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1.
Nat Chem Biol ; 16(3): 267-277, 2020 03.
Article in English | MEDLINE | ID: mdl-31959966

ABSTRACT

A long-standing mystery shrouds the mechanism by which catalytically repressed receptor tyrosine kinase domains accomplish transphosphorylation of activation loop (A-loop) tyrosines. Here we show that this reaction proceeds via an asymmetric complex that is thermodynamically disadvantaged because of an electrostatic repulsion between enzyme and substrate kinases. Under physiological conditions, the energetic gain resulting from ligand-induced dimerization of extracellular domains overcomes this opposing clash, stabilizing the A-loop-transphosphorylating dimer. A unique pathogenic fibroblast growth factor receptor gain-of-function mutation promotes formation of the complex responsible for phosphorylation of A-loop tyrosines by eliminating this repulsive force. We show that asymmetric complex formation induces a more phosphorylatable A-loop conformation in the substrate kinase, which in turn promotes the active state of the enzyme kinase. This explains how quantitative differences in the stability of ligand-induced extracellular dimerization promotes formation of the intracellular A-loop-transphosphorylating asymmetric complex to varying extents, thereby modulating intracellular kinase activity and signaling intensity.


Subject(s)
AAA Domain/physiology , Protein-Tyrosine Kinases/metabolism , Receptor Protein-Tyrosine Kinases/metabolism , AAA Domain/genetics , Catalytic Domain , Dimerization , Enzyme Activation , Humans , Ligands , Phosphorylation , Protein Binding , Protein Conformation , Protein-Tyrosine Kinases/physiology , Receptor Protein-Tyrosine Kinases/genetics , Receptor Protein-Tyrosine Kinases/physiology , Receptor, Fibroblast Growth Factor, Type 1/genetics , Receptor, Fibroblast Growth Factor, Type 1/metabolism , Receptor, Fibroblast Growth Factor, Type 2/genetics , Receptor, Fibroblast Growth Factor, Type 2/metabolism , Receptor, Fibroblast Growth Factor, Type 3/genetics , Receptor, Fibroblast Growth Factor, Type 3/metabolism , Signal Transduction , Structure-Activity Relationship , Tyrosine/chemistry
2.
J Chem Inf Model ; 60(3): 1494-1508, 2020 03 23.
Article in English | MEDLINE | ID: mdl-31995373

ABSTRACT

Modern rational modulator design and structure-function characterization often concentrate on concave regions of biomolecular surfaces, ranging from well-defined small-molecule binding sites to large protein-protein interaction interfaces. Here, we introduce a ß-cluster as a pseudomolecular representation of fragment-centric pockets detected by AlphaSpace [J. Chem. Inf. Model. 2015, 55, 1585], a recently developed computational analysis tool for topographical mapping of biomolecular concavities. By mimicking the shape as well as atomic details of potential molecular binders, this new ß-cluster representation allows direct pocket-to-ligand shape comparison and can be used to guide ligand optimization. Furthermore, we defined the ß-score, the optimal Vina score of the ß-cluster, as an indicator of pocket ligandability and developed an ensemble ß-cluster approach, which allows one-to-one pocket mapping and comparison among aligned protein structures. We demonstrated the utility of ß-cluster representation by applying the approach to a wide variety of problems including binding site detection and comparison, characterization of protein-protein interactions, and fragment-based ligand optimization. These new ß-cluster functionalities have been implemented in AlphaSpace 2.0, which is freely available on the web at http://www.nyu.edu/projects/yzhang/AlphaSpace2.


Subject(s)
Algorithms , Proteins , Binding Sites , Ligands , Models, Molecular , Protein Binding , Proteins/metabolism
3.
Structure ; 27(8): 1308-1315.e3, 2019 08 06.
Article in English | MEDLINE | ID: mdl-31204250

ABSTRACT

An autoinhibitory network of hydrogen bonds located at the kinase hinge (referred to as the "molecular brake") regulates the activity of several receptor tyrosine kinases. The mechanism whereby mutational disengagement of the brake allosterically activates the kinase in human disease is incompletely understood. We used a combination of NMR, bioinformatics, and molecular dynamics simulation to show that mutational disruption of the molecular brake triggers localized conformational perturbations that propagate to the active site. This entails changes in interactions of an isoleucine, one of three hydrophobic residues that lock the phenylalanine of the DFG motif in an inactive conformation. Structural analysis of tyrosine kinases provides evidence that this allosteric control mechanism is shared across the tyrosine kinase family. We also show that highly activating mutations at the brake diminish the enzyme's thermostability, thereby explaining why these mutations cause milder skeletal syndromes compared with less-activating mutations in the activation loop.


Subject(s)
Isoleucine/genetics , Mutation , Protein-Tyrosine Kinases/chemistry , Allosteric Regulation , Catalytic Domain , Humans , Hydrophobic and Hydrophilic Interactions , Magnetic Resonance Spectroscopy , Molecular Dynamics Simulation , Protein Conformation , Protein-Tyrosine Kinases/genetics
4.
J Am Chem Soc ; 141(4): 1788-1796, 2019 01 30.
Article in English | MEDLINE | ID: mdl-30612428

ABSTRACT

Ni(I)-mediated single-electron oxidative activation of alkyl halides has been extensively proposed as a key step in Ni-catalyzed cross-coupling reactions to generate radical intermediates. There are four mechanisms through which this step could take place: oxidative addition, outer-sphere electron transfer, inner-sphere electron transfer, and concerted halogen-atom abstraction. Despite considerable computational studies, there is no experimental study to evaluate all four pathways for Ni(I)-mediated alkyl radical formation. Herein, we report the isolation of a series of (Xantphos)Ni(I)-Ar complexes that selectively activate alkyl halides over aryl halides to eject radicals and form Ni(II) complexes. This observation allows the application of kinetic studies on the steric, electronic, and solvent effects, in combination with DFT calculations, to systematically assess the four possible pathways. Our data reveal that (Xantphos)Ni(I)-mediated alkyl halide activation proceeds via a concerted halogen-atom abstraction mechanism. This result corroborates previous DFT studies on (terpy)Ni(I)- and (py)Ni(I)-mediated alkyl radical formation, and contrasts with the outer-sphere electron transfer pathway observed for (PPh3)4Ni(0)-mediated aryl halide activation. This study of a model system provides insight into the overall mechanism of Ni-catalyzed cross-coupling reactions and offers a basis for differentiating electrophiles in cross-electrophile coupling reactions.

5.
J Am Chem Soc ; 138(14): 4779-86, 2016 Apr 13.
Article in English | MEDLINE | ID: mdl-27005998

ABSTRACT

Ni-catalyzed cross-coupling reactions have found important applications in organic synthesis. The fundamental characterization of the key steps in cross-coupling reactions, including C-C bond-forming reductive elimination, represents a significant challenge. Bimolecular pathways were invoked in early proposals, but the experimental evidence was limited. We present the preparation of well-defined (pyridine-pyrrolyl)Ni monomethyl and monophenyl complexes that allow the direct observation of bimolecular reductive elimination to generate ethane and biphenyl, respectively. The sp(3)-sp(3) and sp(2)-sp(2) couplings proceed via two distinct pathways. Oxidants promote the fast formation of Ni(III) from (pyridine-pyrrolyl)Ni-methyl, which dimerizes to afford a bimetallic Ni(III) intermediate. Our data are most consistent with the subsequent methyl coupling from the bimetallic Ni(III) to generate ethane as the rate-determining step. In contrast, the formation of biphenyl is facilitated by the coordination of a bidentate donor ligand.

6.
J Chem Inf Model ; 55(8): 1585-99, 2015 Aug 24.
Article in English | MEDLINE | ID: mdl-26225450

ABSTRACT

Inhibition of protein-protein interactions (PPIs) is emerging as a promising therapeutic strategy despite the difficulty in targeting such interfaces with drug-like small molecules. PPIs generally feature large and flat binding surfaces as compared to typical drug targets. These features pose a challenge for structural characterization of the surface using geometry-based pocket-detection methods. An attractive mapping strategy--that builds on the principles of fragment-based drug discovery (FBDD)--is to detect the fragment-centric modularity at the protein surface and then characterize the large PPI interface as a set of localized, fragment-targetable interaction regions. Here, we introduce AlphaSpace, a computational analysis tool designed for fragment-centric topographical mapping (FCTM) of PPI interfaces. Our approach uses the alpha sphere construct, a geometric feature of a protein's Voronoi diagram, to map out concave interaction space at the protein surface. We introduce two new features--alpha-atom and alpha-space--and the concept of the alpha-atom/alpha-space pair to rank pockets for fragment-targetability and to facilitate the evaluation of pocket/fragment complementarity. The resulting high-resolution interfacial map of targetable pocket space can be used to guide the rational design and optimization of small molecule or biomimetic PPI inhibitors.


Subject(s)
Drug Discovery/methods , Protein Interaction Mapping/methods , Protein Interaction Maps/drug effects , Proteins/metabolism , Binding Sites/drug effects , Databases, Protein , Humans , Ligands , Models, Molecular , Molecular Targeted Therapy , Protein Binding , Protein Interaction Domains and Motifs/drug effects , Proteins/chemistry , Proto-Oncogene Proteins c-mdm2/chemistry , Proto-Oncogene Proteins c-mdm2/metabolism , Tumor Suppressor Protein p53/chemistry , Tumor Suppressor Protein p53/metabolism
7.
J Phys Chem Lett ; 2012(3): 3503-3508, 2012 Nov 14.
Article in English | MEDLINE | ID: mdl-23205186

ABSTRACT

Fur protein plays key roles in regulating numerous genes in bacteria and is essential for intracellular iron concentration regulation. However, atomic level pictures of the iron binding site and its functional mechanism remain to be established. Here we present results of the first quantum chemical investigation of various first- and second-shell models and experimental Mössbauer data of E. Coli Fur, including 1) the first robust evidence that site 2 is the Fe binding site with a 3His/2Glu ligand set, being the first case in non-heme proteins, with computed Mössbauer data in excellent accord with experiment; 2) the first discovery of a conservative hydrogen bonding interaction in the iron binding site based on X-ray and homology structures; 3) the first atomic level hypothesis of active site reorganization upon iron concentration increase, triggering the conformational change needed for its function. These results shall facilitate structural and functional studies of Fur family proteins.

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