Structural Basis for the Failure of the C1 Domain of Ras Guanine Nucleotide Releasing Protein 2 (RasGRP2) to Bind Phorbol Ester with High Affinity
| Autor: | Elizabeth A. Craft, Derek C. Braun, Nancy E. Lewin, Agnes Czikora, Raymond C. Merritt, Noemi Kedei, Peter M. Blumberg, Adelle Abramovitz, Xiaoling Zhou, Daniel J. Lundberg, Megan L. Peach |
|---|---|
| Rok vydání: | 2016 |
| Předmět: |
Models
Molecular 0301 basic medicine Plasma protein binding Molecular Dynamics Simulation Crystallography X-Ray Biochemistry 03 medical and health sciences chemistry.chemical_compound Guanine Nucleotide Exchange Factors Humans Binding site Molecular Biology Phorbol 12 13-Dibutyrate Protein kinase C C1 domain Diacylglycerol kinase Binding Sites 030102 biochemistry & molecular biology Cell Biology Molecular biology Recombinant Proteins Protein Structure Tertiary Molecular Docking Simulation Kinetics HEK293 Cells 030104 developmental biology Amino Acid Substitution chemistry Protein Structure and Folding Phorbol Mutant Proteins Rap1 Guanine nucleotide exchange factor Protein Binding |
| Zdroj: | Journal of Biological Chemistry. 291:11133-11147 |
| ISSN: | 0021-9258 |
| DOI: | 10.1074/jbc.m116.725333 |
| Popis: | The C1 domain represents the recognition module for diacylglycerol and phorbol esters in protein kinase C, Ras guanine nucleotide releasing protein (RasGRP), and related proteins. RasGRP2 is exceptional in that its C1 domain has very weak binding affinity (Kd = 2890 ± 240 nm for [(3)H]phorbol 12,13-dibutyrate. We have identified four amino acid residues responsible for this lack of sensitivity. Replacing Asn(7), Ser(8), Ala(19), and Ile(21) with the corresponding residues from RasGRP1/3 (Thr(7), Tyr(8), Gly(19), and Leu(21), respectively) conferred potent binding affinity (Kd = 1.47 ± 0.03 nm) in vitro and membrane translocation in response to phorbol 12-myristate 13-acetate in LNCaP cells. Mutant C1 domains incorporating one to three of the four residues showed intermediate behavior with S8Y making the greatest contribution. Binding activity for diacylglycerol was restored in parallel. The requirement for anionic phospholipid for [(3)H]phorbol 12,13-dibutyrate binding was determined; it decreased in going from the single S8Y mutant to the quadruple mutant. The full-length RasGRP2 protein with the mutated C1 domains also showed strong phorbol ester binding, albeit modestly weaker than that of the C1 domain alone (Kd = 8.2 ± 1.1 nm for the full-length protein containing all four mutations), and displayed translocation in response to phorbol ester. RasGRP2 is a guanyl exchange factor for Rap1. Consistent with the ability of phorbol ester to induce translocation of the full-length RasGRP2 with the mutated C1 domain, phorbol ester enhanced the ability of the mutated RasGRP2 to activate Rap1. Modeling confirmed that the four mutations helped the binding cleft maintain a stable conformation. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|