Publications by authors named "Francesca Marassi"

74 Publications

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

Structure 2021 Apr 13. Epub 2021 Apr 13.

Cancer Center, Sanford Burnham Prebys Medical Discovery Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.str.2021.03.018DOI Listing
April 2021

Correlating the Structure and Activity of Y. pestis Ail in a Bacterial Cell Envelope.

Biophys J 2021 02 24;120(3):453-462. Epub 2020 Dec 24.

Cancer Center, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, California. Electronic address:

Understanding microbe-host interactions at the molecular level is a major goal of fundamental biology and therapeutic drug development. Structural biology strives to capture biomolecular structures in action, but the samples are often highly simplified versions of the complex native environment. Here, we present an Escherichia coli model system that allows us to probe the structure and function of Ail, the major surface protein of the deadly pathogen Yersinia pestis. We show that cell surface expression of Ail produces Y. pestis virulence phenotypes in E. coli, including resistance to human serum, cosedimentation of human vitronectin, and pellicle formation. Moreover, isolated bacterial cell envelopes, encompassing inner and outer membranes, yield high-resolution solid-state NMR spectra that reflect the structure of Ail and reveal Ail sites that are sensitive to the bacterial membrane environment and involved in the interactions with human serum components. The data capture the structure and function of Ail in a bacterial outer membrane and set the stage for probing its interactions with the complex milieu of immune response proteins present in human serum.
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http://dx.doi.org/10.1016/j.bpj.2020.12.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7895992PMC
February 2021

Conformational States of the Cytoprotective Protein Bcl-xL.

Biophys J 2020 10 20;119(7):1324-1334. Epub 2020 Aug 20.

Cancer Center, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, California. Electronic address:

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.
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http://dx.doi.org/10.1016/j.bpj.2020.08.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7567986PMC
October 2020

Calcium and hydroxyapatite binding site of human vitronectin provides insights to abnormal deposit formation.

Proc Natl Acad Sci U S A 2020 08 22;117(31):18504-18510. Epub 2020 Jul 22.

Cancer Center, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA 92037;

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.
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http://dx.doi.org/10.1073/pnas.2007699117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7414086PMC
August 2020

Mutually constructive roles of Ail and LPS in Yersinia pestis serum survival.

Mol Microbiol 2020 09 25;114(3):510-520. Epub 2020 Jun 25.

Cancer Center, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA, USA.

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) 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.
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http://dx.doi.org/10.1111/mmi.14530DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7594906PMC
September 2020

Membrane proteins in magnetically aligned phospholipid polymer discs for solid-state NMR spectroscopy.

Biochim Biophys Acta Biomembr 2020 09 1;1862(9):183333. Epub 2020 May 1.

University of California San Diego, La Jolla, CA, USA. Electronic address:

Well-hydrated phospholipid bilayers provide a near-native environment for membrane proteins. They enable the preparation of chemically-defined samples suitable for NMR and other spectroscopic experiments that reveal the structure, dynamics, and functional interactions of the proteins at atomic resolution. The synthetic polymer styrene maleic acid (SMA) can be used to prepare detergent-free samples that form macrodiscs with diameters greater than 30 nm at room temperature, and spontaneously align in the magnetic field of an NMR spectrometer at temperatures above 35 °C. Here we show that magnetically aligned macrodiscs are particularly well suited for solid-state NMR experiments of membrane proteins because the SMA-lipid assembly both immobilizes the embedded protein and provides uniaxial order for oriented sample (OS) solid-state NMR studies. We show that aligned macrodiscs incorporating four different membrane proteins with a wide range of sizes and topological complexity yield high-resolution OS solid-state NMR spectra. The work is dedicated to Michelle Auger who made key contributions to the field of membrane and membrane protein biophysics.
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http://dx.doi.org/10.1016/j.bbamem.2020.183333DOI Listing
September 2020

Structure of human Vitronectin C-terminal domain and interaction with outer membrane protein Ail.

Sci Adv 2019 09 11;5(9):eaax5068. Epub 2019 Sep 11.

Cancer Center, Sanford Burnham Prebys Medical Discovery Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA.

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 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.
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http://dx.doi.org/10.1126/sciadv.aax5068DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6739113PMC
September 2019

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

Methods Mol Biol 2019 ;1877:233-246

Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA, USA.

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.
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http://dx.doi.org/10.1007/978-1-4939-8861-7_16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6599400PMC
May 2019

Lipoprotein Particle Formation by Proapoptotic tBid.

Biophys J 2018 08 26;115(3):533-542. Epub 2018 Jun 26.

Cancer Center, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, California. Electronic address:

The interactions of Bcl-2 family proteins with intracellular lipids are essential for the regulation of apoptosis, a mechanism of programmed cell death that is central to the health and development of multicellular organisms. Bid and its caspase-8 cleavage product, tBid, promote the permeabilization of the mitochondrial outer membrane and sequester antiapoptotic Bcl-2 proteins to counter their cytoprotective activity. Bid and tBid also promote lipid exchange, a characteristic trait of apoptosis. Here, we show that tBid is capable of associating with phospholipids to form soluble, nanometer-sized lipoprotein particles that retain binding affinity for the antiapoptotic protein Bcl-xL. The tBid lipoprotein particles form with a lipid/protein stoichiometry in the range of 20/1 and have a diameter of ∼11.5 nm. Lipoparticle-bound tBid retains an α-helical structure and binds Bcl-xL through its third Bcl-2 homology motif, forming a soluble, lipid-associated heteroprotein complex. The results shed light on the role of lipids in mediating Bcl-2 protein mobility and interactions.
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http://dx.doi.org/10.1016/j.bpj.2018.06.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6084415PMC
August 2018

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

Nat Chem Biol 2018 05 5;14(5):458-465. Epub 2018 Mar 5.

Department of Structural Biology, St. Jude Children's Research Hospital, Memphis, TN, USA.

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.
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http://dx.doi.org/10.1038/s41589-018-0011-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5899648PMC
May 2018

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

J Biomol NMR 2017 Nov 15;69(3):111-121. Epub 2017 Nov 15.

Department of Chemistry and Biochemistry, University of California, San Diego La Jolla, San Diego, CA, 92093-0307, USA.

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.
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http://dx.doi.org/10.1007/s10858-017-0128-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5869024PMC
November 2017

Structural Insights into the Yersinia pestis Outer Membrane Protein Ail in Lipid Bilayers.

J Phys Chem B 2017 08 4;121(32):7561-7570. Epub 2017 Aug 4.

Sanford Burnham Prebys Medical Discovery Institute , 10901 North Torrey Pines Road, La Jolla, California 92037, United States.

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 N transverse relaxation rate. The results provide information about the way in which an outer membrane protein inserts and functions in the bacterial membrane.
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http://dx.doi.org/10.1021/acs.jpcb.7b03941DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5713880PMC
August 2017

Applications of NMR to membrane proteins.

Arch Biochem Biophys 2017 08 18;628:92-101. Epub 2017 May 18.

Sanford Burnham Prebys Medical Discovery Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA. Electronic address:

Membrane proteins present a challenge for structural biology. In this article, we review some of the recent developments that advance the application of NMR to membrane proteins, with emphasis on structural studies in detergent-free, lipid bilayer samples that resemble the native environment. NMR spectroscopy is not only ideally suited for structure determination of membrane proteins in hydrated lipid bilayer membranes, but also highly complementary to the other principal techniques based on X-ray and electron diffraction. Recent advances in NMR instrumentation, spectroscopic methods, computational methods, and sample preparations are driving exciting new efforts in membrane protein structural biology.
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http://dx.doi.org/10.1016/j.abb.2017.05.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5657258PMC
August 2017

High resolution solid-state NMR spectroscopy of the Yersinia pestis outer membrane protein Ail in lipid membranes.

J Biomol NMR 2017 03 26;67(3):179-190. Epub 2017 Feb 26.

Sanford Burnham Prebys Medical Discovery Institute, 10901 North Torrey Pines Road, La Jolla, CA, 92037, USA.

The outer membrane protein Ail (Adhesion invasion locus) is one of the most abundant proteins on the cell surface of Yersinia pestis during human infection. Its functions are expressed through interactions with a variety of human host proteins, and are essential for microbial virulence. Structures of Ail have been determined by X-ray diffraction and solution NMR spectroscopy, but those samples contained detergents that interfere with functionality, thus, precluding analysis of the structural basis for Ail's biological activity. Here, we demonstrate that high-resolution solid-state NMR spectra can be obtained from samples of Ail in detergent-free phospholipid liposomes, prepared with a lipid to protein molar ratio of 100. The spectra, obtained with C or H detection, have very narrow line widths (0.40-0.60 ppm for C, 0.11-0.15 ppm for H, and 0.46-0.64 ppm for N) that are consistent with a high level of sample homogeneity. The spectra enable resonance assignments to be obtained for N, CO, CA and CB atomic sites from 75 out of 156 residues in the sequence of Ail, including 80% of the transmembrane region. The H-detected solid-state NMR H/N correlation spectra obtained for Ail in liposomes compare very favorably with the solution NMR H/N TROSY spectra obtained for Ail in nanodiscs prepared with a similar lipid to protein molar ratio. These results set the stage for studies of the molecular basis of the functional interactions of Ail with its protein partners from human host cells, as well as the development of drugs targeting Ail.
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http://dx.doi.org/10.1007/s10858-017-0094-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5490241PMC
March 2017

High quality NMR structures: a new force field with implicit water and membrane solvation for Xplor-NIH.

J Biomol NMR 2017 01 29;67(1):35-49. Epub 2016 Dec 29.

Sanford-Burnham-Prebys Medical Discovery Institute, 10901 North Torrey Pines Road, La Jolla, CA, 92037, USA.

Structure determination of proteins by NMR is unique in its ability to measure restraints, very accurately, in environments and under conditions that closely mimic those encountered in vivo. For example, advances in solid-state NMR methods enable structure determination of membrane proteins in detergent-free lipid bilayers, and of large soluble proteins prepared by sedimentation, while parallel advances in solution NMR methods and optimization of detergent-free lipid nanodiscs are rapidly pushing the envelope of the size limit for both soluble and membrane proteins. These experimental advantages, however, are partially squandered during structure calculation, because the commonly used force fields are purely repulsive and neglect solvation, Van der Waals forces and electrostatic energy. Here we describe a new force field, and updated energy functions, for protein structure calculations with EEFx implicit solvation, electrostatics, and Van der Waals Lennard-Jones forces, in the widely used program Xplor-NIH. The new force field is based primarily on CHARMM22, facilitating calculations with a wider range of biomolecules. The new EEFx energy function has been rewritten to enable OpenMP parallelism, and optimized to enhance computation efficiency. It implements solvation, electrostatics, and Van der Waals energy terms together, thus ensuring more consistent and efficient computation of the complete nonbonded energy lists. Updates in the related python module allow detailed analysis of the interaction energies and associated parameters. The new force field and energy function work with both soluble proteins and membrane proteins, including those with cofactors or engineered tags, and are very effective in situations where there are sparse experimental restraints. Results obtained for NMR-restrained calculations with a set of five soluble proteins and five membrane proteins show that structures calculated with EEFx have significant improvements in accuracy, precision, and conformation, and that structure refinement can be obtained by short relaxation with EEFx to obtain improvements in these key metrics. These developments broaden the range of biomolecular structures that can be calculated with high fidelity from NMR restraints.
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http://dx.doi.org/10.1007/s10858-016-0082-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5487259PMC
January 2017

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

J Biol Chem 2016 Jul 19;291(27):14072-14084. Epub 2016 Apr 19.

Sanford-Burnham-Prebys Medical Discovery Institute, La Jolla, California 92037,; Roche, Pharma Research and Early Development, Basel 4070, Switzerland. Electronic address:

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.
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http://dx.doi.org/10.1074/jbc.M116.715235DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4933167PMC
July 2016

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

Protein Expr Purif 2016 06 23;122:56-63. Epub 2016 Feb 23.

Sanford Burnham Prebys Medical Discovery Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.pep.2016.02.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4842142PMC
June 2016

Yersinia pestis uses the Ail outer membrane protein to recruit vitronectin.

Microbiology (Reading) 2015 Nov 15;161(11):2174-2183. Epub 2015 Sep 15.

1​ Department of Microbiology and Immunology, University of Miami Miller School of Medicine, Miami, FL 33101, USA.

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.
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http://dx.doi.org/10.1099/mic.0.000179DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4806588PMC
November 2015

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

Biophys J 2015 Aug;109(3):574-85

Sanford-Burnham Medical Research Institute, La Jolla, California. Electronic address:

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.
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http://dx.doi.org/10.1016/j.bpj.2015.06.047DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4572468PMC
August 2015

Backbone structure of Yersinia pestis Ail determined in micelles by NMR-restrained simulated annealing with implicit membrane solvation.

J Biomol NMR 2015 Sep 5;63(1):59-65. Epub 2015 Jul 5.

Sanford-Burnham Medical Research Institute, 10901 North Torrey Pines Road, La Jolla, CA, 92037, USA.

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.
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http://dx.doi.org/10.1007/s10858-015-9963-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4577439PMC
September 2015

Solid-State NMR-Restrained Ensemble Dynamics of a Membrane Protein in Explicit Membranes.

Biophys J 2015 Apr;108(8):1954-62

Department of Molecular Biosciences and Center for Computational Biology, The University of Kansas, Lawrence, Kansas. Electronic address:

Solid-state NMR has been used to determine the structures of membrane proteins in native-like lipid bilayer environments. Most structure calculations based on solid-state NMR observables are performed using simulated annealing with restrained molecular dynamics and an energy function, where all nonbonded interactions are represented by a single, purely repulsive term with no contributions from van der Waals attractive, electrostatic, or solvation energy. To our knowledge, this is the first application of an ensemble dynamics technique performed in explicit membranes that uses experimental solid-state NMR observables to obtain the refined structure of a membrane protein together with information about its dynamics and its interactions with lipids. Using the membrane-bound form of the fd coat protein as a model membrane protein and its experimental solid-state NMR data, we performed restrained ensemble dynamics simulations with different ensemble sizes in explicit membranes. For comparison, a molecular dynamics simulation of fd coat protein was also performed without any restraints. The average orientation of each protein helix is similar to a structure determined by traditional single-conformer approaches. However, their variations are limited in the resulting ensemble of structures with one or two replicas, as they are under the strong influence of solid-state NMR restraints. Although highly consistent with all solid-state NMR observables, the ensembles of more than two replicas show larger orientational variations similar to those observed in the molecular dynamics simulation without restraints. In particular, in these explicit membrane simulations, Lys(40), residing at the C-terminal side of the transmembrane helix, is observed to cause local membrane curvature. Therefore, compared to traditional single-conformer approaches in implicit environments, solid-state NMR restrained ensemble simulations in explicit membranes readily characterize not only protein dynamics but also protein-lipid interactions in detail.
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http://dx.doi.org/10.1016/j.bpj.2015.03.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4407261PMC
April 2015

Conformation of BCL-XL upon Membrane Integration.

J Mol Biol 2015 Jul 27;427(13):2262-70. Epub 2015 Feb 27.

Sanford-Burnham Medical Research Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA. Electronic address:

BCL-XL is an anti-apoptotic BCL-2 family protein found both in the cytosol and bound to intracellular membranes. Structural studies of BCL-XL have advanced by deleting its hydrophobic C-terminus and adding detergents to enhance solubility. However, since the C-terminus is essential for function and detergents strongly affect structure and activity, the molecular mechanisms controlling intracellular localization and cytoprotective activity are incompletely understood. Here we describe the conformations and ligand binding activities of water-soluble and membrane-bound BCL-XL, with its complete C-terminus, in detergent-free environments. We show that the C-terminus interacts with a conserved surface groove in the water-soluble state of the protein and inserts across the phospholipid bilayer in the membrane-bound state. Contrary to current models, membrane binding does not induce a conformational change in the soluble domain and both states bind a known ligand with affinities that are modulated by the specific state of the protein.
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http://dx.doi.org/10.1016/j.jmb.2015.02.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4457587PMC
July 2015

Solid-state NMR of the Yersinia pestis outer membrane protein Ail in lipid bilayer nanodiscs sedimented by ultracentrifugation.

J Biomol NMR 2015 Apr 13;61(3-4):275-86. Epub 2015 Jan 13.

Sanford-Burnham Medical Research Institute, 10901 North Torrey Pines Road, La Jolla, CA, 92037, USA.

Solid-state NMR studies of sedimented soluble proteins has been developed recently as an attractive approach for overcoming the size limitations of solution NMR spectroscopy while bypassing the need for sample crystallization or precipitation (Bertini et al. Proc Natl Acad Sci USA 108(26):10396-10399, 2011). Inspired by the potential benefits of this method, we have investigated the ability to sediment lipid bilayer nanodiscs reconstituted with a membrane protein. In this study, we show that nanodiscs containing the outer membrane protein Ail from Yersinia pestis can be sedimented for solid-state NMR structural studies, without the need for precipitation or lyophilization. Optimized preparations of Ail in phospholipid nanodiscs support both the structure and the fibronectin binding activity of the protein. The same sample can be used for solution NMR, solid-state NMR and activity assays, facilitating structure-activity correlation experiments across a wide range of timescales.
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http://dx.doi.org/10.1007/s10858-014-9893-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4398618PMC
April 2015

Influence of the lipid membrane environment on structure and activity of the outer membrane protein Ail from Yersinia pestis.

Biochim Biophys Acta 2015 Feb 27;1848(2):712-20. Epub 2014 Nov 27.

Sanford-Burnham Medical Research Institute, La Jolla, CA 92037, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.bbamem.2014.11.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4281492PMC
February 2015

Structure of the membrane protein MerF, a bacterial mercury transporter, improved by the inclusion of chemical shift anisotropy constraints.

J Biomol NMR 2014 Sep 8;60(1):67-71. Epub 2014 Aug 8.

Department of Chemistry and Biochemistry, University of California San Diego, 9500 Gilman Drive, La Jolla, CA, 92093-0307, USA.

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http://dx.doi.org/10.1007/s10858-014-9852-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4154067PMC
September 2014

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

Biochim Biophys Acta 2015 Jan 2;1848(1 Pt B):299-306. Epub 2014 May 2.

Sanford-Burnham Medical Research Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.bbamem.2014.04.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4216782PMC
January 2015

A practical implicit solvent potential for NMR structure calculation.

J Magn Reson 2014 Jun 2;243:54-64. Epub 2014 Apr 2.

Sanford-Burnham Medical Research Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.jmr.2014.03.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4037354PMC
June 2014

Membrane protein structure determination in membrana.

Acc Chem Res 2013 Sep 24;46(9):2182-90. Epub 2013 Jun 24.

Sanford-Burnham Medical Research Institute , 10901 North Torrey Pines Road, La Jolla, California 92037, United States.

The two principal components of biological membranes, the lipid bilayer and the proteins integrated within it, have coevolved for specific functions that mediate the interactions of cells with their environment. Molecular structures can provide very significant insights about protein function. In the case of membrane proteins, the physical and chemical properties of lipids and proteins are highly interdependent; therefore structure determination should include the membrane environment. Considering the membrane alongside the protein eliminates the possibility that crystal contacts or detergent molecules could distort protein structure, dynamics, and function and enables ligand binding studies to be performed in a natural setting. Solid-state NMR spectroscopy is compatible with three-dimensional structure determination of membrane proteins in phospholipid bilayer membranes under physiological conditions and has played an important role in elucidating the physical and chemical properties of biological membranes, providing key information about the structure and dynamics of the phospholipid components. Recently, developments in the recombinant expression of membrane proteins, sample preparation, pulse sequences for high-resolution spectroscopy, radio frequency probes, high-field magnets, and computational methods have enabled a number of membrane protein structures to be determined in lipid bilayer membranes. In this Account, we illustrate solid-state NMR methods with examples from two bacterial outer membrane proteins (OmpX and Ail) that form integral membrane β-barrels. The ability to measure orientation-dependent frequencies in the solid-state NMR spectra of membrane-embedded proteins provides the foundation for a powerful approach to structure determination based primarily on orientation restraints. Orientation restraints are particularly useful for NMR structural studies of membrane proteins because they provide information about both three-dimensional structure and the orientation of the protein within the membrane. When combined with dihedral angle restraints derived from analysis of isotropic chemical shifts, molecular fragment replacement, and de novo structure prediction, orientation restraints can yield high-quality three-dimensional structures with few or no distance restraints. Using complementary solid-state NMR methods based on oriented sample (OS) and magic angle spinning (MAS) approaches, one can resolve and assign multiple peaks through the use of (15)N/(13)C labeled samples and measure precise restraints to determine structures.
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http://dx.doi.org/10.1021/ar400041aDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3970975PMC
September 2013

Membrane protein structure determination: back to the membrane.

Methods Mol Biol 2013 ;1063:145-58

Sanford Burnham Medical Research Institute, La Jolla, CA, USA.

NMR spectroscopy enables the structures of membrane proteins to be determined in the native-like environment of the phospholipid bilayer membrane. This chapter outlines the methods for membrane protein structural studies using solid-state NMR spectroscopy with samples of membrane proteins incorporated in proteoliposomes or planar lipid bilayers. The methods for protein expression and purification, sample preparation, and NMR experiments are described and illustrated with examples from OmpX and Ail, two bacterial outer membrane proteins that function in bacterial virulence.
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http://dx.doi.org/10.1007/978-1-62703-583-5_8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4012560PMC
February 2014

NMR-based simulation studies of Pf1 coat protein in explicit membranes.

Biophys J 2013 Aug;105(3):691-8

Department of Molecular Biosciences, The University of Kansas, Lawrence, USA.

As time- and ensemble-averaged measures, NMR observables contain information about both protein structure and dynamics. This work represents a computational study to extract such information for membrane proteins from orientation-dependent NMR observables: solid-state NMR chemical shift anisotropy and dipolar coupling, and solution NMR residual dipolar coupling. We have performed NMR-restrained molecular dynamics simulations to refine the structure of the membrane-bound form of Pf1 coat protein in explicit lipid bilayers using the recently measured chemical shift anisotropy, dipolar coupling, and residual dipolar coupling data. From the simulations, we have characterized detailed protein-lipid interactions and explored the dynamics. All simulations are stable and the NMR restraints are well satisfied. The C-terminal transmembrane (TM) domain of Pf1 finds its optimal position in the membrane quickly (within 6 ns), illustrating efficient solvation of TM domains in explicit bilayer environments. Such rapid convergence also leads to well-converged interaction patterns between the TM helix and the membrane, which clearly show the interactions of interfacial membrane-anchoring residues with the lipids. For the N-terminal periplasmic helix of Pf1, we identify a stable, albeit dynamic, helix orientation parallel to the membrane surface that satisfies the amphiphatic nature of the helix in an explicit lipid bilayer. Such detailed information cannot be obtained solely from NMR observables. Therefore, the present simulations illustrate the usefulness of NMR-restrained MD refinement of membrane protein structure in explicit membranes.
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http://dx.doi.org/10.1016/j.bpj.2013.06.040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3736739PMC
August 2013