NMR studies of [U-13C]cyclosporin A bound to cyclophilin: bound conformation and portions of cyclosporin involved in binding

Cyclosporin A (CsA), a potent immunosuppressant, is known to bind with high specificity to cyclophilin (CyP), a 17.7 kDa protein with peptidyl-prolyl isomerase activity. In order to investigate the three-dimensional structure of the CsA/CyP complex, we have applied a variety of multidimensional NMR methods in the study of uniformly 13C-labeled CsA bound to cyclophilin. The 1H and 13C NMR signals of cyclosporin A in the bound state have been assigned, and from a quantitative interpretation of the 3D NOE data, the bound conformation of CsA has been determined. Three-dimensional structures of CsA calculated from the NOE data by using a distance geometry/simulated appealing protocol were found to be very different from previously determined crystalline and solution conformations of uncomplexed CsA. In addition, from CsA/CyP NOEs, the portions of CsA that interact with cyclophilin were identified. For the most part, those CsA residues with NOEs to cyclophilin were the same residues important for cyclophilin binding and immunosuppressive activity as determined from structure/activity relationships. The structural information derived in this study together with the known structure/activity relationships for CsA analogues may prove useful in the design of improved immunosuppressants. Moreover, the approach that is described for obtaining the structural information is widely applicable to the study of small molecule/large molecule interactions.     

1H, 13C and 15N backbone assignments of cyclophilin when bound to cyclosporin A (CsA) and preliminary structural characterization of the CsA binding site.

The backbone 1H, 13C and 15N chemical shifts of cyclophilin (CyP) when bound to cyclosporin A (CsA) have been assigned from heteronuclear two- and three-dimensional NMR experiments involving selectively 15N- and uniformly 15N- and 15N,13C-labeled cyclophilin. From an analysis of the 1H and 15N chemical shifts of CyP that change upon binding to CsA and from CyP/CsA NOEs, we have determined the regions of cyclophilin involved in binding to CsA.     

Mapping of cyclosporin A binding sites in cyclophilin A by using synthetic peptides

In order to map cyclosporin A (CsA) binding sites of cyclophilin (CyP), we synthesized the complete set of overlapping 157 octapeptides corresponding to human CyP A using the multi-pin peptide synthesis system. The pin-coupled synthetic octapeptides were examined in terms of binding ability to CsA by a modification of the enzyme-linked immunosorbent assay. Significant binding of CsA was detected with 35 synthetic N alpha-acetylated octapeptides possessing the N-terminal amino acids corresponding to the residues in positions 24-26, 42-44, 69-73, 75, 76, 89-91, 102, 116, 124-131, 144-151 and 152 in human CyP A, respectively. Other eight octapeptides showed moderate CsA binding activity. The distinct binding of octapeptides covering the C-terminal region of the CyP A was particularly significant. These data are to be compared with the information provided by X-ray and NMR studies on the CsA binding sites and furnish thus a test of the reported method. The present study also gave added insight into the CsA interaction sites of CyP.     

Cyclophilin A-induced alterations of human immunodeficiency virus type 1 CA protein in vitro.

Cyclophilin A (CyP A), a cellular chaperone with cis-trans prolyl isomerase activity, is required for postassembly events in human immunodeficiency virus type 1 (HIV-1) replication. The requirement for CyP A maps to sequences in the capsid (CA) domain of the structural precursor, Gag. To determine the effects of interaction with CyP A on capsid (CA) protein structure, the binding interaction was investigated in vitro, using recombinant HIV-1 CA protein (which forms oligomers in solution) and human CyP A. The CA and CyP A proteins interacted to form a complex which was detected predominantly as a heterodimer on sodium dodecyl sulfate (SDS)-polyacrylamide gels. Complex formation exhibited a pH optimum of 5. The CA protein in the complex was exclusively in a conformation whereby the N terminus was blocked to Edman degradation. This finding was unexpected since CyP A binds to the central region of the CA protein (residues 85 to 93). Examination of CA protein incubated with CyP A for alterations in structure indicated that CyP A preferentially interacted with the subpopulation of trypsin-susceptible subunits in the oligomers and significantly reduced their sensitivity to proteolysis. Like CA-CyP A complex formation, conversion to trypsin resistance also exhibited a pH optimum of 5. Both complex formation and the changes in tryptic susceptibility were only partially inhibited by cyclosporin A (CsA). This appeared to be due to a CA subpopulation able to bind CyP A despite the presence of CsA. Our results identify specific tryptic sites both proximal and distal to the CyP A binding region that are altered by CyP A binding and/or by CyP A's prolyl isomerase activity. Comparison with the X-ray structure of a complex containing CyP A bound to an amino-terminal fragment of the CA protein (CA1-151) (T.R. Gamble et al., Cell 87:1285-1294, 1996) indicates that the tryptic sites that become inaccessible are among the same residues that lose a significant amount of accessible surface area through CA-CA subunit contacts made in the presence of CyP A.     

Procedure for C2 deuteration of nucleic acids and determination of A psi 31 pseudouridine conformation by nuclear Overhauser effect in yeast tRNAPhe

Nuclear Overhauser effect (NOE) combined with semispecific deuteration provides a general strategy for identification of exchangeable protons in nucleic base pairs, and has been extended to NOEs involving purine C2 protons in tRNA. Deuterated tri-ethyl orthoformate was condensed with 5(4)-amino imidazole 4(5)-carboxamide to yield C2 deuterated hypoxanthine. C2 deuterated hypoxanthine was fed to a purine requiring mutant of yeast and C2 deuterated yeast tRNAPhe was isolated. This C2 deuterated tRNAPhe was used to identify A psi 31 and U8-A14. A psi 31 was found to be bonded through N1H. The utility of C2 deuteration in nucleic acid NMR is thus demonstrated.     

Evaluation of the anti-hepatitis C virus effects of cyclophilin inhibitors, cyclosporin A, and NIM811

Hepatitis C virus (HCV) is a major causative agent of hepatocellular carcinoma. We recently discovered that the immunosuppressant cyclosporin A (CsA) and its analogue lacking immunosuppressive function, NIM811, strongly suppress the replication of HCV in cell culture. Inhibition of a cellular replication cofactor, cyclophilin (CyP) B, is critical for its anti-HCV effects. Here, we explored the potential use of CyP inhibitors for HCV treatment by analyzing the HCV replicon system. Treatment with CsA and NIM811 for 7 days reduced HCV RNA levels by 2-3 logs, and treatment for 3 weeks reduced HCV RNA to undetectable levels. NIM811 exerted higher anti-HCV activity than CsA at lower concentrations. Both CyP inhibitors rapidly reduced HCV RNA levels even further in combination with IFNalpha without modifying the IFNalpha signal transduction pathway. In conclusion, CyP inhibitors may provide a novel strategy for anti-HCV treatment.     

A single Trp121 to Ala121 mutation in human cyclophilin alters cyclosporin A affinity and peptidyl-prolyl isomerase activity

Fluorescence and NMR spectral data have suggested an interaction between the single tryptophan in cyclophilin (CyP) and its high affinity ligand cyclosporin A (CsA). To study this interaction, a site mutation of Trp121 to Ala was introduced into human cyclophilin (CyP) and the encoded protein was expressed in E. coli. The Ala121 mutant was shown to catalyze the peptidyl-prolyl cis-trans isomerase (rotomase) reaction with several peptide substrates, albeit at less than ten percent the rate of the purified recombinant human CyP. Values for the apparent inhibition constant (Ki,app) of cyclosporin A with the human CyP and the Ala121 mutant were determined to be 1.6 +/- 0.4 nM and 640 +/- 90 nM, respectively by tight-binding inhibition analysis. The greater loss of affinity for CsA binding (400-fold) than for rotomase catalysis (20 fold) suggests that the catalytic and CsA binding properties associated with CyP can be decoupled as has been observed with an homologous protein found in E. coli (Liu, J. & Walsh, C.T. (1990) Proc. Natl. Acad. Sci. USA 87, 4028-4032).     

A novel method for the biosynthesis of deuterated proteins with selective protonation at the aromatic rings of Phe,Tyr and Trp

A novel biosynthetic strategy is described for the preparation of deuterated proteins containing protons at the ring carbons of Phe, Tyr and Trp, using the aromatic amino acid precursor shikimic acid. Specific protonation at aromatic side chains, with complete deuteration at C^/positions was achieved in proteins overexpressed in bacteria grown in shikimate-supplemented D₂O medium. Co-expression of a shikimate transporter in prototrophic bacteria resulted in protonation levels of 62–79%, whereas complete labeling was accomplished using shikimate auxotrophic bacteria. Our labeling protocol permits the measurement of important aromatic side chain derived distance restraints in perdeuterated proteins that could be utilized to enhance the accuracy of NMR structures calculated using low densities of NOEs from methyl selectively protonated samples.     

Expression of deuterium-isotope-labelled protein in the yeast Pichia pastoris for NMR studies

Deuterium isotope labelling is important for NMR studies of large proteins and complexes. Many eukaryotic proteins are difficult to express in bacteria, but can be efficiently produced in the methylotrophic yeast Pichia pastoris. In order to facilitate NMR studies of the malaria parasite merozoite surface protein-1 (MSP1) complex and its interactions with antibodies, we have investigated production of the MSP1-19 protein in P. pastoris grown in deuterated media. The resulting deuteration patterns were analyzed by NMR and mass spectrometry. We have compared growth characteristics and levels of heterologous protein expression in cells adapted to growth in deuterated media (95% D₂O), compared with expression in non-adapted cells. We have also compared the relative deuteration levels and the distribution pattern of residual protiation in protein from cells grown either in 95% D₂O medium with protiated methanol as carbon source, or in 95% D₂O medium containing deuterated methanol. A high level of uniform C deuteration was demonstrated, and the consequent reduction of backbone amide signal linewidths in [¹H/¹⁵N]-correlation experiments was measured. Residual protiation at different positions in various amino acid residues, including the distribution of methyl isotopomers, was also investigated. The deuteration procedures examined here should facilitate economical expression of ²H/¹³C/¹⁵N-labelled protein samples for NMR studies of the structure and interactions of large proteins and protein complexes.     

A new strategy for structure determination of large proteins in solution without deuteration

So far high-resolution structure determination by nuclear magnetic resonance (NMR) spectroscopy has been limited to proteins <30 kDa, although global fold determination is possible for substantially larger proteins. Here we present a strategy for assigning backbone and side-chain resonances of large proteins without deuteration, with which one can obtain high-resolution structures from (1)H-(1)H distance restraints. The strategy uses information from through-bond correlation experiments to filter intraresidue and sequential correlations from through-space correlation experiments, and then matches the filtered correlations to obtain sequential assignment. We demonstrate this strategy on three proteins ranging from 24 to 65 kDa for resonance assignment and on maltose binding protein (42 kDa) and hemoglobin (65 kDa) for high-resolution structure determination. The strategy extends the size limit for structure determination by NMR spectroscopy to 42 kDa for monomeric proteins and to 65 kDa for differentially labeled multimeric proteins without the need for deuteration or selective labeling.     

Cyclophilin: distribution and variant properties in normal and neoplastic tissues

The cytosolic concentration, Mr, and isoforms of cyclophilin (CyP), a specific cytosolic binding protein for cyclosporin A (CsA), were determined in normal and neoplastic human tissues as well as tissues from species of diverse phylogeny. CyP was present in all tissues examined; however, concentrations varied significantly among different tissue types. The CyP concentration was highest in lymphoblasts from a patient with T cell acute lymphocytic leukemia (1.15 micrograms/mg protein) and Hodgkin's and non-Hodgkin's lymphomas. CyP concentration in colon adenocarcinomas was twofold to threefold greater than that found in adjacent normal tissue. CyP from all normal and neoplastic human tissues examined had an apparent Mr of 17,000 determined by gel filtration HPLC. Major (pI 8.6 to 8.7) and minor (pI 6.7 to 6.9) CyP isoforms were identified in all human and murine tissue extracts by column sucrose gradient isoelectrofocusing; however, the ratio of the major to minor isoform varied widely. Among other species examined, significant concentrations of CyP were detected in cytosol extracts from sponges (Microciona prolifera), yeast (Saccharomyces cerevisiae), mushrooms, the giant cockroach (Blaberus discoidalis), and a trematode (Schistosoma mansoni). By contrast, CyP was not detectable in extracts of Escherichia coli. A twofold to threefold elevation in the CyP content of murine splenocytes was detected 72 hr after Con A stimulation. A survey of a variety of natural products, synthetic compounds, and immunoregulating agents has failed thus far to identify compounds capable of competing with CsA for binding to CyP. The broad tissue and phylogenetic distribution of CyP, its highly conserved structure, and its increased content after mitogenic stimulation suggest a fundamental role in cellular metabolism.     

Regulation of Ca2+-dependent desensitization in the vanilloid receptor TRPV1 by calcineurin and cAMP-dependent protein kinase

The vanilloid receptor TRPV1 is a polymodal nonselective cation channel of nociceptive sensory neurons involved in the perception of inflammatory pain. TRPV1 exhibits desensitization in a Ca2+-dependent manner upon repeated activation by capsaicin or protons. The cAMP-dependent protein kinase (PKA) decreases desensitization of TRPV1 by directly phosphorylating the channel presumably at sites Ser116 and Thr370. In the present study we investigated the influence of protein phosphatase 2B (calcineurin) on Ca2+-dependent desensitization of capsaicin- and proton-activated currents. By using site-directed mutagenesis, we generated point mutations at PKA and protein kinase C consensus sites and studied wild type (WT) and mutant channels transiently expressed in HEK293t or HeLa cells under whole cell voltage clamp. We found that intracellular application of the cyclosporin A.cyclophilin A complex (CsA.CyP), a specific inhibitor of calcineurin, significantly decreased desensitization of capsaicin- or proton-activated TRPV1-WT currents. This effect was similar to that obtained by extracellular application of forskolin (FSK), an indirect activator of PKA. Simultaneous applications of CsA.CyP and FSK in varying concentrations suggested that these substances acted independently from each other. In mutation T370A, application of CsA.CyP did not reduce desensitization of capsaicin-activated currents as compared with WT and to mutant channels S116A and T144A. In a double mutation at candidate protein kinase C phosphorylation sites, application of CsA.CyP or FSK decreased desensitization of capsaicin-activated currents similar to WT channels. We conclude that Ca2+-dependent desensitization of TRPV1 might be in part regulated through channel dephosphorylation by calcineurin and channel phosphorylation by PKA possibly involving Thr370 as a key amino acid residue.     

Uniform and Residue-specific 15N-labeling of Proteins on a Highly Deuterated Background

A general method for stable-isotope labeling of large proteins is introduced and applied for studies of the E. coli GroE chaperone proteins by solution NMR. In addition to enabling the residue-specific (15)N-labeling of proteins on a highly deuterated background, it is also an efficient approach for uniform labeling. The method meets the requirements of high-level deuteration, minimal cross-labeling and high protein yield, which are crucial for NMR studies of structures with sizes above 150 kDa. The results obtained with the new protocol are compared to other strategies for protein labeling, and evaluated with regard to the influence of external factors on the resulting isotope labeling patterns. Applications with the GroE system show that these strategies are efficient tools for studies of structure, dynamics and intermolecular interactions in large supramolecular complexes, when combined with TROSY- and CRINEPT-based experimental NMR schemes.     

Biosynthetic preparation of selectively deuterated phosphatidylcholine in genetically modified <Emphasis Type="Italic">Escherichia coli</Emphasis>

Phosphatidylcholine (PC) is a major component of eukaryotic cell membranes and one of the most commonly used phospholipids for reconstitution of membrane proteins into carrier systems such as lipid vesicles, micelles and nanodiscs. Selectively deuterated versions of this lipid have many applications, especially in structural studies using techniques such as NMR, neutron reflectivity and small-angle neutron scattering. Here we present a comprehensive study of selective deuteration of phosphatidylcholine through biosynthesis in a genetically modified strain of Escherichia coli. By carefully tuning the deuteration level in E. coli growth media and varying the deuteration of supplemented carbon sources, we show that it is possible to achieve a controlled deuteration for three distinct parts of the PC lipid molecule, namely the (a) lipid head group, (b) glycerol backbone and (c) fatty acyl tail. This biosynthetic approach paves the way for the synthesis of specifically deuterated, physiologically relevant phospholipid species which remain difficult to obtain through standard chemical synthesis.     

Automated protein fold determination using a minimal NMR constraint strategy

Determination of precise and accurate protein structures by NMR generally requires weeks or even months to acquire and interpret all the necessary NMR data. However, even medium-accuracy fold information can often provide key clues about protein evolution and biochemical function(s). In this article we describe a largely automatic strategy for rapid determination of medium-accuracy protein backbone structures. Our strategy derives from ideas originally introduced by other groups for determining medium-accuracy NMR structures of large proteins using deuterated, (13)C-, (15)N-enriched protein samples with selective protonation of side-chain methyl groups ((13)CH(3)). Data collection includes acquiring NMR spectra for automatically determining assignments of backbone and side-chain (15)N, H(N) resonances, and side-chain (13)CH(3) methyl resonances. These assignments are determined automatically by the program AutoAssign using backbone triple resonance NMR data, together with Spin System Type Assignment Constraints (STACs) derived from side-chain triple-resonance experiments. The program AutoStructure then derives conformational constraints using these chemical shifts, amide (1)H/(2)H exchange, nuclear Overhauser effect spectroscopy (NOESY), and residual dipolar coupling data. The total time required for collecting such NMR data can potentially be as short as a few days. Here we demonstrate an integrated set of NMR software which can process these NMR spectra, carry out resonance assignments, interpret NOESY data, and generate medium-accuracy structures within a few days. The feasibility of this combined data collection and analysis strategy starting from raw NMR time domain data was illustrated by automatic analysis of a medium accuracy structure of the Z domain of Staphylococcal protein A.     

Accessibility of the functional groups of chitosan aerogel probed by FT-IR-monitored deuteration

Transmission FT-IR spectroscopy allowed us to monitor the deuteration of wafers of chitosan aerogel and xerogel by D2O vapor at room temperature. The complete deuteration of the alcohol and amine groups of the aerogel (surface area 175 m2 g(-1) as measured by N2 volumetry) confirmed the high accessibility of the functional groups of the polymer. The xerogel (surface area 5 m2 g(-1)) was only partially deuterated in more severe conditions. The isotopic shift of the deuterated groups allowed us to confirm or revise some attributions of infrared bands of chitosan.     

Deuterium labeling of diethylstilbestrol and analogues

E-2,2,3',3″,5,5,5',5″-octadeuteriodiethylstilbestrol (DES-d8) and Z-2,3',3″,4,5,5,5',5″-octadeuterio-3,4-bis(p-hydroxyphenyl)-2-hexene (ψ-DES-d8) were synthesized from E-diethylstilbestrol (DES) by hydrogen/ deuterium exchange in a mixture of methanol-d and deuterium chloride in deuterium oxide. The structures, isotopic purity, and positions of up-take of deuterium were determined by nuclear magnetic resonance (NMR) and mass spectrometry (MS). Additional confirmation of the positions of deuterium exchange in stilbestrols was obtained from an analysis of the oxidation of DES-d8 to Z,Z-2,3',3″,5,5',5″-hexadeuteriodienestrol (β-DIES-d6) and of the hydrogen/deuterium exchange reaction of hexestrol (HEX) to 3',3″,5',5″-hexestrol (HEX-d4). Structural analysis and the determination of isotopic purity of the latter two compounds were also carried out by NMR and MS. The uptake of eight deuterium atoms by DES is postulated to proceed via two different reactions occurring simultaneously: 1. acid catalyzed deuteration of all four phenolic ortho-positions (3',3″,5',5″); 2. acid catalyzed deuteration of the olefin bridge with subsequent formation of deuterated ψ-DES (3 or 4). Due to the equilibration between DES, ψ-DES, and Z-diethylstilbestrol (cis-DES) in the acidic reaction mixture at 85°C, the deuterated ψ-DES is thought to rapidly rearrange to deuterated DES. Repeated deuteration will eventually form DES-d8 fully labeled in the 2,2,5,5 methylene positions.     

The synthesis of deuterionucleosides

The synthesis of deuterionucleosides for site-specific incorporation into oligo-DNA or -RAA is herein reviewed for NMR or biological studies. The review covers the following aspects: (i) deuteration of the aglycone; (ii) single-site chemical deuteration of the sugar residues; (iii) multiple-site chemical deuteration of the sugar residues; (iv) enzymatic synthesis of deuterated nucleosides or nucleotides; and (v) synthesis of labelled nucleosides with multiple isotopes     

Expression, purification, characterization, and NMR studies of highly deuterated recombinant cytochrome c peroxidase

Two forms of extensively deuterated S. cerevisiae cytochrome c peroxidase (CcP; EC 1.11.1.5) have been overexpressed in E. coli by growth in highly deuterated medium. One of these ferriheme enzyme forms (recDCcP) was produced using >97% deuterated growth medium and was determined to be approximately 84% deuterated. The second form [recD(His)CcP] was grown in the same highly deuterated medium that had been supplemented with excess histidine (at natural hydrogen isotope abundance) and was also approximately 84% deuterated. This resulted in direct histidine incorporation without isotope scrambling. Both of these enzymes along with the corresponding recombinant native CcP (recNATCcP), which was expressed in a standard medium with normal hydrogen isotope abundance, consisted of 294 amino acid polypeptide chains having the identical sequence to the yeast-isolated enzyme, without any N-terminal modifications. Comparative characterizations of all three enzymes have been carried out for the resting-state, high-spin forms and in the cyanide-ligated, low-spin forms. The primary physical methods employed were electrophoresis, UV-visible spectroscopy, hydrogen peroxide reaction kinetics, mass spectrometry, and (1)H NMR spectroscopy. The results indicate that high-level deuteration does not significantly alter CcP's reactivity or spectroscopy. As an example of potential NMR uses, recDCcPCN and recD(His)CcPCN have been used to achieve complete, unambiguous, stereospecific (1)H resonance assignments for the heme hyperfine-shifted protons, which also allows the heme side chain conformations to be assessed. Assigning these important active-site protons has been an elusive goal since the first NMR spectra on this enzyme were reported 18 years ago, due to a combination of the enzyme's comparatively large size, paramagnetism, and limited thermal stability.