Structural basis for the association of PLEKHA7 with membrane-embedded phosphatidylinositol lipids.

PLEKHA7 (pleckstrin homology domain containing family A member 7) plays key roles in intracellular signaling, cytoskeletal organization, and cell adhesion, and is associated with multiple human cancers. The interactions of its pleckstrin homology (PH) domain with membrane phosphatidyl-inositol-phosphate (PIP) lipids are critical for proper cellular localization and function, but little is known about how PLEKHA7 and other PH domains interact with membrane-embedded PIPs. Here we describe the structural basis for recognition of membrane-bound PIPs by PLEHA7. Using X-ray crystallography, nuclear magnetic resonance, molecular dynamics simulations, and isothermal titration calorimetry, we show that the interaction of PLEKHA7 with PIPs is multivalent, distinct from a discrete one-to-one interaction, and induces PIP clustering. Our findings reveal a central role of the membrane assembly in mediating protein-PIP association and provide a roadmap for understanding how the PH domain contributes to the signaling, adhesion, and nanoclustering functions of PLEKHA7.

Conformational States of the Cytoprotective Protein Bcl-xL.

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 anticancer therapy and for studies aimed at understanding its structure and activity. Although 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 the conformation of full-length Bcl-xL. Using NMR spectroscopy, molecular dynamics simulations, and isothermal titration calorimetry, we show how the three structural elements affect the protein's structure, dynamics, and ligand-binding activity in both its soluble and membrane-anchored states. The combined data provide information about the molecular basis for the protein's functionality and a view of its complex molecular mechanisms.

Reconstitution and Characterization of BCL-2 Family Proteins in Lipid Bilayer Nanodiscs.

The BCL-2 family proteins are key regulators of programmed cell death or apoptosis, and represent important targets for the development of anticancer drugs. Because their functions are intimately connected with intracellular membranes, it is important to perform structural and activity studies in precisely characterized samples that include phospholipids and capture the features of the native physiological environment as closely as possible. NMR studies and activity assays based on lipid bilayer nanodiscs are ideally suited for this purpose: they enable the conformations and interactions of these proteins to be probed at atomic resolution in their membrane-associated states. Here we describe detailed protocols for generating the protein components and the reconstituted nanodisc samples suitable for NMR studies and functional assays. The protocols focus on the BCL-2 family protein BCL-XL, a dominant inhibitor of programmed cell death and a major anticancer drug target. The protocols are relatively straightforward. Provided care is taken to ensure protein integrity and sample homogeneity, BCL-XL can be readily reconstituted in nanodiscs, with its hydrophobic C-terminal tail anchored through the nanodisc lipid bilayer, and its folded N-terminal head and ligand binding pocket exposed to the aqueous solution. We anticipate that BCL-2 samples prepared with these protocols will advance structural and mechanistic studies for this important protein family.

Regulation of apoptosis by an intrinsically disordered region of Bcl-xL.

Intrinsically disordered regions (IDRs) of proteins often regulate function upon post-translational modification (PTM) through interactions with folded domains. An IDR linking two α-helices (α1-α2) of the antiapoptotic protein Bcl-xL experiences several PTMs that reduce antiapoptotic activity. Here, we report that PTMs within the α1-α2 IDR promote its interaction with the folded core of Bcl-xL that inhibits the proapoptotic activity of two types of regulatory targets, BH3-only proteins and p53. This autoregulation utilizes an allosteric pathway whereby, in one direction, the IDR induces a direct displacement of p53 from Bcl-xL coupled to allosteric displacement of simultaneously bound BH3-only partners. This pathway operates in the opposite direction when the BH3-only protein PUMA binds to the BH3 binding groove of Bcl-xL, directly displacing other bound BH3-only proteins, and allosterically remodels the distal site, displacing p53. Our findings show how an IDR enhances functional versatility through PTM-dependent allosteric regulation of a folded protein domain.

Structure of monomeric Interleukin-8 and its interactions with the N-terminal Binding Site-I of CXCR1 by solution NMR spectroscopy.

The structure of monomeric human chemokine IL-8 (residues 1-66) was determined in aqueous solution by NMR spectroscopy. The structure of the monomer is similar to that of each subunit in the dimeric full-length protein (residues 1-72), with the main differences being the location of the N-loop (residues 10-22) relative to the C-terminal α-helix and the position of the side chain of phenylalanine 65 near the truncated dimerization interface (residues 67-72). NMR was used to analyze the interactions of monomeric IL-8 (1-66) with ND-CXCR1 (residues 1-38), a soluble polypeptide corresponding to the N-terminal portion of the ligand binding site (Binding Site-I) of the chemokine receptor CXCR1 in aqueous solution, and with 1TM-CXCR1 (residues 1-72), a membrane-associated polypeptide that includes the same N-terminal portion of the binding site, the first trans-membrane helix, and the first intracellular loop of the receptor in nanodiscs. The presence of neither the first transmembrane helix of the receptor nor the lipid bilayer significantly affected the interactions of IL-8 with Binding Site-I of CXCR1.

Orphan Nuclear Receptor NR4A1 Binds a Novel Protein Interaction Site on Anti-apoptotic B Cell Lymphoma Gene 2 Family Proteins.

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.

Characterization of the membrane-inserted C-terminus of cytoprotective BCL-XL.

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). 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 Escherichia 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. Nevertheless, 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.

A Practical Implicit Membrane Potential for NMR Structure Calculations of Membrane Proteins.

The highly anisotropic environment of the lipid bilayer membrane imposes significant constraints on the structures and functions of membrane proteins. However, NMR structure calculations typically use a simple repulsive potential that neglects the effects of solvation and electrostatics, because explicit atomic representation of the solvent and lipid molecules is computationally expensive and impractical for routine NMR-restrained calculations that start from completely extended polypeptide templates. Here, we describe the extension of a previously described implicit solvation potential, eefxPot, to include a membrane model for NMR-restrained calculations of membrane protein structures in XPLOR-NIH. The key components of eefxPot are an energy term for solvation free energy that works together with other nonbonded energy functions, a dedicated force field for conformational and nonbonded protein interaction parameters, and a membrane function that modulates the solvation free energy and dielectric screening as a function of the atomic distance from the membrane center, relative to the membrane thickness. Initial results obtained for membrane proteins with structures determined experimentally in lipid bilayer membranes show that eefxPot affords significant improvements in structural quality, accuracy, and precision. Calculations with eefxPot are straightforward to implement and can be used to both fold and refine structures, as well as to run unrestrained molecular-dynamics simulations. The potential is entirely compatible with the full range of experimental restraints measured by various techniques. Overall, it provides a useful and practical way to calculate membrane protein structures in a physically realistic environment.

Structure of the Na,K-ATPase regulatory protein FXYD2b in micelles: implications for membrane-water interfacial arginines.

FXYD2 is a membrane protein responsible for regulating the function of the Na,K-ATPase in mammalian kidney epithelial cells. Here we report the structure of FXYD2b, one of two splice variants of the protein, determined by NMR spectroscopy in detergent micelles. Solid-state NMR characterization of the protein embedded in phospholipid bilayers indicates that several arginine side chains may be involved in hydrogen bond interactions with the phospholipid polar head groups. The structure and the NMR data suggest that FXYD2b could regulate the Na,K-ATPase by modulating the effective membrane surface electrostatics near the ion binding sites of the pump.

A practical implicit solvent potential for NMR structure calculation.

The benefits of protein structure refinement in water are well documented. However, performing structure refinement with explicit atomic representation of the solvent molecules is computationally expensive and impractical for NMR-restrained structure calculations that start from completely extended polypeptide templates. Here we describe a new implicit solvation potential, EEFx (Effective Energy Function for XPLOR-NIH), for NMR-restrained structure calculations of proteins in XPLOR-NIH. The key components of EEFx are an energy term for solvation energy that works together with other nonbonded energy functions, and a dedicated force field for conformational and nonbonded protein interaction parameters. The initial results obtained with EEFx show that significant improvements in structural quality can be obtained. EEFx is computationally efficient and can be used both to fold and refine structures. Overall, EEFx improves the quality of protein conformation and nonbonded atomic interactions. Moreover, such benefits are accompanied by enhanced structural precision and enhanced structural accuracy, reflected in improved agreement with the cross-validated dipolar coupling data. Finally, implementation of EEFx calculations is straightforward and computationally efficient. Overall, EEFx provides a useful method for the practical calculation of experimental protein structures in a physically realistic environment.

Structure of the chemokine receptor CXCR1 in phospholipid bilayers.

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.

FXYD proteins reverse inhibition of the Na+-K+ pump mediated by glutathionylation of its beta1 subunit.

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 ß(1) subunit glutathionylation induced by oxidative signals causes pump inhibition in a previous study. In the present study, we found that ß(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 ß(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 ß(1) subunit, FXYD proteins reverse oxidative inhibition of the Na(+)-K(+) pump and play a dynamic role in its regulation.

BAX and BAK caught in the act.

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.

Effects of PKA phosphorylation on the conformation of the Na,K-ATPase regulatory protein FXYD1.

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.

Competitive interactions of collagen and a jararhagin-derived disintegrin peptide with the integrin alpha2-I domain.

Integrin alpha2beta1 is a major receptor required for activation and adhesion of platelets, through the specific recognition of collagen by the alpha2-I domain (alpha2-I), which binds fibrillar collagen via Mg(2+)-bridged interactions. The crystal structure of a truncated form of the alpha2-I domain, bound to a triple helical collagen peptide, revealed conformational changes suggestive of a mechanism where the ligand-bound I domain can initiate and propagate conformational change to the full integrin complex. Collagen binding by alpha2-I and fibrinogen-dependent platelet activity can be inhibited by snake venom polypeptides. Here we describe the inhibitory effect of a short cyclic peptide derived from the snake toxin metalloprotease jararhagin, with specific amino acid sequence RKKH, on the ability of alpha2-I to bind triple helical collagen. Isothermal titration calorimetry measurements showed that the interactions of alpha2-I with collagen or RKKH peptide have similar affinities, and NMR chemical shift mapping experiments with (15)N-labeled alpha2-I, and unlabeled RKKH peptide, indicate that the peptide competes for the collagen-binding site of alpha2-I but does not induce a large scale conformational rearrangement of the I domain.

Structures of the FXYD regulatory proteins in lipid micelles and membranes.

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.

Nuclear magnetic resonance structural studies of membrane proteins in micelles and bilayers.

Nuclear magnetic resonance (NMR) spectroscopy enables determination of membrane protein structures in lipid environments, such as micelles and bilayers. This chapter outlines the steps for membrane-protein structure determination using solution NMR with micelle samples, and solid-state NMR with oriented lipid-bilayer samples. The methods for protein expression and purification, sample preparation, and NMR experiments are described and illustrated with examples from gamma and CHIF, two membrane proteins that function as regulatory subunits of the Na+- and K+-ATPase.

Mapping the specific cytoprotective interaction of humanin with the pro-apoptotic protein bid.

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.

Structure of the Na,K-ATPase regulatory protein FXYD1 in micelles.

FXYD1 is a major regulatory subunit of the Na,K-ATPase and the principal substrate of hormone-regulated phosphorylation by c-AMP dependent protein kinases A and C in heart and skeletal muscle sarcolemma. It is a member of an evolutionarily conserved family of membrane proteins that regulate the function of the enzyme complex in a tissue-specific and physiological-state-specific manner. Here, we present the three-dimensional structure of FXYD1 determined in micelles by NMR spectroscopy. Structure determination was made possible by measuring residual dipolar couplings in weakly oriented micelle samples of the protein. This allowed us to obtain the relative orientations of the helical segments and information about the protein dynamics. The structural analysis was further facilitated by the inclusion of distance restraints, obtained from paramagnetic spin label relaxation enhancements, and by refinement with a micelle depth restraint, derived from paramagnetic Mn line broadening effects. The structure of FXYD1 provides the foundation for understanding its intra-membrane association with the Na,K-ATPase alpha subunit and 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.

NMR of membrane proteins in micelles and bilayers: the FXYD family proteins.

Determining the atomic resolution structures of membrane proteins is of particular interest in contemporary structural biology. Helical membrane proteins constitute one-third of the expressed proteins encoded in a genome, many drugs have membrane-bound proteins as their receptors, and mutations in membrane proteins result in human diseases. Although integral membrane proteins provide daunting technical challenges for all methods of protein structure determination, nuclear magnetic resonance (NMR) spectroscopy can be an extremely versatile and powerful method for determining their structures and characterizing their dynamics, in lipid environments that closely mimic the cell membranes. Once milligram amounts of isotopically labeled protein are expressed and purified, micelle samples can be prepared for solution NMR analysis, and lipid bilayer samples can be prepared for solid-state NMR analysis. The two approaches are complementary and can provide detailed structural and dynamic information. This paper describes the steps for membrane protein structure determination using solution and solid-state NMR. The methods for protein expression and purification, sample preparation and NMR experiments are described and illustrated with examples from the FXYD proteins, a family of regulatory subunits of the Na,K-ATPase.