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1.
Structure ; 2024 Jun 09.
Artigo em Inglês | MEDLINE | ID: mdl-38870939

RESUMO

Cyclin dependent kinase 7 (CDK7) is an important therapeutic kinase best known for its dual role in cell cycle regulation and gene transcription. Here, we describe the application of protein engineering to generate constructs leading to high resolution crystal structures of human CDK7 in both active and inactive conformations. The active state of the kinase was crystallized by incorporation of an additional surface residue mutation (W132R) onto the double phosphomimetic mutant background (S164D and T170E) that yielded the inactive kinase structure. A novel back-soaking approach was developed to determine crystal structures of several clinical and pre-clinical inhibitors of this kinase, demonstrating the potential utility of the crystal system for structure-based drug design (SBDD). The crystal structures help to rationalize the mode of inhibition and the ligand selectivity profiles versus key anti-targets. The protein engineering approach described here illustrates a generally applicable strategy for structural enablement of challenging molecular targets.

2.
Sci Signal ; 11(543)2018 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-30108182

RESUMO

Hsp72 is a member of the 70-kDa heat shock family of molecular chaperones (Hsp70s) that comprise a nucleotide-binding domain (NBD) and a substrate-binding domain (SBD) connected by a linker that couples the exchange of adenosine diphosphate (ADP) for adenosine triphosphate (ATP) with the release of the protein substrate. Mitotic phosphorylation of Hsp72 by the kinase NEK6 at Thr66 located in the NBD promotes the localization of Hsp72 to the mitotic spindle and is required for efficient spindle assembly and chromosome congression and segregation. We determined the crystal structure of the Hsp72 NBD containing a genetically encoded phosphoserine at position 66. This revealed structural changes that stabilized interactions between subdomains within the NBD. ATP binding to the NBD of unmodified Hsp72 resulted in the release of substrate from the SBD, but phosphorylated Hsp72 retained substrate in the presence of ATP. Mutations that prevented phosphorylation-dependent subdomain interactions restored the connection between ATP binding and substrate release. Thus, phosphorylation of Thr66 is a reversible mechanism that decouples the allosteric connection between nucleotide binding and substrate release, providing further insight into the regulation of the Hsp70 family. We propose that phosphorylation of Hsp72 on Thr66 by NEK6 during mitosis promotes its localization to the spindle by stabilizing its interactions with components of the mitotic spindle.


Assuntos
Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Choque Térmico HSP72/metabolismo , Fuso Acromático/metabolismo , Regulação Alostérica , Sítios de Ligação/genética , Cristalografia por Raios X , Proteínas de Choque Térmico HSP72/química , Proteínas de Choque Térmico HSP72/genética , Células HeLa , Humanos , Mitose/genética , Modelos Moleculares , Mutação , Quinases Relacionadas a NIMA/genética , Quinases Relacionadas a NIMA/metabolismo , Fosforilação , Domínios Proteicos , Fuso Acromático/genética , Treonina/genética , Treonina/metabolismo
3.
EMBO J ; 37(8)2018 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-29510984

RESUMO

Aurora-A regulates the recruitment of TACC3 to the mitotic spindle through a phospho-dependent interaction with clathrin heavy chain (CHC). Here, we describe the structural basis of these interactions, mediated by three motifs in a disordered region of TACC3. A hydrophobic docking motif binds to a previously uncharacterized pocket on Aurora-A that is blocked in most kinases. Abrogation of the docking motif causes a delay in late mitosis, consistent with the cellular distribution of Aurora-A complexes. Phosphorylation of Ser558 engages a conformational switch in a second motif from a disordered state, needed to bind the kinase active site, into a helical conformation. The helix extends into a third, adjacent motif that is recognized by a helical-repeat region of CHC, not a recognized phospho-reader domain. This potentially widespread mechanism of phospho-recognition provides greater flexibility to tune the molecular details of the interaction than canonical recognition motifs that are dominated by phosphate binding.


Assuntos
Aurora Quinase A/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Fuso Acromático/metabolismo , Linhagem Celular , Humanos , Proteínas Associadas aos Microtúbulos/genética , Conformação Proteica em alfa-Hélice
4.
Mol Cell ; 62(1): 121-36, 2016 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-26949039

RESUMO

HECT-family E3 ligases ubiquitinate protein substrates to control virtually every eukaryotic process and are misregulated in numerous diseases. Nonetheless, understanding of HECT E3s is limited by a paucity of selective and potent modulators. To overcome this challenge, we systematically developed ubiquitin variants (UbVs) that inhibit or activate HECT E3s. Structural analysis of 6 HECT-UbV complexes revealed UbV inhibitors hijacking the E2-binding site and activators occupying a ubiquitin-binding exosite. Furthermore, UbVs unearthed distinct regulation mechanisms among NEDD4 subfamily HECTs and proved useful for modulating therapeutically relevant targets of HECT E3s in cells and intestinal organoids, and in a genetic screen that identified a role for NEDD4L in regulating cell migration. Our work demonstrates versatility of UbVs for modulating activity across an E3 family, defines mechanisms and provides a toolkit for probing functions of HECT E3s, and establishes a general strategy for systematic development of modulators targeting families of signaling proteins.


Assuntos
Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/metabolismo , Animais , Domínio Catalítico , Linhagem Celular , Movimento Celular , Cães , Células HCT116 , Humanos , Células Madin Darby de Rim Canino , Modelos Moleculares , Organoides/citologia , Organoides/metabolismo , Biblioteca de Peptídeos , Ubiquitina/química , Ubiquitina/genética
5.
J Biol Chem ; 289(37): 25611-23, 2014 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-25074933

RESUMO

Hakai, an E3 ubiquitin ligase, disrupts cell-cell contacts in epithelial cells and is up-regulated in human colon and gastric adenocarcinomas. Hakai acts through its phosphotyrosine-binding (HYB) domain, which bears a dimeric fold that recognizes the phosphotyrosine motifs of E-cadherin, cortactin, DOK1, and other Src substrates. Unlike the monomeric nature of the SH2 and phosphotyrosine-binding domains, the architecture of the HYB domain consists of an atypical, zinc-coordinated tight homodimer. Here, we report a C-terminal truncation mutant of the HYB domain (HYB(ΔC)), comprising amino acids 106-194, which exists as a monomer in solution. The NMR structure revealed that this deletion mutant undergoes a dramatic structural change caused by a rearrangement of the atypical zinc-coordinated unit in the C terminus of the HYB domain to a C2H2-like zinc finger in HYB(ΔC). Moreover, using isothermal titration calorimetry, we show that dimerization of HYB(ΔC) can be induced using a phosphotyrosine substrate peptide. This ligand-induced dimerization of HYB(ΔC) is further validated using analytical ultracentrifugation, size-exclusion chromatography, NMR relaxation studies, dynamic light scattering, and circular dichroism experiments. Overall, these observations suggest that the dimeric architecture of the HYB domain is essential for the phosphotyrosine-binding property of Hakai.


Assuntos
Caderinas/química , Células Epiteliais/química , Ubiquitina-Proteína Ligases/química , Domínios de Homologia de src , Sítios de Ligação , Caderinas/metabolismo , Cristalografia por Raios X , Células Epiteliais/metabolismo , Humanos , Ligantes , Espectroscopia de Ressonância Magnética , Fosfotirosina/química , Ligação Proteica , Conformação Proteica , Soluções/química , Ubiquitina-Proteína Ligases/metabolismo
6.
EMBO J ; 31(5): 1308-19, 2012 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-22252131

RESUMO

Phosphotyrosine-binding domains, typified by the SH2 (Src homology 2) and PTB domains, are critical upstream components of signal transduction pathways. The E3 ubiquitin ligase Hakai targets tyrosine-phosphorylated E-cadherin via an uncharacterized domain. In this study, the crystal structure of Hakai (amino acids 106-206) revealed that it forms an atypical, zinc-coordinated homodimer by utilizing residues from the phosphotyrosine-binding domain of two Hakai monomers. Hakai dimerization allows the formation of a phosphotyrosine-binding pocket that recognizes specific phosphorylated tyrosines and flanking acidic amino acids of Src substrates, such as E-cadherin, cortactin and DOK1. NMR and mutational analysis identified the Hakai residues required for target binding within the binding pocket, now named the HYB domain. ZNF645 also possesses a HYB domain but demonstrates different target specificities. The HYB domain is structurally different from other phosphotyrosine-binding domains and is a potential drug target due to its novel structural features.


Assuntos
Caderinas/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo , Sequência de Aminoácidos , Cristalografia por Raios X , Análise Mutacional de DNA , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Multimerização Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína
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