Jordan H. Chill

NMR study of the tetrameric KcsA potassium channel in detergent micelles

Nuclear magnetic resonance (NMR) studies of large membrane‐associated proteins are limited by the difficulties in preparation of stable protein–detergent mixed micelles and by line broadening, which is typical of these macroassemblies. We have used the 68‐kDa homotetrameric KcsA, a thermostable N‐terminal deletion mutant of a bacterial potassium channel from Streptomyces lividans, as a model system for applying NMR methods to membrane proteins. Optimization of measurement conditions enabled us to perform the backbone assignment of KcsA in SDS micelles and establish its secondary structure, which was found to closely agree with the KcsA crystal structure. The C‐terminal cytoplasmic domain, absent in the original structure, contains a 14‐residue helix that could participate in tetramerization by forming an intersubunit four‐helix bundle. A quantitative estimate of cross‐ relaxation between detergent and KcsA backbone amide protons, together with relaxation and light scattering data, suggests SDS–KcsA mixed micelles form an oblate spheroid with ∼180 SDS molecules per channel. K+ ions bind to the micelle‐solubilized channel with a KD of 3 ± 0.5 mM, resulting in chemical shift changes in the selectivity filter. Related pH‐induced changes in chemical shift along the “outer” transmembrane helix and the cytoplasmic membrane interface hint at a possible structural explanation for the observed pH‐gating of the potassium channel.

The human type I interferon receptor. NMR structure reveals the molecular basis of ligand binding.

The potent antiviral and antiproliferative activities of human type I interferons (IFNs) are mediated by a single receptor comprising two subunits, IFNAR1 and IFNAR2. The structure of the IFNAR2 IFN binding ectodomain (IFNAR2-EC), the first helical cytokine receptor structure determined in solution, reveals the molecular basis for IFN binding. The atypical perpendicular orientation of its two fibronectin domains explains the lack of C domain involvement in ligand binding. A model of the IFNAR2-EC/IFNalpha2 complex based on double mutant cycle-derived constraints uncovers an extensive and predominantly aliphatic hydrophobic patch on the receptor that interacts with a matching hydrophobic surface of IFNalpha2. An adjacent motif of alternating charged side chains guides the two proteins into a tight complex. The binding interface may account for crossreactivity and ligand specificity of the receptor. This molecular description of IFN binding should be invaluable for study and design of IFN-based biomedical agents.

The Mechanism for Acetylcholine Receptor Inhibition by α-Neurotoxins and Species-Specific Resistance to α-Bungarotoxin Revealed by NMR

Abstract The structure of a peptide corresponding to residues 182–202 of the acetylcholine receptor α1 subunit in complex with α-bungarotoxin was solved using NMR spectroscopy. The peptide contains the complete sequence of the major determinant of AChR involved in α-bungarotoxin binding. One face of the long β hairpin formed by the AChR peptide consists of exposed nonconserved residues, which interact extensively with the toxin. Mutations of these receptor residues confer resistance to the toxin. Conserved AChR residues form the opposite face of the β hairpin, which creates the inner and partially hidden pocket for acetylcholine. An NMR-derived model for the receptor complex with two α-bungarotoxin molecules shows that this pocket is occupied by the conserved α-neurotoxin residue R36, which forms cation-π interactions with both α W149 and γ W55/ δ W57 of the receptor and mimics acetylcholine.

Determination of the human type I interferon receptor binding site on human interferon-α2 by cross saturation and an NMR-based model of the complex

Type I interferons (IFNs) are a family of homologous helical cytokines that exhibit pleiotropic effects on a wide variety of cell types, including antiviral activity and antibacterial, antiprozoal, immunomodulatory, and cell growth regulatory functions. Consequently, IFNs are the human proteins most widely used in the treatment of several kinds of cancer, hepatitis C, and multiple sclerosis. All type I IFNs bind to a cell surface receptor consisting of two subunits, IFNAR1 and IFNAR2, associating upon binding of interferon. The structure of the extracellular domain of IFNAR2 (R2‐EC) was solved recently. Here we study the complex and the binding interface of IFNα2 with R2‐EC using multidimensional NMR techniques. NMR shows that IFNα2 does not undergo significant structural changes upon binding to its receptor, suggesting a lock‐and‐key mechanism for binding. Cross saturation experiments were used to determine the receptor binding site upon IFNα2. The NMR data and previously published mutagenesis data were used to derive a docking model of the complex with an RMSD of 1 Å, and its well‐defined orientation between IFNα2 and R2‐EC and the structural quality greatly improve upon previously suggested models. The relative ligand–receptor orientation is believed to be important for interferon signaling and possibly one of the parameters that distinguish the different IFN I subtypes. This structural information provides important insight into interferon signaling processes and may allow improvement in the development of therapeutically used IFNs and IFN‐like molecules.

Exclusively Heteronuclear 13C-Detected Amino-Acid-Selective NMR Experiments for the Study of Intrinsically Disordered Proteins (IDPs)

Carbon‐13 direct‐detection NMR methods have proved to be very useful for the characterization of intrinsically disordered proteins (IDPs). Here we present a suite of experiments in which amino‐acid‐selective editing blocks are encoded in CACON‐ and CANCO‐type sequences to give 13C‐detected spectra containing correlations arising from a particular type or group of amino acid(s). These two general types of experiments provide the complementary intra‐ and inter‐residue correlations necessary for sequence‐specific assignment of backbone resonance frequencies. We demonstrate the capabilities of these experiments on two IDPs: fully reduced Cox17 and WIPC. The proposed approach constitutes an independent strategy to simplify crowded spectra as well as to perform sequence‐specific assignment, thereby demonstrating its potential to study IDPs.

A solution NMR view of protein dynamics in the biological membrane

Structure determination of membrane-associated proteins (MPs) represents a frontier of structural biology that is characterized by unique challenges in sample preparation and data acquisition. No less important is our ability to study the dynamics of MPs, since MP flexibility and characteristic motions often make sizeable contributions to their function. This review focuses on solution state NMR methods to characterize dynamics of MPs in the membrane environment. NMR approaches to study molecular motions on a wide range of time-scales and their application to membrane proteins are described. Studies of polytopic and bitopic MPs demonstrating the power of such methods to characterize the dynamic behavior of MPs and their interaction with the membrane-mimicking surroundings are presented. Attempts are made to place the dynamic conclusions into a biological context. The importance and limitations of such investigations guarantee that further developments in this field will be actively pursued.

NMR determines transient structure and dynamics in the disordered C-terminal domain of WASp interacting protein.

WASp-interacting protein (WIP) is a 503-residue proline-rich polypeptide expressed in human T cells. The WIP C-terminal domain binds to Wiskott-Aldrich syndrome protein (WASp) and regulates its activation and degradation, and the WIP-WASp interaction has been shown to be critical for actin polymerization and implicated in the onset of WAS and X-linked thrombocytopenia. WIP is predicted to be an intrinsically disordered protein, a class of polypeptides that are of great interest because they violate the traditional structure-function paradigm. In this first (to our knowledge) study of WIP in its unbound state, we used NMR to investigate the biophysical behavior of WIP(C), a C-terminal domain fragment of WIP that includes residues 407-503 and contains the WASp-binding site. In light of the poor spectral dispersion exhibited by WIP(C) and the high occurrence (25%) of proline residues, we employed 5D-NMR(13)C-detected NMR experiments with nonuniform sampling to accomplish full resonance assignment. Secondary chemical-shift analysis, (15)N relaxation rates, and protection from solvent exchange all concurred in detecting transient structure located in motifs that span the WASp-binding site. Residues 446-456 exhibited a propensity for helical conformation, and an extended conformation followed by a short, capped helix was observed for residues 468-478. The (13)C-detected approach allows chemical-shift assignment in the WIP(C) polyproline stretches and thus sheds light on their conformation and dynamics. The effects of temperature on chemical shifts referenced to a denatured sample of the polypeptide demonstrate that heating reduces the structural character of WIP(C). Thus, we conclude that the disordered WIP(C) fragment is comprised of regions with latent structure connected by flexible loops, an architecture with implications for binding affinity and function.

Triple-Color FRET Analysis Reveals Conformational Changes in the WIP-WASp Actin-Regulating Complex

Two interaction sites provide a mechanism to finely balance the activity and degradation of an actin-regulating protein complex. WhIPping WASp into Shape Changes in the actin cytoskeleton in T cells in response to T cell receptor (TCR) activation are mediated by a complex consisting of WIP, an actin-binding protein, and WASp, a protein that promotes actin nucleation. Through a fluorescence resonance energy transfer–based study that visualized protein-protein interactions in live cells, Fried et al. revealed a two-way, end-to-end interaction between WIP and WASp. In response to TCR stimulation, one of the interactions between WIP and WASp was lost, but the other interaction remained, resulting in a conformation-induced increase in activity that stimulated actin polymerization. The conformational change also enabled the ubiquitylation and degradation of WASp to inhibit activity of the complex. Thus, the conformational change functioned as both the on and off switch. Wiskott-Aldrich syndrome protein (WASp) is a key regulator of the actin cytoskeletal machinery. Binding of WASp-interacting protein (WIP) to WASp modulates WASp activity and protects it from degradation. Formation of the WIP-WASp complex is crucial for the adaptive immune response. We found that WIP and WASp interacted in cells through two distinct molecular interfaces. One interaction occurred between the WASp-homology-1 (WH1) domain of WASp and the carboxyl-terminal domain of WIP that depended on the phosphorylation status of WIP, which is phosphorylated by protein kinase C θ (PKCθ) in response to T cell receptor activation. The other interaction occurred between the verprolin homology, central hydrophobic region, and acidic region (VCA) domain of WASp and the amino-terminal domain of WIP. This latter interaction required actin, because it was inhibited by latrunculin A, which sequesters actin monomers. With triple-color fluorescence resonance energy transfer (3FRET) technology, we demonstrated that the WASp activation mechanism involved dissociation of the first interaction, while leaving the second interaction intact. This conformation exposed the ubiquitylation site on WASp, leading to degradation of WASp. Together, these data suggest that the activation and degradation of WASp are delicately balanced and depend on the phosphorylation state of WIP. Our molecular analysis of the WIP-WASp interaction provides insight into the regulation of actin-dependent processes.

Multifunctional Cyclic d,l-α-Peptide Architectures Stimulate Non-Insulin Dependent Glucose Uptake in Skeletal Muscle Cells and Protect Them Against Oxidative Stress

Oxidative stress directly correlates with the early onset of vascular complications and the progression of peripheral insulin resistance in diabetes. Accordingly, exogenous antioxidants augment insulin sensitivity in type 2 diabetic patients and ameliorate its clinical signs. Herein, we explored the unique structural and functional properties of the abiotic cyclic D,L-α-peptide architecture as a new scaffold for developing multifunctional agents to catalytically decompose ROS and stimulate glucose uptake. We showed that His-rich cyclic D,L-α-peptide 1 is very stable under high H2O2 concentrations, effectively self-assembles to peptide nanotubes, and increases the uptake of glucose by increasing the translocation of GLUT1 and GLUT4. It also penetrates cells and protects them against oxidative stress induced under hyperglycemic conditions at a much lower concentration than α-lipoic acid (ALA). In vivo studies are now required to probe the mode of action and efficacy of these abiotic cyclic D,L-α-peptides as a novel class of antihyperglycemic compounds.

Conformational flexibility in the binding surface of the potassium channel blocker ShK.

ShK is a 35‐residue peptide that binds with high affinity to human voltage‐gated potassium channels through a conserved K‐Y dyad. Here we have employed NMR measurements of backbone‐amide 15N spin‐relaxation rates to investigate motions of the ShK backbone. Although ShK is rigid on the ps to ns timescale, increased linewidths observed for 11 backbone‐amide 15N resonances identify chemical or conformational exchange contributions to the spin relaxation. Relaxation dispersion profiles indicate that exchange between major and minor conformers occurs on the sub‐millisecond timescale. Affected residues are mostly clustered around the central helix‐kink‐helix structure and the critical K22–Y23 motif. We suggest that the less structured minor conformer increases the exposure of Y23, known to contribute to binding affinity and selectivity, thereby facilitating its interaction with potassium channels. These findings have potential implications for the design of new channel blockers based on ShK.