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1.
J Med Chem ; 41(11): 1894-908, 1998 May 21.
Article in English | MEDLINE | ID: mdl-9599239

ABSTRACT

The design, synthesis, and evaluation of dipeptide analogues as ligands for the pp60c-src SH2 domain are described. The critical binding interactions between Ac-Tyr-Glu-N(n-C5H11)2 (2) and the protein are established and form the basis for our structure-based drug design efforts. The effects of changes in both the C-terminal (11-27) and N-terminal (51-69) portions of the dipeptide are explored. Analogues with reduced overall charge (92-95) are also investigated. We demonstrate the feasibility of pairing structurally diverse subunits in a modest dipeptide framework with the goal of increasing the druglike attributes without sacrificing binding affinity.


Subject(s)
Dipeptides/pharmacology , Enzyme Inhibitors/pharmacology , Proto-Oncogene Proteins pp60(c-src)/antagonists & inhibitors , src Homology Domains , Crystallography, X-Ray , Dipeptides/chemical synthesis , Dipeptides/chemistry , Dipeptides/metabolism , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , Enzyme-Linked Immunosorbent Assay , Humans , Ligands , Models, Molecular , Molecular Conformation , Proto-Oncogene Proteins pp60(c-src)/metabolism , Structure-Activity Relationship
2.
Bioorg Med Chem Lett ; 8(10): 1189-94, 1998 May 19.
Article in English | MEDLINE | ID: mdl-9871733

ABSTRACT

The X-ray crystal structure of the src SH2 domain revealed the presence of a thiol residue (Cys 188) located proximal to the phosphotyrosine portion of a dipeptide ligand. An aldehyde bearing ligand (1) was designed to position an electrophilic carbonyl group in the vicinity of the thiol. X-ray crystallographic and NMR examination of the complex formed between (1) and the src SH2 domain revealed a hemithioacetal formed by addition of the thiol to the aldehyde group with an additional stabilizing hydrogen bond between the acetal hydroxyl and a backbone carbonyl.


Subject(s)
Dipeptides/chemistry , Protein Conformation , Proteins/chemistry , src Homology Domains , Aldehydes , Amino Acid Sequence , Binding Sites , Crystallography, X-Ray , Cysteine , Dipeptides/chemical synthesis , Hydrogen Bonding , Ligands , Magnetic Resonance Spectroscopy , Models, Molecular , Phosphotyrosine
3.
Biochemistry ; 36(21): 6283-93, 1997 May 27.
Article in English | MEDLINE | ID: mdl-9174343

ABSTRACT

Thermodynamic measurements, structural determinations, and molecular computations were applied to a series of peptide ligands of the pp60(c-src) SH2 domain in an attempt to understand the critical binding determinants for this class of molecules. Isothermal titration calorimetry (ITC) measurements were combined with structural data derived from X-ray crystallographic studies on 12 peptide-SH2 domain complexes. The peptide ligands studied fall into two general classes: (1) dipeptides of the general framework N-acetylphosphotyrosine (or phosphotyrosine replacement)-Glu or methionine (or S-methylcysteine)-X, where X represents a hydrophobic amine, and (2) tetra- or pentapeptides of the general framework N-acetylphosphotyrosine-Glu-Glu-Ile-X, where X represents either Glu, Gln, or NH2. Dipeptide analogs which featured X as either hexanolamine or heptanolamine were able to pick up new hydrogen bonds involving their hydroxyl groups within a predominantly lipophilic surface cavity. However, due to internal strain as well as the solvent accessibility of the new hydrogen bonds formed, no net increase in binding affinity was observed. Phosphatase-resistant benzylmalonate and alpha,alpha-difluorobenzyl phosphonate analogs of phosphotyrosine retained some binding affinity for the pp60(c-src) SH2 domain but caused local structural perturbations in the phosphotyrosine-binding site. In the case where a reversible covalent thiohemiacetal was formed between a formylated phosphotyrosine analog and the thiol side chain of Cys-188, deltaS was 25.6 cal/(mol K) lower than for the nonformylated phosphotyrosine parent. Normal mode calculations show that the dramatic decrease in entropy observed for the covalent thiohemiacetal complex is due to the inability of the phosphotyrosine moiety to transform lost rotational and translational degrees of freedom into new vibrational modes.


Subject(s)
Peptides/chemistry , Protein Structure, Tertiary , Proto-Oncogene Proteins pp60(c-src)/chemistry , src Homology Domains , Amino Acid Sequence , Crystallography, X-Ray , Dipeptides/chemistry , Dipeptides/metabolism , Kinetics , Ligands , Oligopeptides/chemistry , Oligopeptides/metabolism , Peptides/metabolism , Phosphotyrosine/chemistry , Phosphotyrosine/metabolism , Protein Binding , Proto-Oncogene Proteins pp60(c-src)/metabolism , Structure-Activity Relationship , Thermodynamics
4.
Science ; 270(5243): 1838-41, 1995 Dec 15.
Article in English | MEDLINE | ID: mdl-8525381

ABSTRACT

The crystal structure of a nucleic acid triplex reveals a helix, designated P-form, that differs from previously reported nucleic acid structures. The triplex consists of one polypurine DNA strand complexed to a polypyrimidine hairpin peptide nucleic acid (PNA) and was successfully designed to promote Watson-Crick and Hoogsteen base pairing. The P-form helix is underwound, with a base tilt similar to B-form DNA. The bases are displaced from the helix axis even more than in A-form DNA. Hydrogen bonds between the DNA backbone and the Hoogsteen PNA backbone explain the observation that polypyrimidine PNA sequences form highly stable 2:1 PNA-DNA complexes. This structure expands the number of known stable helical forms that nucleic acids can adopt.


Subject(s)
DNA/chemistry , Nucleic Acid Conformation , Oligopeptides/chemistry , Amino Acid Sequence , Base Sequence , Crystallography, X-Ray , Models, Molecular , Molecular Sequence Data , Oligodeoxyribonucleotides/chemistry , Protein Conformation
6.
Biochemistry ; 33(27): 8207-17, 1994 Jul 12.
Article in English | MEDLINE | ID: mdl-8031754

ABSTRACT

Collagenase is a member of the matrix metalloproteinase (MMP) family of enzymes. Aberrant regulation of this family has been implicated in pathologies such as arthritis and metastasis. Two crystal forms of the catalytic (19-kDa) domain of human fibroblast collagenase have been determined using collagenase complexed with a peptide-based inhibitor (CPLX) as a starting model [Lovejoy et al. (1994) Science 263, 375]. The first crystal form (CF1) contains one molecule in the asymmetric unit and has been determined at 1.9-A resolution with an R factor of 19.8%. The second crystal form (CF2) contains two molecules (A and B) in the asymmetric unit and has been determined at 2.1-A resolution with an R factor of 19.7%. The catalytic domain of collagenase is spherical with an active site cleft that contains a ligated catalytic zinc ion. Collagenase shares some structural homology with the bacterial zinc proteinase, thermolysin [Matthews et al. (1972) Nature, New Biol. 238, 37], and the crayfish digestive peptidase, astacin [Bode et al. (1992) Nature 358, 164]. The amino terminus (Leu 102 to Gly 105) of CF1 and CF2 molecules A and B differs from the conformation found in CPLX by bending away from the molecule and interacting with the active site cleft of symmetry-related molecules. In this alternative conformation, both the mainchain nitrogen and carbonyl oxygen of Leu 102 ligate the symmetry-related catalytic zinc. Although there are structural differences in the active site clefts of CF1, CF2, and CPLX, a number of complex-stabilizing interactions are conserved. The structure of collagenase will be useful for developing compounds that selectively inhibit individual members of the closely related matrix metalloproteinase family.


Subject(s)
Collagenases/chemistry , Fibroblasts/enzymology , Amino Acid Sequence , Binding Sites , Calcium/pharmacology , Collagenases/metabolism , Crystallization , Crystallography, X-Ray , Humans , Hydrogen Bonding , Mass Spectrometry , Matrix Metalloproteinase Inhibitors , Models, Molecular , Molecular Sequence Data , Molecular Structure , Protein Conformation , Recombinant Proteins/chemistry , Thermolysin/antagonists & inhibitors , Thermolysin/chemistry , Thermolysin/metabolism , Zinc/pharmacology
8.
J Mol Biol ; 236(5): 1410-2, 1994 Mar 11.
Article in English | MEDLINE | ID: mdl-8126730

ABSTRACT

Crystals of the catalytic domain of human fibroblast collagenase have been grown in the presence and absence of an inhibitor. Crystals of the inhibitor complex grew from 0.2 M ammonium sulfate and 15 to 30% PEG 8000 at 22 degrees C as bipyramids in the space group P6(2) or P6(4). Crystals of the unligated enzyme grew as rods in the space group P4(1)2(1)2 or P4(3)2(1)2 from 1.0 to 2.0 M sodium formate at 4 degrees C. Both crystal forms grew quite slowly over a period of months, but ultimately yielded crystals that diffracted beyond 2.5 A. The collagenase samples used in these studies were heterogeneous at the amino terminus. Three major species (full length, N-1 and N-2) were identified by mass spectrometry and Edman sequencing. Analysis of dissolved crystals revealed the native crystal form selectively crystallized as the N-2 species; however, no selectivity of N-terminal forms was observed for crystals of the inhibitor complex.


Subject(s)
Collagenases/ultrastructure , Crystallography, X-Ray , Fibroblasts/enzymology , Humans , Macromolecular Substances , Mass Spectrometry , Matrix Metalloproteinase Inhibitors , Molecular Weight , Recombinant Proteins
9.
Nature ; 363(6425): 172-6, 1993 May 13.
Article in English | MEDLINE | ID: mdl-8483502

ABSTRACT

Interleukin-5 (IL-5) is a lineage-specific cytokine for eosinophilpoiesis and plays an important part in diseases associated with increased eosinophils, such as asthma. Human IL-5 is a disulphide-linked homodimer with 115 amino-acid residues in each chain. The crystal structure at 2.4 A resolution reveals a novel two-domain structure, with each domain showing a striking similarity to the cytokine fold found in granulocyte macrophage and macrophage colony-stimulating factors, IL-2 (ref. 5), IL-4 (ref. 6), and human and porcine growth hormones. IL-5 is unique in that each domain requires the participation of two chains. The IL-5 structure consists of two left-handed bundles of four helices laid end to end and two short beta-sheets on opposite sides of the molecule. Surprisingly, the C-terminal strand and helix of one chain complete a bundle of four helices and a beta-sheet with the N-terminal three helices and one strand of the other chain. The structure of IL-5 provides a molecular basis for the design of antagonists and agonists that would delineate receptor recognition determinants critical in signal transduction. This structure determination extends the family of the cytokine bundle of four helices and emphasizes its fundamental significance and versatility in recognizing its receptor.


Subject(s)
Interleukin-5/chemistry , Animals , Binding Sites , Computer Simulation , Cytokines/chemistry , Humans , Mice , Models, Molecular , Protein Conformation , Protein Structure, Secondary , Sequence Alignment , X-Ray Diffraction
10.
Eur J Biochem ; 212(3): 751-5, 1993 Mar 15.
Article in English | MEDLINE | ID: mdl-8462547

ABSTRACT

Interleukin-5 (IL-5) plays a key role in the proliferation and differentiation of eosinophils. To aid the solution of the crystallographic three-dimensional structure, we have expressed large quantities of recombinant human IL-5 (hIL-5) in a methionine auxotroph strain of Escherichia coli (DL41) grown on an enriched seleno-DL-methionine-containing medium. Cell densities of A650 = 10 have been achieved. The selenomethionyl-labelled hIL-5 (Se-hIL-5) has been purified and found to contain 3.6 selenium atoms/dimer, and 0.4 methionine residues/dimer. In a B-cell growth factor assay, the Se-hIL-5 is significantly more active than the non-labelled hIL-5. Electrospray mass spectrometry shows two major peaks, with relative molecular masses of 26,326 +/- 6 and 26,280 +/- 8 corresponding to the 4Se and 3Se/1S forms of hIL-5. Unlike the methionine-containing hIL-5, the N-terminal selenomethionine is neither oxidised nor carbamoylated and can only be resolved into two species in isoelectric focusing gel electrophoresis. Se-hIL-5 crystallises in the same space group and unit cell as hIL-5. Difference Fourier calculations identify two of the selenomethionines corresponding to Met107 in the dimer. However, the N-terminal is disordered in the crystal, and the N-terminal selenomethionines are not resolved in the difference Fourier.


Subject(s)
Interleukin-5/analogs & derivatives , Interleukin-5/isolation & purification , Recombinant Proteins/isolation & purification , Selenomethionine/analogs & derivatives , Animals , B-Lymphocytes , Cell Division/drug effects , Chromatography, Gel , Chromatography, Ion Exchange , Crystallization , Electrophoresis, Polyacrylamide Gel , Escherichia coli/genetics , Humans , Interleukin-5/chemistry , Interleukin-5/pharmacology , Mice , Recombinant Proteins/chemistry , Recombinant Proteins/pharmacology , Selenomethionine/chemistry , Selenomethionine/isolation & purification , Selenomethionine/pharmacology
11.
J Mol Biol ; 229(4): 1150-2, 1993 Feb 20.
Article in English | MEDLINE | ID: mdl-8445640

ABSTRACT

Recombinant human interleukin-5 (rhIL-5) has been crystallized by the hanging drop vapor diffusion method using 0.1 M-Tris.HCl buffer (pH 8.5) containing 0.2 to 0.25 M-sodium acetate and 26 to 30% PEG 4000 at 22 degrees C. The parallel-piped crystals belong to the space group C2 with unit cell dimensions of a = 122.1 A, b = 36.11 A, c = 56.42 A, beta = 98.59 degrees. They diffract to at least 2.0 A resolution on a rotating anode X-ray source. The molecular mass weight of the protein and the volume of the unit cell suggest that the asymmetric unit contains one intermolecular disulfide-bonded homodimer.


Subject(s)
Interleukin-5/chemistry , Crystallization , Escherichia coli , Humans , Mass Spectrometry , Recombinant Proteins/chemistry , X-Ray Diffraction
12.
J Med Chem ; 35(5): 847-58, 1992 Mar 06.
Article in English | MEDLINE | ID: mdl-1548676

ABSTRACT

Antifolate inhibitors of thymidylate synthase (TS) have primarily been based on the structure of folic acid. This paper describes the identification and development of novel 6,7-imidazotetrahydroquinoline TS inhibitors by iterative ligand design, synthesis, and crystallographic analysis of protein-inhibitor complexes. Beginning with a high-resolution crystal structure of E. coli TS (TS, EC 2.1.1.45), an imidazotetrahydroquinoline inhibitor was designed de novo to occupy the folate binding pocket. Structural modifications of the initial compound 1h (Ki approximately 5 microM human/E. coli TS) were then made on the basis of feedback from additional cocrystal structures and activity data. An amino group in the 2-position of the imidazole was found to increase the potency of the series by 1-2 orders of magnitude. Other substitutions on the imidazole ring (1-CH3, 2-CH3, 2-NHCH3, 2-SCH3) generally led to weaker inhibition. Additional improvements in activity were obtained by modification of the substituents on the tetrahydroquinoline nitrogen, bringing the Ki of three of the compounds below 15 nM against the human TS enzyme. The compounds were tested for cytotoxicity and were shown to inhibit the growth of three tumor cell lines in vitro.


Subject(s)
Drug Design , Imidazoles/chemical synthesis , Quinolines/chemical synthesis , Thymidylate Synthase/antagonists & inhibitors , Adenocarcinoma/pathology , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/pharmacology , Binding Sites , Cell Division/drug effects , Crystallization , Escherichia coli/enzymology , Folic Acid/metabolism , Humans , Imidazoles/metabolism , Imidazoles/pharmacology , Leukemia/pathology , Leukemia L1210/pathology , Mice , Molecular Structure , Quinolines/metabolism , Quinolines/pharmacology , Thymidylate Synthase/metabolism , Tumor Cells, Cultured
13.
Science ; 252(5002): 88-95, 1991 Apr 05.
Article in English | MEDLINE | ID: mdl-1707186

ABSTRACT

The crystal structure of the ribonuclease (RNase) H domain of HIV-1 reverse transcriptase (RT) has been determined at a resolution of 2.4 A and refined to a crystallographic R factor of 0.20. The protein folds into a five-stranded mixed beta sheet flanked by an asymmetric distribution of four alpha helices. Two divalent metal cations bind in the active site surrounded by a cluster of four conserved acidic amino acid residues. The overall structure is similar in most respects to the RNase H from Escherichia coli. Structural features characteristic of the retroviral protein suggest how it may interface with the DNA polymerase domain of p66 in the mature RT heterodimer. These features also offer insights into why the isolated RNase H domain is catalytically inactive but when combined in vitro with the isolated p51 domain of RT RNase H activity can be reconstituted. Surprisingly, the peptide bond cleaved by HIV-1 protease near the polymerase-RNase H junction of p66 is completely inaccessible to solvent in the structure reported here. This suggests that the homodimeric p66-p66 precursor of mature RT is asymmetric with one of the two RNase H domains at least partially unfolded.


Subject(s)
Endoribonucleases/ultrastructure , HIV-1/enzymology , RNA-Directed DNA Polymerase/ultrastructure , Amino Acid Sequence , Binding Sites , Computer Graphics , Crystallography , Endoribonucleases/chemistry , Escherichia coli/enzymology , Manganese/metabolism , Models, Molecular , Molecular Sequence Data , Protein Conformation , RNA-Directed DNA Polymerase/chemistry , Ribonuclease H , Structure-Activity Relationship , X-Ray Diffraction
14.
Science ; 247(4947): 1210-3, 1990 Mar 09.
Article in English | MEDLINE | ID: mdl-2315694

ABSTRACT

Comparison of a lambda repressor-operator complex and a 434 repressor-operator complex reveals that three conserved residues in the helix-turn-helix (HTH) region make similar contacts in each of the crystallographically determined structures. These conserved residues and their interactions with phosphodiester oxygens help establish a frame of reference within which other HTH residues make contacts that are critical for site-specific recognition. Such "positioning contacts" may be important conserved features within families of HTH proteins. In contrast, the structural comparisons appear to rule out any simple "recognition code" at the level of detailed side chain-base pair interactions.


Subject(s)
DNA-Binding Proteins , Operator Regions, Genetic , Repressor Proteins/metabolism , Transcription Factors/metabolism , Amino Acid Sequence , Asparagine , Base Composition , Base Sequence , Binding Sites , Glutamine , Hydrogen Bonding , Molecular Sequence Data , Molecular Structure , Protein Conformation , Viral Proteins , Viral Regulatory and Accessory Proteins
16.
Science ; 242(4880): 893-9, 1988 Nov 11.
Article in English | MEDLINE | ID: mdl-3187530

ABSTRACT

The crystal structure of a complex containing the DNA-binding domain of lambda repressor and a lambda operator site was determined at 2.5 A resolution and refined to a crystallographic R factor of 24.2 percent. The complex is stabilized by an extensive network of hydrogen bonds between the protein and the sugar-phosphate backbone. Several side chains form hydrogen bonds with sites in the major groove, and hydrophobic contacts also contribute to the specificity of binding. The overall arrangement of the complex is quite similar to that predicted from earlier modeling studies, which fit the protein dimer against linear B-form DNA. However, the cocrystal structure reveals important side chain-side chain interactions that were not predicted from the modeling or from previous genetic and biochemical studies.


Subject(s)
DNA-Binding Proteins , DNA/metabolism , Operator Regions, Genetic , Repressor Proteins/metabolism , Transcription Factors/metabolism , Base Composition , Base Sequence , Binding Sites , Chemical Phenomena , Chemistry , Crystallization , Glutamine/metabolism , Hydrogen Bonding , Molecular Sequence Data , Molecular Structure , Nucleic Acid Conformation , Protein Binding , Protein Conformation , Repressor Proteins/genetics , Sugar Phosphates/metabolism , Viral Proteins , Viral Regulatory and Accessory Proteins
17.
Science ; 230(4732): 1383-5, 1985 Dec 20.
Article in English | MEDLINE | ID: mdl-3906896

ABSTRACT

Crystals have been grown that contain the operator-binding domain of the lambda repressor and the lambda operator site OL1. Crystallization conditions were tested with a set of DNA fragments, ranging in length from 17 to 23 base pairs. The best crystals were grown with a 20-base pair DNA fragment. These crystals have space-group symmetry P2I, with unit cell dimensions a = 37.1 A, b = 68.8 A, c = 56.8 A, and a beta angle of 91.5 degrees. They diffracted to at least 2.5 A resolution. High resolution data from these crystals should allow the direct determination of how a repressor recognizes its operator site.


Subject(s)
DNA, Bacterial/genetics , Gene Expression Regulation , Repressor Proteins/genetics , Transcription Factors/genetics , Base Sequence , Chromatography, High Pressure Liquid , Crystallization , Escherichia coli/genetics , Nucleic Acid Conformation , Protein Conformation , Repressor Proteins/isolation & purification , Structure-Activity Relationship , X-Ray Diffraction
18.
J Mol Biol ; 185(2): 445-6, 1985 Sep 20.
Article in English | MEDLINE | ID: mdl-4057251

ABSTRACT

Arc, a repressor from Salmonella phage P22 has been crystallized from ammonium phosphate at pH 8.0. The crystals form in space group P212121 with a = 90.26 A, b = 52.88 A and c = 47.58 A. The crystals diffract to 2.2 A resolution.


Subject(s)
Repressor Proteins , Salmonella Phages/genetics , Transcription Factors , Viral Proteins , Crystallography , Viral Regulatory and Accessory Proteins
19.
J Biomol Struct Dyn ; 1(4): 1039-49, 1983 Dec.
Article in English | MEDLINE | ID: mdl-6400903

ABSTRACT

The ways in which amino acid side chains could make a pair of hydrogen bonds within the major groove of B DNA are systematically analyzed. Hydrogen bond donors within the major groove are characterized by determining the idealized position of the hydrogen bond acceptors that they might bond with. It appears that an amino acid side chain could, at most, contact a pair of base pairs. The ten possible pairs of base pairs are analyzed to determine how they could be recognized by the amino acid side chains.


Subject(s)
Amino Acids , DNA , Base Composition , Binding Sites , Hydrogen Bonding , Molecular Structure , Nucleic Acid Conformation
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