Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 6 de 6
Filter
Add more filters










Database
Language
Publication year range
1.
J Med Chem ; 54(7): 2359-67, 2011 Apr 14.
Article in English | MEDLINE | ID: mdl-21417343

ABSTRACT

ABL2 (also known as ARG (ABL related gene)) is closely related to the well-studied Abelson kinase cABL. ABL2 is involved in human neoplastic diseases and is deregulated in solid tumors. Oncogenic gene translocations occur in acute leukemia. So far no structural information for ABL2 has been reported. To elucidate structural determinants for inhibitor interaction, we determined the cocrystal structure of ABL2 with the oncology drug imatinib. Interestingly, imatinib not only interacted with the ATP binding site of the inactive kinase but was also bound to the regulatory myristate binding site. This structure may therefore serve as a tool for the development of allosteric ABL inhibitors. In addition, we determined the structures of ABL2 in complex with VX-680 and with an ATP-mimetic type I inhibitor, which revealed an interesting position of the DFG motif intermediate between active and inactive conformations, that may also serve as a template for future inhibitor design.


Subject(s)
Piperazines/metabolism , Protein-Tyrosine Kinases/chemistry , Protein-Tyrosine Kinases/metabolism , Pyrimidines/metabolism , Triazoles/metabolism , Triazoles/pharmacology , Amino Acid Motifs , Benzamides , Crystallography, X-Ray , Humans , Imatinib Mesylate , Models, Molecular , Protein Binding , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/metabolism , Protein Kinase Inhibitors/pharmacology , Protein Structure, Tertiary , Protein-Tyrosine Kinases/antagonists & inhibitors , Triazoles/chemistry
2.
Cell ; 136(2): 352-63, 2009 Jan 23.
Article in English | MEDLINE | ID: mdl-19167335

ABSTRACT

Protein tyrosine phosphatases (PTPs) play a critical role in regulating cellular functions by selectively dephosphorylating their substrates. Here we present 22 human PTP crystal structures that, together with prior structural knowledge, enable a comprehensive analysis of the classical PTP family. Despite their largely conserved fold, surface properties of PTPs are strikingly diverse. A potential secondary substrate-binding pocket is frequently found in phosphatases, and this has implications for both substrate recognition and development of selective inhibitors. Structural comparison identified four diverse catalytic loop (WPD) conformations and suggested a mechanism for loop closure. Enzymatic assays revealed vast differences in PTP catalytic activity and identified PTPD1, PTPD2, and HDPTP as catalytically inert protein phosphatases. We propose a "head-to-toe" dimerization model for RPTPgamma/zeta that is distinct from the "inhibitory wedge" model and that provides a molecular basis for inhibitory regulation. This phosphatome resource gives an expanded insight into intrafamily PTP diversity, catalytic activity, substrate recognition, and autoregulatory self-association.


Subject(s)
Protein Tyrosine Phosphatases/chemistry , Amino Acid Sequence , Crystallography, X-Ray , Dimerization , Humans , Models, Molecular , Protein Structure, Tertiary , Protein Tyrosine Phosphatases/genetics , Protein Tyrosine Phosphatases/metabolism , Sequence Alignment , Structure-Activity Relationship
3.
Mol Cell Endocrinol ; 301(1-2): 199-204, 2009 Mar 25.
Article in English | MEDLINE | ID: mdl-19013211

ABSTRACT

Two members of the human aldo-keto reductase (AKR) superfamily participate in the biosynthesis of bile acids by catalyzing the NADP(H) dependent reduction of 3-keto groups (AKR1C4) and Delta4 double bonds (AKR1D1) of oxysterol precursors. Structure determination of human AKR1C4 and homology modelling of AKR1D1 followed by docking experiments were used to explore active site geometries. Substrate docking resulted in ligand poses satisfying catalytic constraints, and indicates a critical role for Trp227/230 in positioning the substrate in a catalytically competent orientation. Based on the evidence gathered from our docking experiments and experimental structures, this tryptophan residue emerges as a major determinant governing substrate specificity of a subset of enzymes belonging to the AKR1 subfamily.


Subject(s)
Bile Acids and Salts/biosynthesis , Oxidoreductases/chemistry , Oxidoreductases/metabolism , Catalytic Domain , Crystallography, X-Ray , Humans , Ligands , Models, Molecular , Structural Homology, Protein , Structure-Activity Relationship , Tryptophan/metabolism
4.
J Mol Biol ; 371(4): 1007-21, 2007 Aug 24.
Article in English | MEDLINE | ID: mdl-17599355

ABSTRACT

Inorganic long-chain polyphosphate is a ubiquitous linear polymer in biology, consisting of many phosphate moieties linked by phosphoanhydride bonds. It is synthesized by polyphosphate kinase, and metabolised by a number of enzymes, including exo- and endopolyphosphatases. The Saccharomyces cerevisiae gene PPX1 encodes for a 45 kDa, metal-dependent, cytosolic exopolyphosphatase that processively cleaves the terminal phosphate group from the polyphosphate chain, until inorganic pyrophosphate is all that remains. PPX1 belongs to the DHH family of phosphoesterases, which includes: family-2 inorganic pyrophosphatases, found in Gram-positive bacteria; prune, a cyclic AMPase; and RecJ, a single-stranded DNA exonuclease. We describe the high-resolution X-ray structures of yeast PPX1, solved using the multiple isomorphous replacement with anomalous scattering (MIRAS) technique, and its complexes with phosphate (1.6 A), sulphate (1.8 A) and ATP (1.9 A). Yeast PPX1 folds into two domains, and the structures reveal a strong similarity to the family-2 inorganic pyrophosphatases, particularly in the active-site region. A large, extended channel formed at the interface of the N and C-terminal domains is lined with positively charged amino acids and represents a conduit for polyphosphate and the site of phosphate hydrolysis. Structural comparisons with the inorganic pyrophosphatases and analysis of the ligand-bound complexes lead us to propose a hydrolysis mechanism. Finally, we discuss a structural basis for substrate selectivity and processivity.


Subject(s)
Acid Anhydride Hydrolases/chemistry , Acid Anhydride Hydrolases/metabolism , Cytosol/enzymology , Saccharomyces cerevisiae/enzymology , Acid Anhydride Hydrolases/genetics , Acid Anhydride Hydrolases/isolation & purification , Amino Acid Sequence , Binding Sites , Cloning, Molecular , Cobalt/chemistry , Computational Biology , Crystallography, X-Ray , Gene Expression , Molecular Sequence Data , Phosphates/chemistry , Protein Structure, Quaternary , Protein Structure, Secondary , Protein Structure, Tertiary , Protein Subunits/chemistry , Protein Subunits/genetics , Protein Subunits/metabolism , Saccharomyces cerevisiae/genetics , Sequence Alignment , Substrate Specificity
5.
J Biol Chem ; 282(13): 9302-9311, 2007 Mar 30.
Article in English | MEDLINE | ID: mdl-17215253

ABSTRACT

Yeast exopolyphosphatase (scPPX) processively splits off the terminal phosphate group from linear polyphosphates longer than pyrophosphate. scPPX belongs to the DHH phosphoesterase superfamily and is evolutionarily close to the well characterized family II pyrophosphatase (PPase). Here, we used steady-state kinetic and binding measurements to elucidate the metal cofactor requirement for scPPX catalysis over the pH range 4.2-9.5. A single tight binding site for Mg(2+) (K(d) of 24 microm) was detected by equilibrium dialysis. Steady-state kinetic analysis of tripolyphosphate hydrolysis revealed a second site that binds Mg(2+) in the millimolar range and modulates substrate binding. This step requires two protonated and two deprotonated enzyme groups with pK(a) values of 5.0-5.3 and 7.6-8.2, respectively. The catalytic step requiring two deprotonated groups (pK(a) of 4.6 and 5.6) is modulated by ionization of a third group (pK(a) of 8.7). Conservative mutations of Asp(127), His(148), His(149) (conserved in scPPX and PPase), and Asn(35) (His in PPase) reduced activity by a factor of 600-5000. N35H and D127E substitutions reduced the Mg(2+) affinity of the tight binding site by 25-60-fold. Contrary to expectations, the N35H variant was unable to hydrolyze pyrophosphate, but markedly altered metal cofactor specificity, displaying higher catalytic activity with Co(2+) bound to the weak binding site versus the Mg(2+)- or Mn(2+)-bound enzyme. These results provide an initial step toward understanding the dynamics of scPPX catalysis and reveal significant functional differences between structurally similar scPPX and family II PPase.


Subject(s)
Acid Anhydride Hydrolases/genetics , Acid Anhydride Hydrolases/metabolism , Amino Acid Substitution/genetics , Cytosol/enzymology , Saccharomyces cerevisiae/enzymology , Sequence Analysis, Protein , Acid Anhydride Hydrolases/biosynthesis , Acid Anhydride Hydrolases/chemistry , Acid Anhydride Hydrolases/isolation & purification , Amino Acid Sequence , Kinetics , Saccharomyces cerevisiae/genetics , Sequence Homology, Amino Acid
6.
Biochem J ; 395(3): 483-91, 2006 May 01.
Article in English | MEDLINE | ID: mdl-16441242

ABSTRACT

Protein tyrosine phosphatases PTPN5, PTPRR and PTPN7 comprise a family of phosphatases that specifically inactivate MAPKs (mitogen-activated protein kinases). We have determined high-resolution structures of all of the human family members, screened them against a library of 24000 compounds and identified two classes of inhibitors, cyclopenta[c]quinolinecarboxylic acids and 2,5-dimethylpyrrolyl benzoic acids. Comparative structural analysis revealed significant differences within this conserved family that could be explored for the design of selective inhibitors. PTPN5 crystallized, in two distinct crystal forms, with a sulphate ion in close proximity to the active site and the WPD (Trp-Pro-Asp) loop in a unique conformation, not seen in other PTPs, ending in a 3(10)-helix. In the PTPN7 structure, the WPD loop was in the closed conformation and part of the KIM (kinase-interaction motif) was visible, which forms an N-terminal aliphatic helix with the phosphorylation site Thr66 in an accessible position. The WPD loop of PTPRR was open; however, in contrast with the structure of its mouse homologue, PTPSL, a salt bridge between the conserved lysine and aspartate residues, which has been postulated to confer a more rigid loop structure, thereby modulating activity in PTPSL, does not form in PTPRR. One of the identified inhibitor scaffolds, cyclopenta[c]quinoline, was docked successfully into PTPRR, suggesting several possibilities for hit expansion. The determined structures together with the established SAR (structure-activity relationship) propose new avenues for the development of selective inhibitors that may have therapeutic potential for treating neurodegenerative diseases in the case of PTPRR or acute myeloblastic leukaemia targeting PTPN7.


Subject(s)
Intracellular Signaling Peptides and Proteins/chemistry , Intracellular Signaling Peptides and Proteins/metabolism , Mitogen-Activated Protein Kinases/metabolism , Protein Tyrosine Phosphatases/chemistry , Protein Tyrosine Phosphatases/metabolism , Amino Acid Sequence , Binding Sites , Catalytic Domain , Crystallography, X-Ray , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Humans , Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Intracellular Signaling Peptides and Proteins/classification , Ions/chemistry , Ions/metabolism , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Models, Molecular , Molecular Sequence Data , Protein Structure, Tertiary , Protein Tyrosine Phosphatases/antagonists & inhibitors , Protein Tyrosine Phosphatases/classification , Protein Tyrosine Phosphatases, Non-Receptor , Receptor-Like Protein Tyrosine Phosphatases, Class 7 , Sequence Alignment , Sequence Homology, Amino Acid , Structural Homology, Protein , Structure-Activity Relationship , Substrate Specificity
SELECTION OF CITATIONS
SEARCH DETAIL
...