Biomedical subjects

D K Stammers

Publications and source records attributed to D K Stammers.

At least 55 records · Page 3Linked to original sources

PubMed1994

Crystals of HIV-1 reverse transcriptase diffracting to 2.2 A resolution.

Reverse transcriptase (RT) from the human immunodeficiency virus type 1 has been crystallized in four closely related forms, the best of which diffract X-rays to 2.2 A resolution. The RT was crystallized as a complex with a non-nucleoside inhibitor, either nevirapine or a nevirapine analogue. Crystals grew from 6% PEG 3400 buffered at pH 5. These were of space group P2(1)2(1)2(1) with unit cell parameters a = 147 A, b = 112 A, c = 79 A (form A), with one RT heterodimer in the asymmetric unit. Changes in unit cell parameters and degree of crystalline order were observed on soaking pregrown crystals in various solutions, giving three further sets of unit cells. These were a = 143 A, b = 112, A, c = 79 A (form B), a = 141 A, b = 111 A, c = 73 A (form C), a = 143 A, b = 117 A, c = 66.5 A (form D). The last two forms diffract X-rays to 2.2 A resolution. Structure determinations of these latter crystal forms of RT should give a detailed atomic model for this therapeutically important drug target.

Crystallography, X-Ray↗

PubMed1994

Inhibition of HIV-1 reverse transcriptase by defined template/primer DNA oligonucleotides: effect of template length and binding characteristics.

The interaction of partially double stranded DNA oligonucleotides with HIV-1 RT was studied by investigating their ability to inhibit the homopolymeric poly(rC) directed (dG) synthesis reaction. A 20/18mer oligonucleotide, with a sequence based on the Lys3-tRNA primer region, showed stronger inhibition of the homopolymeric RT reaction than a G/C rich oligonucleotide series lacking or possessing a hairpin moiety. Interaction of the enzyme with the G/C rich oligonucleotides, as determined by IC50 measurements, was insensitive to the extent of the unpaired template region at the 3' or 5' position. Addition of a hairpin moiety, composed of four thymidine bases, onto G/C rich oligonucleotides increase their inhibitory potency (at least six times) and shifted the mode of inhibition of RT to competitive with respect to poly (rC).(dG), which was otherwise mixed (competitive/noncompetitive) for the linear G/C rich and 20/18mer oligonucleotides. The results indicate that interaction of the enzyme with the primer/template stem, but not with the unpaired template region, is an important step in complex formation.

Binding Sites↗

PubMed1993

Preliminary crystallographic data for Pneumocystis carinii dihydrofolate reductase.

Dihydrofolate reductase from Pneumocystis carinii has been crystallized in a form suitable for high resolution X-ray diffraction studies. Recombinant enzyme that had been refolded following solubilization in guanidinium hydrochloride was crystallized as both a ternary complex with the cofactor NADPH and the inhibitor trimethoprim as well as a binary complex with NADPH. The two types of complex crystallized isomorphously from polyethylene glycol using sitting-drop vapour diffusion. The crystals were of space group P2(1) with unit cell parameters, a = 69.9 A, b = 43.6 A, c = 37.6 A, beta = 117.7 degrees, with one molecule per asymmetric unit. The crystals diffracted to 1.8 A resolution.

AIDS-Related Opportunistic Infections↗

PubMed1993

Refolding of recombinant Pneumocystis carinii dihydrofolate reductase and characterization of the enzyme.

The isolation of dihydrofolate reductase (DHFR) cDNA sequences from the messenger RNA of Pneumocystis carinii using the polymerase chain reaction is described. The 206-amino acid P. carinii DHFR was expressed to high levels in Escherichia coli inclusion bodies using the T7 promoter expression system. Solubilization of the inclusion bodies in 4 M guanidine hydrochloride and refolding of the recombinant protein in the presence of 0.5% polyethylene glycol 1450 yielded correctly folded DHFR which was purified to homogeneity by methotrexate-Sepharose affinity chromatography. The refolded enzyme was readily crystallized as a ternary complex with NADPH and various inhibitors. The enzyme exhibited a sharp pH optimum with maximum activity at pH 7.0 (turnover number = 6500 min-1). Km values for dihydrofolate (DHF) and NADPH were 2.3 and 3.0 microM, respectively, in 0.1 m imidazole buffer, pH 7. Folate did not act as a substrate. Comparison of the kinetic properties of the refolded enzyme with soluble P. carinii DHFR expressed at low levels in the T7 expression system showed similar pH-activity profiles, Km values for DHF and NADPH, and IC50 values for several known antifolates which were tested as inhibitors of the enzyme.

Amino Acid Sequence↗

PubMed1993

Isolation and characterization of monoclonal antibodies raised against the reverse transcriptase of human immunodeficiency virus type 2 and cross-reactivity with that of type 1.

Monoclonal antibodies to human immunodeficiency virus (HIV)-2 reverse transcriptase have been raised with the ultimate goal of generating Fab fragments for future co-crystallization studies. A number of mouse monoclonal antibodies to recombinant HIV-2 reverse transcriptase have been obtained and characterized in terms of the possible epitopes they recognise together with cross-reactivity with a related reverse transcriptase. The antibodies were shown to fall into three groups that recognize different regions of the reverse transcriptase enzyme. One antibody, which recognizes part of the RNase H domain, demonstrated cross-reactivity between the HIV-1 and HIV-2 reverse transcriptase.

Amino Acid Sequence↗

PubMed1992

Formation of heterodimers of human-immunodeficiency-virus-type-1 reverse transcriptase by recombination of separately purified subunits.

Human-immunodeficiency-virus-type-1 reverse transcriptase exists in virions as a heterodimer of a M(r) 66,000 subunit and its C-terminally truncated form of M(r) 51,000, but, when expressed as a recombinant M(r) 66,000 protein, a mixture of heterodimers and homodimers results which co-purify by most conventional techniques. We describe a method of hydrophobic chromatography which gives baseline separation of these two forms of the protein. This method has been applied to purify heterodimers formed by recombination of separately expressed and purified M(r) 66,000 and 51,000 subunits, resulting in significantly more homogeneous heterodimer preparations. The recombined heterodimer showed similar kinetic properties and RNase H activity to the standard heterodimer and a specific activity significantly higher than the original homodimer of the M(r) 66,000 protein. Heterodimers having greater asymmetry have also been prepared by recombining Mr 66,000 subunits containing single-point or deletion mutations, with wild-type M(r) 51,000 subunits, and the resulting heterodimers analysed.

Chromatography, Gel↗

PubMed1992

The multifunctional folic acid synthesis fas gene of Pneumocystis carinii appears to encode dihydropteroate synthase and hydroxymethyldihydropterin pyrophosphokinase.

We describe the cloning of a multifunctional folic acid synthesis (fas) gene from Pneumocystis carinii. The nucleotide sequence contains an open reading frame interrupted by three introns, that encodes a protein of 740 amino acids with an Mr of 97,278. The predicted Fas protein has homology to two enzyme domains, dihydropteroate synthase and 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase, both of which are involved in folate synthesis, and at least one other region of unknown function.

Amino Acid Sequence↗

PubMed1991

Use of the Glu-Glu-Phe C-terminal epitope for rapid purification of the catalytic domain of normal and mutant ras GTPase-activating proteins.

The C-terminal catalytic domain (residues 704-1047) of the human ras GTPase-activating protein (GAP) has been engineered so as to incorporate the tripeptide, Glu-Glu-Phe, at its C terminus. This motif is recognized by the commercially available YL1/2 monoclonal antibody to alpha-tubulin and has previously been used for the immunoaffinity purification of HIV enzymes engineered to contain this epitope (Stammers, D. K., Tisdale, M., Court, S., Parmar, V., Bradley, C., and Ross, C. K. (1991) FEBS Lett. 283, 298-302). The engineered GAP catalytic domain (GAP-344) was obtained in high yield and purity from Escherichia coli extracts by means of a single affinity column of immobilized YL1/2, eluted under mild conditions with the dipeptide, Asp-Phe. The protein had similar activity to that previously described for full-length GAP, suggesting that the addition of the epitope did not grossly affect the activity. R903K and L902I mutants of GAP-344 were constructed, and the immunoaffinity purification procedure allowed their rapid characterization. The R903K mutant had less than 3% the activity of the normal protein, whereas the L902I substitution had less than 0.5% of normal activity, suggesting an important role for Leu-902 and Arg-903, residues absolutely conserved among GAP-related proteins. This work exemplifies the general utility of the C-terminal Glu-Glu-Phe motif for the rapid purification of proteins whose function is not altered by C-terminal modification.

Amino Acid Sequence↗

PubMed1991

Rapid purification and characterisation of HIV-1 reverse transcriptase and RNaseH engineered to incorporate a C-terminal tripeptide alpha-tubulin epitope.

The C-termini of p66 and p51 forms of HIV-1 reverse transcriptase have been engineered to contain a Glu-Glu-Phe sequence recognized by a monoclonal antibody to alpha-tubulin, YL1/2. Mutated RTs were purified in a single step using peptide elution from columns of immobilized YL1/2. The known sequence requirements of the YL1/2 epitope are consistent with protein eluting from the column with an intact C-terminus. Kinetic parameters of these mutated RTs are essentially unchanged from wild-type enzyme. The p15 RNaseH domain has been purified using this method and shown to have low enzyme activity compared to the parental p66 subunit.

Amino Acid Sequence↗

PubMed1991

The dynamic instability of microtubules is not modulated by alpha-tubulin tyrosinylation.

The tyrosinylation of chick brain alpha-tubulin and the effects of the tyrosinylation status on the assembly and dynamic instability of chick brain MAP2:tubulin microtubule protein have been examined. Each of the eight major alpha-isotypes can be tyrosinylated in vitro, irrespective of whether a C-terminal tyrosine is genetically encoded. The extent of tyrosinylation is however limited to congruent to 0.3 mol.mol-1. The tyrosinylation status (0 vs. 0.3 mol.mol-1) has no effect on either the assembly kinetics of chick brain microtubule protein or on the rate of length redistribution following assembly and shearing. It is therefore unlikely that the tyrosinylation status directly affects the intrinsic stability of assembled microtubules since the rate of length redistribution is both a sensitive assay and a function of the kinetic parameters governing dynamic instability.

Animals↗

PubMed1989

Structural characterization of HIV reverse transcriptase: a target for the design of specific virus inhibitors.

The reverse transcriptase (RT) of HIV is an important target for chemotherapy as demonstrated by the effective treatment of AIDS patients with zidovudine, a potent inhibitor of RT. Structural studies of HIV RT were therefore undertaken with a view to designing more effective inhibitors. To obtain sufficient quantities of enzyme for these studies the reverse transcriptase gene of HIV was cloned into a high level expression plasmid yielding reverse transcriptase at a level of 10% of the total Escherichia coli proteins. Monoclonal antibodies to RT were raised in mice and have been used to purify the enzyme by immunoaffinity chromatography. Crystallization of the enzyme has been achieved and studies are underway to determine its three-dimensional structure. In addition, carboxy-terminal truncated mutants were prepared by inserting stop codons into the gene at appropriate sites. The proteins expressed were analysed for RT and RNase H activity and used for mapping RT epitopes. This, together with previous data on site-directed mutagenesis of conserved regions of HIV RT has helped to map some of the structural and functional regions of the enzyme.

Animals↗

PubMed1988

HIV-1 reverse transcriptase: crystallization and analysis of domain structure by limited proteolysis.

Bacterially expressed recombinant HIV-1 reverse transcriptase is active as both a homodimer of Mr 66,000 subunits and a heterodimer of Mr 66,000 and 51,000 subunits. The heterodimer is formed by cleavage of a C-terminal fragment from one Mr 66,000 polypeptide, which occurs during purification and crystallization of reverse transcriptase. Thus, crystals obtained from purified Mr 66,000 polypeptide preparations consisted of an apparently equimolar mixture of Mr 66,000 and 51,000 polypeptides, which were apparently analogous to the Mr 66,000 and 51,000 polypeptides detected in HIV-infected cells and in virions. Limited proteolysis of the homodimer with alpha-chymotrypsin also resulted in cleavage to a stable Mr 66,000/51,000 mixture, and proteolysis with trypsin resulted in the transient formation of some Mr 51,000 polypeptide. These results are consistent with the reverse transcriptase molecule having a protease-sensitive linker region following a structured domain of Mr 51,000. Further digestion with trypsin resulted in cleavage of the Mr 51,000 polypeptide after residue 223, yielding peptides of apparent Mr 29,000 and 30,000. A minor peptide of Mr 40,000 was also produced by cleavage of the Mr 66,000 polypeptide after residue 223. About half the original Mr 66,000 polypeptides remained resistant to proteolysis and existed in complex with the above peptides in solution. During both chymotrypsin and trypsin digestion there was an increase in the reverse transcriptase activity caused by a doubling of Vmax with little change in Km for dTTP.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1987

Comparison of angiotensinogen and tetradecapeptide as substrates for human renin. Substrate dependence of the mode of inhibition of renin by a statine-containing hexapeptide.

The kinetic properties of two different substrates for human renin, a synthetic tetradecapeptide and the natural substrate human angiotensinogen, have been compared. While the Vmax was similar for the two substrates, the Km values differed by a factor of 10, i.e., 11.7 +/- 0.7 microM (tetradecapeptide) and 1.0 +/- 0.1 microM (angiotensinogen). The mode of inhibition of renin by a statine (Sta)-containing hexapeptide, BW897C, that is a close structural analog of residues 8-13 of human angiotensinogen (Phe-His-Sta-Val-Ile-His-OMe), was determined for the two substrates. Competitive inhibition was observed when tetradecapeptide was the substrate (Ki = 2.0 +/- 0.2 microM), but a more complex mixed inhibition mode (Ki = 1.7 +/- 0.1 microM, Ki' = 3.0 +/- 0.23 microM) was found with angiotensinogen as substrate. This mixed inhibition probably results from the formation of an enzyme-inhibitor-substrate or enzyme-inhibitor-product complex and reflects the more extensive interactions that the protein angiotensinogen, as opposed to the small tetradecapeptide substrate, can make with renin. We conclude that the mixed inhibition observed when angiotensinogen is used as renin substrate could be important in the clinical application of renin inhibitors because it is less readily reversed by increased concentrations of substrate than is simple competitive inhibition.

Amino Acids↗

PubMed1987

The structure of mouse L1210 dihydrofolate reductase-drug complexes and the construction of a model of human enzyme.

The structure of mouse L1210 dihydrofolate reductase (DHFR) complexed with NADPH and trimethoprim has been refined at 2.0 A resolution. The analogous complex with NADPH and methotrexate has been refined at 2.5 A resolution. These structures reveal for the first time details of drug interactions with a mammalian DHFR, which are compared with those observed from previous X-ray investigations of DHFR/inhibitor complexes. The refined L1210 structure has been used as the basis for the construction of a model of the human enzyme. There are only twenty-one sequence differences between mouse L1210 and human DHFRs, and all but two of these are located close to the molecular surface: a strong indication that the active sites are essentially identical in these two mammalian enzymes.

Animals↗

PubMed1986

Reduction in potency and reversal of left-shifting activity of BW12C with the major and minor components of chicken hemoglobin.

The effects of the left-shifting, anti-sickling compound BW12C (5-(2-formyl-3-hydroxyphenoxy)pentanoic acid) on the oxygen saturation curve of whole chicken blood and the isolated major (AII) and minor (AI) components of chicken hemoglobin have been studied. The results support the postulated major binding mode for BW12C to human hemoglobin of bridging between the alpha-chain terminal amino groups in the oxy conformation with an important hydrophobic component contributed mainly by Pro 77 alpha residues. In chicken AII (Pro 77 alpha----Ser) BW12C still left-shifts at high concentrations but its potency is greatly reduced (at least 10-fold). In chicken AI (Pro 77 alpha----Ser and Val 1 alpha----Met) BW12C is a right-shifter with a potency comparable to that of 2,3-diphosphoglycerate suggesting that binding at the beta-chain termini in the deoxy conformation is now dominant with alpha-chain binding no longer significant.

Aldehydes↗

PubMed1986

Characterization of the binding of the anti-sickling compound, BW12C, to haemoglobin.

The anti-sickling agent BW12C [Beddell, Goodford, Kneen, White, Wilkinson & Wootton (1984) Br. J. Pharmacol. 82, 397-407] was designed to left-shift the oxygen saturation curve of haemoglobin (HbA) by preferential binding to the oxy conformation at a single site between the terminal amino groups of the alpha-chains through Schiff's base formation, ionic and hydrophobic interactions. In the present work, Schiff's base linkages formed with [14C]BW12C were reduced with NaBH4 and the alpha- and beta-globin chains separated. Under oxy conditions at a molar ratio of 2:1, the covalently bound BW12C is localized almost exclusively on a single alpha-chain; tryptic digestion confirms the terminal amino group (alpha 1-valine) as the reaction site, in accord with the design hypothesis. However, about half the labelled BW12C is released on tetramer disruption, suggesting the presence of additional non-covalent binding. Under deoxy conditions, alpha- and beta-chains are labelled approximately equally, and at higher molar ratios additional binding in both oxy and deoxy conditions is seen. Isoelectric-focusing studies under oxy conditions show a complex pattern of modified bands for both HbA and HbA1c (blocked beta-terminal amino groups) but no modification for HbA carbamylated at both alpha- and beta-terminal amino groups or at the alpha-chains only, again confirming the alpha-terminal amino region as the main interaction site. Equilibrium dialysis measurements under oxy conditions indicate two strong binding sites with a binding constant of less than 10(-6) M and a number of weaker binding sites. The present data thus confirm that BW12C binds at the intended locus but reveal additional non-covalent binding at an undefined site, and weaker binding through Schiff's base formation with other amino groups.

Aldehydes↗

PubMed1986

Crystallographic investigation of the cooperative interaction between trimethoprim, reduced cofactor and dihydrofolate reductase.

The structure of the complex between E. coli (RT500) form I dihydrofolate reductase, the antibacterial trimethoprim and NADPH has been determined by X-ray crystallography. The inhibitor and cofactor are in mutual contact. A flexible chain segment which includes Met 20 is in contact with the inhibitor in the presence of NADPH, but more distant in its absence. By contrast, the inhibitor conformation is little changed with NADPH present. We discuss these observations with regard to the mutually cooperative binding of these ligands to the protein, and to the associated enhancement of inhibitory selectivity shown by trimethoprim for bacterial as opposed to vertebrate enzyme.

Binding Sites↗

PubMed1986

Human renin: a new class of inhibitors.

A new class of human renin inhibitor is described, containing a novel analogue of the peptide bond. High inhibitory potency was observed for octapeptide-length substrate analogues but inhibition progressively weakened as the molecule was shortened from the amino terminal end.

Amino Acid Sequence↗