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BACKGROUND: Cyclic AMP binding domains possess common structural features yet are diversely coupled to different signaling modules. Each cAMP binding domain receives and transmits a cAMP signal; however, the signaling networks differ even within the same family of regulatory proteins as evidenced by the long-standing biochemical and physiological differences between type I and type II regulatory subunits of cAMP-dependent protein kinase. RESULTS: We report the first type II regulatory subunit crystal structure, which we determined to 2.45 A resolution and refined to an R factor of 0.176 with a free R factor of 0.198. This new structure of the type II beta regulatory subunit of cAMP-dependent protein kinase demonstrates that the relative orientations of the two tandem cAMP binding domains are very different in the type II beta as compared to the type I alpha regulatory subunit. Each structural unit for binding cAMP contains the highly conserved phosphate binding cassette that can be considered the "signature" motif of cAMP binding domains. This motif is coupled to nonconserved regions that link the cAMP signal to diverse structural and functional modules. CONCLUSIONS: Both the diversity and similarity of cAMP binding sites are demonstrated by this new type II regulatory subunit structure. The structure represents an intramolecular paradigm for the cooperative triad that links two cAMP binding sites through a domain interface to the catalytic subunit of cAMP-dependent protein kinase. The domain interface surface is created by the binding of only one cAMP molecule and is enabled by amino acid sequence variability within the peptide chain that tethers the two domains together.

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Protein kinases are important targets for designing therapeutic drugs. This paper illustrates a computational approach to extend the usefulness of a single protein-inhibitor structure in aiding the design of protein kinase inhibitors. Using the complex structure of the catalytic subunit of PKA (cPKA) and balanol as a guide, we have analyzed and compared the distribution of amino acid types near the protein-ligand interface for nearly 400 kinases. This analysis has identified a number of sites that are more variable in amino acid types among the kinases analyzed, and these are useful sites to consider in designing specific protein kinase inhibitors. On the other hand, we have found kinases whose protein-ligand interfaces are similar to that of the cPKA-balanol complex and balanol can be a useful lead compound for developing effective inhibitors for these kinases. Generally, this approach can help us discover new drug targets for an existing class of compounds that have already been well characterized pharmacologically. The relative significance of the charge/polarity of residues at the protein-ligand interface has been quantified by carrying out computational sensitivity analysis in which the charge/polarity of an atom or functional group was turned off/on, and the resulting effects on binding affinity have been examined. The binding affinity was estimated by using an implicit-solvent model in which the electrostatic contributions were obtained by solving the Poisson equation and the hydrophobic effects were accounted for by using surface-area dependent terms. The same sensitivity analysis approach was applied to the ligand balanol to develop a pharmacophoric model for searching new drug leads from small-molecule libraries. To help evaluate the binding affinity of designed inhibitors before they are made, we have developed a semiempirical approach to improve the predictive reliability of the implicit-solvent binding model.

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To elucidate the structural basis for important differences between types I and II regulatory subunit isoforms (RI and RII) of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase, the full-length RII beta isoform and five RII beta deletion mutants were constructed, expressed, purified, and screened for crystallization. Only one of these six proteins yielded diffraction quality crystals. Crystals were grown of the RII beta deletion mutant (delta 1-111) monomer potentially in complex with two cAMP molecules. X-ray diffraction quality data were obtained only after significant modification to existing purification procedures. Modifications required a Sepharose, not agarose, support for cAMP affinity chromatography followed by rapid, quantitative removal of free cAMP by size-exclusion chromatography under reducing conditions. Data to 2.4 A resolution were collected at 29 degrees C using synchrotron radiation on a single crystal measuring 0.2 x 0.3 x 1.2 mm(3). Data were 99% complete. The hexagonal crystal belonged to space group P6((1)) or P6((5)) with unit cell dimensions a = b = 161.62 A and c = 39.66 A.

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The structural basis for the effect of the S96P mutation in chicken triosephosphate isomerase (cTIM) has been analyzed using a combination of X-ray crystallography and Fourier transform infrared spectroscopy. The X-ray structure is that of the enzyme complexed with phosphoglycolohydroxamate (PGH), an intermediate analogue, solved at a resolution of 1.9 A. The S96P mutation was identified as a second-site reverent when catalytically crippled mutants, E165D and H95N, were subjected to random mutagenesis. The presence of the second mutation leads to enhanced activity over the single mutation. However, the effect of the S96P mutation alone is to decrease the catalytic efficiency of the enzyme. The crystal structures of the S96P double mutants show that this bulky proline side chain alters the water structure within the active-site cavity (E165D; ref 1) and prevents nonproductive binding conformations of the substrate (H95N; ref 2). Comparison of the S96P single mutant structure with those of the wild-type cTIM, those of the single mutants (E165D and H95N), and those of the double mutants (E165D/S96P and H95N/S96P) begins to address the role of the conserved serine residue at this position. The results indicate that the residue positions the catalytic base E165 optimally for polarization of the substrate carbonyl, thereby aiding in proton abstraction. In addition, this residue is involved in positioning critical water molecules, thereby affecting the way in which water structure influences activity.

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Endogenous protein kinase inhibitors are essential for a wide range of physiological functions. These endogenous inhibitors may mimic peptide substrates as in the case of the heat-stable protein kinase inhibitor (PKI), or they may mimic nucleotide triphosphates. Natural product inhibitors, endogenous to the unique organisms producing them, can be potent exogenous inhibitors against foreign protein kinases. Balanol is a natural product inhibitor exhibiting low nanomolar Ki values against serine and threonine specific kinases, while being ineffective against protein tyrosine kinases. To elucidate balanol's specific inhibitory effects and provide a basis for understanding inhibition-regulated biological processes, a 2.1 A resolution crystal structure of balanol in complex with cAMP-dependent protein kinase (cAPK) was determined. The structure reveals conserved binding regions and displays extensive complementary interactions between balanol and conserved cAPK residues. This report describes the structure of a protein kinase crystallized with a natural ATP mimetic in the absence of metal ions and peptide inhibitor.

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Three crystal forms of Naja naja naja phospholipase A2 were discovered through random crystallization screening, including two heretofore uncharacterized forms. The crystallization conditions for both of these novel crystal forms are Ca(2+)-free whereas previously reported conditions include Ca2+. One of the new crystal forms has a cubic lattice in the space group P2(1)3 (a = b = c = 69.24 A), the other has an orthorhombic lattice in the space group P2(1)2(1)2(1) (a = 67.22 A, b = 73.48 A, c = 87.52 A) and a previously characterized crystal belong to the tetragonal space group P4(3)2(1)2 (a = b = 88.6 A, c = 107.4 A). The structure from the cubic crystal form has been determined to 1.8 A and refined to an R-factor of 17% while the structure from the orthorhombic form has been determined to 2.65 A and has been refined to an R-factor of 21%. The determination of the cubic structure extends the resolution to which structures of this molecule have been determined from 2.3 A to 1.8 A. The two newly determined structures, in combination with the previously determined structure, generate an informative structural ensemble from which structural changes due to Ca2+, which is required for catalysis, and the effect of crystal contacts on side-chain conformations and oligomeric association can be inferred. Both of the newly determined structures reveal a trimeric oligomer as observed in the tetragonal structure; this appears to be a unique feature of the Naja naja naja enzyme.

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A two-dimensional application specific integrated circuit (ASIC) based detector, designed for X-ray protein crystallography, has been tested to determine its suitability as a direct electron detector for TEM imaging in the voltage range of 20-400 keV. Several markedly different properties of this device distinguish it from the charge coupled device (CCD) detectors: (1) the ASIC detector can be used directly under electron bombardment in the voltage range stated above, therefore requiring no scintillator screen; (2) each active pixel of the device is an electron counter and generates digital output independently; (3) the readout of the device is frameless and event driven; (4) the device can be operated at the room temperature and is nearly noise free; and (5) the counting dynamic range of the device is virtually unlimited. It appears that an imaging system based on this type of device would be ideal for low-dose TEM imaging and online diffraction observation and recording, as well as more conventional imaging, providing the many advantages of direct digital readout for almost all applications.

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X-ray diffraction-quality crystals of the unliganded mouse recombinant catalytic subunit of cAMP-dependent protein kinase were grown by the hanging-drop vapour-diffusion technique using 2-methyl-2,4-pentanediol as precipitant. The crystals belong to the monoclinic space group P21 with unit-cell parameters a = 48.9, b = 147.4, c = 54.2 A, beta = 110.2 degrees. A data set to 3.0 A resolution with 92% completeness has been collected using synchrotron radiation. The unit cell contains four molecules of molecular weight 40 kDa with a corresponding volume solvent content of 45%.

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Spo0F is a secondary messenger in the "two-component" system controlling the sporulation of Bacillus subtilis. Spo0F, like the chemotaxis protein CheY, is a single-domain protein homologous to the N-terminal activator domain of the response regulators. We recently reported the crystal structure of a phosphatase-resistant mutant Y13S of Spo0F with Ca2+ bound in the active site. The crystal structure of wild-type Spo0F in the absence of a metal ion is presented here. A comparison of the two structures reveals that the cation induces significant changes in the active site. In the present wild-type structure, the carboxylate of Asp11 points away from the center of the active site, whereas when coordinated to the Ca2+, as in the earlier structure, it points toward the active site. In addition, Asp54, the site of phosphorylation, is blocked by a salt bridge interaction of an Arg side chain from a neighboring molecule. From fluorescence quenching studies with Spo0F Y13W, we found that only the amino acid Arg binds to Spo0F in a saturable manner (Kd = 15 mM). This observation suggests that a small molecule with a shape complementary to the active site and having a guanidinium group might inhibit phosphotransfer between response regulators and their cognate histidine kinases.

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The structural basis for the 3000-fold decrease in catalytic efficiency of the H95N mutant chicken triosephosphate isomerase and the 60-fold regain of catalytic efficiency in the double mutant, H95N.S96P, have been analyzed. The results from a combination of X-ray crystallography and Fourier transform infrared spectroscopy experiments indicate that the predominant defect in the H95N mutant isomerase appears to be its inability to bind the substrate in a coplanar, cis conformation. The structures of each mutant isomerase were determined from X-ray crystallography of the complex of phosphoglycolohydroxamate (PGH), an intermediate analog with the isomerase, and each was solved to a resolution of 1.9 A. The PGH appeared to be in two different conformations in which the enediol-mimicking atoms, O2-N2-C1-O1, of the PGH were not coplanar. No density was observed that would correspond to the coplanar conformation. Two bands are observed for the dihydroxyacetone phosphate carbonyl in the H95N mutant FTIR spectrum, and these can be explained if the O1 of DHAP, like the O1 of PGH in the crystal structure, is in two different positions. Two ordered water molecules are located between O1 of PGH and N delta of N95. Comparison of the structure of the pseudorevertant, H95N.S96P with that for the H95N single mutant, shows that S96P mutation causes the double mutant to regain the ability to bind PGH predominantly in the coplanar, cis conformation. Electron density for a single ordered water molecule bridging the N95 amide side chain and the O2 of PGH is observed, but the density was weak, perhaps indicating that the water molecule is somewhat disordered. Whether or not a water molecule is hydrogen bonded to O2 of PGH may explain the two carbonyl stretching frequencies observed for the GAP carbonyl. Together, the crystal structures and the FTIR data allow a complete explanation of the catalytic properties of these two mutant isomerases.

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Fibronectin is a large cell adhesion molecule that is composed of several functional domains. The cell-binding domain that binds to cell surface integrins consists of repeated homologous type III modules. In this study, recombinant fragments from the cell-binding domain of human fibronectin that participate in a newly characterized fibronectin-fibronectin interaction with FNIII1 were crystallized. In each case, the crystals had more than one fibronectin fragment in the asymmetric unit. Crystals of FNIII10-11 grew in the space group C2 with a = 117.1 A, b = 38.6 A, c = 80.6 A, beta = 97.2 degrees, and two molecules in the asymmetric unit. These crystals diffracted to 2.5 A resolution. Fragment FNIII8-11 and a shorter fragment, FNIII8-10, crystallized in hexagonal space groups with large unit cells and two to four molecules per asymmetric unit. Even very large crystals of these fragments did not diffract beyond 4 A. The crystal packing for this collection of fibronectin fragments suggests conformational flexibility between linked type III modules. The functional relevance of this flexibility for elongated versus compact models of the cell-binding domain of fibronectin is discussed.

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BACKGROUND: Spo0F, a phosphotransferase containing an aspartyl pocket, is involved in the signaling pathway (phosphorelay) controlling sporulation in Bacillus subtilis. It belongs to the superfamily of bacterial response regulatory proteins, which are activated upon phosphorylation of an invariant aspartate residue. This phosphorylation is carried out in a divalent cation dependent reaction catalyzed by cognate histidine kinases. Knowledge of the Spo0F structure would provide valuable information that would enable the elucidation of its function as a secondary messenger in a system in which a phosphate is donated from Spo0F to Spo0B, the third of four main proteins that constitute the phosphorelay. RESULTS: We have determined the crystal structure of a Rap phosphatase resistant mutant, Spo0F Tyr13-->Ser, at 1.9 A resolution. The structure was solved by single isomorphous replacement and anomalous scattering techniques. The overall structural fold is (beta/alpha)5 and contains a central beta sheet. The active site of the molecule is formed by three aspartate residues and a lysine residue which come together at the C terminus of the beta sheet. The active site accommodates a calcium ion. CONCLUSIONS: The structural analysis reveals that the overall topology and metal-binding coordination at the active site are similar to those of the bacterial chemotaxis response regulator CheY. Structural differences between Spo0F and CheY in the vicinity of the active site provide an insight into how similar molecular scaffolds can be adapted to perform different biological roles by the alteration of only a few amino acid residues. These differences may contribute to the observed stability of the phosphorylated species of Spo0F, a feature demanded by its role as a secondary messenger within the phosphorelay system which controls sporulation.

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Spo0F, a member of a superfamily of bacterial response regulatory proteins, is crucial to the regulation of sporulation in Bacillus subtilis. As there were difficulties in reproducing crystals of wild-type Spo0F, we report here the crystallization and preliminary studies of a mutant, Y13S protein, which gave well diffracting reproducible crystals. The crystals of the mutant obtained by the hanging-drop method belong to the tetragonal space group P4(1)2(1)2 (P4(3)2(1)2) a = b = 105.1, c = 85.9 A. Diffraction data were collected at 2.8 A at the laboratory source and subsequently 2.05. A data were collected upon flash freezing the crystal at the Stanford Synchrotron Radiation Laboratory. This mutant participates in the phosphorelay in a similar manner to the wild-type protein. The presence of divalent cations are essential for wild-type phosphorylation and the present mutant crystal form is obtained in the presence of calcium.

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Nine single genetic mutants of rat dihydropteridine reductase (EC 1.6.99.7), D37I, W86I, Y146F, Y146H, K150Q, K150I, K150M, N186A, and A133S and one double mutant, Y146F/K150Q, have been engineered, overexpressed in Escherichia coli and their proteins purified. Of these, five, W86I, Y146F, Y146H, Y146F/K150Q, and A133S, have been crystallized and structurally characterized. Kinetic constants for each of the mutant enzyme forms, except N186A, which was too unstable to isolate in a homogeneous form, have been derived and in the five instances where structures are available the altered activities have been interpreted by correlation with these structures. It is readily apparent that specific interactions of the apoenzyme with the cofactor, NADH, are vital to the integrity of the total protein tertiary structure and that the generation of the active site requires bound cofactor in addition to a suitably placed W86. Thus when the three major centers for hydrogen bonding to the cofactor are mutated, i.e. 37, 150, and 186, an unstable partially active enzyme is formed. It is also apparent that tyrosine 146 is vital to the activity of the enzyme, as the Y146F mutant is almost inactive having only 1.1% of wild-type activity. However, when an additional mutation, K150Q, is made, the rearrangement of water molecules in the vicinity of Lys150 is accompanied by the recovery of 50% of the wild-type activity. It is suggested that the involvement of a water molecule compensates for the loss of the tyrosyl hydroxyl group. The difference between tyrosine and histidine groups at 146 is seen in the comparably unfavorable geometry of hydrogen bonds exhibited by the latter to the substrate, reducing the activity to 15% of the wild type. The mutant A133S shows little alteration in activity; however, its hydroxyl substituent contributes to the active site by providing a possible additional proton sink. This is of little value to dihydropteridine reductase but may be significant in the sequentially analogous short chain dehydrogenases/reductases, where a serine is the amino acid of choice for this position.

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In the molecular scheme of living organisms, adenosine 3',5'-monophosphate (cyclic AMP or cAMP) has been a universal second messenger. In eukaryotic cells, the primary receptors for cAMP are the regulatory subunits of cAMP-dependent protein kinase. The crystal structure of a 1-91 deletion mutant of the type I alpha regulatory subunit was refined to 2.8 A resolution. Each of the two tandem cAMP binding domains provides an extensive network of hydrogen bonds that buries the cyclic phosphate and the ribose between two beta strands that are linked by a short alpha helix. Each adenine base stacks against an aromatic ring that lies outside the beta barrel. This structure provides a molecular basis for understanding how cAMP binds cooperatively to its receptor protein, thus mediating activation of the kinase.

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A binary complex of dihydropteridine reductase and NADH crystallizes in the space group C2, with a = 222.2, b = 46.5, c = 95.3 A and beta = 101.1 degrees. There are two dimers in the asymmetric unit. The structure was solved by molecular-replacement techniques and refined with 2.6 A data to a crystallographic R factor of 16.8%. Each dimer has twofold non-crystallographic symmetry and the four individual monomers in the asymmetric unit have the same overall molecular conformation.

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Phenylketonuria (PKU) is a debilitating hereditary disorder related to an individual's inability to convert phenylalanine to its usual tyrosine product. The genetic errors occur in three regions: in the cooperative enzymes phenylalanine hydroxylase (PAH) and dihydropteridine reductase (DHPR), and in the biosynthetic pathway from GTP to the hydroxylation cofactor, tetrahydrobiopterin (BH4). Many instances of naturally occurring defects in DHPR metabolism have been identified, and in most cases the error has been equated with an altered enzyme gene sequence. Using computer graphics, this report analyses the altered structural characteristics of eight of the enzymes encoded by mutant gene sequence and provides logical explanations for their diminished enzyme activities. In one instance, that of a threonine insertion, a mutant construct of the rat analog has been expressed in Escherichia coli and the DHPR isolated and characterised, confirming the marked changes this insert can create.

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Mirabilis anti-viral protein (MAP) is a ribosome-inactivating protein from Mirabilis jalapa L. Since MAP is effective over a broad spectrum of species, the protein is difficult to express in heterologous hosts such as Escherichia coli. Recently, we obtained a MAP mutant, Y72F which exhibits a lower (1/100) activity against E. coli ribosomes while retaining almost full activity against mammalian cells [Habuka, Miyano, Kataoka, Tsuge & Noma (1992). J. Biol. Chem. 267, 7758-7760]. For the crystallographic studies, the Y72F MAP expression vector with an OmpA leading sequence was constructed and expressed in E. coli. The Y72F MAP mutant was then isolated and purified from the cell culture medium. Crystals were grown using the crystallization conditions for the native MAP crystals [Miyano et al. (1992). J. Mol. Biol. 226, 281-283]: 50% ammonium sulfate containing 50 mM ammonium citrate and 2 mM adenine sulfate, pH 5.4. The crystals belong to space group P3(1)21 (or P3(2)21) with a = b = 104.1 and c = 134.3 A. The crystals are isomorphous with the wild-type crystals but diffract to higher resolution. Imaging-plate photographs of the Y72F mutant showed sharp intense spots without the streaking observed in the native crystals.

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Dihydropteridine reductase (EC 1.6.99.7) is a member of the recently identified family of proteins known as short-chain dehydrogenases. When the x-ray structure of dihydropteridine reductase is correlated with conserved amino acid sequences characteristic of this enzyme class, two important common structural regions can be identified. One is close to the protein N terminus and serves as the cofactor binding site, while a second conserved feature makes up the inner surface of an alpha-helix in which a tyrosine side chain is positioned in close proximity to a lysine residue four residues downstream in the sequence. The main function of this Tyr-Lys couple may be to facilitate tyrosine hydroxyl group participation in proton transfer. Thus, it appears that there is a distinctive common mechanism for this group of short-chain or pyridine dinucleotide-dependent oxidoreductases that is different from their higher molecular weight counterparts.

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The crystal structure of the cell adhesion module of fibronectin (FNIII10) has been determined at 1.8 A resolution. A recombinant fragment corresponding to the tenth type III module of human fibronectin was crystallized in space group P2(1) with a = 30.7, b = 35.1 and c = 37.7 A and beta = 107 degrees. The structure was determined by molecular replacement and refined by least squares methods. The crystallographic R-factor for the final model of the 91 amino acid module plus 56 solvent atoms is 0.18 for 10 to 1.8 A data. The module consists of two layers of beta-sheet, one with three antiparallel strands and the other with four antiparallel strands. The beta-sheets enclose a hydrophobic core of 24 amino acid side-chains. The module contains the RGD cell recognition sequence in a flexible loop connecting two beta-strands. The tertiary structure of the FNIII10 module has been used to develop a structure-based sequence alignment of 17 type III modules in fibronectin based on the striking conservation of homologous hydrophobic residues. A similar pattern of homologous alternating hydrophobic residues is also evident in a comparison of type III modules in proteins unrelated to fibronectin such as cytokine receptors and muscle proteins.

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