxed with forskolin and then homogenized. Proteins were separated on SDS gels and the intensity of immunoreactivity to the two PLM antibodies determined on immunoblots. Results: The antipeptide antibody ‘C2’ primarily reacted with unphosphorylated PLM, and the antipeptide antibody ‘CP68’ detected S68 PLM phosphorylation. Histamine stimulation of intact swine carotid artery induced a contraction, increased the CP68 PLM antibody signal and reduced the C2 PLM antibody signal. High extracellular [K+] depolarization induced a contraction without altering the C2 or CP68 PLM signal. Forskolin-induced relaxation of histamine or extracellular [K+] contracted arteries correlated with an increased CP68 signal. Nitroglycerin-induced relaxation was not associated with changes in the C2 or CP68 PLM signal. Conclusions: These data suggest that a contractile agonist increased S68 PLM phosphorylation. Agents that increase [cAMP], but not agents that increase [cGMP], increased S68 PLM phosphorylation. S68 PLM phosphorylation may be involved in cAMP-dependent regulation of smooth muscle force.

Apoptosis, a mechanism for programmed cell death, has key roles in human health and disease. Many signals for cellular life and death are regulated by the BCL-2 family proteins and converge at mitochondria, where cell fate is ultimately decided. The BCL-2 family includes both pro-life (e.g. BCL-XL) and pro-death (e.g. BAX, BAK) proteins. Previously, it was thought that a balance between these opposing proteins, like a simple ‘rheostat', could control the sensitivity of cells to apoptotic stresses. Later, this rheostat concept had to be extended, when it became clear that BCL-2 family proteins regulate each other through a complex network of bimolecular interactions, some transient and some relatively stable. Now, studies have shown that the apoptotic circuitry is even more sophisticated, in that BCL-2 family interactions are spatially dynamic, even in nonapoptotic cells. For example, BAX and BCL-XL can shuttle between the cytoplasm and the mitochondrial outer membrane (MOM). Upstream signaling pathways can regulate the cytoplasmic–MOM equilibrium of BAX and thereby adjust the sensitivity of cells to apoptotic stimuli. Thus, we can view the MOM as the central locale of a dynamic life–death rheostat. BAX invariably forms extensive homo-oligomers after activation in membranes. However, recent studies, showing that activated BAX monomers determine the kinetics of MOM permeabilization (MOMP), perturb the lipid bilayer and form nanometer size pores, pose questions about the role of the oligomerization. Other lingering questions concern the molecular mechanisms of BAX redistribution between MOM and cytoplasm and the details of BAX/BAK–membrane assemblies. Future studies need to delineate how BCL-2 family proteins regulate MOMP, in concert with auxiliary MOM proteins, in a dynamic membrane environment. Technologies aimed at elucidating the structure and function of the full-length proteins in membranes are needed to illuminate some of these critical issues.

Rv0899 is a membrane protein encoded by an operon that is required for fast ammonia secretion, rapid pH neutralization and growth of M. tuberculosis in acidic environments. The gene is restricted to pathogenic mycobacteria and, thus, is an attractive candidate for the development of anti-tuberculosis chemotherapy. Rv0899 contains three independently structured domains. The N-terminus (Rv0899-M; residues 1-80) includes a membrane-anchoring sequence of 20 hydrophobic amino acids (residues 28-50) that is required for membrane translocation. The central region (Rv0899-B; residues 81-194) contains two consecutive repeats of the BON (Bacterial OsmY and Nodulation) domain (pfam04972), a conserved, putative lipid binding sequence that is found in some bacterial osmotic shock protection proteins. Finally, the C-terminus (Rv0899-C; residues 195-326) contains an OmpA-like domain (pfam00691), a periplasmic peptidoglycan-binding structure found in several types of bacterial membrane proteins. Using NMR spectroscopy, we determined the high-resolution structures of Rv0899-B and Rv0899-C. Rv0899-B does not form a transmembrane beta barrel and has an undocumented mixed alpha/beta fold. Furthermore, we found Rv0899-B has a specific zinc binding site and associates with lipid vesicles in a pH dependent manner. The structure of Rv0899-C adopts the characteristic OmpA-like family fold. It exhibits pH-dependent conformational dynamics, with significant disorder at neutral pH and a more ordered structure at acidic pH. Rv0899-C associates tightly with polymeric peptidoglycan isolated from M. tuberculosis and also associates with a soluble peptide intermediate of peptidoglycan biosynthesis. This enabled us to characterize the peptidoglycan binding site where five highly conserved Rv0899-C residues, including two key arginines, establish the specificity for DAP- but not Lys-type peptidoglycan. The three-dimensional structure of Rv0899 suggests alternative possible modes of membrane association and provides a molecular basis for the screening of small molecules with anti-TB potential.

The three Yersinia species that are pathogenic for humans each cause distinctive diseases, ranging from gastroenteritis (Y. pseudotuberculosis, Y. enterocolitica) to plague (Y. pestis), one of the most deadly human infectious diseases, classified as a Category A Biothreat Agent. In all three, host recognition and resistance to the complement system are associated with the bacterial outer membrane protein Ail (Attachment invasion locus) and its interactions with the human host proteins Fn (Fibronectin) and Vn (Vitronectin), which play important functions in adhesion, and C4BP (complement component 4b binding protein), which plays important functions in immunity.Ail belongs to the Ail/Lom family (pfam PF06316) of outer membrane proteins, whose members share amino acid sequence homology in the membrane-spanning segments, but vary widely in the sequences of the extracellular loops. E. coli OmpX is regarded as the prototypical member of this family because it is the only one for which the three-dimensional structure, a transmembrane eight-stranded β-barrel, has been determined. However, while Ail has marked adhesion/invasion activity and is essential for virulence, OmpX has no identified function and is not essential. The four extracellular loops of Ail have completely different amino acid sequences than those of OmpX and are thought to be responsible for function. Here we present NMR results for Ail reconstituted in lipid bilayers and lipid micelles as well as binding assays aimed at characterizing the interactions of ail with its human host partners.

Mycobacterium tuberculosis ArfA (Rv0899) is a membrane protein encoded by an operon that is required for supporting bacterial growth in acidic environments. Its C-terminal domain (C domain) shares significant sequence homology with the OmpA-like family of peptidoglycan-binding domains, suggesting that its physiological function in acid stress protection may be related to its interaction with the mycobacterial cell wall. Previously, we showed that ArfA forms three independently structured modules, and we reported the structure of its central domain (B domain). Here, we describe the high-resolution structure and dynamics of the C domain, we identify ArfA as a peptidoglycan-binding protein and we elucidate the molecular basis for its specific recognition of diaminopimelate-type peptidoglycan. The C domain of ArfA adopts the characteristic fold of the OmpA-like family. It exhibits pH-dependent conformational dynamics (with significant heterogeneity at neutral pH and a more ordered structure at acidic pH), which could be related to its acid stress response. The C domain associates tightly with polymeric peptidoglycan isolated from M. tuberculosis and also associates with a soluble peptide intermediate of peptidoglycan biosynthesis. This enabled us to characterize the peptidoglycan binding site where five highly conserved ArfA residues, including two key arginines, establish the specificity for diaminopimelate- but not Lys-type peptidoglycan. ArfA is the first peptidoglycan-binding protein to be identified in M. tuberculosis. Its functions in acid stress protection and peptidoglycan binding suggest a link between the acid stress response and the physicochemical properties of the mycobacterial cell wall.

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 β-sheet conformation in lipids.

The FXYD membrane proteins constitute a family of conserved auxiliary subunits of the Na,K-ATPase, and have been the focus of recent attention due to their ability to finely regulate the activity of the enzyme complex in various physiological settings. In this review we describe the structures of the proteins, as well as their dynamics and their associations with the lipid bilayer membrane, which we have recently determined by NMR spectroscopy. Although the proteins are relatively small, their genes contain as many as six to nine small exons, and the coincidence of structured protein segments with their genetic elements suggests assembly from discrete structural modules through exon shuffling. The three-dimensional structures and backbone dynamics provide the foundation for understanding their intra-membrane association with the Na,K-ATPase alpha subunit, and the structure of FXYD1 suggests a mechanism whereby the phosphorylation of conserved Ser residues, by protein kinases A and C, could induce a conformational change in the cytoplasmic domain of the protein, to modulate its interaction with the alpha subunit.

Humanin is a short endogenous peptide, which can provide protection from cell death through its association with various receptors, including the pro-apoptotic Bcl-2 family proteins Bid, Bim, and Bax. By using NMR chemical shift mapping experiments, we demonstrate that the interaction between Humanin-derived peptides and Bid is specific, and we localize the binding site to a region on the surface of Bid, which includes residues from the conserved helical BH3 domain of the protein. The BH3 domain mediates the association of Bid with other Bcl-2 family members and is essential for the protein's cytotoxic activity. The data suggest that Humanin exerts its cytoprotective activity by engaging the Bid BH3 domain; this would hinder the association of Bid with other Bcl-2 family proteins, thereby mitigating its toxicity. The identification of a Humanin-specific binding site on the surface of Bid reinforces its importance as a direct modulator of programmed cell death, and suggests a strategy for the design of cytoprotective peptide inhibitors of Bid.

Fred Naider, Boris Arshava, Haibo Xie, Shi-feng Liu, Woei Y. Eng, Shu-Hua Wang, Kathleen Valentine, Gianluigi Veglia, Francesca Marassi, Stanley J. Opella, and Jeffrey M. Becker 1 College of Staten Island, CUNY, S.I., NY 10314, U.S.A.; 2 University of Pennsylvania, Philadelphia, PA 19104, U.S.A.; Wistar Institute, Philadelphia, PA 19104, U.S.A.; and University of Tennessee, Knoxville, TN 37996, U.S.A.

Tuberculosis is a deadly disease with a death toll of 1.5 million people every year and very recently an outbreak in Jacksonville, FL was devastating. All frontline antibiotics are failed for the multidrug resistant (MDR) bacilli and the key to disease control is to inhibit bacterial cell division and stabilize the bacilli in its dormant stage. Here we present the structure function studies of a novel drug target membrane protein CrgA in M. tuberculosis peptidoglycan polymerization complex. We show that CrgA interacts strongly with FtsZ, FtsQ, CwsA and the class B penicillin binding proteins such as FtsI and PBPA in M. smegmatis. CwsA a binding partner of CrgA binds to Wag31 another key protein in peptidoglycan synthesis. Co-localization of CrgA with FtsZ in the mid cell region facilitates the Z-ring formation during the cell division process. Thus structural characterization of CrgA in a lipid bilayer is essential. Full length CrgA has 2 transmembrane (TM) helices, a 32 residue N terminal domain, a very short C terminal domain, and a 10 residue inter-helical loop for a total of 93 residues and an 11.4 kDa molecular weight. Full length 15N, 13C uniform and amino acid specific labeled CrgA was over-expressed and purified from E. coli with a yield of 35 mg/L. To mimic the membrane environment, isotope labeled CrgA was reconstituted in POPC:POPG liposomes. Orientation dependent 2D Polarization Inversion Spin Exchange at Magic Angle (PISEMA) and 3D Magic Angle Spinning (MAS) solid state NMR experiments were performed to obtain structural restraints. For all ssNMR experiments Low E probes in 600 and 900 MHz magnets were used at the NHMFL. CrgA TMD structure was calculated by CS-Rosetta and XPLOR-NIH. Structure refinement was enhanced by restrained molecular dynamics in a lipid bilayer environment.

The Bcl-2 family proteins are key regulators of programmed cell death, in health and major human diseases, including cancer. Their pro- or anti-apoptotic functions are regulated by subcellular location, as the proteins cycle between soluble and membrane-bound forms; by dimerization with other Bcl-2 family members; by binding to other non-homologous proteins; and by formation of membrane pores that are believed to regulate apoptosis by perturbing mitochondrial physiology. The solution structures of several Bcl-2 family proteins are very similar despite their antagonistic activities, however, the structures of the membrane-associated proteins are not known and may be key to their opposing functions. The goals of this project are: (1) to determine the structures of the membrane-associated Bcl-2 proteins; and (2) to determine their mechanism of apoptosis regulation. The research strategy combines NMR structure determination in lipid environments with biological assays carried out in parallel with structure determination.

Solid-state NMR spectroscopy is being used to determine the structures of membrane proteins involved in the regulation of apoptosis and ion transport. The Bcl-2 family includes pro- and anti-apoptotic proteins that play a major regulatory role in mitochondrion-dependent apoptosis or programmed cell death. The NMR data obtained for 15N-labeled anti-apoptotic Bcl-xL in lipid bilayers are consistent with membrane association through insertion of the two central hydrophobic α-helices that are also required for channel formation and cytoprotective activity. The FXYD family proteins regulate ion flux across membranes, through interaction with the Na+, K+-ATPase, in tissues that perform fluid and solute transport or that are electrically excitable. We have expressed and purified three FXYD family members, Mat8 (mammary tumor protein), CHIF (channel-inducing factor) and PLM (phospholemman), for structure determination by NMR in lipids. The solid-state NMR spectra of Bcl-2 and FXYD proteins, in uniaxially oriented lipid bilayers, give the first view of their membrane-associated architectures. Copyright © 2004 John Wiley & Sons, Ltd.

A solid-state NMR approach for simultaneous resonance assignment and three-dimensional structure determination of a membrane protein in lipid bilayers is described. The approach is based on the scattering, hence the descriptor "shotgun," of (15)N-labeled amino acids throughout the protein sequence (and the resulting NMR spectra). The samples are obtained by protein expression in bacteria grown on media in which one type of amino acid is labeled and the others are not. Shotgun NMR short-circuits the laborious and time-consuming process of obtaining complete sequential assignments prior to the calculation of a protein structure from the NMR data by taking advantage of the orientational information inherent to the spectra of aligned proteins. As a result, it is possible to simultaneously assign resonances and measure orientational restraints for structure determination. A total of five two-dimensional (1)H/(15)N PISEMA (polarization inversion spin exchange at the magic angle) spectra, from one uniformly and four selectively (15)N-labeled samples, were sufficient to determine the structure of the membrane-bound form of the 50-residue major pVIII coat protein of fd filamentous bacteriophage. Pisa (polarity index slat angle) wheels are an essential element in the process, which starts with the simultaneous assignment of resonances and the assembly of isolated polypeptide segments, and culminates in the complete three-dimensional structure of the protein with atomic resolution. The principles are also applicable to weakly aligned proteins studied by solution NMR spectroscopy. [The structure we determined for the membrane-bound form of the Fd bacteriophage pVIII coat protein has been deposited in the Protein Data Bank as PDB file 1MZT.]

Rv0899 (OmpATb) is a 326-residue membrane-associated virulence factor of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis. It is essential for the adaptation of Mtb to acidic environments and has been identified as an outer membrane protein. The M domain (residues 1-72) contains a 20-residue hydrophobic sequence that may form a membrane-anchoring helix. The B domain (residues 73-195) has been described as pore-forming. It shares sequence homology with the “BON” domain of Bacterial Osmotic-shock-resistance, Nodulation-specificity and lipid-binding proteins. The C domain (residue 196-326) shares significance homology with other bacterial peptidoglycan-binding domains, including the C-terminus of the E. coli outer membrane protein OmpA, after which Rv0899 was originally named. Using NMR spectroscopy, we show that residues 73-326, spanning the joint B and C domains, adopt a well-defined three-dimensional structure in water solution, however, the individual B and C domains fold independently and interact with each other to a minimal extent. The B domain adopts a rigid and stable structure, that forms a six-stranded β-sheet protected on one side by three α-helices. No comparable arrangement of secondary structure elements could be found in the databases, suggesting that is a new fold. The structure of the B domain is suggestive of a lipid binding or structural function, and provides insights to the role of Rv0899 in Mtb virulence.(This research was supported by the National Institutes of Health (NIH), and utilized the Burnham Institute NMR Facility supported by the NIH National Cancer Institute).

In this issue of Molecular Cell, Kim et al. (2009) describe the steps involved in the direct activation of the proapoptotic proteins BAX and BAK by their BH3-only partners, resolving the controversy regarding direct versus indirect activation of these proteins.

The BCL-2 family proteins are major regulators of mitochondrion-dependent programmed cell death. They includes both pro-death and pro-life proteins, which exert their activities through physical interactions with each other with other non-homologous proteins, and with intracellular membranes. The BH3-only cytotoxic protein BID is activated by caspase-8 cleavage upon engagement of cell surface death receptors. The resulting C-terminal fragment, tBID, translocates to mitochondria, triggering the release of cytotoxic molecules and cell death. The activity of tBID is regulated by its interactions with pro-survival BCL-XL and pro-death BAX, both in the cytosol and at the mitochondrial membrane. Using NMR spectroscopy we show that full length BCL-XL is soluble and monomeric in aqueous solution. Its hydrophobic C-terminal tail, which is predicted to form a transmembrane helix in lipid membranes, folds back to interact with the hydrophobic pocket known to bind the BH3 domains of pro-apoptotic proteins. The presence of the C terminus reduces the binding affinity of BCL-XL for BH3 domains 4-fold, compared to the affinity of truncated BCL-XL. The activated pro-apoptotic protein tBID adopts an α-helical but dynamically disordered conformation in solution. However, its three-dimensional conformation is stabilized when tBID engages its BH3 domain in the BH3-binding hydrophobic groove of BCL-XL to form a stable heterodimeric complex. Studies in lipid micelles show that the proteins' conformations and interactions are dramatically different in the presence of lipids.[This research was supported by the National Institutes of Health (NIH), and utilized the Burnham Institute Structural Biology Facility supported by the NIH National Cancer Institute.]

Antimicrobial peptides are found in a large variety of organisms throughout all branches of cellular life: bacteria, archaea, and eukarya. They provide an evolutionarily ancient mechanism of defense against microbial invasion and play a fundamental role in the innate immunity of animals and humans. The initial discovery of Cecropin peptides in silk moth pupae, where they are expressed in response to insect injury and infection and during development (1), and the subsequent isolation of Magainin peptides from the skin of the African clawed frog, Xenopus laevis (2), established their importance as potent antibiotics (3). Early electrophysiological studies showed that Magainins, and other related peptides, cause highly selective and rapid lysis of bacteria in a concentration-dependent manner. The peptides are capable of forming pores that rapidly disrupt the electrical potential across membranes of bacterial cells, isolated mitochondria, and synthetic vesicles (3). Furthermore, the biological activities of antimicrobial peptides are highly synergistic; strong synergism is observed in their antimicrobial activity as well as in their cytotoxicity for other cell types, including transformed tumor cells (3). The finding that antimicrobial peptides synthesized from all D amino acids retain their full biological activity provided the first evidence that their function does not involve interaction of the peptide with a membrane receptor but instead relies on interaction of the peptide with the lipid bilayer membrane (4). This is consistent with the highly positively charged amphiphilic structures common to all peptides, which confer specificity for the predominantly electronegative membranes of bacteria. However, the functional mechanism of antimicrobial peptide activity has been a subject of intense discussion. In this issue, Strandberg et al. (5) describe the subtle effects of phospholipid structure on the interactions of antimicrobial peptides with membranes. Their results provide an explanation for the peptides' selective cytotoxicity for bacteria. Strandberg et al. (5) demonstrate that the interactions of both individual and synergized antimicrobial peptides with membranes depend not only on the electrostatic attraction between a cationic peptide and the anionic membrane surface, but also, very significantly, on the fundamental chemical structures of the phospholipids that influence their predisposition for membrane curvature. Negative curvature predisposition is observed in lipids with unsaturated hydrocarbon chains, while more ordered, saturated chains typically result in positive curvature (Fig. 1). Figure 1 Antimicrobial peptide (AMP) surface association is promoted by lipids with negative curvature predisposition. Lipids are shown with anionic headgroups (red) and hydrocarbon chains (yellow). The amphiphilic helical peptide, viewed down its helix axis, ... While, in some cases, antimicrobial peptides can insert across lipid bilayer membranes, their predominant conformation as membrane surface-associated α-helices was demonstrated early on by solid-state NMR structural studies (6,7). Membrane surface association has been elegantly rationalized as an essential early step in the functional mechanism of antimicrobial peptides, leading to massive pore formation and bacterial cell death. Rather than forming a classic channel with peptide helices inserted across the lipid bilayer membrane, the accumulation of peptides at the membrane surface is thought to cause a strain in the lipid bilayer, resulting in the formation of toroidal pores where the essential alignment of the peptide helices relative to the geometry of individual lipids is maintained (8–10). In a series of elegant solid-state NMR experiments Strandberg et al. (5) probe the membrane-associated conformations of two synergistic Magainin peptides, MAG2 and PGLa, as a function of phospholipid bilayer composition. The data unequivocally show that the membrane-surface-associated states of both isolated and combined MAG2 and PGLa are stabilized by phospholipids whose structures predispose them to negative membrane curvature. MAG2 and PGLa do not insert in lipid bilayers composed of negative curvature lipids with unsaturated hydrocarbon chains, regardless of the hydrocarbon chain length. By contrast, phospholipids with fully saturated dimyristoyl and dipalmitoyl hydrocarbon chains and a predisposition for positive curvature allow transmembrane insertion of PGLa and antagonize the surface-bound state. Notably, such lipids also fail to elicit appreciable membrane depolarization by the peptides (3), suggesting that the membrane inserted state may not be critical for function. The results obtained for membranes composed of bacterial phospholipids are particularly revealing. In the presence of bacterial lipids, with palmitoyl and oleoyl hydrocarbon chains and cardiolipin, phosphoethanolamine, and phospho-glycerol headgroups, both MAG2 and PGLa helices always align parallel to the membrane surface. These lipids have a marked propensity for negative membrane curvature and are enriched in bacterial membranes against which Magainin peptides exhibit their highest biological activity. Thus, the data suggest that membrane surface association of the peptides, promoted by negative curvature lipids, is a key aspect of their antimicrobial cytotoxicity. Recently, cardiolipin microdomains have been shown to localize to negatively curved regions of Escherichia coli membranes (11) and a clear phenomenological link has been demonstrated between anionic lipid clustering and the bacterial species specificity of several antimicrobial agents (12). The results of Strandberg et al. (5) help explain how lipid clustering could result in bacterial toxicity: the anionic and negative curvature properties of bacterial lipid clusters could conspire to recruit the accumulation of antimicrobial peptides and maintain them in a membrane surface orientation up to the critical concentration beyond which membrane disruption and massive pore formation are inevitable and commit the cell to death. Interestingly, the mitochondrial trans-acylating enzyme Tafazzin was recently shown to interact with specific pools of lipids that possess negative curvature properties (13). Just as proposed for Tafazzin, the data of Strandberg et al. (5) do not demonstrate that the peptides interact with curved membrane regions but, instead, how they interact with lipids that have a natural tendency to induce negative curvature in membranes. Antimicrobial peptides have been the focus of a large number of biophysical and biological studies aimed at understanding the molecular basis for their activity. Now, Strandberg et al. (5) provide significant new insights that can advance their development as effective therapeutics.

Ail (Attachment invasion locus) is a virulence factor outer membrane protein of Yersinia pestis, a devastating human pathogen that causes plague. Ail plays key roles in mediating adhesion and providing resistance to human complement, making it a prime candidate target for drug development. It interacts with several human host proteins, including fibronectin, to regulate adhesion and innate immunity. Ail belongs to the Ail/Lom family (pfam PF06316) of outer membrane proteins, whose members share amino acid sequence homology in the membrane-spanning segments, but vary widely in the sequences of the extracellular loops. E. coli OmpX is regarded as the prototypical member of this family, with transmembrane eight-stranded β-barrel and four extracellular loops. However, while Ail has marked adhesion/invasion activity and is essential for virulence, OmpX has no identified function and is not essential. The four extracellular loops of Ail are thought to be responsible for function. Here we present NMR structural data obtained for Ail in detergent micelles, phospholipid vesicles and phospholipid nanodiscs. Since Ail is an integral membrane protein, understanding the molecular mechanism underpinning its function requires structure determination within the phospholipid bilayer membrane. NMR spectroscopy is unique in its ability to provide high-resolution information in lipid environments that closely resemble the cellular membranes. The NMR results are correlated with functional studies that characterize the interactions of Ail with its human protein partners essential for its function in cell adhesion.

Integral membrane proteins regulate major cellular processes in health and disease, including transport, signaling, secretion, adhesion, pathogenesis, and apoptosis, and therefore, represent important targets for structural and functional characterization. Membrane protein structures and functions are regulated by their physical interactions with the surrounding lipids, and NMR is unique in its ability to provide high-resolution information in lipid environments that closely resemble the cellular membranes. Solid-state NMR experiments with proteins in oriented bilayers, and solution NMR experiments with proteins in weakly oriented micelles, provide high-resolution orientation-dependent restraints, which can be combined for protein structure determination and refinement. As previously observed for helical membrane proteins, the NMR spectra of outer membrane barrels in lipid bilayers exhibit characteristic patterns that reflect both protein structure and intra-membrane orientation. Results are presented for mammalian and bacterial α-helical and β-stranded membrane proteins. The NMR structures characterized in lipids provide insights to their distinct functions.(This research was supported by the National Institutes of Health.)

Tuberculosis is the seventh most common cause of death globally. The complete genome sequence of Mycobacterium tuberculosis, its causative agent, has allowed identification of several stress response genes that contribute to pathogenicity. Among these, the membrane protein Rv0899 is a virulence factor that confers adaptation of M. tuberculosis to the acidic environment of the phagosome. Its gene is restricted to pathogenic mycobacteria associated with TB and other TB-related diseases and, thus, is an attractive candidate for the development of anti-TB chemotherapeutic agents. We have determined the three dimensional structure and dynamics of residues 73-326 of this 326-residue protein. In contrast to the original predictions, residues 73-326 form a globular structure, which encompasses two independently folded domains, with mixed α/β-secondary structure, connected by a flexible linker. The central B domain (residues 80-195) reveals for the first time the fold of a BON homology domain associated with bacterial osmotic shock resistance, nodulation-specificity and lipid-binding proteins. The C-terminal domain (residues 205-326) adopts the typical fold of peptidoglycan-binding domains, and also binds peptidoglycan suggesting a periplasmic localization for this part of the protein. Residues 1-73 contain a 20-residue hydrophobic sequence that forms a transmembrane anchor. The overall architecture of the protein, its ligands, and the unexpected structure of the B domain make it difficult to reconcile a porin activity with its central domain but suggest alternative modes of membrane association.[This research was supported by the National Institutes of Health (AI074805, GM075917). It utilized the NMR Facilities at the Sanford Burnham Medical Research Institute and at the University of California, San Diego, supported by grants from the National Institutes of Health (CA030199, EB002031, RR23773)].

Yersinia pestis, the causative agent of plague, is a highly pathogenic organism that spreads rapidly and causes extremely high human mortality. Y. pestis is sensitive to a restricted panel of antibiotics, however, it is classified as a Tier 1 Select Agent due to its extraordinary pathogenicity, the potential weaponization of aerolized bacteria with bio-engineered antibiotic resistance, and the lack of an effective vaccine. The outer membrane protein Ail (attachment invasion locus) is a Y. pestis virulence factor and a prime candidate for therapeutic development due to its two principal activities in mediating bacterial adhesion to host cells and promoting resistance to human complement. Here we present the backbone structure of Ail, determined by NMR spectroscopy in detergent micelles. We also describe activity assays that provide information about the interactions of Ail with its human ligands. Furthermore, we present the results of NMR and activity studies performed with Ail incorporated in phospholipid liposomes and nanodiscs. Overall the data highlight the importance of obtaining structural and functional data in the native-like heterogeneous environment of the lipid bilayer membrane, where the extracellular loops of the protein can be solvated by water and the transmembrane barrel is embedded in the hydrophobic membrane core.This research is supported by a grant from the National Institutes of Health (GM100265). It utilized the NIH-supported NMR Facilities at the Sanford-Burnham Medical Research Institute (CA030199) and at the University of California San Diego (EB002031).

CXCR1 is a receptor for the chemokine interleukin-8 (IL-8), a mediator of immune and inflammatory responses. Strategically located in the cell membrane, CXCR1 binds to IL-8 with high affinity, and subsequently transduces a signal across the membrane bilayer to a G-protein activated second messenger system. Here, we describe the three-dimensional structure of human CXCR1 determined in phospholipid bilayers under physiological conditions by solid-state NMR spectroscopy. We use a five-step approach: (1) prepare a sample of a uniformly 13C/15N labeled CXCR1 in proteoliposomes by bacterial expression, purification, and refolding. (2) Resolve individual signals with MAS solid-state NMR experiments. (3) Assign each signal to a specific residue. (4) Measure two or more orientation-dependent frequencies for each residue and some distances. (5) Calculate de novo three-dimensional structure. The availability of a method for determining the structures of unmodified GPCRs in their native environment of phospholipid bilayers under physiological conditions has the potential to transform the field of structural determination of membrane proteins, and to accelerate the discovery of drugs that interact with membrane-associated receptors. Progress towards determining the structure and function of IL-8 bound to CXCR1 in membrane environments will be described.