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Apo Form (apo + form)

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Chemistry100%

Selected Abstracts

Molecular determinants of ligand specificity in family 11 carbohydrate binding modules , an NMR, X-ray crystallography and computational chemistry approach

FEBS JOURNAL, Issue 10 2008
Aldino Viegas
The direct conversion of plant cell wall polysaccharides into soluble sugars is one of the most important reactions on earth, and is performed by certain microorganisms such as Clostridium thermocellum (Ct). These organisms produce extracellular multi-subunit complexes (i.e. cellulosomes) comprising a consortium of enzymes, which contain noncatalytic carbohydrate-binding modules (CBM) that increase the activity of the catalytic module. In the present study, we describe a combined approach by X-ray crystallography, NMR and computational chemistry that aimed to gain further insight into the binding mode of different carbohydrates (cellobiose, cellotetraose and cellohexaose) to the binding pocket of the family 11 CBM. The crystal structure of C. thermocellum CBM11 has been resolved to 1.98 Å in the apo form. Since the structure with a bound substrate could not be obtained, computational studies with cellobiose, cellotetraose and cellohexaose were carried out to determine the molecular recognition of glucose polymers by CtCBM11. These studies revealed a specificity area at the CtCBM11 binding cleft, which is lined with several aspartate residues. In addition, a cluster of aromatic residues was found to be important for guiding and packing of the polysaccharide. The binding cleft of CtCBM11 interacts more strongly with the central glucose units of cellotetraose and cellohexaose, mainly through interactions with the sugar units at positions 2 and 6. This model of binding is supported by saturation transfer difference NMR experiments and linebroadening NMR studies. [source]

Triad of polar residues implicated in pH specificity of acidic mammalian chitinase

PROTEIN SCIENCE, Issue 3 2009
Andrea M. Olland
Abstract Acidic mammalian chitinase (AMCase) is a mammalian chitinase that has been implicated in allergic asthma. One of only two active mammalian chinases, AMCase, is distinguished from other chitinases by several unique features. Here, we present the novel structure of the AMCase catalytic domain, both in the apo form and in complex with the inhibitor methylallosamidin, determined to high resolution by X-ray crystallography. These results provide a structural basis for understanding some of the unique characteristics of this enzyme, including the low pH optimum and the preference for the ,-anomer of the substrate. A triad of polar residues in the second-shell is found to modulate the highly conserved chitinase active site. As a novel target for asthma therapy, structural details of AMCase activity will help guide the future design of specific and potent AMCase inhibitors. [source]

Comparison of negative and positive ion electrospray ionization mass spectra of calmodulin and its complex with trifluoperazine

RAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 15 2005
Stephen J. Watt
The protein calmodulin (apoCaM) undergoes a conformational change when it binds calcium. This structure of the protein (Ca4CaM) is a dumbbell-shaped molecule that undergoes a further profound conformational change on binding of the antipsychotic drug trifluoperazine (TFP). Experimental conditions were developed to prepare samples of apoCaM, Ca4CaM and Ca4CaM/TFP that were substantially free of sodium. The effects of the conformational changes of calmodulin on the charge-state distributions observed in positive ion and negative ion electrospray ionization (ESI) mass spectra were examined. Conversion of apoCaM into Ca4CaM was concomitant with a change in the negative ion ESI mass spectrum whereby the 16, ion was the most abundant ion observed for the apo form and the 8, ion was the most abundant for the complex. In contrast, in the positive ion ESI mass spectra of apoCaM and Ca4CaM, the most abundant species in each case was the 8+ ion. When a complex of Ca4CaM with TFP was prepared, the most abundant species was the 5+ ion. This is consistent with a conformational change of Ca4CaM that rendered some basic sites inaccessible to ionization in the ESI process. Using the same Ca4CaM/TFP mixture, no complex with TFP was observed in negative ion ESI mass spectra. These observations are discussed in the context of the structural changes that are known to occur in calmodulin, and suggestions are made to explain the apparently conflicting data. The results reported here reflect on the validity of using differences in charge-state distributions observed in ESI mass spectra to assess conformational changes in proteins. Copyright © 2005 John Wiley & Sons, Ltd. [source]

Structures of the nucleotide-binding domain of the human ABCB6 transporter and its complexes with nucleotides

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 9 2010
Matthias Haffke
The human ATP-binding cassette (ABC) transporter ABCB6 is involved in haem-precursor transport across the mitochondrial membrane. The crystal structure of its nucleotide-binding domain (NBD) has been determined in the apo form and in complexes with ADP, with ADP and Mg2+ and with ATP at high resolution. The overall structure is L-shaped and consists of two lobes, consistent with other reported NBD structures. Nucleotide binding is mediated by the highly conserved Tyr599 and the Walker A motif, and induces notable structural changes. Structural comparison with other structurally characterized NBDs and full-length ABC transporters gives the first insight into the possible catalytic mechanism of ABCB6 and the role of the N-terminal helix ,1 in full-length ABCB6. [source]

Structural analysis of mycobacterial branched-chain aminotransferase: implications for inhibitor design

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 5 2010
Alina Castell
The branched-chain aminotransferase (BCAT) of Mycobacterium tuberculosis has been characterized as being essential to the survival of the bacterium. The enzyme is pyridoxal 5,-phosphate-dependent and belongs to the aminotransferase IIIa subfamily, to which the human BCATs also belong. The overall sequence similarity is high within the subfamily and the sequence identity among the active-site residues is high. In order to identify structurally unique features of M. tuberculosis BCAT, X-ray structural and functional analyses of the closely related BCAT from M. smegmatis were carried out. The crystal structures include the apo form at 2.2,Å resolution and a 1.9,Å structure of the holo form cocrystallized with the inhibitor O -benzylhydroxylamine (Obe). The analyses highlighted the active-site residues Tyr209 and Gly243 as being structurally unique characteristics of the mycobacterial BCATs relative to the human BCATs. The inhibitory activities of Obe and ammonium sulfate were verified in an inhibition assay. Modelling of the inhibitor Obe in the substrate pocket indicated potential for the design of a mycobacterial-specific inhibitor. [source]

Structures of the PKC-, kinase domain in its ATP-bound and apo forms reveal defined structures of residues 533,551 in the C-terminal tail and their roles in ATP binding

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 5 2010
Tetsuo Takimura
Protein kinase C (PKC) plays an essential role in a wide range of cellular functions. Although crystal structures of the PKC-,, PKC-, and PKC-,II kinase domains have previously been determined in complexes with small-molecule inhibitors, no structure of a PKC,substrate complex has been determined. In the previously determined PKC-, complex, residues 533,551 in the C-terminal tail were disordered. In the present study, crystal structures of the PKC-, kinase domain in its ATP-bound and apo forms were determined at 2.1 and 2.0,Å resolution, respectively. In the ATP complex, the electron density of all of the C-terminal tail residues was well defined. In the structure, the side chain of Phe543 protrudes into the ATP-binding pocket to make van der Waals interactions with the adenine moiety of ATP; this is also observed in other AGC kinase structures such as binary and ternary substrate complexes of PKA and AKT. In addition to this interaction, the newly defined residues around the turn motif make multiple hydrogen bonds to glycine-rich-loop residues. These interactions reduce the flexibility of the glycine-rich loop, which is organized for ATP binding, and the resulting structure promotes an ATP conformation that is suitable for the subsequent phosphoryl transfer. In the case of the apo form, the structure and interaction mode of the C-terminal tail of PKC-, are essentially identical to those of the ATP complex. These results indicate that the protein structure is pre-organized before substrate binding to PKC-,, which is different from the case of the prototypical AGC-branch kinase PKA. [source]

Structures of the apo and holo forms of formate dehydrogenase from the bacterium Moraxella sp.

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 12 2009
C-1: towards understanding the mechanism of the closure of the interdomain cleft
NAD+ -dependent formate dehydrogenase (FDH) catalyzes the oxidation of formate ion to carbon dioxide coupled with the reduction of NAD+ to NADH. The crystal structures of the apo and holo forms of FDH from the methylotrophic bacterium Moraxella sp. C-1 (MorFDH) are reported at 1.96 and 1.95,Å resolution, respectively. MorFDH is similar to the previously studied FDH from the bacterium Pseudomonas sp. 101 in overall structure, cofactor-binding mode and active-site architecture, but differs in that the eight-residue-longer C-terminal fragment is visible in the electron-density maps of MorFDH. MorFDH also differs in the organization of the dimer interface. The holo MorFDH structure supports the earlier hypothesis that the catalytic residue His332 can form a hydrogen bond to both the substrate and the transition state. Apo MorFDH has a closed conformation of the interdomain cleft, which is unique for an apo form of an NAD+ -dependent dehydrogenase. A comparison of the structures of bacterial FDH in open and closed conformations allows the differentiation of the conformational changes associated with cofactor binding and domain motion and provides insights into the mechanism of the closure of the interdomain cleft in FDH. The C-terminal residues 374,399 and the substrate (formate ion) or inhibitor (azide ion) binding are shown to play an essential role in the transition from the open to the closed conformation. [source]

Structure of the methyltransferase domain from the Modoc virus, a flavivirus with no known vector

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 8 2009
Anna M. Jansson
The Modoc virus (MODV) is a flavivirus with no known vector (NKV). Evolutionary studies have shown that the viruses in the MODV group have evolved in association with mammals (bats, rodents) without transmission by an arthropod vector. MODV methyltransferase is the first enzyme from this evolutionary branch to be structurally characterized. The high-resolution structure of the methyltransferase domain of the MODV NS5 protein (MTaseMODV) was determined. The protein structure was solved in the apo form and in complex with its cofactor S -adenosyl- l -methionine (SAM). Although it belongs to a separate evolutionary branch, MTaseMODV shares structural characteristics with flaviviral MTases from the other branches. Its capping machinery is a relatively new target in flaviviral drug development and the observed structural conservation between the three flaviviral branches indicates that it may be possible to identify a drug that targets a range of flaviviruses. The structural conservation also supports the choice of MODV as a possible model for flavivirus studies. [source]

Structures of mannose-6-phosphate isomerase from Salmonella typhimurium bound to metal atoms and substrate: implications for catalytic mechanism

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 7 2009
S. R. Sagurthi
Mannose-6-phosphate isomerase (MPI) catalyzes the interconversion of mannose 6-phosphate and fructose 6-phosphate. X-ray crystal structures of MPI from Salmonella typhimurium in the apo form (with no metal bound) and in the holo form (with bound Zn2+) and two other structures with yttrium bound at an inhibitory site and complexed with Zn2+ and fructose 6-phosphate (F6P) were determined in order to gain insights into the structure and the isomerization mechanism. Isomerization involves acid/base catalysis with proton transfer between the C1 and C2 atoms of the substrate. His99, Lys132, His131 and Asp270 are close to the substrate and are likely to be the residues involved in proton transfer. The interactions observed at the active site suggest that the ring-opening step is probably catalyzed by His99 and Asp270. An active-site loop consisting of residues 130,133 undergoes conformational changes upon substrate binding. Zn2+ binding induces structural order in the loop consisting of residues 50,54. The metal atom appears to play a role in substrate binding and is probably also important for maintaining the architecture of the active site. Isomerization probably follows the previously suggested cis -enediol mechanism. [source]

Use of an in-house approach to study the three-dimensional structures of various outer membrane proteins: structure of the alcaligin outer membrane transporter FauA from Bordetella pertussis

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 4 2009
Karl Brillet
Bordetella pertussis is the bacterial agent of whooping cough in humans. Under iron-limiting conditions, it produces the siderophore alcaligin. Released to the extracellular environment, alcaligin chelates iron, which is then taken up as a ferric alcaligin complex via the FauA outer membrane transporter. FauA belongs to a family of TonB-dependent outer membrane transporters that function using energy derived from the proton motive force. Using an in-house protocol for membrane-protein expression, purification and crystallization, FauA was crystallized in its apo form together with three other TonB-dependent transporters from different organisms. Here, the protocol used to study FauA is described and its three-dimensional structure determined at 2.3,Å resolution is discussed. [source]

The 1.9,Å resolution structure of Mycobacterium tuberculosis 1-deoxy- d -xylulose 5-­phosphate reductoisomerase, a potential drug target

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 7 2006
Lena M. Henriksson
1-Deoxy- d -xylulose 5-phosphate reductoisomerase catalyzes the NADPH-dependent rearrangement and reduction of 1-­deoxy- d -xylulose 5-phosphate to form 2- C -methyl- d -­erythritol 4-phosphate, as the second step of the deoxyxylulose 5-phosphate/methylerythritol 4-phosphate pathway found in many bacteria and plants. The end product, isopentenyl diphosphate, is the precursor of various isoprenoids vital to all living organisms. The pathway is not found in humans; the mevalonate pathway is instead used for the formation of isopentenyl diphosphate. This difference, combined with its essentiality, makes the reductoisomerase an excellent drug target in a number of pathogenic organisms. The structure of 1-deoxy- d -xylulose 5-phosphate reductoisomerase from Mycobacterium tuberculosis (Rv2870c) was solved by molecular replacement and refined to a resolution of 1.9,Å. The enzyme exhibited an estimated kcat of 5.3,s,1 and Km and kcat/Km values of 7.2,µM and 7.4 × 105,M,1,s,1 for NADPH and 340,µM and 1.6 × 104,M,1,s,1 for 1-deoxy- d -­xylulose 5-phosphate. In the structure, a sulfate is bound at the expected site of the phosphate moiety of the sugar substrate. The M. tuberculosis enzyme displays a similar fold to the previously published structures from Escherichia coli and Zymomonas mobilis. Comparisons offer suggestions for the design of specific drugs. Furthermore, the new structure represents an intermediate conformation between the open apo form and the closed holo form observed previously, giving insights into the conformational changes associated with catalysis. [source]

Structure of Escherichia coli tryptophanase

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 7 2006
Shao-Yang Ku
Pyridoxal 5,-phosphate (PLP) dependent tryptophanase has been isolated from Escherichia coli and its crystal structure has been determined. The structure shares the same fold with and has similar quaternary structure to Proteus vulgaris tryptophanase and tyrosine-phenol lyase, but is found in a closed conformation when compared with these two enzymes. The tryptophanase structure, solved in its apo form, does not have covalent PLP bound in the active site, but two sulfate ions. The sulfate ions occupy the phosphoryl-binding site of PLP and the binding site of the ,-carboxyl of the natural substrate tryptophan. One of the sulfate ions makes extensive interactions with both the transferase and PLP-binding domains of the protein and appears to be responsible for holding the enzyme in its closed conformation. Based on the sulfate density and the structure of the P. vulgaris enzyme, PLP and the substrate tryptophan were modeled into the active site. The resulting model is consistent with the roles of Arg419 in orienting the substrate to PLP and acidifying the ,-­proton of the substrate for ,-elimination, Lys269 in the formation and decomposition of the PLP quinonoid intermediate, Arg230 in orienting the substrate,PLP intermediates in the optimal conformation for catalysis, and His463 and Tyr74 in determining substrate specificity and suggests that the closed conformation observed in the structure could be induced by substrate binding and that significant conformational changes occur during catalysis. A catalytic mechanism for tryptophanase is proposed. Since E. coli tryptophanase has resisted forming diffraction-quality crystals for many years, the molecular surface of tryptophanase has been analyzed in various crystal forms and it was rationalized that strong crystal contacts occur on the flat surface of the protein and that the size of crystal contact surface seems to correlate with the diffraction quality of the crystal. [source]

Overproduction, crystallization and preliminary diffraction data of ADP-ribose pyrophosphatase from Thermus thermophilus HB8

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 10 2003
Sachiko Yoshiba
An ADP-ribose pyrophosphatase from Thermus thermophilus HB8 was overproduced in Escherichia coli and purified. Gel-filtration chromatography showed the protein to be in a dimeric state. This protein catalyses the Mg2+ - or Zn2+ -dependent hydrolysis of ADP-ribose to AMP and ribose-5,-phosphate. It was crystallized in the absence and the presence of ADP-ribose by the hanging-drop vapour-diffusion method. Complete data sets were collected to 1.50,Å resolution from the apo form using synchrotron radiation and to 2.0,Å resolution from the complexed form. Both crystals belong to space group P3121 or P3221 and contain one molecule in the asymmetric unit. [source]

Crystallization and preliminary X-ray analysis of a recombinant Fab fragment in complex with 17,-­oestradiol

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 12 2000
Urpo Lamminmäki
The recombinant Fab fragment of the anti-17,-oestradiol antibody 57-­2 has been a target for several protein-engineering experiments. A method for production, purification and crystallization of the Fab fragment alone (apo form) and in complex with the major female sex hormone 17,-oestradiol is reported here. Diffracting apo-form crystals were only obtained with microseeding; crystals of the Fab,steroid complex were produced by co-crystallization in the presence of oestradiol and cross-seeding with the apo-form crystals. The crystals were grown using vapour-diffusion methods with reservoir solutions containing 10,14% PEG 4000 or 8,12% PEG 8000 and Tris,HCl buffer at high pH (9.0,9.5). Both the apo and complex crystals belong to space group P212121 and diffract to 2.0,Å resolution. High-resolution X-ray data sets suitable for structure determination were collected from flash-cooled crystals using 25% glycerol as the cryoprotectant. [source]

Purification, crystallization and preliminary X-ray analysis of apo glyceraldehyde-3-phosphate dehydrogenase 1 (GAP1) from methicillin-resistant Staphylococcus aureus (MRSA252)

ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 5 2010
Somnath Mukherjee
Glyceraldehyde-3-phosphate dehydrogenase 1 (GAP1) from methicillin-resistant Staphylococcus aureus (MRSA252) has been purified to homogeneity in the apo form. The protein was crystallized using the hanging-drop vapour-diffusion method. The crystals belonged to space group P21, with unit-cell parameters a = 69.95, b = 93.68, c = 89.05,Å, , = 106.84°. X-ray diffraction data have been collected and processed to a maximum resolution of 2.2,Å. The presence of one tetramer in the asymmetric unit gives a Matthews coefficient (VM) of 1.81,Å3,Da,1 with a solvent content of 32%. The structure has been solved by molecular replacement and structure refinement is now in progress. [source]

Crystallization and preliminary crystallographic analysis of nosiheptide-resistance methyltransferase from Streptomyces actuosus in complex with SAM

ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 5 2010
Huirong Yang
Nosiheptide-resistance methyltransferase (NSR) methylates 23S rRNA at the nucleotide adenosine 1067 in Escherichia coli and thus contributes to resistance against nosiheptide, a sulfur-containing peptide antibiotic. Here, the expression, purification and crystallization of NSR from Streptomyces actuosus are reported. Diffracting crystals were grown by the hanging-drop vapour-diffusion method in reservoir solution consisting of 0.35,M ammonium chloride, 24%(w/v) PEG 3350, 0.1,M MES pH 5.7 at 293,K. Native data have been collected from the apo enzyme and a SAM complex, as well as apo SeMet SAD data. The diffraction patterns of the apo form of NSR, of NSR complexed with SAM and of SeMet-labelled NSR crystals extended to 1.90, 1.95 and 2.25,Å resolution, respectively, using synchrotron radiation. All crystals belonged to space group P21, with approximate unit-cell parameters a = 64.6, b = 69.6, c = 64.9,Å, , = 117.8°. [source]

Cloning, expression and crystallization of dihydrodipicolinate reductase from methicillin-resistant Staphylococcus aureus

ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 1 2010
Sudhir Dommaraju
Dihydrodipicolinate reductase (DHDPR; EC 1.3.1.26) catalyzes the nucleotide (NADH/NADPH) dependent second step of the lysine-biosynthesis pathway in bacteria and plants. Here, the cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of DHDPR from methicillin-resistant Staphylococcus aureus (MRSA-DHDPR) are presented. The enzyme was crystallized in a number of forms, predominantly with ammonium sulfate as a precipitant, with the best crystal form diffracting to beyond 3.65,Å resolution. Crystal structures of the apo form as well as of cofactor (NADPH) bound and inhibitor (2,6-pyridinedicarboxylate) bound forms of MRSA-DHDPR will provide insight into the structure and function of this essential enzyme and valid drug target. [source]

Crystallization and crystallographic analysis of the apo form of the orange protein (ORP) from Desulfovibrio gigas.

ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 8 2009
Corrigendum
A correction is made to the name of one of the authors in the article by Najmudin et al. [Acta Cryst. (2009). F65, 730732]. [source]

Crystallization and crystallographic analysis of the apo form of the orange protein (ORP) from Desulfovibrio gigas

ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 7 2009
Shabir Najmudin
The orange-coloured protein (ORP) from Desulfovibrio gigas is a 12,kDa protein that contains a novel mixed-metal sulfide cluster of the type [S2MoS2CuS2MoS2]. Diffracting crystals of the apo form of ORP have been obtained. Data have been collected for the apo form of ORP to 2.25,Å resolution in-house and to beyond 2.0,Å resolution at ESRF, Grenoble. The crystals belonged to a trigonal space group, with unit-cell parameters a = 43, b = 43, c = 106,Å. [source]

Structure of the apo form of the catabolite control protein A (CcpA) from Bacillus megaterium with a DNA-binding domain

ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 4 2007
Rajesh Kumar Singh
Crystal structure determination of catabolite control protein A (CcpA) at 2.6,Å resolution reveals for the first time the structure of a full-length apo-form LacI-GalR family repressor protein. In the crystal structures of these transcription regulators, the three-helix bundle of the DNA-binding domain has only been observed in cognate DNA complexes; it has not been observed in other crystal structures owing to its mobility. In the crystal packing of apo-CcpA, the protein,protein contacts between the N-terminal three-helix bundle and the core domain consisted of interactions between the homodimers that were similar to those between the corepressor protein HPr and the CcpA N-subdomain in the ternary DNA complex. In contrast to the DNA complex, the apo-CcpA structure reveals large subdomain movements in the core, resulting in a complete loss of contacts between the N-subdomains of the homodimer. [source]

Crystallization, X-ray diffraction analysis and phasing of 17,-hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus

ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 12 2005
Alberto Cassetta
17,-Hydroxysteroid dehydrogenase from the filamentous fungus Cochliobolus lunatus (17,-HSDcl) is an NADP(H)-dependent enzyme that preferentially catalyses the oxidoreduction of oestrogens and androgens. The enzyme belongs to the short-chain dehydrogenase/reductase superfamily and is the only fungal hydroxysteroid dehydrogenase known to date. 17,-HSDcl has recently been characterized and cloned and has been the subject of several functional studies. Although several hypotheses on the physiological role of 17,-HSDcl in fungal metabolism have been formulated, its function is still unclear. An X-ray crystallographic study has been undertaken and the optimal conditions for crystallization of 17,-HSDcl (apo form) were established, resulting in well shaped crystals that diffracted to 1.7,Å resolution. The space group was identified as I4122, with unit-cell parameters a = b = 67.14, c = 266.77,Å. Phasing was successfully performed by Patterson search techniques. A catalytic inactive mutant Tyr167Phe was also engineered, expressed, purified and crystallized for functional and structural studies. [source]

Crystallization and preliminary crystallographic analysis of the nickel-responsive regulator NikR from Pyrococcus horikoshii

ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 1 2005
Tomoe Kitao
The nickel-responsive repressor from Pyrococcus horikoshii OT3 (PhNikR) has been crystallized in the apo form (PhNikR-apo) and two nickel-bound forms (PhNikR-Ni-1 and PhNikR-Ni-2). The PhNikR-apo crystals belong to space group P21, with unit-cell parameters a = 75.78, b = 54.32, c = 77.28,Å, , = 116.07°, and diffract to 2.2,Å. The PhNikR-Ni-1 crystals belong to space group P41212, with unit-cell parameters a = b = 99.89, c = 97.98,Å, and diffract to 3.0,Å and the PhNikR-Ni-2 crystals belong to space group P3121 or P3221, with unit-cell parameters a = b = 109.95, c = 79.0,Å, and diffract to 2.1,Å. The crystals obtained were suitable for detailed structural studies. [source]

Site-Specific Investigation of the Steady-State Kinetics and Dynamics of the Multistep Binding of Bile Acid Molecules to a Lipid Carrier Protein

CHEMISTRY - A EUROPEAN JOURNAL, Issue 37 2010
Dr. Clelia Cogliati
Abstract The investigation of multi-site ligand,protein binding and multi-step mechanisms is highly demanding. In this work, advanced NMR methodologies such as 2D 1H,15N line-shape analysis, which allows a reliable investigation of ligand binding occurring on micro- to millisecond timescales, have been extended to model a two-step binding mechanism. The molecular recognition and complex uptake mechanism of two bile salt molecules by lipid carriers is an interesting example that shows that protein dynamics has the potential to modulate the macromolecule,ligand encounter. Kinetic analysis supports a conformational selection model as the initial recognition process in which the dynamics observed in the apo form is essential for ligand uptake, leading to conformations with improved access to the binding cavity. Subsequent multi-step events could be modelled, for several residues, with a two-step binding mechanism. The protein in the ligand-bound state still exhibits a conformational rearrangement that occurs on a very slow timescale, as observed for other proteins of the family. A global mechanism suggesting how bile acids access the macromolecular cavity is thus proposed. [source]

Structures of the PKC-, kinase domain in its ATP-bound and apo forms reveal defined structures of residues 533,551 in the C-terminal tail and their roles in ATP binding

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 5 2010
Tetsuo Takimura
Protein kinase C (PKC) plays an essential role in a wide range of cellular functions. Although crystal structures of the PKC-,, PKC-, and PKC-,II kinase domains have previously been determined in complexes with small-molecule inhibitors, no structure of a PKC,substrate complex has been determined. In the previously determined PKC-, complex, residues 533,551 in the C-terminal tail were disordered. In the present study, crystal structures of the PKC-, kinase domain in its ATP-bound and apo forms were determined at 2.1 and 2.0,Å resolution, respectively. In the ATP complex, the electron density of all of the C-terminal tail residues was well defined. In the structure, the side chain of Phe543 protrudes into the ATP-binding pocket to make van der Waals interactions with the adenine moiety of ATP; this is also observed in other AGC kinase structures such as binary and ternary substrate complexes of PKA and AKT. In addition to this interaction, the newly defined residues around the turn motif make multiple hydrogen bonds to glycine-rich-loop residues. These interactions reduce the flexibility of the glycine-rich loop, which is organized for ATP binding, and the resulting structure promotes an ATP conformation that is suitable for the subsequent phosphoryl transfer. In the case of the apo form, the structure and interaction mode of the C-terminal tail of PKC-, are essentially identical to those of the ATP complex. These results indicate that the protein structure is pre-organized before substrate binding to PKC-,, which is different from the case of the prototypical AGC-branch kinase PKA. [source]