Structure determination of the three disulfide bond isomers of alpha-conotoxin GI: A model for the role of disulfide bonds in structural stability

The three possible disulfide bonded isomers of alpha-conotoxin GI have been selectively synthesised and their structures determined by H-1 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 Angstrom for a family of 35 structures, and comprises primarily a distorted 3(10),, 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) < GI(2-13;3-7) < GI(2-3;7-13). It was found that the peptide bond joining Cys2 to Cys3 in GI(2-3;7-13) is predominantly trans, rather than cis as theoretically predicted. These structural data are used to interpret the varying nAChR binding of the non-native forms. A model for the binding of native GI(2-7;3-13) to the mammalian nAChR is proposed, with an alpha-subunit binding face made up of Cys2, Asn4, Pro5, Ala6 and Cys7 and a selectivity face, comprised of Arg9 and His10. These two faces orient the molecule between the alpha and delta subunits of the receptor. The structure of the CCNPAC sequence of the native GI(2-7;3-13) is compared to the structure of the identical sequence from the toxic domain of heat-stable enterotoxins, which forms part of the receptor binding region of the enterotoxins, but which has a different disulfide connectivity. (C) 1998 Academic Press Limited.

A new level of conotoxin diversity, a non-native disulfide bond connectivity in alpha-conotoxin AuIB reduces structural definition but increases biological activity

alpha-Conotoxin AuIB and a disulfide bond variant of AuIB have been synthesized to determine the role of disulfide bond connectivity on structure and activity. Both of these peptides contain the 15 amino acid sequence GCCSYPPCFATNPDC, with the globular (native) isomer having the disulfide connectivity Cys(2-8 and 3-15) and the ribbon isomer having the disulfide connectivity Cys(2-15 and 3-8). The solution structures of the peptides were determined by NAIR spectroscopy, and their ability to block the nicotinic acetylcholine receptors on dissociated neurons of the rat parasympathetic ganglia was examined. The ribbon disulfide isomer, although having a less well defined structure, is surprisingly found to have approximately 10 times greater potency than the native peptide. To our knowledge this is the first demonstration of a non-native disulfide bond isomer of a conotoxin exhibiting greater biological activity than the native isomer.

Determination of the intramolecular disulfide bond arrangement and biochemical identification of the glycosylation sites of the nonstructural protein NS1 of Murray Valley encephalitis virus

The 12 cysteine residues in the flavivirus NS1 protein are strictly conserved, suggesting that they form disulfide bonds that are critical for folding the protein into a functional structure. In this study, we examined the intramolecular disulfide bond arrangement of NS1 of Murray Valley encephalitis virus and elucidated three of the six cysteine-pairing arrangements. Disulfide linkages were identified by separating tryptic-digested NS1 by reverse-phase high pressure liquid chromatography and analysing the resulting peptide peaks by protein sequencing, amino acid analysis and/or electrospray mass spectrometry. The pairing arrangements between the six amino-terminal cysteines were identified as follows: Cys(4)-Cys(15), Cys(55)-Cys(143) and Cys(179)-Cys(223). Although the pairing arrangements between the six carboxyterminal cysteines were not determined, we were able to eliminate several cysteine-pairing combinations. Furthermore, we demonstrated that all three putative N-linked glycosylation sites of NS1 are utilized and that the Asn(207) glycosylation site contains a mannose-rich glycan.

Assignment strategies for large proteins by magic-angle spinning NMR: the 21-kDa disulfide-bond-forming enzyme DsbA.

We present strategies for chemical shift assignments of large proteins by magic-angle spinning solid-state NMR, using the 21-kDa disulfide-bond-forming enzyme DsbA as prototype. Previous studies have demonstrated that complete de novo assignments are possible for proteins up to approximately 17 kDa, and partial assignments have been performed for several larger proteins. Here we show that combinations of isotopic labeling strategies, high field correlation spectroscopy, and three-dimensional (3D) and four-dimensional (4D) backbone correlation experiments yield highly confident assignments for more than 90% of backbone resonances in DsbA. Samples were prepared as nanocrystalline precipitates by a dialysis procedure, resulting in heterogeneous linewidths below 0.2 ppm. Thus, high magnetic fields, selective decoupling pulse sequences, and sparse isotopic labeling all improved spectral resolution. Assignments by amino acid type were facilitated by particular combinations of pulse sequences and isotopic labeling; for example, transferred echo double resonance experiments enhanced sensitivity for Pro and Gly residues; [2-(13)C]glycerol labeling clarified Val, Ile, and Leu assignments; in-phase anti-phase correlation spectra enabled interpretation of otherwise crowded Glx/Asx side-chain regions; and 3D NCACX experiments on [2-(13)C]glycerol samples provided unique sets of aromatic (Phe, Tyr, and Trp) correlations. Together with high-sensitivity CANCOCA 4D experiments and CANCOCX 3D experiments, unambiguous backbone walks could be performed throughout the majority of the sequence. At 189 residues, DsbA represents the largest monomeric unit for which essentially complete solid-state NMR assignments have so far been achieved. These results will facilitate studies of nanocrystalline DsbA structure and dynamics and will enable analysis of its 41-kDa covalent complex with the membrane protein DsbB, for which we demonstrate a high-resolution two-dimensional (13)C-(13)C spectrum.

The role of the disulfide bond in amyloid-like fibrillogenesis in a model peptide system

The role of the disulfide bond in amyloid-like fibrillogenesis in a model peptide system Apurba Kumar Das,(a) Michael G. B. Drew,(b) Debasish Haldar(a) and Arindam Banerjee*(a) (a)Department of Biological Chemistry, Indian Association for the Cultivation of Science, Jadavpur, Kolkata 700 032, India. E-mail: bcab@mahendra.iacs.res.in; Fax: +91-33-2473-2805 b School of Chemistry, The University of Reading, Whiteknights, Reading, UK RG6 6AD Received 28th June 2005, Accepted 20th July 2005 First published as an Advance Article on the web 11th August 2005

Tuning of intramolecular electron transfer between Ru(II) and the disulfide bond

Reaction of 2,2'-dithiodipyridine (DTDP) with cis-Ru(bpy)(2)Cl-2 (bpy = 2,2'-bipyridine) and cis-Ru(phen)(2)Cl-2 (phen = 1,10-phenanthroline) respectively yields the dicationic species [Ru(bpy) (2)(DTDP)](2+) and [Ru(phen)(2) (DTDP)](2+) in which the S-S bond of DTDP remains intact. The S-S bond undergoes a reductive cleavage when DTDP is reacted with cis-Ru(bisox)(2)Cl-2 (bisox = 4,4,4',4'-tetramethyl-2,2'-bisoxazoline) under identical conditions to generate the monocationic species [Ru(bisox)(2)(2-thiolatopyridine)]. The intramolecular electron transfer between the metal and the S-S bond is found to be subtly controlled by the crystal field strength of the ancillary bidentate N-donor ligands.

Automated NMR structure determination and disulfide bond identification of the myotoxin crotamine from Crotalus durissus terrificus

Crotamine is one of four major components of the venom of the South American rattlesnake Crotalus durissus terrificus. Similar to its counterparts in the family of the myotoxins, it induces myonecrosis of skeletal muscle cells. This paper describes a new NMR structure determination of crotamine in aqueous solution at pH 5.8 and 20 degrees C, using standard homonuclear (1)H NMR spectroscopy at 900 MHz and the automated structure calculation software ATNOS/CANDID/DYANA. The automatic NOESY spectral analysis included the identification of a most likely combination of the six cysteines into three disulfide bonds, i.e. Cys4-Cys36, Cys11-Cys30 and Cys18-Cys37; thereby a generally applicable new computational protocol is introduced to determine unknown disulfide bond connectivities in globular proteins. A previous NMR structure determination was thus confirmed and the structure refined. Crotamine contains an alpha-helix with residues 1-7 and a two-stranded anti-parallel beta-sheet with residues 9-13 and 34-38 as the only regular secondary structures. These are connected with each other and the remainder of the polypeptide chain by the three disulfide bonds, which also form part of a central hydrophobic core. A single conformation was observed, with Pro13 and Pro21 in the trans and Pro20 in the cis-form. The global fold and the cysteine-pairing pattern of crotamine are similar to the beta-defensin fold, although the two proteins have low sequence homology, and display different biological activities. (c) 2005 Elsevier Ltd. All rights reserved.

An engineered disulfide bond reversibly traps the IgE-Fc3-4 in a closed, nonreceptor binding conformation

IgE antibodies interact with the high affinity IgE Fc receptor, FcεRI, and activate inflammatory pathways associated with the allergic response. The IgE-Fc region, comprising the C-terminal domains of the IgE heavy chain, binds FcεRI and can adopt different conformations ranging from a closed form incompatible with receptor binding to an open, receptor-bound state. A number of intermediate states are also observed in different IgE-Fc crystal forms. To further explore this apparent IgE-Fc conformational flexibility and to potentially trap a closed, inactive state, we generated a series of disulfide bond mutants. Here we describe the structure and biochemical properties of an IgE-Fc mutant that is trapped in the closed, non-receptor binding state via an engineered disulfide at residue 335 (Cys-335). Reduction of the disulfide at Cys-335 restores the ability of IgE-Fc to bind to its high affinity receptor, FcεRIα. The structure of the Cys-335 mutant shows that its conformation is within the range of previously observed, closed form IgE-Fc structures and that it retains the hydrophobic pocket found in the hinge region of the closed conformation. Locking the IgE-Fc into the closed state with the Cys-335 mutation does not affect binding of two other IgE-Fc ligands, omalizumab and DARPin E2_79, demonstrating selective blocking of the high affinity receptor binding.

Activating mutations in the extracellular domain of the fibroblast growth factor receptor 2 function by disruption of the disulfide bond in the third immunoglobulin-like domain

Multiple human skeletal and craniosynostosis disorders, including Crouzon, Pfeiffer, Jackson–Weiss, and Apert syndromes, result from numerous point mutations in the extracellular region of fibroblast growth factor receptor 2 (FGFR2). Many of these mutations create a free cysteine residue that potentially leads to abnormal disulfide bond formation and receptor activation; however, for noncysteine mutations, the mechanism of receptor activation remains unclear. We examined the effect of two of these mutations, W290G and T341P, on receptor dimerization and activation. These mutations resulted in cellular transformation when expressed as FGFR2/Neu chimeric receptors. Additionally, in full-length FGFR2, the mutations induced receptor dimerization and elevated levels of tyrosine kinase activity. Interestingly, transformation by the chimeric receptors, dimerization, and enhanced kinase activity were all abolished if either the W290G or the T341P mutation was expressed in conjunction with mutations that eliminate the disulfide bond in the third immunoglobulin-like domain (Ig-3). These results demonstrate a requirement for the Ig-3 cysteine residues in the activation of FGFR2 by noncysteine mutations. Molecular modeling also reveals that noncysteine mutations may activate FGFR2 by altering the conformation of the Ig-3 domain near the disulfide bond, preventing the formation of an intramolecular bond. This allows the unbonded cysteine residues to participate in intermolecular disulfide bonding, resulting in constitutive activation of the receptor.

Respiratory chain is required to maintain oxidized states of the DsbA-DsbB disulfide bond formation system in aerobically growing Escherichia coli cells

DsbA, the disulfide bond catalyst of Escherichia coli, is a periplasmic protein having a thioredoxin-like Cys-30-Xaa-Xaa-Cys-33 motif. The Cys-30–Cys-33 disulfide is donated to a pair of cysteines on the target proteins. Although DsbA, having high oxidizing potential, is prone to reduction, it is maintained essentially all oxidized in vivo. DsbB, an integral membrane protein having two pairs of essential cysteines, reoxidizes DsbA that has been reduced upon functioning. It is not known, however, what might provide the overall oxidizing power to the DsbA–DsbB disulfide bond formation system. We now report that E. coli mutants defective in the hemA gene or in the ubiA-menA genes markedly accumulate the reduced form of DsbA during growth under the conditions of protoheme deprivation as well as ubiquinone/menaquinone deprivation. Disulfide bond formation of β-lactamase was impaired under these conditions. Intracellular state of DsbB was found to be affected by deprivation of quinones, such that it accumulates first as a reduced form and then as a form of a disulfide-linked complex with DsbA. This is followed by reduction of the bulk of DsbA molecules. These results suggest that the respiratory electron transfer chain participates in the oxidation of DsbA, by acting primarily on DsbB. It is remarkable that a cellular catalyst of protein folding is connected to the respiratory chain.

An in vivo pathway for disulfide bond isomerization in Escherichia coli

Biochemical studies have shown that the periplasmic protein disulfide oxidoreductase DsbC can isomerize aberrant disulfide bonds. Here we present the first evidence for an in vivo role of DsbC in disulfide bond isomerization. Furthermore, our data suggest that the enzymes DsbA and DsbC play distinct roles in the cell in disulfide bond formation and isomerization, respectively. We have shown that mutants in dsbC display a defect in disulfide bond formation specific for proteins with multiple disulfide bonds. The defect can be complemented by the addition of reduced dithiothreitol to the medium, suggesting that absence of DsbC results in accumulation of misoxidized proteins. Mutations in the dipZ and trxA genes have similar phenotypes. We propose that DipZ, a cytoplasmic membrane protein with a thioredoxin-like domain, and thioredoxin, the product of the trxA gene, are components of a pathway for maintaining DsbC active as a protein disulfide bond isomerase.

Roles of a conserved arginine residue of DsbB in linking protein disulfide-bond-formation pathway to the respiratory chain of Escherichia coli

The active-site cysteines of DsbA, the periplasmic disulfide-bond-forming enzyme of Escherichia coli, are kept oxidized by the cytoplasmic membrane protein DsbB. DsbB, in turn, is oxidized by two kinds of quinones (ubiquinone for aerobic and menaquinone for anaerobic growth) in the electron-transport chain. We describe the isolation of dsbB missense mutations that change a highly conserved arginine residue at position 48 to histidine or cysteine. In these mutants, DsbB functions reasonably well aerobically but poorly anaerobically. Consistent with this conditional phenotype, purified R48H exhibits very low activity with menaquinone and an apparent Michaelis constant (Km) for ubiquinone seven times greater than that of the wild-type DsbB, while keeping an apparent Km for DsbA similar to that of wild-type enzyme. From these results, we propose that this highly conserved arginine residue of DsbB plays an important role in the catalysis of disulfide bond formation through its role in the interaction of DsbB with quinones.

An isolated, surface-expressed I domain of the integrin αLβ2 is sufficient for strong adhesive function when locked in the open conformation with a disulfide bond

We introduced disulfide bonds to lock the integrin αLβ2 I domain in predicted open, ligand binding or closed, nonbinding conformations. Transfectants expressing αLβ2 heterodimers containing locked-open but not locked-closed or wild-type I domains constitutively adhered to intercellular adhesion molecule-1 (ICAM-1) substrates. Locking the I domain closed abolished constitutive and activatable adhesion. The isolated locked-open I domain bound as well as the activated αLβ2 heterodimer, and binding was abolished by reduction of the disulfide. Lovastatin, which binds under the conformationally mobile C-terminal α-helix of the I domain, inhibited binding to ICAM-1 by αLβ2 with wild-type, but not locked-open I domains. These data establish the importance of conformational change in the αL I domain for adhesive function and show that this domain is sufficient for full adhesive activity.

Structure and function in rhodopsin: Mass spectrometric identification of the abnormal intradiscal disulfide bond in misfolded retinitis pigmentosa mutants

Retinitis pigmentosa (RP) point mutations in both the intradiscal (ID) and transmembrane domains of rhodopsin cause partial or complete misfolding of rhodopsin, resulting in loss of 11-cis-retinal binding. Previous work has shown that misfolding is caused by the formation of a disulfide bond in the ID domain different from the native Cys-110–Cys-187 disulfide bond in native rhodopsin. Here we report on direct identification of the abnormal disulfide bond in misfolded RP mutants in the transmembrane domain by mass spectrometric analysis. This disulfide bond is between Cys-185 and Cys-187, the same as previously identified in misfolded RP mutations in the ID domain. The strategy described here should be generally applicable to identification of disulfide bonds in other integral membrane proteins.

Reversibly locking a protein fold in an active conformation with a disulfide bond: Integrin αL I domains with high affinity and antagonist activity in vivo

The integrin αLβ2 has three different domains in its headpiece that have been suggested to either bind ligand or to regulate ligand binding. One of these, the inserted or I domain, has a fold similar to that of small G proteins. The I domain of the αM and α2 subunits has been crystallized in both open and closed conformations; however, the αL I domain has been crystallized in only the closed conformation. We hypothesized that the αL domain also would have an open conformation, and that this would be the ligand binding conformation. Therefore, we introduced pairs of cysteine residues to form disulfides that would lock the αL I domain in either the open or closed conformation. Locking the I domain open resulted in a 9,000-fold increase in affinity to intercellular adhesion molecule-1 (ICAM-1), which was reversed by disulfide reduction. By contrast, the affinity of the locked closed conformer was similar to wild type. Binding completely depended on Mg2+. Orders of affinity were ICAM-1 > ICAM-2 > ICAM-3. The kon, koff, and KD values for the locked open I domain were within 1.5-fold of values previously determined for the αLβ2 complex, showing that the I domain is sufficient for full affinity binding to ICAM-1. The locked open I domain antagonized αLβ2-dependent adhesion in vitro, lymphocyte homing in vivo, and firm adhesion but not rolling on high endothelial venules. The ability to reversibly lock a protein fold in an active conformation with dramatically increased affinity opens vistas in therapeutics and proteomics.