Mutational analysis reveals a single binding interface between RhoA and its effector, PRK1.


:Protein kinase C-related kinases (PRKs) are serine/threonine kinases that are members of the protein kinase C superfamily and can be activated by binding to members of the Rho family of small G proteins via a Rho binding motif known as an HR1 domain. The PRKs contain three tandem HR1 domains at their N-termini. The structure of the HR1a domain from PRK1 in complex with RhoA [Maesaki, R., et al. (1999) Mol. Cell 4, 793-803] identified two potential contact interfaces between the G protein and the HR1a domain. In this work, we have used an alanine scanning mutagenesis approach to identify whether both contact sites are used when the two proteins interact in solution and also whether HR1b, the second HR1 domain from PRK1, plays a role in binding to RhoA. The mutagenesis identified just one contact site as being relevant for binding of RhoA and HR1a in solution, and the HR1b domain was found not to contribute to RhoA binding. The folded state and thermal stability of the HR1a and HR1b domains were also investigated. HR1b was found to be more thermally stable than HR1a, and it is hypothesized that the differences in the biophysical properties of these two domains govern their interaction with small G proteins.






Hutchinson CL,Lowe PN,McLaughlin SH,Mott HR,Owen D




Has Abstract


2011-04-12 00:00:00












  • A test of discreteness-of-charge effects in phospholipid vesicles: measurements using paramagnetic amphiphiles.

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    pub_type: 杂志文章


    authors: Hartsel SC,Cafiso DS

    更新日期:1986-12-16 00:00:00

  • Site of action and biphasic effect of neutral salts in the phosphorylase kinase reaction.

    abstract::The inhibition of phosphorylase kinase catalytic activity by 0.1 M neutral salts was predicted by the Hofmeister series of anions. The site of action of the salts was determined by the following evidence to be on the phosphorylase kinase molecule directly, rather than on its protein substrate. (1) Nonactivated kinase ...


    pub_type: 杂志文章


    authors: Carlson GM,Graves DJ

    更新日期:1976-10-05 00:00:00

  • Analysis of folding and unfolding reactions of cytochrome b5.

    abstract::The guanidine hydrochloride- (GuHCl-) induced unfolding and refolding of a recombinant domain of bovine microsomal cytochrome b(5) containing the first 104 amino acid residues has been characterized by both transient and equilibrium spectrophotometric methods. The soluble domain is reversibly unfolded and the equilibr...


    pub_type: 杂志文章


    authors: Manyusa S,Mortuza G,Whitford D

    更新日期:1999-10-26 00:00:00

  • Purification and characteristics of photoreceptor light-activated guanosinetriphosphatase.

    abstract::We describe a reconstitution of light-activated vertebrate photoreceptor GTPase and a purification of the GTP-binding protein (G protein), which is a component of the GTPase and modulates the light-activated phosphodiesterase (PDE) enzyme system. Rod outer segments (ROS) of bull frogs were treated with ethylenediamine...


    pub_type: 杂志文章


    authors: Shinozawa T,Bitensky MW

    更新日期:1981-12-08 00:00:00

  • Post-translational phosphorylation of serine 74 of human deoxycytidine kinase favors the enzyme adopting the open conformation making it competent for nucleoside binding and release.

    abstract::Deoxycytidine kinase (dCK) uses either ATP or UTP as a phosphoryl donor to catalyze the phosphorylation of nucleoside acceptors. The kinetic properties of human dCK are modulated in vivo by phosphorylation of serine 74. This residue is a part of the insert region and is distant from the active site. Replacing the seri...


    pub_type: 杂志文章


    authors: Hazra S,Szewczak A,Ort S,Konrad M,Lavie A

    更新日期:2011-04-12 00:00:00

  • Mechanistic studies of the methyltransferase from Clostridium thermoaceticum: origin of the pH dependence of the methyl group transfer from methyltetrahydrofolate to the corrinoid/iron-sulfur protein.

    abstract::A methyltetrahydrofolate:corrinoid/iron-sulfur protein methyltransferase (MeTr) from Clostridium thermoaceticum catalyzes the transfer of the N5 methyl group from (6S)-methyltetrahydrofolate (CH3-H4folate) to the cobalt center of a corrinoid/iron-sulfur protein (C/Fe-SP). The methylcobamide product is the first in a s...


    pub_type: 杂志文章


    authors: Zhao S,Roberts DL,Ragsdale SW

    更新日期:1995-11-21 00:00:00

  • The BARD1 C-terminal domain structure and interactions with polyadenylation factor CstF-50.

    abstract::The BARD1 N-terminal RING domain binds BRCA1 while the BARD1 C-terminal ankyrin and tandem BRCT repeat domains bind CstF-50 to modulate mRNA processing and RNAP II stability in response to DNA damage. Here we characterize the BARD1 structural biochemistry responsible for CstF-50 binding. The crystal structure of the B...


    pub_type: 杂志文章


    authors: Edwards RA,Lee MS,Tsutakawa SE,Williams RS,Nazeer I,Kleiman FE,Tainer JA,Glover JN

    更新日期:2008-11-04 00:00:00

  • The CD4 determinant for downregulation by HIV-1 Nef directly binds to Nef. Mapping of the Nef binding surface by NMR.

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    pub_type: 杂志文章


    authors: Grzesiek S,Stahl SJ,Wingfield PT,Bax A

    更新日期:1996-08-13 00:00:00

  • Cold adaptation of enzyme reaction rates.

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    pub_type: 杂志文章


    authors: Bjelic S,Brandsdal BO,Aqvist J

    更新日期:2008-09-23 00:00:00

  • An unusual catalytic subunit for the cAMP-dependent protein kinase of Dictyostelium discoideum.

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    pub_type: 杂志文章


    authors: Anjard C,Etchebehere L,Pinaud S,Véron M,Reymond CD

    更新日期:1993-09-21 00:00:00

  • Sortase activity is controlled by a flexible lid in the pilus biogenesis mechanism of gram-positive pathogens.

    abstract::Pili are surface-linked virulence factors that play key roles in infection establishment in a variety of pathogenic species. In Gram-positive pathogens, pilus formation requires the action of sortases, dedicated transpeptidases that covalently associate pilus building blocks. In Streptococcus pneumoniae, a major human...


    pub_type: 杂志文章


    authors: Manzano C,Izoré T,Job V,Di Guilmi AM,Dessen A

    更新日期:2009-11-10 00:00:00

  • New class of bacterial membrane oxidoreductases.

    abstract::A new class of bacterial multisubunit membrane-bound electron-transfer complexes has been identified based on biochemical and bioinformatic data. It contains subunits homologous to the three-subunit molybdopterin oxidoreductases and four additional subunits, two of which are c-type cytochromes. The complex was purifie...


    pub_type: 杂志文章


    authors: Yanyushin MF,del Rosario MC,Brune DC,Blankenship RE

    更新日期:2005-08-02 00:00:00

  • Three-state denaturation of DnaK induced by guanidine hydrochloride. Evidence for an expandable intermediate.

    abstract::The denaturation of the heat shock protein DnaK induced by guanidine hydrochloride (Gdn-HCl) was investigated by circular dichroism, fluorescence, size-exclusion HPLC, and dynamic light scattering. DnaK unfolding takes place in two discrete steps. The midpoint (Cm) of the first transition (0.5 M) was shifted to higher...


    pub_type: 杂志文章


    authors: Palleros DR,Shi L,Reid KL,Fink AL

    更新日期:1993-04-27 00:00:00

  • Role of guanine nucleotides in the vinblastine-induced self-association of tubulin: effects of guanosine alpha,beta-methylenetriphosphate and guanosine alpha,beta-methylenediphosphate.

    abstract::It is now well established that guanine nucleotides are allosteric effectors of the vinca alkaloid-induced self-association of tubulin. GDP enhances self-association for vinblastine-, vincristine- and vinorelbine-induced spiral assembly relative to GTP by 0.90 +/- 0.17 kcal/mol [Lobert et al. (1996) Biochemistry 35, 6...


    pub_type: 杂志文章


    authors: Vulevic B,Lobert S,Correia JJ

    更新日期:1997-10-21 00:00:00

  • Selective labeling of the erythrocyte hexose carrier with a maleimide derivative of glucosamine: relationship of an exofacial sulfhydryl to carrier conformation and structure.

    abstract::Sulfhydryl-reactive derivatives of glucosamine were synthesized as potentially transportable affinity labels of the human erythrocyte hexose carrier. N-Maleoylglycyl derivatives of either 6- or 2-amino-2-deoxy-D-glucopyranose were the most potent inhibitors of 3-O-methylglucose uptake, with concentrations of half-maxi...


    pub_type: 杂志文章


    authors: May JM

    更新日期:1989-02-21 00:00:00

  • Ionization equilibria for side-chain carboxyl groups in oxidized and reduced human thioredoxin and in the complex with its target peptide from the transcription factor NF kappa B.

    abstract::The pH dependence of the 13C chemical shifts of the side-chain carboxyl carbons of all Asp and Glu residues in the reduced and oxidized states of human thioredoxin and in a mixed disulfide complex of human thioredoxin with a target peptide from the transcription factor NF kappa B has been investigated by multidimensio...


    pub_type: 杂志文章


    authors: Qin J,Clore GM,Gronenborn AM

    更新日期:1996-01-09 00:00:00

  • Characterization of the macrolide P-450 hydroxylase from Streptomyces venezuelae which converts narbomycin to picromycin.

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    pub_type: 杂志文章


    authors: Betlach MC,Kealey JT,Ashley GW,McDaniel R

    更新日期:1998-10-20 00:00:00

  • Design of a selective insulin receptor tyrosine kinase inhibitor and its effect on glucose uptake and metabolism in intact cells.

    abstract::An inhibitor of the insulin receptor tyrosine kinase (IRTK), (hydroxy-2-naphthalenyl-methyl) phosphonic acid, was designed and synthesized and was shown to be an inhibitor of the biological effects of insulin in vitro. With a wheat germ purified human placental insulin receptor preparation, this compound inhibited the...


    pub_type: 杂志文章


    authors: Saperstein R,Vicario PP,Strout HV,Brady E,Slater EE,Greenlee WJ,Ondeyka DL,Patchett AA,Hangauer DG

    更新日期:1989-06-27 00:00:00

  • Changes in protein conformation and stability accompany complex formation between human C1 inhibitor and C1-s.

    abstract::The fluorescence spectrum of C1 inhibitor (C1-Inh) in aqueous buffer has a maximum at 324 nm which shifts to 358 nm in 6.0 M guanidinium chloride (GdmC1), indicating that fluorescent tryptophans are buried in the native protein. When titrated with GdmC1, the fluorescence intensity, polarization, and emission maximum o...


    pub_type: 杂志文章


    authors: Lennick M,Brew SA,Ingham KC

    更新日期:1985-05-07 00:00:00

  • Engineering of papain: selective alteration of substrate specificity by site-directed mutagenesis.

    abstract::The S2 subsite specificity of the plant protease papain has been altered to resemble that of mammalian cathepsin B by site-directed mutagenesis. On the basis of amino acid sequence alignments for papain and cathepsin B, a double mutant (Val133Ala/Ser205Glu) was produced where Val133 and Ser205 are replaced by Ala and ...


    pub_type: 杂志文章


    authors: Khouri HE,Vernet T,Ménard R,Parlati F,Laflamme P,Tessier DC,Gour-Salin B,Thomas DY,Storer AC

    更新日期:1991-09-17 00:00:00

  • Studies into the identity of the sites of insulin-stimulated insulin receptor serine phosphorylation. Characterization of synthetic peptide substrates for the insulin-stimulated insulin receptor serine kinase.

    abstract::The identity of the sites of insulin-stimulated serine phosphorylation in the human insulin receptor was examined by synthesizing peptides that together encompassed all the serine residues of the cytosolic portion of the beta-subunit and testing them as substrates for phosphorylation by a preparation of human insulin ...


    pub_type: 杂志文章


    authors: Carter WG,Asamoah KA,Sale GJ

    更新日期:1995-07-25 00:00:00

  • NMR characterization of three-disulfide variants of lysozyme, C64A/C80A, C76A/C94A, and C30A/C115A--a marginally stable state in folded proteins.

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    pub_type: 杂志文章


    authors: Yokota A,Hirai K,Miyauchi H,Iimura S,Noda Y,Inoue K,Akasaka K,Tachibana H,Segawa S

    更新日期:2004-06-01 00:00:00

  • Importance of hydrophobic matching for spontaneous insertion of a single-spanning membrane protein.

    abstract::In this study, we have investigated the effect of hydrophobic mismatch between the thickness of the membrane and a transmembrane segment of a protein that directly inserts into the membrane bilayer. For this purpose we used mutants of the single-spanning Pf3 coat protein that can spontaneously insert into Escherichia ...


    pub_type: 杂志文章


    authors: Ridder AN,van de Hoef W,Stam J,Kuhn A,de Kruijff B,Killian JA

    更新日期:2002-04-16 00:00:00

  • Formation of hybrid luciferases from subunits of different species of Photobacterium.

    abstract::Enzyme divergence within three species of the genus Photobacterium (P. fischeri, P. leiognathi, and P. phosphoreum) was studied by comparing the catalytic characteristics and quaternary interactions of bacterial luciferases isolated from each species. Each luciferase was composed of two subunits of different molecular...


    pub_type: 杂志文章


    authors: Ruby EG,Hastings JW

    更新日期:1980-10-28 00:00:00

  • Generation of a free alpha-amino group by Raney nickel after 2-nitro-5-thiocyanobenzoic acid cleavage at cysteine residues: application to automated sequencing.

    abstract::The selective reaction of SH containing proteins and peptides with NTCB (2-nitro-5-thiocyanobenzoic acid) has been reported (Degani, Y., & Patchornick, A. (1974) Biochemistry 13, 1; Jacobson, G.A., Schaffer, M.H., Stark, G.R., & Vanaman, T.C. (1973) J. Biol. Chem. 248, 6583). With this reagent, cysteinyl peptide bonds...


    pub_type: 杂志文章


    authors: Otieno S

    更新日期:1978-12-12 00:00:00

  • Indoleglycerol phosphate synthase-phosphoribosyl anthranilate isomerase: comparison of the bifunctional enzyme from Escherichia coli with engineered monofunctional domains.

    abstract::Putative domain--domain interactions of the monomeric bifunctional enzyme indoleglycerol phosphate synthase:phosphoribosyl anthranilate isomerase from Escherichia coli were probed by separating the domains on the gene level and expressing them as monofunctional proteins. The engineered monofunctional enzymes were foun...


    pub_type: 杂志文章


    authors: Eberhard M,Tsai-Pflugfelder M,Bolewska K,Hommel U,Kirschner K

    更新日期:1995-04-25 00:00:00

  • Effects of metal ligation and oxygen on the reversibility of the thermal denaturation of Pseudomonas aeruginosa azurin.

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    pub_type: 杂志文章


    authors: Sandberg A,Leckner J,Shi Y,Schwarz FP,Karlsson BG

    更新日期:2002-01-22 00:00:00

  • Structure and function of DNA photolyase.

    abstract::Cyclobutane pyrimidine dimers (Pyr < > Pyr) are the major DNA photoproducts induced by the UV component of solar radiation. Photoreactivating enzyme (DNA photolyase) repairs DNA by utilizing the energy of visible light to break the cyclobutane ring of the dimer. Photolyases are monomeric proteins of 50-60 kDa with sto...


    pub_type: 杂志文章,评审


    authors: Sancar A

    更新日期:1994-01-11 00:00:00

  • Study of hydrogen exchange in hemoglobin as a function of fractional ligand saturation.

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    pub_type: 杂志文章


    authors: Hallaway PE,Hallaway BE,Rosenberg A

    更新日期:1984-01-17 00:00:00

  • Low-frequency dynamics of Caldariomyces fumago chloroperoxidase probed by femtosecond coherence spectroscopy.

    abstract::Ultrafast laser spectroscopy techniques are used to measure the low-frequency vibrational coherence spectra and nitric oxide rebinding kinetics of Caldariomyces fumago chloroperoxidase (CPO). Comparisons of the CPO coherence spectra with those of other heme species are made to gauge the protein-specific nature of the ...


    pub_type: 杂志文章


    authors: Gruia F,Ionascu D,Kubo M,Ye X,Dawson J,Osborne RL,Sligar SG,Denisov I,Das A,Poulos TL,Terner J,Champion PM

    更新日期:2008-05-06 00:00:00