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Biomedical subjects

M R Sanderson

Publications and source records attributed to M R Sanderson.

At least 19 recordsLinked to original sources
PubMed1999

NMR assignments and secondary structure of the UvrC binding domain of UvrB.

The 55 residue C-terminal domain of UvrB that interacts with UvrC during excision repair in Escherichia coli has been expressed and purified as a (His)6 fusion construct. The fragment forms a stable folded domain in solution. Heteronuclear NMR experiments were used to obtain extensive 15N, 13C and 1H NMR assignments. NOESY and chemical shift data showed that the protein comprises two helices from residues 630 to 648 and from 652 to 670. 15N relaxation data also show that the first 11 and last three residues are unstructured. The effective rotational correlation time within the structured region is not consistent with a monomer. This oligomerisation may be relevant to the mode of dimerisation of UvrB with the homologous domain of UvrC.

Amino Acid Sequence↗
PubMed1999

Structure to 1.9 A resolution of a complex with herpes simplex virus type-1 thymidine kinase of a novel, non-substrate inhibitor: X-ray crystallographic comparison with binding of aciclovir.

Treatment of herpes infections with nucleoside analogues requires as an initial step the activation of the compounds by thymidine kinase. As an aid to developing more effective chemotherapy, both for treatment of recurrent herpes infection and in gene therapy systems where thymidine kinase is expressed, two high-resolution X-ray structures of thymidine kinase have been compared: one with the relatively poor substrate aciclovir (Zovirax), the other with a synthetic inhibitor having an N2-substituted guanine. Both compounds have similar binding modes in spite of their size difference and apparently distinct ligand properties.

Acyclovir↗
PubMed1998

5-Substituted pyrimidines with a 1,5-anhydro-2, 3-dideoxy-D-arabino-hexitol moiety at N-1: synthesis, antiviral activity, conformational analysis, and interaction with viral thymidine kinase.

A new series of anhydrohexitol nucleosides are described. These compounds have a pyrimidine base moiety substituted in the 5-position with a chloro (1b), trifluoromethyl (1c), vinyl (1d), 2-thienyl (1e), ethynyl (1f) or propynyl (1g) substituent. The vinyl, propynyl, and, in particular, the 5-trifluoromethyl analogue showed potent activity against herpes simplex virus (HSV), 1c with a selectivity index of >16000 against HSV-1 and >1000 against HSV-2. Conformational analysis of anhydrohexitol nucleosides using computational methods indicates that these nucleosides occur in an equilibrium between the C1 and 1C form with a DeltaE of 5.9 kJ/mol. When the anhydrohexitol nucleoside is cocrystallized with the HSV-1 thymidine kinase it adopts a 1C conformation, which is opposite to the conformation found for the small molecule alone. The enzyme, apparently, induces a conformational change, and conformational flexibility of an anhydrohexitol nucleoside may be advantageous for recognition by viral enzymes.

Antiviral Agents↗
PubMed1998

Exploring the active site of herpes simplex virus type-1 thymidine kinase by X-ray crystallography of complexes with aciclovir and other ligands.

Antiherpes therapies are principally targeted at viral thymidine kinases and utilize nucleoside analogs, the triphosphates of which are inhibitors of viral DNA polymerase or result in toxic effects when incorporated into DNA. The most frequently used drug, aciclovir (Zovirax), is a relatively poor substrate for thymidine kinase and high-resolution structural information on drugs and other molecules binding to the target is therefore important for the design of novel and more potent chemotherapy, both in antiherpes treatment and in gene therapy systems where thymidine kinase is expressed. Here, we report for the first time the binary complexes of HSV-1 thymidine kinase (TK) with the drug molecules aciclovir and penciclovir, determined by X-ray crystallography at 2.37 A resolution. Moreover, from new data at 2.14 A resolution, the refined structure of the complex of TK with its substrate deoxythymidine (R = 0.209 for 96% of all data) now reveals much detail concerning substrate and solvent interactions with the enzyme. Structures of the complexes of TK with four halogen-containing substrate analogs have also been solved, to resolutions better than 2.4 A. The various TK inhibitors broadly fall into three groups which together probe the space of the enzyme active site in a manner that no one molecule does alone, so giving a composite picture of active site interactions that can be exploited in the design of novel compounds.

Acyclovir↗
PubMed1995

Crystal structures of the thymidine kinase from herpes simplex virus type-1 in complex with deoxythymidine and ganciclovir.

The crystal structures of thymidine kinase from herpes simplex virus type-1 complexed with its natural substrate deoxythymidine (dT) and complexed with the guanosine analogue Ganciclovir have been solved. Both structures are in the C222(1) crystal form with two molecules per asymmetric unit related by a non-crystallographic two-fold axis. The present models have been refined to 2.8 A and 2.2 A, with crystallographic R factors of 24.1% and 23.3% for the dT and Ganciclovir complexes respectively, without the inclusion of any solvent molecules. The core of the molecule exhibits high structural homology with adenylate kinase and other nucleotide binding proteins. These structural similarities provide an insight into the mechanism of nucleoside phosphorylation by thymidine kinase.

Adenylate Kinase↗
PubMed1994

Crystal structure of a disulfide-linked "trefoil" motif found in a large family of putative growth factors.

Porcine pancreatic spasmolytic polypeptide (PSP) belongs to a large family of homologous growth factor-like polypeptides characterized by a disulfide-linked "trefoil motif," duplicated and conserved in various family members. PSP contains two trefoil motifs, has several pharmacological actions on the gut, and has growth factor properties on epithelial cells in vitro. The human PSP analogue, human spasmolytic polypeptide, appears to be involved in many regenerative situations and, especially, in healing gastrointestinal ulcers. One member of the trefoil family, pS2, is secreted in approximately 50% of estrogen-dependent human breast carcinomas, which has led to its use as a tumor prognostic marker. Both pS2 and human spasmolytic polypeptide are also widely expressed in chronic gastrointestinal ulcerative conditions such as Crohn disease. Here we report the three-dimensional structure at 2.6-A resolution of a trefoil-containing protein, namely PSP, purified from porcine pancreas. The structure shows two homologous domains that share a supersecondary structure and disulfide bond pattern. The two domains pack asymmetrically giving rise to a number of protruding loops, exposed clefts, and an unusual electrostatic surface potential. Knowledge of the structure of PSP should allow the design of mutants to investigate further the function of PSP and other trefoil-containing peptides.

Amino Acid Sequence↗
PubMed1992

Crystal structure of a berenil-d(CGCAAATTTGCG) complex. An example of drug-DNA recognition based on sequence-dependent structural features.

The AT-selective drug berenil has been co-crystallized with the dodecanucleotide sequence d(CGCAAATTTGCG)2. The crystal structure has been solved to a resolution of 2.0 A and an R factor of 18.3%, with the location of 65 water molecules. The drug is symmetrically bound in the 5'-AATT region of the minor groove, with its amidinium groups hydrogen-bonding to O-2 atoms of the thymine base at each end of the binding site. This arrangement is distinct from that previously found for berenil with the sequence d(CGCGAATTCGCG)2, which has the drug bound to the sequencing 5'-ATT via hydrogen bonds to adenine N-3 atoms with the involvement of a bridging water molecule at one end of the binding site. The reasons for these differences are discussed in terms of changes in helical parameters; in particular propeller twist and base-pair roll are considered to be important. The conformational and base-pair geometry of the dodecanucleotide in the structure reported here, is closely similar to that for the native structure, suggesting that the 5'-AAATTT sequence does not significantly alter during drug binding, either because of its inflexibility or because its geometry is nearly ideal for berenil binding.

Base Sequence↗
PubMed1990

The crystal structure of the catalytic domain of the site-specific recombination enzyme gamma delta resolvase at 2.7 A resolution.

The crystal structure of the catalytic domain of the site-specific recombination enzyme gamma delta resolvase has been determined at 2.7 A resolution. Its first 120 amino acids form a central five-stranded, beta-pleated sheet surrounded by five alpha helices. In one of the four dyad-related dimers, the two active site Ser-10 residues are 19 A apart, perhaps close enough to contact and become covalently linked to the DNA at the recombination site. This dimer also forms the only closely packed tetramer found in the crystal. The subunit interface at a second dyad-related dimer is more extensive and more highly conserved among the homologous recombinases; however, its active site Ser-10 residues are more than 30 A apart. Side chains, identified by mutations that eliminate catalysis but not DNA binding, are located on the subunit surface near the active site serine and at the interface between a third dyad-related pair of subunits of the tetramer.

Binding Sites↗
PubMed1990

Crystal and solution structures of the oligonucleotide d(ATGCGCAT)2: a combined X-ray and NMR study.

A combined crystal-structure determination and NMR analysis of the octanucleotide d(ATGCGCAT)2 is reported. The X-ray analysis shows that the structure is A-form duplex in crystal state. The NMR study shows that in solution this sequence is B-type. The conformational results from each technique are presented in detail. The implications of these findings in terms of conformational flexibility and ligand binding are discussed.

Base Sequence↗
PubMed1990

Crystal structure of a berenil-dodecanucleotide complex: the role of water in sequence-specific ligand binding.

The three-dimensional structure of a complex between the dodecanucleotide d(CGCGAATTCGCG) and the anti-trypanocidal drug berenil, has been determined to a resolution of 2.5 A. The structure has been solved by molecular replacement and refined to an R factor of 0.177. A total of 49 water molecules have been located. The drug is bound at the 5'-AAT-3' region of the oligonucleotide. At one end of the drug the amidinium group is in hydrogen-bonded contact with N3 of the adenine base complementary to the thymine of the AAT. The other amidinium group does not make direct interactions with the DNA. Instead, a water molecule mediates between them. This is in hydrogen-bonded contact with an amidinium nitrogen atom, N3 of the 5' end adenine base and the ring oxygen atom of an adjacent deoxyribose. Molecular mechanics calculations have been performed on this complex, with the drug at various positions along the sequence. These show that the observed position is only 0.8 kcal/mol higher in energy than the best position. It is suggested that there is a broad energy well in the AATT region for this drug, and that water molecules as well as the neighbouring sequence, will determine precise positioning. More general aspects of minor groove binding are discussed.

Amidines↗
PubMed1990

Crystal structure and sequence-dependent conformation of the A.G mispaired oligonucleotide d(CGCAAGCTGGCG).

The crystal structure of the dodecanucleotide d(CGCAAGCTGGCG) has been determined to a resolution of 2.5 A and refined to an R factor of 19.3% for 1710 reflections. The sequence crystallizes as a B-type double helix, with two G(anti).A(syn) base pairs. These are stabilized by three-center hydrogen bonds to pyrimidines that induce perturbations in base-pair geometry. The central AGCT region of the helix has a wide (greater than 6 A) minor groove.

Adenine↗
PubMed1989

Preparation of heavy-atom derivatives using site-directed mutagenesis. Introduction of cysteine residues into gamma delta resolvase.

The ability to determine protein structures by X-ray crystallography is often thwarted by the difficulty of finding isomorphous heavy-atom derivatives. The crystal structure of the site-specific recombinase, resolvase, has been difficult to determine for this reason. We have overcome this problem by introducing 13 single cysteine substitutions into the resolvase catalytic domain using oligonucleotide mutagenesis. The mutant proteins were screened for their ability to crystallize into the orthorhombic form and bind mercury ions isomorphously. Two mutant proteins provided excellent heavy-atom derivatives. This approach should be of general use and particularly helpful in cases where traditional methods have failed to produce a derivative.

Amino Acid Sequence↗
PubMed1989

Preliminary crystallographic data for NAD(P)H quinone reductase isolated from the Walker 256 rat carcinoma cell line.

An NAD(P)H quinone reductase isolated from Walker rat 256 carcinoma cells has been crystallized in a form suitable for high-resolution structural analysis. The crystals belong to orthorhombic space group P2(1)2(1)2(1) with cell parameters a = 168.15 A, b = 105.09 A and c = 67.38 A and contain four monomeric or two dimeric enzyme molecules per asymmetric unit. Diffraction extends beyond 2.3 A resolution.

Animals↗