RESUMO
14-3-3 Proteins bind phosphorylated sequences in proteins and regulate multiple cellular functions. For the first time, we show that pure recombinant human 14-3-3 ζ, γ, ε and τ isofoms hydrolyze ATP with similar Km and kcat values. In sharp contrast the sigma isoform has no detectable activity. Docking studies identify two putative binding pockets in 14-3-3 zeta. Mutation of D124A in the amphipathic pocket enhances binding affinity and catalysis. Mutation of a critical Arg (R55A) at the dimer interface in zeta reduces binding and decreases catalysis. These experimental results coincide with a binding pose at the dimer interface. This newly identified function could be a moon lighting function in some of these isoforms.
Assuntos
Proteínas 14-3-3/metabolismo , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas 14-3-3/química , Proteínas 14-3-3/genética , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Trifosfato de Adenosina/química , Sequência de Aminoácidos , Sítios de Ligação/genética , Western Blotting , Humanos , Hidrólise , Cinética , Modelos Moleculares , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Estrutura Molecular , Mutagênese , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Homologia de Sequência de AminoácidosRESUMO
BACKGROUND: Coiled-coils are found in different proteins like transcription factors, myosin tail domain, tropomyosin, leucine zippers and kinesins. Analysis of various structures containing coiled-coils has revealed the importance of electrostatic and hydrophobic interactions. In such domains, regions of different strength of interactions need to be identified since they could be biologically relevant. FINDINGS: We have updated our coiled-coil validation webserver, now called COILCHECK+, where new features were added to efficiently identify the strength of interaction at the interface region and measure the density of charged residues and hydrophobic residues. We have examined charged residues and hydrophobic ladders, using a new algorithm called CHAHO, which is incorporated within COILCHECK + server. CHAHO permits the identification of spatial charged residue patches and the continuity of hydrophobic ladder which stabilizes and destabilizes the coiled-coil structure. CONCLUSIONS: The availability of such computational tools should be useful to understand the importance of spatial clustering of charged residues and the continuity of hydrophobic residues at the interface region of coiled-coil dimers. COILCHECK + is a structure based tool to validate coiled-coil stability; it can be accessed at http://caps.ncbs.res.in/coilcheckplus.