Biomedical subjects

D Suck

Publications and source records attributed to D Suck.

At least 37 records · Page 2Linked to original sources

PubMed1995

Three-dimensional structure of the bifunctional protein PCD/DCoH, a cytoplasmic enzyme interacting with transcription factor HNF1.

The bifunctional protein pterin-4a-carbinolamine dehydratase (PCD)/dimerization cofactor of HNF1 (DCoH) is a cytoplasmic enzyme involved in the tetrahydrobiopterin regeneration and is found in complex with the transcription factor HNF1 in liver cell nuclei. An atypical hyperphenylalaninemia and the depigmentation disorder vitiligo are related to a deficiency of PCD/DCoH activity. The crystal structure of PCD/DCoH was solved by multiple isomorphous replacement and refined to a crystallographic R-factor of 20.5% at 2.7 A resolution. The single domain monomer comprises three alpha-helices packed against one side of a four-stranded, antiparallel beta-sheet. The functional enzyme is a homo-tetramer of 222 symmetry where each of the monomers contributes one helix to a central four helix bundle. In the tetramer two monomers form an eight-stranded, antiparallel beta-sheet with six helices packing against it from one side. The concave, hydrophobic surface of the eight-stranded beta-sheet with its two protruding loops at either end is reminiscent of the saddle-like shape seen in the TATA-box binding protein. PCD/DCoH binds as a dimer to the helical dimerization domain of dimeric HNF1 forming a hetero-tetramer possibly through a mixed four helix bundle.

Amino Acid Sequence↗

PubMed1995

Sequence-dependent bending propensity of DNA as revealed by DNase I: parameters for trinucleotides.

Structural parameters characterizing the bending propensity of trinucleotides were deduced from DNase I digestion data using simple probabilistic models. In contrast to dinucleotide-based models of DNA bending and/or bendability, the trinucleotide parameters are in good agreement with X-ray crystallographic data on bent DNA. This improvement may be due to the fact that the trinucleotide model incorporates more sequence context information than do dinucleotide-based descriptions.

Base Sequence↗

PubMed1995

Crystallization and preliminary crystallographic studies of recombinant dimerization cofactor of transcription factor HNF1/pterin-4 alpha-carbinolamine dehydratase from liver.

The bi-functional protein dimerization cofactor of HNF1 (DCoH)/pterin-4 alpha-carbinolamine dehydratase (PCD) is found in liver cell nuclei bound to the transcription factor hepatocyte nuclear factor 1 (HNF1) as well as in the cytoplasm acting as an enzyme involved in the phenylalanine hydroxylation system. Deficiency of DCoH/PCD activity in liver causes an atypical hyperphenylalaninemia and deficiency in human epidermis is related to the depigmentation disorder vitiligo. DCoH/PCD from rat liver, which is identical to the human protein, was expressed in E. coli, purified to homogeneity and crystallized. The crystals belong to the trigonal space group P3(1)21 (or P3(2)21) with unit cell dimensions of a = b = 106.2 A, c = 197.1 A. Native crystals diffract to a resolution of 2.5 A.

Animals↗

PubMed1995

Trinucleotide models for DNA bending propensity: comparison of models based on DNaseI digestion and nucleosome packaging data.

DNaseI digestion studies (Brukner et al, EMBO J 14, 1812-1818 1995) and nucleosomebinding data (Satchwell et al, J. Mol. Biol. 191, 639-659 1986, Goodsell and Dickerson, Nucleic trinucleotides. A detailed comparison of the two models suggests that while both of them represent improvements with respect to dinucleotide based descriptions, the individual trinucleotide parameters are not highly correlated (linear correlation coefficient is 0.53), and a number of motifs such as TA-elements and CCA/TGG motifs are more realistically described in the DNaseI-based model. This may be due to the fact that the DNaseI-based model does not rely on a static geometry but rather captures a dynamic ability of ds DNA to bend towards the major grove. Future refinement of both models of both models on larger experimental data sets is expected to further improve the prediction of macroscopic DNA-curvature.

DNA↗

PubMed1995

Mutational analysis of DNase I-DNA interactions: design, expression and characterization of a DNase I loop insertion mutant with altered sequence selectivity.

A mutant of bovine pancreatic DNase I containing two additional residues in a loop next to C173 has been expressed in Escherichia coli, purified and characterized biochemically. Modelling studies suggest that the inserted arginine and glutamate side chains of the modified loop sequence C173-R-E-G-T-V176 could contact the bases 3' to the cleaved bond in the major groove of a bound DNA, and that up to 10 bp could interact with the enzyme and potentially influence its cutting rate. The loop insertion mutant has an 800-fold lower specific activity than wild-type and shows overall cleavage characteristics similar to bovine pancreatic DNase I. Compared with the wild-type enzyme, the mutant shows a strongly enhanced preference for cutting the inverted repeat: (formula: see text) or close variants thereof. Unexpectedly for a minor groove binding protein, the preferred cutting sites in opposite strands are staggered by 1 bp in the 5' direction, causing the cleavage of a TA and a TT step, respectively. This finding demonstrates that the sequence context is relatively more important for the cutting frequency than the nature of the dinucleotide step of the cleaved bond, and clearly shows that base recognition is involved in determining the sequence selectivity of the mutant. The importance of the sequence 5' to the cleaved bond for the cutting rate suggests that the additional major groove contacts may require a distortion of the DNA associated with a higher energy barrier, resulting in an increased selectivity for flexible DNA sequences and a lower overall activity of the mutant enzyme.

Animals↗

PubMed1994

DNA-protein interactions. Flip out and modify.

The crystal structure of a complex between a methyltransferase and DNA shows that, remarkably, the target cytosine base is swung out of the double helix and located next to the enzyme's S-adenosyl-L-homocysteine cofactor.

Base Sequence↗

PubMed1994

DNA recognition by DNase I.

Bovine pancreatic DNase I shows a strong preference for double-stranded substrates and cleaves DNA with strongly varying cutting rates suggesting that the enzyme recognises sequence-dependent structural variations of the DNA double helix. The complicated cleavage pattern indicates that several local as well as global helix parameters influence the cutting frequency of DNase I at a given bond. The high resolution crystal structures of two DNase I-DNA complexes showed that the enzyme binds tightly in the minor groove, and to the sugar-phosphate backbones of both strands, and thereby induces a widening of the minor groove and a bending towards the major groove. In agreement with biochemical data this suggests that flexibility and minor groove geometry are major parameters determining the cutting rate of DNase I. Experimental observations showing that the sequence environment of a dinucleotide step strongly affects its cleavage efficiency can be rationalized by the fact that six base pairs are in contact with the enzyme. Mutational analysis based on the structural results has identified critical residues for DNA binding and cleavage and has lead to a proposal for the catalytic mechanism.

Animals↗

PubMed1993

Preliminary crystallographic studies on the D15 5' to 3' exonuclease from phage T5.

The D15 exonuclease from phage T5 has been crystallized from 35% (w/v) ammonium sulfate by the hanging drop vapor diffusion technique. The crystals grow in tetragonal space group P4(1)22 or P4(3)22 with cell dimensions a = b = 79.2 A and c = 138.0 A. The crystals diffract to 2.5 A and are suitable for X-ray structure determination.

Crystallization↗

PubMed1993

Evidence for opposite groove-directed curvature of GGGCCC and AAAAA sequence elements.

The repetitive sequence (AGGGCCCTAGAGGGGCCC-TAG)n was previously shown to be curved by gel mobility assays. Here we show, using hydroxy radical/DNase I digestion and differential helical phasing experiments that the curvature is directed towards the major groove and is located in the GGGCCC, but not the CTAGAG segments. The effect of the GC step in the context of the GGGCCC motif is apparently about as large as that of AA/TT, i.e. enough to cancel the macroscopic curvature of helically phased A-tracts. These data are in agreement with positive roll-like curvature of the GCC/GGC motif, predicted from nucleosome packing data and the 3D structure of the GGGGCCCC octamer, but they are not in agreement with the dinucleotide-based roll angle values predicted for AG/CT, TA, GG/CC and GC steps. Our results thus indicate the importance of interactions beyond the dinucleotide steps in predictive models of DNA curvature.

Base Sequence↗

PubMed1993

X-ray structures of two single-residue mutants of DNase I: H134Q and Y76A.

The structures of the single-residue mutants H134Q and Y76A of bovine pancreatic DNase I have been determined and refined including data to 2.3 and 2.4 A resolution respectively, by X-ray crystallography. H134 is an essential catalytic residue, while Y76 contributes to the binding of DNA by providing a large van der Waals contact area that stabilizes the wide minor groove seen in DNase I-DNA complexes. The mutant proteins, which show strongly reduced activities of 0.001% (H134Q) and 0.3% (Y76A), were expressed in E. coli and both crystallize in space-group C2 with almost identical unit cells. The crystal packing scheme is different from that found in wild type crystals grown under very similar conditions, presumably due to the absence of the carbohydrate moiety. In both mutants the conformation of the protein is nearly identical to that of the wild type enzyme and changes are confined to surface loops involved in packing. The disruption of the hydrogen bonds between H134, E78 and Y76 in both mutants leads to an increased mobility and positional shifts in the DNA-binding loop, mainly around residue Y76. This in turn may further reduce DNA-binding affinity and, thus, contribute to the low activity. In contrast, symmetry contacts involving residues 97-108 lead to a stabilization of the flexible loop compared to wild type DNase I.

Animals↗

PubMed1993

The X-ray structure of an atypical homeodomain present in the rat liver transcription factor LFB1/HNF1 and implications for DNA binding.

The transcription factor LFB1/HNF1 from rat liver nuclei is a 628 amino acid protein that functions as a dimer binding to the inverted palindrome GTTAATN-ATTAAC consensus site. We have crystallized a 99 residue protein containing the homeodomain portion of LFB1, and solved its structure using X-ray diffraction data to 2.8 A resolution. The topology and orientation of the helices is essentially the same as that found in the engrailed, MAT alpha 2 and Antennapedia homeodomains, even though the LFB1 homeodomain contains 21 more residues. The 21 residue insertion is found in an extension of helix 2 and consequent lengthening of the connecting loop between helix 2 and helix 3. Comparison with the engrailed homeodomain-DNA complex indicates that the mode of interaction with DNA is similar in both proteins, with a number of conserved contacts in the major groove. The extra 21 residues of the LFB1 homeodomain are not involved in DNA binding. Binding of the LFB1 dimer to a B-DNA palindromic consensus sequence requires either a conformational change of the DNA (presumably bending), or a rearrangement of the subunits relative to the DNA.

Amino Acid Sequence↗

PubMed1992

X-ray structure of the DNase I-d(GGTATACC)2 complex at 2.3 A resolution.

The crystal structure of a complex between DNase I and the self-complementary octamer duplex d(GGTATACC)2 has been solved using the molecular replacement method and refined to a crystallographic R-factor of 18.8% for all data between 6.0 and 2.3 A resolution. In contrast to the structure of the DNase I-d(GCGATCGC)2 complex solved previously, the DNA remains uncleaved in the crystal. The general architecture of the two complexes is highly similar. DNase I binds in the minor groove of a right-handed DNA duplex, and to the phosphate backbones on either side over five base-pairs, resulting in a widening of the minor groove and a concurrent bend of the DNA away from the bound enzyme. There is very little change in the structure of the DNase I on binding the substrate. Many other features of the interaction are conserved in the two complexes, in particular the stacking of a deoxyribose group of the DNA onto the side-chain of a tyrosine residue (Y76), which affects the DNA conformation and the binding of an arginine side-chain in the minor groove. Although the structures of the DNA molecules appear at first sight rather similar, detailed analysis reveals some differences that may explain the relative resistance of the d(GGTATACC)2 duplex to cleavage by DNase I: whilst some backbone parameters are characteristic of a B-conformation, the spatial orientation of the base-pairs in the d(GGTATACC)2 duplex is close to that generally observed in A-DNA. These results further support the hypothesis that the minor-groove width and depth and the intrinsic flexibility of DNA are the most important parameters affecting the interaction. The disposition of residues around the scissile phosphate group suggests that two histidine residues, H134 and H252, are involved in catalysis.

Base Composition↗

PubMed1991

DNase I-induced DNA conformation. 2 A structure of a DNase I-octamer complex.

The structure of a complex between DNase I and d(GCGATCGC)2 has been solved by molecular replacement and refined to an R-factor of 0.174 for all data between 6 and 2 A resolution. The nicked octamer duplexes have lost a dinucleotide from the 3' ends of one strand and are hydrogen-bonded across a 2-fold axis to form a quasi-continuous double helix of 14 base-pairs. DNase I is bound in the minor groove of the B-type DNA duplex forming contacts in and along both sides of the minor groove extending over a total of six base-pairs. As a consequence of binding of DNase I to the DNA-substrate the minor groove opens by about 3 A and the duplex bends towards the major groove by about 20 degrees. Apart from these more global distortions the bound duplex also shows significant deviations in local geometry. A major cause for the observed perturbations in the DNA conformation seems to be the stacking type interaction of a tyrosine ring (Y76) with a deoxyribose. In contrast, the enzyme structure is nearly unchanged compared to free DNase I (0.49 A root-mean-square deviations for main-chain atoms) thus providing a rigid framework to which the DNA substrate has to adapt on binding. These results confirm the hypothesis that groove width and stiffness are major factors determining the global sequence dependence of the enzyme's cutting rates. The nicked octamer present in the crystals did not allow us to draw detailed conclusions about the catalytic mechanism but confirmed the location of the active site near H134 on top of the central beta-sheets. A second cut of the DNA induced by diffusion of Mn2+ into the crystals may suggest the presence of a secondary active site in DNase I.

Amino Acid Sequence↗

PubMed1991

Crystal structure of Penicillium citrinum P1 nuclease at 2.8 A resolution.

P1 nuclease from Penicillium citrinum is a zinc dependent glyco-enzyme consisting of 270 amino acid residues which cleaves single-stranded RNA and DNA into 5'-mononucleotides. The X-ray structure of a tetragonal crystal form of the enzyme with two molecules per asymmetric unit has been solved at 3.3 and refined at 2.8 A resolution to a crystallographic R-factor of 21.6%. The current model consists of 269 amino acid residues, three Zn ions and two N-acetyl glucosamines per subunit. The enzyme is folded very similarly to phospholipase C from Bacillus cereus, with 56% of the structure displaying an alpha-helical conformation. The three Zn ions are located at the bottom of a cleft and appear to be rather inaccessible for any phosphate group in double-stranded RNA or DNA substrates. A crystal soaking experiment with a dinucleotide gives clear evidence for two mononucleotide binding sites separated by approximately 20 A. One site shows binding of the phosphate group to one of the zinc ions. At both sites there is a hydrophobic binding pocket for the base, but no direct interaction between the protein and the deoxyribose. A cleavage mechanism is proposed involving nucleophilic attack by a Zn activated water molecule.

Acetylglucosamine↗

PubMed1990

Crystallisation and preliminary crystallographic analysis of P1 nuclease from Penicillium citrinum.

P1 nuclease, a zinc-dependent single-strand specific endonuclease from Penicillium citrinum, has been crystallized in three different space groups using either ammonium sulphate or polyethylene glycol 4000 as the precipitating agent. The crystals diffract to between 3 A and 2.2 A. A 4.5 A electron density map has been calculated for a tetragonal crystal form, based on a platinum derivative, and was improved by solvent flattening. The boundaries of the two molecules in the asymmetric unit are clearly visible in most regions and the presence of rod-like density features are indicative of a rather high alpha-helix content. The highest density peaks in the map were identified as a trinuclear zinc cluster present in each monomer by a difference Fourier of an EDTA-soaked crystal.

Crystallography↗

PubMed1990

Atomic structure of the actin:DNase I complex.

The atomic models of the complex between rabbit skeletal muscle actin and bovine pancreatic deoxyribonuclease I both in the ATP and ADP forms have been determined by X-ray analysis at an effective resolution of 2.8 A and 3A, respectively. The two structures are very similar. The actin molecule consists of two domains which can be further subdivided into two subdomains. ADP or ATP is located in the cleft between the domains with a calcium ion bound to the beta- or beta- and gamma-phosphates, respectively. The motif of a five-stranded beta sheet consisting of a beta meander and a right handed beta alpha beta unit appears in each domain suggesting that gene duplication might have occurred. These sheets have the same topology as that found in hexokinase.

Actins↗

PubMed1988

Structure refined to 2A of a nicked DNA octanucleotide complex with DNase I.

The cutting rates of bovine pancreatic deoxyribonuclease I (DNase I) vary along a given DNA sequence, indicating that the enzyme recognizes sequence-dependent structural variations of the DNA double-helix. In an attempt to define the helical parameters determining this sequence-dependence, we have co-crystallized a complex of DNase I with a self-complementary octanucleotide and refined the crystal structure at 2 A resolution. This structure confirms the basic features of an early model, namely that an exposed loop of DNase I binds in the minor groove of B-type DNA and that interactions do occur with the backbone of both strands. Nicked octamer duplexes that have lost a dinucleotide from the 3'-end of one strand are hydrogen-bonded across a two-fold axis in the crystal to form a quasi-continuous double helix of 14 base pairs. The DNA 14-mer has a B-type conformation and shows substantial distortion of both local and overall helix parameters, induced mainly by the tight interaction of Y73 and R38 in the unusually wide minor groove. Directly coupled to the widening of the groove by approximately 3A is a 21.5 degree bend of the DNA away from the bound enzyme towards the major groove, suggesting that both DNA stiffness and groove width are important in determining the sequence-dependence of the enzyme cutting rate. A second cut of the DNA which is induced by diffusion of Mn2+ into the co-crystals suggests that there are two active sites in DNase I separated by more than 15A.

Deoxyribonuclease I↗

PubMed1986

Crystallographic refinement and structure of DNase I at 2 A resolution.

The structure of bovine pancreatic deoxyribonuclease I (DNase I) has been refined at 2 A resolution using the restrained parameter, reciprocal least-squares procedure of Hendrickson and Konnert. The conventional R-factor for 16,104 reflections with I greater than or equal to 3 sigma (I) from 6.0 to 2.0 A resolution is 0.157. Bond lengths and angles of the refined structure are close to ideal values with root-mean-square (r.m.s.) deviations of 0.023 A and 1.4 degrees, respectively. The r.m.s. deviation of short non-bonded contacts from the sum of van der Waals' radii is 0.18 A. The orientation of side-chains shows a clear trimodal distribution of chi 1-angles at -60 degrees, 180 degrees, 60 degrees (in the order of preference) corresponding to staggered conformations. The chemically determined sequence was corrected at four positions, the major correction being an insertion of the tripeptide Ile-Val-Arg between Arg27 and Arg28. Extended hydrophobic regions in between, and on either side of, the two central six-stranded beta-pleated sheets are mainly responsible for the low average isotropic temperature factor of 11.9 A2 for the 2033 protein atoms. Besides the flexible loop region between Gly97 and Gly102 (Glu99 and Ser100 are disordered) and the carbohydrate side-chain, which both extend into a large solvent channel, only the exposed loop Arg70 to Lys74 shows elevated thermal mobility. The longest of the eight helices in DNase I, together representing 26% of the structure, has a 22 degree kink and consists of two alpha-helical segments (residues 136 to 144 and 145 to 155) separated by a 3(10)-helical turn. DNase I fragments 1 to 120 and 121 to 257 can be superimposed by an approximate 2-fold axis (r.m.s. deviation 1.49 A for 61 equivalent C alpha positions), suggesting that the enzyme might be the result of gene duplication. The two Ca2+ bound to DNase I under crystallization conditions are important for its structural integrity by stabilizing the surface loop Asp198 to Thr204 and limiting the region of high thermal mobility in the flexible loop to residues Gly97 to Gly102. The N-linked carbohydrate side-chain attached to Asn18 is of the high-mannose type with a branching point at the mannose residue in position 3.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗