Time-resolved X-ray crystallographic study of the conformational change in Ha-Ras p21 protein on GTP hydrolysis

Schlichting, Ilme; Almo, Steven C.; Rapp, Gert; Wilson, K.S.; Petratos, Kyriacos; Lentfer, Arno; Wittinghofer, Alfred; Kabsch, Wolfgang; Pai, E.F.; Petsko, Gregory A.; Goody, Roger S.

doi:10.1038/345309a0

Cited by 499 publications

(339 citation statements)

References 27 publications

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“…Two of these motifs, Walker A and B (7), are thought to form a portion of a nucleotide-binding pocket. The conserved lysine in Walker A is predicted to interact with the phosphoryl moiety of the bound nucleotide (8,9) and the aspartic acid in Walker B to coordinate Mg2+ of the MgATP complex (10,11 pocket to other domains of the protein (12,13). The importance of this putative linker motif to CFTR function is evidenced by the seven CF missense mutations reported within this region of NBF1 and NBF2 (14).…”

Section: -Isobutyl-l-methylxanthine In Xenopus Oocytes Expressingmentioning

confidence: 99%

Functional roles of the nucleotide-binding folds in the activation of the cystic fibrosis transmembrane conductance regulator.

Smit

Wilkinson

Mansoura

et al. 1993

Proc. Natl. Acad. Sci. U.S.A.

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The cystic fibrosis transmembrane conductance regulator (CFTR), a member of the traffic ATPase superfamily, possesses two putative nudeotide-binding folds (NBFs). The NBFs are sufficiently similar that sequence alignment of highly conserved regions can be used to identify analogous residues in the two domains. To determine whether this structural homology is paralleled in function, we compared the activation of chloride conductance by forskolin and 3-isobutyl-l-methylxanthine in Xenopus oocytes expressing CFTRs bearing mutations in NBF1 or NBF2. Mutation of a conserved glycine in the putative linker domain in either NBF produced virtually identical changes in the sensitivity of chloride conductance to activating conditions, and mutation of this site in both NBFs produced additive effects, suggesting that in the two NBFs this region plays a similar and critical role in the activation process. In contrast, amino acid substitutions in the Walker A and B motifs, thought to form an integral part of the nucleotide-binding pockets, produced strikingly different effects in NBF1 and NBF2. Substitutions for the conserved lysine (Walker A) or aspartate (Walker B) in NBF1 resulted in a marked decrease in sensitivity to activation, whereas the same changes in NBF2 produced an increase in sensitivity. These results are consistent with a model for the activation of CFTR in which both NBF1 and NBF2 are required for normal function but in which either the nature or the exact consequences of nucleotide binding differ for the two domains.

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Section: -Isobutyl-l-methylxanthine In Xenopus Oocytes Expressingmentioning

confidence: 99%

Functional roles of the nucleotide-binding folds in the activation of the cystic fibrosis transmembrane conductance regulator.

Smit

Wilkinson

Mansoura

et al. 1993

Proc. Natl. Acad. Sci. U.S.A.

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“…In these studies Laue data have been used in conjunction with difference-Fourier techniques where the initial structure of the protein of interest is available. The studies have demonstrated that Laue data can be used (i) to locate substrates and metal ions bound to proteins (Farber et al, 1988) (ii) for crystallographic refinement and (iii) for time-resolved studies of enzyme reactions (Schlichting et al, 1990).…”

Section: Introductionmentioning

confidence: 99%

“…Using one of the currently available synchrotron sources, the intensity of the incident radiation permits data sets to be collected on the millisecond to second timescale and experiments (Szebenyi, Bilderback, LeGrand, Moffat, Schildkamp & Teny, 1988) have already demonstrated that exposure times of micro-to picoseconds are possible in the future. In favorable systems, this reduction in data-collection time may allow complex enzymesubstrate reactions to be investigated crystallographically (Hajdu, Acharya, Stuart, Barford & Johnson, 1988;Schlichting et al, 1990). Turkey eggwhite lysozyme (TEWL) is potentially such a system.…”

Section: Introductionmentioning

confidence: 99%

Structure determination of turkey egg-white lysozyme using Laue diffraction data

Howell

Almo²,

Parsons³

et al. 1992

Acta Crystallogr Sect B

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The three-dimensional structure of turkey egg-white lysozyme (TEWL) has been solved and refined at 2.5 A~ resolution using X-ray data collected by the Laue method. This is the first protein structure determination undertaken using Laue diffraction data. A re-examination of the existing structure of TEWL was necessary when attempts to refine an atomic model based on the C,~ positions in the Protein Data Bank (entry 1LZ2) failed. The correct orientation and position of the turkey lysozyme molecules within the crystallographic unit cell were determined by molecular replacement using a refined model of the homologous hen egg-white lysozyme crystal structure. After modification of the model to reflect the differences in amino-acid sequence between the chicken and turkey enzymes, the structure was subjected to crystallographic refinement using the simulated-annealing refinement technique and conventional least-squares refinement. This yielded a final residual of R = 20.7%. This crystal form is of potential interest for time-resolved crystallographic studies since the amino-acid residues involved in

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“…The application of this to study the laser light stimulated photodissociation of carbon monoxide bound to the haem in myoglobin is thus underway (Bourgeois et al 1996), essentially probing then the nanosecond behaviour of this protein structure complex. The emergence of the synchrotron Laue method for rapid data collection from protein crystals (reviewed in Cassetta et al (1993)) has stimulated a strong interest to study enzyme reactions in a crystal in real time, with examples including the protein p2i in guanosine triphosphate (GTP) hydrolysis (Schlichting et al 1990;Scheidig et al 1994), trypsin (Singer et al 1993) and isocitrate dehydrogenase (Bolduc et al 1995) as well as photostimulable systems like CO myoglobin (Bourgeois et al 1996).…”

Section: Io Dynamical Studies Of Proteins In Crystalsmentioning

confidence: 99%

Trends and Challenges in Experimental Macromolecular Crystallography

Chayen

Boggon

Cassetta

et al. 1996

Quart. Rev. Biophys.

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Macromolecular X-ray crystallography underpins the vigorous field of structural molecular biology having yielded many protein, nucleic acid and virus structures in fine detail. The understanding of the recognition by these macromolecules, as receptors, of their cognate ligands involves the detailed study of the structural chemistry of their molecular interactions. Also these structural details underpin the rational design of novel inhibitors in modern drug discovery in the pharmaceutical industry. Moreover, from such structures the functional details can be inferred, such as the biological chemistry of enzyme reactivity. There is then a vast number and range of types of biological macromolecules that potentially could be studied. The completion of the protein primary sequencing of the yeast genome, and the human genome sequencing project comprising some 105 proteins that is underway, raises expectations for equivalent three dimensional structural databases.

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Time-resolved X-ray crystallographic study of the conformational change in Ha-Ras p21 protein on GTP hydrolysis

Cited by 499 publications

References 27 publications

Functional roles of the nucleotide-binding folds in the activation of the cystic fibrosis transmembrane conductance regulator.

Functional roles of the nucleotide-binding folds in the activation of the cystic fibrosis transmembrane conductance regulator.

Structure determination of turkey egg-white lysozyme using Laue diffraction data

Trends and Challenges in Experimental Macromolecular Crystallography

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