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 crossrelaxation 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 K D of 3 6 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.

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 IFNa2 with R2-EC using multidimensional NMR techniques. NMR shows that IFNa2 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 IFNa2. 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 IFNa2 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.Keywords: interferons; protein-protein docking; protein-protein interactions; multidimensional NMR; cross saturation Type I Interferons (IFNs) are a family of homologous helical cytokines initiating strong antiviral and antiproliferative activity. Since IFNs are at the forefront of defense against viral infection and promote a variety of biological effects, they are essential for the survival of higher vertebrates (Stark et al. 1998;Biron 2001). Not surprisingly, IFNs are the human proteins most widely used as therapeutics for the treatment of several kinds of cancer and viral diseases (e.g., Perry and Jarvis 2001; Kirkwood 2002). Human type I interferons include 13 IFNa isotypes (and allelic forms) and single forms of IFNb, IFNe, IFNk, and IFNv (Pestka et al. 2004). Sequence homology between all IFNa isotypes is high, with ;80% identity, and the identity of the IFNa isotypes to v, b, e, and k subtypes is 50%, 31%, 28%, and 27%, respectively. IFNg is the only known type II interferon (Pestka et al. 1987), and it shares only 10% identity with IFNa. The threedimensional structures of several type I IFNs have been solved, and a high resolution NMR structure of human IFNa2a (Klaus et al. 1997) and the X-ray structures of IFNa2b (Karpusas et al. 1997) and IFNb (Radhakrishnan et al. 1996) are available.Reprint requests to: Jacob Anglister, Department of Structural Biology, Weizmann Institute of Science, 76100 Rehovot, Israel; e-mail: jacob.anglister@wei...

A set of TROSY-HNCO (tHNCO)-based 3D experiments is presented for measuring (15)N relaxation parameters in large, membrane-associated proteins, characterized by slow tumbling times and significant spectral overlap. Measurement of backbone (15)N R (1), R (1rho), (15)N-{(1)H} NOE, and (15)N CSA/dipolar cross correlation is demonstrated and applied to study the dynamic behavior of the homotetrameric KcsA potassium channel in SDS micelles under conditions where this channel is in the closed state. The micelle-encapsulated transmembrane domain, KcsA(TM), exhibits a high degree of order, tumbling as an oblate ellipsoid with a global rotational correlation time, tau(c) = 38 +/- 2.5 ns, at 50 degrees C and a diffusion anisotropy, Dparallel/Dperpendicular = 0.79+/-0.05, corresponding to an aspect ratio a/b >/= 1.4. The N- and C-terminal intracellular segments of KcsA exhibit considerable internal dynamics (S (2) values in the 0.2-0.45 range), but are distinctly more ordered than what has been observed for unstructured random coils. Relaxation behavior in these domains confirms the position of the C-terminal helix, and indicates that in SDS micelles, this amphiphilic helix does not associate into a stable homotetrameric helical bundle. The relaxation data indicate the absence of elevated backbone dynamics on the ps-ns time scale for the 5-residue selectivity filter, which selects K(+) ions to enter the channel.

The structure of a peptide corresponding to residues 182-202 of the acetylcholine receptor alpha1 subunit in complex with alpha-bungarotoxin was solved using NMR spectroscopy. The peptide contains the complete sequence of the major determinant of AChR involved in alpha-bungarotoxin binding. One face of the long beta 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 beta hairpin, which creates the inner and partially hidden pocket for acetylcholine. An NMR-derived model for the receptor complex with two alpha-bungarotoxin molecules shows that this pocket is occupied by the conserved alpha-neurotoxin residue R36, which forms cation-pi interactions with both alphaW149 and gammaW55/deltaW57 of the receptor and mimics acetylcholine.