The filmless hospital--a legal challenge]

Rapid inactivation of voltage-gated K+ (Kv) channels is mediated by an N-terminal domain (inactivating ball domain) which blocks the open channel from the cytoplasmic side. Inactivating ball domains of various Kv channels are also biologically active when synthesized separately and added as a peptide to the solution. Synthetic inactivating ball domains from different Kv channels with hardly any sequence homology mediate quite similar effects even on unrelated Kv channel subtypes whose inactivation domain has been deleted. The solution structure of the inactivating ball peptide from Shaker (Sh-P22) was analyzed with NMR spectroscopy. The NMR data indicate a non-random structure in an aqueous environment. However, while other inactivating ball peptides showed well-defined three-dimensional structures under these conditions, Sh-P22 does not have a unique, compactly folded structure in solution.     

NMR structure of inactivation gates from mammalian voltage-dependent potassium channels

The electrical signalling properties of neurons originate largely from the gating properties of their ion channels. N-type inactivation of voltage-gated potassium (Kv) channels is the best-understood gating transition in ion channels, and occurs by a 'ball-and-chain' type mechanism. In this mechanism an N-terminal domain (inactivation gate), which is tethered to the cytoplasmic side of the channel protein by a protease-cleavable chain, binds to its receptor at the inner vestibule of the channel, thereby physically blocking the pore. Even when synthesized as a peptide, ball domains restore inactivation in Kv channels whose inactivation domains have been deleted. Using high-resolution nuclear magnetic resonance (NMR) spectroscopy, we analysed the three-dimensional structure of the ball peptides from two rapidly inactivating mammalian K. channels (Raw3 (Kv3.4) and RCK4 (Kv1.4)). The inactivation peptide of Raw3 (Raw3-IP) has a compact structure that exposes two phosphorylation sites and allows the formation of an intramolecular disulphide bridge between two spatially close cysteine residues. Raw3-IP exhibits a characteristic surface charge pattern with a positively charged, a hydrophobic, and a negatively charged region. The RCK4 inactivation peptide (RCK4-IP) shows a similar spatial distribution of charged and uncharged regions, but is more flexible and less ordered in its amino-terminal part.     

Adoption of beta structure by the inactivating "ball" peptide of the Shaker B potassium channel

The conformation of the inactivating peptide of the Shaker B K+ channel (ShB peptide) and that of a noninactivating mutant (ShBL7E peptide) have been studied. Under all experimental conditions explored, the mutant peptide remains in a predominantly nonordered conformation. On the contrary, the inactivating ShB peptide has a great tendency to adopt a highly stable beta structure, particularly when challenged "in vitro" by anionic phospholipid vesicles. Because the putative peptide binding elements at the inner mouth of the channel comprise a ring of anionic residues and a hydrophobic pocket, we hypothesize that the conformational restrictions imposed on the ShB peptide by its interaction with the anionic lipid vesicles could partly imitate those imposed by the above ion channel elements. Thus, we propose that adoption of beta structure by the inactivating peptide may also occur during channel inactivation. Moreover, the difficulties encountered by the noninactivating ShBL7E peptide mutant to adopt beta structure and the observation that trypsin hydrolysis of the ShB peptide prevent both structure formation and channel inactivation lend further support to the hypothesis that adoption of beta structure by the inactivating peptide in a hydrophobic environment is important in determining channel blockade.     

Structure-activity relationships of the Kvbeta1 inactivation domain and its putative receptor probed using peptide analogs of voltage-gated potassium channel alpha- and beta-subunits

Certain beta-subunits exert profound effects on the kinetics of voltage-gated (Kv) potassium channel inactivation through an interaction between the amino-terminal "inactivation domain" of the beta-subunit and a "receptor" located at or near the cytoplasmic mouth of the channel pore. Here we used a bacterial random peptide library to examine the structural requirements for this interaction. To identify peptides that bind Kv1.1 we screened the library against a synthetic peptide corresponding to the predicted S4-S5 cytoplasmic loop of the Kv1.1 alpha-subunit (residues 313-328). Among the highest affinity interactors were peptides with significant homology to the amino terminus of Kvbeta1. We performed a second screen using a peptide from the amino terminus of Kvbeta1 (residues 2-31) as "bait" and identified peptide sequences with significant homology to the S4-S5 loop of Kv1.1. A series of synthetic peptides containing mutations of the wild-type Kvbeta1 and Kv1.1 sequences were examined for their ability to inhibit Kvbeta1/Kv1.1 binding. Amino acids Arg20 and Leu21 in Kvbeta1 and residues Arg324 and Leu328 in Kv1.1 were found to be important for the interaction. Taken together, these data provide support for the contention that the S4-S5 loop of the Kv1.1 alpha subunit is the likely acceptor for the Kvbeta1 inactivation domain and provide information about residues that may underlie the protein-protein interactions responsible for beta-subunit mediated Kv channel inactivation.     

Structure of segments of a G protein-coupled receptor: CD and NMR analysis of the Saccharomyces cerevisiae tridecapeptide pheromone receptor

Peptides representing both loop and the sixth transmembrane regions of the alpha-factor receptor of Saccharomyces cerevisiae were synthesized by solid-phase procedures and purified to near homogeneity. CD, nmr, and modeling analysis indicated that in aqueous media the first extracellular loop peptide E1(107-125), the third intracellular loop peptide I3(231-243), and the carboxyl terminus peptide I4(350-372) were mostly disordered. In contrast, the second extracellular loop peptide E2(191-206) assumed a well-defined structure in aqueous medium and the sixth transmembrane domain peptide receptor M6(252-269, C252A) was highly helical in trifluoroethanol/water (4:1), exhibiting a kink at Pro258. A synthetic peptide containing a sequence similar to that of the sixth transmembrane domain of a constitutively active alpha-factor receptor M6(252-269, C252A, P258L) in which Leu replaces Pro258 exhibited significantly different biophysical properties than the wild-type sequence. In particular, this peptide had very low solubility and gave CD resembling that of a beta-sheet structure in hexafluoroacetone/water (1:1) whereas the wild-type peptide was partially helical under identical conditions. These results would be consistent with the hypothesis that the constitutive activity of the mutant receptor is linked to a conformational change in the sixth transmembrane domain. The study of the receptor segments also indicate that peptides corresponding to loops of the alpha-factor receptor do not appear to assume turn structures.     

ShK-Dap22, a potent Kv1.3-specific immunosuppressive polypeptide

The voltage-gated potassium channel in T lymphocytes, Kv1.3, is an important molecular target for immunosuppressive agents. A structurally defined polypeptide, ShK, from the sea anemone Stichodactyla helianthus inhibited Kv1.3 potently and also blocked Kv1.1, Kv1.4, and Kv1.6 at subnanomolar concentrations. Using mutant cycle analysis in conjunction with complementary mutagenesis of ShK and Kv1.3, and utilizing the structure of ShK, we determined a likely docking configuration for this peptide in the channel. Based upon this topological information, we replaced the critical Lys22 in ShK with the positively charged, non-natural amino acid diaminopropionic acid (ShK-Dap22) and generated a highly selective and potent blocker of the T-lymphocyte channel. ShK-Dap22, at subnanomolar concentrations, suppressed anti-CD3 induced human T-lymphocyte [3H]thymidine incorporation in vitro. Toxicity with this mutant peptide was low in a rodent model, with a median paralytic dose of approximately 200 mg/kg body weight following intravenous administration. The overall structure of ShK-Dap22 in solution, as determined from NMR data, is similar to that of native ShK toxin, but there are some differences in the residues involved in potassium channel binding. Based on these results, we propose that ShK-Dap22 or a structural analogue may have use as an immunosuppressant for the prevention of graft rejection and for the treatment of autoimmune diseases.     

Disulfide bond exchange in rhodopsin

Rhodopsin contains two cysteines (Cys110 and Cys187) that are highly conserved among members of the G protein coupled receptor family and that form a disulfide bond connecting helixes 3 and 4 on the extracellular side of the protein. However, recent work on a rhodopsin mutant split in the cytoplasmic loop connecting helixes 3 and 4 has shown that the amino- and carboxy-terminal fragments of this split protein do not comigrate on nonreducing SDS-PAGE gels, suggesting that the native Cys110-Cys187 disulfide bond is not present in this mutant [Ridge et al. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 3204-3208; Yu et al. (1995) Biochemistry 34, 14963-14969]. We show here that the inability to observe the disulfide bond on SDS gels is the result of a disulfide bond exchange reaction which occurs when this split rhodopsin is denatured in preparation for SDS-PAGE. Cys185 reacts with the native disulfide, displacing Cys110 and forming a new disulfide with Cys187. If the sulfhydryl-specific reagent N-ethylmaleimide is included in the sample during preparation for electrophoresis or if Cys185 is changed to Ser, the two fragments do comigrate with full-length rhodopsin on SDS gels and, therefore, are connected by the native Cys110-Cys187 disulfide bond. In related experiments, we find no evidence that the Cys110-Cys187 disulfide bond is broken upon formation of the active intermediate metarhodopsin II.     

Nonparametric estimation of covariance structure in longitudinal data

The three possible disulfide bonded isomers of alpha-conotoxin GI have been selectively synthesised and their structures determined by 1H NMR spectroscopy. alpha-Conotoxin GI derives from the venom of Conus geographus and is a useful neuropharmacological tool as it selectively binds to the nicotinic acetylcholine receptor (nAChR), a ligand-gated ion channel involved in nerve signal transmission. The peptide has the sequence ECCNPACGRHYSC-NH2, and the three disulfide bonded isomers are referred to as GI(2-7;3-13), GI(2-13;3-7) and GI(2-3;7-13). The NMR structure for the native isomer GI(2-7;3-13) is of excellent quality, with a backbone pairwise RMSD of 0.16 A for a family of 35 structures, and comprises primarily a distorted 310 helix between residues 5 to 11. The two non-native isomers exhibit multiple conformers in solution, with the major populated forms being different in structure both from each other and from the native form. Structure-activity relationships for the native GI(2-7;3-13) as well as the role of the disulfide bonds on folding and stability of the three isomers are examined. It is concluded that the disulfide bonds in alpha-conotoxin GI play a crucial part in determining both the structure and stability of the peptide. A trend for increased conformational heterogeneity was observed in the order of GI(2-7;3-13)相似文献   

Conformations of primary amphipathic carrier peptides in membrane mimicking environments

Two peptides designed for drug delivery were generated by the combination of a signal peptide with a nuclear localization sequence and are shown to facilitate the cellular internalization of small molecules which are covalently linked to these peptides. In order to understand the mechanism of internalization, the conformations of the peptides were investigated through different approaches both in solution and in membrane-mimicking environments. These peptides are highly versatile and adopt different conformational states depending on their environment. While in a disordered form in water, they adopt an alpha-helical structure in TFE and in the presence of micelles of SDS or DPC. The structured domain encompasses the hydrophobic part of the peptides, whereas the charged C-termini remain unstructured. In contrast, in the presence of lipids and whatever the nature of the phosphate headgroup, the two peptides mainly adopt an antiparallel beta-sheet form and embed in the lipidic cores. This result suggests that the beta-sheet is responsible for the translocation through the cellular membranes but also questions the conformational state of signal peptides when associated to hydrophilic sequences.     

Roles for the two cysteine residues of AhpC in catalysis of peroxide reduction by alkyl hydroperoxide reductase from Salmonella typhimurium

The catalytic properties of cysteine residues Cys46 and Cys165, which form intersubunit disulfide bonds in the peroxidatic AhpC protein of the alkyl hydroperoxide reductase (AhpR) system from Salmonella typhimurium, have been investigated. The AhpR system, composed of AhpC and a flavoprotein reductase, AhpF, catalyzes the pyridine nucleotide-dependent reduction of organic hydroperoxides and hydrogen peroxide. Amino acid sequence analysis of the disulfide-containing tryptic peptide demonstrated the presence of two identical disulfide bonds per dimer of oxidized AhpC located between Cys46 on one subunit and Cys165 on the other. Mutant AhpC proteins containing only one (C46S and C165S) or no (C46,165S) cysteine residues were purified and shown by circular dichroism studies to exhibit no major disruptions in secondary structure. In NADH-dependent peroxidase assays in the presence of AhpF, the C165S mutant was fully active in comparison with wild-type AhpC, while C46S and C46,165S displayed no peroxidatic activity. In addition, only C165S was oxidized by 1 equiv of hydrogen peroxide, giving a species that was stoichiometrically reducible by NADH in the presence of a catalytic amount of AhpF. Oxidized C165S also reacted rapidly with a stoichiometric amount of the thiol-containing reagent 2-nitro-5-thiobenzoic acid to generate a mixed disulfide, and was susceptible to inactivation by hydrogen peroxide, strongly supporting its identification as a cysteine sulfenic acid (Cys46-SOH). The lack of reactivity of the C46S mutant toward peroxides was not a result of inaccessibility of the remaining thiol as demonstrated by its modification with 5, 5'-dithiobis(2-nitrobenzoic acid), but could be due to the lack of a proximal active-site base which would support catalysis through proton donation to the poor RO- leaving group. Our results clearly identify Cys46 as the peroxidatic center of AhpC and Cys165 as an important residue for preserving the activity of wild-type AhpC by reacting with the nascent sulfenic acid of the oxidized protein (Cys46-SOH) to generate a stable disulfide bond, thus preventing further oxidation of Cys46-SOH by substrate.     

Evidence for interaction between transmembrane segments in assembly of Kv1.3

Previously, we showed that the N-terminal recognition domain (T1) of Kv1.3 was not required for assembly of functional channels [Tu et al. (1996) J. Biol. Chem. 271, 18904-18911]. Moreover, specific Kv1.3 peptide fragments including regions of the central core are able to inhibit expression of current produced from a channel lacking the T1 domain, Kv1.3(T1-). To elucidate the mechanism whereby Kv1.3 peptide fragments suppress Kv1.3(T1-) current, we have studied the ability of peptide fragments containing the transmembrane segments S1, S1-S2, or S1-S2-S3 to physically associate with the Kv1.3(T1-) polypeptide subunit in vitro in microsomal membranes. Using c-myc (9E10) epitope-labeled peptide fragments and anti-myc antibody as well as antisera to the Kv1.3 C-terminus, we now demonstrate specific association of these peptide fragments with Kv1.3(T1-). Association of peptide fragments with Kv1.3(T1-) was correlated with integration of both proteins into the membrane. Furthermore, the relative strength and kinetics of this association directly correlated with the ability of fragments to suppress Kv1.3(T1-) current. The rate-limiting step in the sequential synthesis, integration, and formation of a complex was the association of integrated polypeptides within the plane of the lipid bilayer. These results strongly suggest that the physical association of transmembrane segments provides the basis for suppression of K+ channel function by K+ channel peptide fragments in vivo. Moreover, the S1-S2-S3 peptide fragment potently suppressed full-length Kv1.3, thus implicating a role for the S1-S2-S3 region of Kv1.3 in the assembly of the Kv1.3 channel. We refer to these putative association sites as IMA (intramembrane association) sites.     

A mammalian transient type K+ channel, rat Kv1.4, has two potential domains that could produce rapid inactivation

The "ball and chain" model has been shown to be suitable for explaining the rapid inactivation of voltage-dependent K+ channels. For the Drosophila Shaker K+ channel (ShB), the first 20 residues of the amino terminus have been identified as the inactivation ball that binds to the open channel pore and blocks ion flow (Hoshi, T., Zagotta, W. N., and Aldrich, R. W. (1990) Science 250, 533-538; Zagotta, W. N., Hoshi, T., and Aldrich, R. W. (1990) Science 250, 568-571). We studied the structural elements responsible for rapid inactivation of a mammalian transient type K+ channel (rat Kv1.4) by constructing various mutants in the amino terminus and expressing them in Xenopus oocytes. Although it has been reported that the initial 37 residues might form the inactivation ball for rat Kv1.4 (Tseng-Crank, J., Yao, J.-A., Berman M. F., and Tseng, G.-N. (1993) J. Gen. Physiol. 102, 1057-1083), we found that not only the initial 37 residues, but also the following region, residues 40-68, could function independently as an inactivation gate. Like the Shaker inactivation ball, both potential inactivation domains have a hydrophobic amino-terminal region and a hydrophilic carboxyl-terminal region having net positive charge, which is essential for the domains to function as an inactivation gate.     

Aberrant intermolecular disulfide bonding in a mutant HLA-DM molecule: implications for assembly, maturation, and function

HLA-DM (abbreviated DM) is an MHC-encoded glycoprotein that catalyzes the selective release of peptides, including class II-associated invariant chain peptides, from MHC class II molecules. To perform its function, DM must assemble in the endoplasmic reticulum (ER), travel to endosomes, and interact productively with class II molecules. We have described previously an EBV-transformed B cell line, 7.12.6, which displays a partial Ag presentation defect and expresses a mutated DM beta-chain with Cys79 replaced by Tyr. In this study, we show that HLA-DR molecules in 7.12.6 have a defect in peptide loading and accumulate class II-associated invariant chain peptides (CLIP). Peptide loading is restored by transfection of wild-type DMB. The mutant DM molecules exit the ER slowly and are degraded rapidly, resulting in greatly reduced levels of mutant DM in post-Golgi compartments. Whereas wild-type DM forms noncovalent alphabeta dimers, such dimers form inefficiently in 7.12.6; many mutant DM beta-chains instead form a disulfide-bonded dimer with DM alpha. Homodimers of DM beta are also detected in 7.12.6 and in the alpha-chain defective mutant, 2.2.93. We conclude that during folding of wild-type DM, the native conformation is stabilized by a conserved disulfide bond involving Cys79beta and by noncovalent contacts with DM alpha. Without these interactions, DM beta can form malfolded structures containing interchain disulfide bonds; malfolding is correlated with ER retention and accelerated degradation.     

Susceptibility of cloned K+ channels to reactive oxygen species

Free radical-induced oxidant stress has been implicated in a number of physiological and pathophysiological states including ischemia and reperfusion-induced dysrhythmia in the heart, apoptosis of T lymphocytes, phagocytosis, and neurodegeneration. We have studied the effects of oxidant stress on the native K+ channel from T lymphocytes and on K+ channels cloned from cardiac, brain, and T-lymphocyte cells and expressed in Xenopus oocytes. The activity of three Shaker K+ channels (Kv1.3, Kv1.4, and Kv1.5), one Shaw channel (Kv3.4), and one inward rectifier K+ channel (IRK3) was drastically inhibited by photoactivation of rose bengal, a classical generator of reactive oxygen species. Other channel types (such as Shaker K+ channel Kv1.2, Shab channels Kv2.1 and Kv2.2, Shal channel Kv4.1, inward rectifiers IRK1 and ROMK1, and hIsK) were completely resistant to this treatment. On the other hand tert-butyl hydroperoxide, another generator of reactive oxygen species, removed the fast inactivation processes of Kv1.4 and Kv3.4 but did not alter other channels. Xanthine/xanthine oxidase system had no effect on all channels studied. Thus, we show that different types of K+ channels are differently modified by reactive oxygen species, an observation that might be of importance in disease states.     

Rapidly evolving hepatitis C virus-related cirrhosis in a human immunodeficiency virus-infected patient receiving triple antiretroviral therapy

The Tyr92-Pro93 peptide group of bovine pancreatic ribonuclease A (RNase A) exists in the cis conformation in the native state. From unfolding/refolding kinetic studies of the disulfide-intact wild-type protein and of a variant in which Pro93 had been replaced by Ala, it had been suggested that the Tyr92-Ala93 peptide group also exists in the cis conformation in the native state. Here, we report the crystal structure of the P93A variant. Although there is disorder in the region of residues 92 and 93, the best structural model contains a cis peptide at this position, lending support to the results of the kinetics experiments. We also report the crystal structure of the C[40, 95]A variant, which is an analog of the major rate-determining three-disulfide intermediate in the oxidative folding of RNase A, missing the 40-95 disulfide bond. As had been detected by NMR spectroscopy, the crystal structure of this analog shows disorder in the region surrounding the missing disulfide. However, the global chain fold of the remainder of the protein, including the disulfide bond between Cys65 and Cys72, appears to be unaffected by the mutation.     

Solution structure of bromelain inhibitor IV from pineapple stem: structural similarity with Bowman-Birk trypsin/chymotrypsin inhibitor from soybean

Bromelain inhibitor VI from pineapple stem (BI-VI) is a unique double-chain inhibitor with an 11-residue light chain and a 41-residue heavy chain by disulfide bonds and inhibits the cysteine proteinase bromelain competitively. The structure of BI-VI in aqueous solution was determined using nuclear magnetic resonance spectroscopy and simulated annealing-based calculations. Its three-dimensional structure was shown to be composed of two distinct domains, each of which is formed by a three-stranded antiparallel beta-sheet. Unexpectedly, BI-VI was found to share a similar folding and disulfide bond connectivities not with cystatin superfamily inhibitors which inhibit the same cysteine proteinases but with the Bowman-Birk trypsin/chymotrypsin inhibitor from soybean (BBI-I). BBI-I is a 71-residue inhibitor which has two independent inhibitory sites toward the serine proteinases trypsin and chymotrypsin. These structural similarities with BBI-I suggest that they have evolved from a common ancestor and differentiated in function during a course of molecular evolution.     

Alternate amino terminal processing of surfactant protein A results in cysteinyl isoforms required for multimer formation

The biological functions of rat surfactant protein A (SP-A), an oligomer composed of 18 polypeptide subunits derived from a single gene, are dependent on intact disulfide bonds. Reducible and collagenase-reversible covalent linkages of as many as six or more subunits in the molecule indicate the presence of at least two NH2-terminal interchain disulfide bonds. However, the reported primary structure of rat SP-A predicts that only Cys6 in this region is available for interchain disulfide formation. Direct evidence for a second disulfide bridge was obtained by analyses of a set of three mutant SP-As with telescoping deletions from the reported NH2-terminus. Two of the truncated recombinant proteins formed reducible dimers despite deletion of the domain containing Cys6. Edman degradation revealed that each mutant protein was a mixture of two isoforms with and without an isoleucine-lysine-cysteine (IKC) extension at the NH2-terminus, which was derived from the COOH-terminal end of the reported signal peptide. Large variations in the abundance of the IKC isoforms between truncated SP-As suggested that the amino acid sequences located downstream from the signal peptide modulated alternate-site cleavage by signal peptidase. Elution of the newly identified cysteine in the position of DiPTH-Cys indicated participation in disulfide linkage, which was interchain based on the direct correlation between prevalence of the IKC variant and the extent of dimerization for each truncated protein. Sequencing of both native rat SP-A and human SP-A also revealed isoforms with disulfide-forming NH2-terminal extensions. The extended rat SP-A isoforms were enriched in the more fully glycosylated and multimeric SP-A species separated on SDS-PAGE gels. Thus, a novel post translational modification results in naturally occurring cysteinyl isoforms of rat SP-A, which are essential for multimer formation.     

Metal binding properties and secondary structure of the zinc-binding domain of Nup475

Nup475 is a nuclear zinc-binding protein of unknown function that is induced in mammalian cells by growth factor mitogens. Nup475 contains two tandemly repeated sequences YKTELCX8CX5CX3H (Cys3His repeats) that are thought to be zinc-bindin domains. Similar sequences have been found in a number of proteins from various species of eukaryotes. To determine the metal binding properties and secondary structure of the putative zinc-binding domains of Nup475, we have used synthetic or recombinant peptides that contain one or two domain sequences. The peptide with a single domain bound 1.0 +/- 0.1 equivalents of Co2+, and the peptide with two domains bound 1.7 +/- 0.4 equivalents of Co2+. Both peptides bound Co2+ and Zn2+ with affinities similar to those of classical zinc finger peptides. In each case, the Co2+ complex exhibited strong d-d transitions characteristic of tetrahedral coordination. For structural studies by nuclear magnetic resonance spectroscopy, we used a more soluble two-domain peptide that had a single amino acid substitution in a nonconserved amino acid residue in the second Cys3His repeat. The mutant peptide unexpectedly showed loss of one of its metal binding sites and displayed ordered structure for only the first Cys3His sequence. On the basis of the nuclear magnetic resonance data, we propose a structure for the Nup475 metal-binding domain in which the zinc ion is coordinated by the conserved cysteines and histidine, and the conserved YKTEL motif forms a parallel sheet-like structure with the C terminus of this domain. This structure is unlike that of any previously described class of metal binding domain.