Calcium-induced structural rearrangements release autoinhibition in the Rap-GEF CalDAG-GEFI

• Type of Material: Article
• Authors: Aaron A. Cook, University of North Carolina; Wei Deng, Emory University; Jinqi Ren, University of North Carolina; Renhao Li, Emory University; John Sondek, University of North Carolina; Wolfgang Bergmeier, University of North Carolina
• Language: English
• Date: 2018-06-01
• Publisher: American Society for Biochemistry and Molecular Biology
• Publication Version: Final Published Version
• Copyright Statement: © 2018 Cook et al. Published under exclusive license by The American Society for Biochemistry and Molecular Biology, Inc.
• Final Published Version (URL): http://dx.doi.org/10.1074/jbc.RA118.002712
• Title of Journal or Parent Work: Journal of Biological Chemistry
• ISSN: 0021-9258
• Volume: 293
• Issue: 22
• Start Page: 8521
• End Page: 8529
• Grant/Funding Information: This work was supported by NHLBI; National Institutes of Health Grants R01 HL130404 and R01 HL121650 and NIGMS; National Institutes of Health Grant P01GM103723.
• Keywords: calcium; conformational dynamics; RASGRP2; PROTEINS; autoinhibition; Life Sciences & Biomedicine; platelet; MUTATIONS; EF hand; GTPase; Biochemistry & Molecular Biology; AFFINITY; ACTIVATION; PARVALBUMIN; guanine nucleotide exchange factor (GEF); Science & Technology; PLATELET DYSFUNCTION; DOMAIN; structural model
• Research Categories: Chemistry, Biochemistry; Biology, Molecular

Abstract

Platelets are recruited to sites of vascular injury, where they are activated and aggregate to form a hemostatic plug. This process requires the activation of the small GTPase Rap1B by its cognate guanine nucleotide exchange factor CalDAG-GEFI. Studies on platelet function suggest that CalDAG-GEFI activity is regulated by changes in cytosolic calcium, but the exact molecular mechanism is poorly understood. Here we show that purified CalDAG-GEFI is autoinhibited and directly regulated by calcium. Substitutions of putative calcium-binding residues within the canonical EF hands of CalDAG-GEFI diminish its capacity to activate Rap1B. Structural differences between active (WT) and inactive (EF hand variant) CalDAG-GEFI protein were determined by hydrogen– deuterium exchange MS. The highest differential rates of deuterium uptake in WT over EF hand variant CalDAG-GEFI were observed in regions within the catalytic Cdc25 domain and a putative autoinhibitory linker connecting the Cdc25 and EF hand domains. Exchange activity in the EF hand variant was fully restored by an additional substitution, valine 406 to glutamate, which is thought to disrupt the interface between the autoinhibitory linker and the Cdc25 domain. Overall, our results suggest a model for how CalDAG-GEFI remains in an autoinhibited state when levels of cytosolic calcium in resting platelets are low. In response to cellular stimulation, calcium mobilization and binding to the EF hands causes conformational rearrangements within CalDAG-GEFI, including the autoinhibitory linker that frees the catalytic surface of CalDAG-GEFI to engage and activate Rap1B. The data from this study are the first evidence linking CalDAG-GEFI activity directly to calcium.