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R Ishima

Publications and source records attributed to R Ishima.

At least 19 recordsLinked to original sources
PubMed2001

Folded monomer of HIV-1 protease.

The mature human immunodeficiency virus type 1 protease rapidly folds into an enzymatically active stable dimer, exhibiting an intricate interplay between structure formation and dimerization. We now show by NMR and sedimentation equilibrium studies that a mutant protease containing the R87K substitution (PR(R87K)) within the highly conserved Gly(86)-Arg(87)-Asn(88) sequence forms a monomer with a fold similar to a single subunit of the dimer. However, binding of the inhibitor DMP323 to PR(R87K) produces a stable dimer complex. Based on the crystal structure and our NMR results, we postulate that loss of specific interactions involving the side chain of Arg(87) destabilizes PR(R87K) by perturbing the inner C-terminal beta-sheet (residues 96-99 from each monomer), a region that is sandwiched between the two beta-strands formed by the N-terminal residues (residues 1-4) in the mature protease. We systematically examined the folding, dimerization, and catalytic activities of mutant proteases comprising deletions of either one of the terminal regions (residues 1-4 or 96-99) or both. Although both N- and C-terminal beta-strands were found to contribute to dimer stability, our results indicate that the inner C-terminal strands are absolutely essential for dimer formation. Knowledge of the monomer fold and regions critical for dimerization may aid in the rational design of novel inhibitors of the protease to overcome the problem of drug resistance.

Amino Acid Sequence↗
PubMed2001

Comparison of methyl rotation axis order parameters derived from model-free analyses of (2)H and (13)C longitudinal and transverse relaxation rates measured in the same protein sample.

Recombinant HIV-1 protease was obtained from bacteria grown on a 98% D(2)O medium containing 3-(13)C pyruvic acid as the sole source of (13)C and (1)H. The purified protein is highly deuterated at non-methyl carbons, but contains significant populations of (13)CHD(2) and (13)CH(2)D methyl isotopomers. This pattern of isotope labeling permitted measurements of (1)H and (13)C relaxation rates of (13)CHD(2) isotopomers and (2)H (D) relaxation rates of (13)CH(2)D isotopomers using a single sample. The order parameters S(axis)(2), which characterize the motions of the methyl rotation axes, were derived from model-free analyses of R(1) and R(2) data sets measured for (13)C and (2)H spins. Our primary goal was to compare the S(axis)(2) values derived from the two independent types of data sets to test our understanding of the relaxation mechanisms involved. However, S(axis)(2) values derived from the analyses depend strongly on the geometry of the methyl group, the sizes of the quadrupolar and dipolar couplings, and the effects of bond vibrations and librations on these couplings. Therefore uncertainties in these basic physical parameters complicate comparison of the order parameters. This problem was circumvented by using an experimental relationship, between the methyl quadrupolar, (13)C-(13)C and (13)C-(1)H dipolar couplings, derived from independent measurements of residual static couplings of weakly aligned proteins by Ottiger and Bax (J. Am. Chem. Soc. 1999, 121, 4690-4695) and Mittermaier and Kay (J. Am. Chem. Soc. 1999, 121, 10608-10613). This approach placed a tight experimental restraint on the values of the (2)H quadrupolar and (13)C-(1)H dipolar interactions and greatly facilitated the accurate comparison of the relative values of the order parameters. When applied to our data this approach yielded satisfactory agreement between the S(axis)(2) values derived from the (13)C and (2)H data sets.

Amino Acids↗
PubMed2001

Characterization of two hydrophobic methyl clusters in HIV-1 protease by NMR spin relaxation in solution.

Nearly 50 % of the amino acid residues of HIV-1 protease contain methyl side-chains, most of which appear to be organized into two clusters: the inner cluster that nearly surrounds the active site and the outer cluster that contains the hydrophobic core which stabilizes the inhibitor-free protease structure. NMR relaxation experiments sensitive to motions of methyl groups on the sub-nanosecond and the milli-microsecond time-scales revealed flexible methyl groups in residues that link the two clusters, the methyl groups of L10, L23, V75, and L76. We hypothesize that flexibility at the junctions of these clusters allows the protease to minimize conformational changes upon drug-binding. The two-methyl cluster motif appears to be a common structural feature among retroviral proteases and may play a similar role throughout this family of enzymes.

Amino Acid Substitution↗
PubMed2001

Optimized labeling of 13CHD2 methyl isotopomers in perdeuterated proteins: potential advantages for 13C relaxation studies of methyl dynamics of larger proteins.

13CHD2 methyl isotopomers are particularly useful to study methyl dynamics in proteins because, as compared with other methyl isotopomers, the 13C relaxation mechanism for this isotopomer is straightforward. However, in the case of proteins, where (omega tau)2 >> 1, the refocused INEPT pulse sequence does not completely suppress unwanted 13CH3 signals. The presence of weak 13CH3 peaks is usually not a serious problem for smaller proteins because there are relatively few methyl signals and they are sharp; however, signal overlap becomes more common as the size of the protein increases. We overcome this problem by preparing a protein using a 98% D2O cell culture medium containing 3-(13)C pyruvic acid, 50-60% deuterated at the 3-position, and 4-(13)C 2-ketobutyric acid, 98% and 62% deuterated at the 3- and 4-positions, respectively. This approach significantly reduces the population of the CH3 isotopomer while optimizing the production of 13CHD2, the isotopomer desired for 13C relaxation measurements. In larger proteins where the deuterium T2 may be too short to measure accurately, we also suggest the alternative measurement of the proton T2 of the 13CH2D methyl isotopomer, because these protons are well-isolated from other protons in these highly deuterated samples.

Carbon Isotopes↗
PubMed2000

Protein dynamics from NMR.

This review surveys recent investigations of conformational fluctuations of proteins in solution using NMR techniques. Advances in experimental methods have provided more accurate means of characterizing fast and slow internal motions as well as overall diffusion. The information obtained from NMR dynamics experiments provides insights into specific structural changes or configurational energetics associated with function. A variety of applications illustrate that studies of protein dynamics provide insights into protein-protein interactions, target recognition, ligand binding, and enzyme function.

Amides↗
PubMed1999

Flap opening and dimer-interface flexibility in the free and inhibitor-bound HIV protease, and their implications for function.

BACKGROUND: (1)H and (15)N transverse relaxation measurements on perdeuterated proteins are ideally suited for detecting backbone conformational fluctuations on the millisecond-microsecond timescale. The identification of conformational exchange on this timescale by measuring the relaxation of both (1)H and (15)N holds great promise for the elucidation of functionally relevant conformational changes in proteins. RESULTS: We measured the transverse (1)H and (15)N relaxation rates of backbone amides of HIV-1 protease in its free and inhibitor-bound forms. An analysis of these rates, obtained as a function of the effective rotating frame field, provided information about the timescale of structural fluctuations in several regions of the protein. The flaps that cover the active site of the inhibitor-bound protein undergo significant changes of backbone (φ,psi) angles, on the 100 micros timescale, in the free protein. In addition, the intermonomer beta-sheet interface of the bound form, which from protease structure studies appears to be rigid, was found to fluctuate on the millisecond timescale. CONCLUSIONS: We present a working model of the flap-opening mechanism in free HIV-1 protease which involves a transition from a semi-open to an open conformation that is facilitated by interaction of the Phe53 ring with the substrate. We also identify a surprising fluctuation of the beta-sheet intermonomer interface that suggests a structural requirement for maturation of the protease. Thus, slow conformational fluctuations identified by (1)H and (15)N transverse relaxation measurements can be related to the biological functions of proteins.

Azepines↗
PubMed1999

Transverse 1H cross relaxation in 1H-15N correlated 1H CPMG experiments.

Transverse 1H cross relaxation was observed in Carr-Purcell-Meiboom-Gill (CPMG) experiments by recording 15N-1H correlated spectra of amides in HIV protease that was perdeuterated at nonexchangeable sites. Perdeuteration suppresses 1H-1H J coupling and improves spectral resolution and sensitivity. Measurements of cross-peak intensities, arising from cross relaxation, were made as a function of (i) Deltaf, the frequency difference between the spins, and (ii) tauCPMG, one-half of the duration between CPMG pi pulses. Cross peaks were observed when tauCPMG was less than 1/(2Deltaf), in agreement with theoretical calculations.

Amides↗
PubMed1999

Estimating the time scale of chemical exchange of proteins from measurements of transverse relaxation rates in solution.

Chemical (conformational) exchange on the ms--microsecond time scale is reliably identified by the observation of transverse relaxation rates, Rex, that depend upon the strength of the effective field (omega 1eff = gamma B1eff) used in spin lock or CPMG experiments. In order to determine if the exchange correlation time, tau ex, is the fast or slow limit, measurements of (i) signal line shape and (ii) temperature dependence of Rex have been commonly used in studies of stable, small molecules. However, these approaches are often not applicable to proteins, because sample stability and solubility, respectively, limit the temperature range and signal sensitivity of experiments. Herein we use a complex, but general, two-site exchange equation to show when the simple fast exchange equations for Rex are good approximations, in the case of proteins. We then present a simple empirical equation that approximately predicts Rex in all exchange regimes, and explains these results in a clear, straightforward manner. Finally we show how one can reliably determine whether tau ex is in the fast or slow exchange limit.

Animals↗
PubMed1999

Solution structure of the homodimeric core domain of Escherichia coli histidine kinase EnvZ.

Escherichia coli osmosensor EnvZ is a protein histidine kinase that plays a central role in osmoregulation, a cellular adaptation process involving the His-Asp phosphorelay signal transduction system. Dimerization of the transmembrane protein is essential for its autophosphorylation and phosphorelay signal transduction functions. Here we present the NMR-derived structure of the homodimeric core domain (residues 223-289) of EnvZ that includes His 243, the site of autophosphorylation and phosphate transfer reactions. The structure comprises a four-helix bundle formed by two identical helix-turn-helix subunits, revealing the molecular assembly of two active sites within the dimeric kinase.

Amino Acid Sequence↗
PubMed1998

NMR structure of the histidine kinase domain of the E. coli osmosensor EnvZ.

Bacteria live in capricious environments, in which they must continuously sense external conditions in order to adjust their shape, motility and physiology. The histidine-aspartate phosphorelay signal-transduction system (also known as the two-component system) is important in cellular adaptation to environmental changes in both prokaryotes and lower eukaryotes. In this system, protein histidine kinases function as sensors and signal transducers. The Escherichia coli osmosensor, EnvZ, is a transmembrane protein with histidine kinase activity in its cytoplasmic region. The cytoplasmic region contains two functional domains: domain A (residues 223-289) contains the conserved histidine residue (H243), a site of autophosphorylation as well as transphosphorylation to the conserved D55 residue of response regulator OmpR, whereas domain B (residues 290-450) encloses several highly conserved regions (G1, G2, F and N boxes) and is able to phosphorylate H243. Here we present the solution structure of domain B, the catalytic core of EnvZ. This core has a novel protein kinase structure, distinct from the serine/threonine/tyrosine kinase fold, with unanticipated similarities to both heatshock protein 90 and DNA gyrase B.

Amino Acid Sequence↗
PubMed1998

Solution structure of a TBP-TAF(II)230 complex: protein mimicry of the minor groove surface of the TATA box unwound by TBP.

General transcription factor TFIID consists of TATA box-binding protein (TBP) and TBP-associated factors (TAF(II)s), which together play a central role in both positive and negative regulation of transcription. The N-terminal region of the 230 kDa Drosophila TAF(II) (dTAF(II)230) binds directly to TBP and inhibits TBP binding to the TATA box. We report here the solution structure of the complex formed by dTAF(II)230 N-terminal region (residues 11-77) and TBP. dTAF(II)230(11-77) comprises three alpha helices and a beta hairpin, forming a core that occupies the concave DNA-binding surface of TBP. The TBP-binding surface of dTAF(II)230 markedly resembles the minor groove surface of the partially unwound TATA box in the TBP-TATA complex. This protein mimicry of the TATA element surface provides the structural basis of the mechanism by which dTAF(II)230 negatively controls the TATA box-binding activity within the TFIID complex.

Amino Acid Sequence↗
PubMed1998

Human general transcription factor TFIIB: conformational variability and interaction with VP16 activation domain.

Human TFIIB, an essential factor in transcription of protein-coding genes by RNA polymerase II, consists of an amino-terminal zinc binding domain (TFIIBn) connected by a linker of about 60 residues to a carboxy-terminal core domain (TFIIBc). The TFIIB core domain has two internally repeated motifs, each comprising five alpha-helices arranged as in the cyclin box. Compared to the crystal structure of TFIIBc in complex with TBP and a TATA-containing oligonucleotide, the NMR-derived solution structure of free TFIIBc is more compact, with a different repeat-repeat orientation and a significantly shorter first helix in the second repeat. Analysis of backbone 15N relaxation parameters indicates the presence of relatively large amplitude, nanosecond time-scale motions in the TFIIBc interrepeat linker and structural fluctuations throughout the backbone. Interaction of TFIIBc with the acidic activation domain of VP16 or with TFIIBn induces 1H-15N chemical shift and line width changes concentrated in the first repeat, interrepeat linker and the first helix of the second repeat. These results suggest that TFIIB is somewhat pliable and that the conformation of the C-terminal core domain can be modulated by interaction with the N-terminal zinc binding domain. Furthermore, binding of the VP16 activation domain may promote TFIIBc conformations primed for binding to a TBP-DNA complex.

Binding Sites↗
PubMed1997

Molecular mechanics of calcium-myristoyl switches.

Many eukaryotic cellular and viral proteins have a covalently attached myristoyl group at the amino terminus. One such protein is recoverin, a calcium sensor in retinal rod cells, which controls the lifetime of photoexcited rhodopsin by inhibiting rhodopsin kinase. Recoverin has a relative molecular mass of 23,000 (M[r] 23K), and contains an amino-terminal myristoyl group (or related acyl group) and four EF hands. The binding of two Ca2+ ions to recoverin leads to its translocation from the cytosol to the disc membrane. In the Ca2+-free state, the myristoyl group is sequestered in a deep hydrophobic box, where it is clamped by multiple residues contributed by three of the EF hands. We have used nuclear magnetic resonance to show that Ca2+ induces the unclamping and extrusion of the myristoyl group, enabling it to interact with a lipid bilayer membrane. The transition is also accompanied by a 45-degree rotation of the amino-terminal domain relative to the carboxy-terminal domain, and many hydrophobic residues are exposed. The conservation of the myristoyl binding site and two swivels in recoverin homologues from yeast to humans indicates that calcium-myristoyl switches are ancient devices for controlling calcium-sensitive processes.

Calcium↗
PubMed1997

An in vitro 1H-MRS model of oncogene transfection. The spectral feature of c-erbB-2 and c-Ha-ras transfected NIH3T3 fibroblast cells.

PURPOSE: Malignancy is an abnormality of cell division and differentiation based on abnormal expression of oncogenes. This note describes the in vitro 1H-NMR spectral features of oncogene-transfected NIH3T3 fibroblast cells compared to non-transfected cells. MATERIAL AND METHODS: 1H-NMR spectra of cultured NIH3T3 cells and c-erbB-2 or c-Ha-ras gene-transfected cells were obtained by 400 MHz high resolution NMR. The peaks were assigned by 2D HOHAHA spectra of the cell suspension and the spectral changes were evaluated in 1D and 1D differential spectra. RESULTS: The 1H spectra obtained from both transfected cell lines were broadened over all peaks, suggesting reduced mobility in plasma membrane lipid molecules. No other differential spectra for characterizing metabolic change was detected. CONCLUSION: Broadened 1H spectra observed after c-erbB-2 or c-Ha-ras transfection suggest changes of plasma membrane viscosity, which may be related to the oncogene expression.

3T3 Cells↗
PubMed1996

1H-magnetic resonance spectroscopic observation of cultured malignant cells pharmacologically induced to different phenotypes.

RATIONALE AND OBJECTIVES: We evaluated the 1H nuclear magnetic resonance spectra of malignant cells after the administration of drugs that cause morphologic changes. METHODS: 1H spectra of a human lung adenocarcinoma cell line cultured with interferon gamma, dexamethasone, or sodium butyrate were obtained. The peaks were assigned by two-dimensional homonuclear Hartmann-Hahn spectroscopy spectra of the cells and their perchloric acid extracts. Differential spectra were used to evaluate relative changes in the peaks. RESULTS: In the control culture, choline/phosphocholine peaks were increased in the cell-growth phase, and the 1.26-ppm peak was increased in the confluent state. Treatment by interferon gamma and dexamethasone induced reproducible changes in the peaks of differential spectra corresponding to 1.26 ppm, choline/phosphocholine, and glutamate/glutamine. Dexamethasone treatment broadened lipid peaks. Changes after treatment with sodium butyrate were obscure. Microscopically, cells were induced to morphologically different phenotypes by each drug. CONCLUSION: Cells induced to exhibit morphologically different phenotypes present different 1H spectra.

Adenocarcinoma↗
PubMed1995

Protein backbone dynamics revealed by quasi spectral density function analysis of amide N-15 nuclei.

Spectral density functions J(0), J(omega N), and J(omega H + omega N) of individual amide N-15 nuclei in proteins were approximated by a quasi spectral density function (QSDF). Using this function, the backbone dynamics were analyzed for seven protein systems on which data have been published. We defined J(0; omega N) as the difference between the J(0) and the J(omega N) values, which describes motions slower than 50 (or 60) MHz, and J(omega N; omega H+N) as the difference between the J(omega N) and the J(omega H + omega N) values, which describes motions slower than 450 (or 540) MHz. The QSDF analysis can easily extract the J(0; omega N) of protein backbones, which have often some relation to biologically relevant reactions. Flexible N-terminal regions in eglin c and glucose permease IIA and a loop region in eglin c showed smaller values of both the J(0; omega N) and the J(omega N; omega H+N) as compared with the other regions, indicating increases in motions faster than nanosecond. The values of the J(0; omega N) for the backbone of the FK506 binding protein showed a large variation in the apoprotein but fell in a very narrow range after the binding of FK506. Characteristic increase or decrease in the values of J(0) and J(omega N) was observed in two or three residues located between secondary structures.

Calmodulin↗
PubMed1995

Three-dimensional structure of gurmarin, a sweet taste-suppressing polypeptide.

The solution structure of gurmarin was studied by two-dimensional proton NMR spectroscopy at 600 MHz. Gurmarin, a 35-amino acid residue polypeptide recently discovered in an Indian-originated tree Gymnema sylvestre, selectively suppresses the neural responses of rat to sweet taste stimuli. Sequence-specific resonance assignments were obtained for all backbone protons and for most of the side-chain protons. The three-dimensional solution structure was determined by simulated-annealing calculations on the basis of 135 interproton distance constraints derived from NOEs, six distance constraints for three hydrogen bonds and 16 dihedral angle constraints derived from coupling constants. A total of 10 structures folded into a well-defined structure with a triple-stranded antiparallel beta-sheet. The average rmsd values between any two structures were 1.65 +/- 0.39 A for the backbone atoms (N, C alpha, C) and 2.95 +/- 0.27 A for all heavy atoms. The positions of the three disulfide bridges, which could not be determined chemically, were estimated to be Cys3-Cys18, Cys10-Cys23 and Cys17-Cys33 on the basis of the NMR distance constraints. This disulfide bridge pattern in gurmarin turned out to be analogous to that in omega-conotoxin and Momordica charantia trypsin inhibitor-II, and the topology of folding was the same as that in omega-conotoxin.

Amino Acid Sequence↗