Solid state NMR spectral editing of histidine, arginine and lysine using Hadamard encoding.

The NMR signals from protein sidechains are rich in information about intra- and inter-molecular interactions, but their detection can be complicated due to spectral overlap as well as conformational and hydrogen exchange. In this work, we demonstrate a protocol for multi-dimensional solid-state NMR spectral editing of signals from basic sidechains based on Hadamard matrix encoding. The Hadamard method acquires multi-dimensional experiments in such a way that both the backbone and under-sampled sidechain signals can be decoded for unambiguous editing in the N spectral frequency dimension.

A pH-sensitive motif in an outer membrane protein activates bacterial membrane vesicle production.

Outer membrane vesicles (OMVs) produced by Gram-negative bacteria have key roles in cell envelope homeostasis, secretion, interbacterial communication, and pathogenesis. The facultative intracellular pathogen Salmonella Typhimurium increases OMV production inside the acidic vacuoles of host cells by changing expression of its outer membrane proteins and modifying the composition of lipid A. However, the molecular mechanisms that translate pH changes into OMV production are not completely understood.

Solid-state NMR MAS CryoProbe enables structural studies of human blood protein vitronectin bound to hydroxyapatite.

The low sensitivity of nuclear magnetic resonance (NMR) is a major bottleneck for studying biomolecular structures of complex biomolecular assemblies. Cryogenically cooled probe technology overcomes the sensitivity limitations enabling NMR applications to challenging biomolecular systems. Here we describe solid-state NMR studies of the human blood protein vitronectin (Vn) bound to hydroxyapatite (HAP), the mineralized form of calcium phosphate, using a CryoProbe designed for magic angle spinning (MAS) experiments.

Calcium-induced environmental adaptability of the blood protein vitronectin.

The adaptability of proteins to their work environments is fundamental for cellular life. Here, we describe how the hemopexin-like domain of the multifunctional blood glycoprotein vitronectin binds Ca to adapt to excursions of temperature and shear stress. Using X-ray crystallography, molecular dynamics simulations, NMR, and differential scanning fluorimetry, we describe how Ca and its flexible hydration shell enable the protein to perform conformational changes that relay beyond the calcium-binding site and alter the number of polar contacts to enhance conformational stability.

A periplasmic cinched protein is required for siderophore secretion and virulence of Mycobacterium tuberculosis.

Iron is essential for growth of Mycobacterium tuberculosis, the causative agent of tuberculosis. To acquire iron from the host, M. tuberculosis uses the siderophores called mycobactins and carboxymycobactins.

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.

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

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.

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.

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

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.

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

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.

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.

Lipoprotein Particle Formation by Proapoptotic tBid.

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.

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.

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.

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

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.

Applications of NMR to membrane proteins.

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.

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

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.

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

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.

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.

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.