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

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.

Subcellular localization of Rap1 GTPase activator CalDAG‐GEFI is orchestrated by interaction of its atypical C1 domain with membrane phosphoinositides

“…The CDGI/Rap1 signaling module plays a central role in this process . Critical to CDGI catalytic activity regulation is a pair of EF hands in its regulatory domain, which have high affinity for calcium . In this study, we demonstrate that an atypical C1 regulatory domain controls CDGI subcellular localization by interacting with phosphoinositides in cellular membranes.…”

“…These studies suggest a critical role for the C1 domain in CDGI function. To test whether deletion of the C1 domain affects the catalytic activity of CDGI, we next tested purified CDGI protein containing all functional domains (consisting of residues 1‐551, referred to simply as CDGI) and the C1‐truncated protein (consisting of residues 1‐499, referred to as CDGIΔC1) for catalytic activity toward nucleotide exchange in Rap1B . CDG1ΔC1 lacking the C1 domain showed similar nucleotide exchange activity toward Rap1B when compared with CDG1 (Figure D).…”

“…Nucleotide exchange was measured using a fluorescence‐based assay as previously described . Purified Rap1B (1 µmol/L) and BODIPY‐GDP (1 µmol/L; Life Technologies) were incubated in 20 mmol/L Tris‐HCl (pH 7.5), 150 mmol/L NaCl, 5 mmol/L MgCl 2 , 1 mmol/L DTT, 5% glycerol, and 0.004% NP‐40 before addition of 400 nmol/L CDGI proteins for a final volume of 100 μL using a black bottom 96‐well plate (Corning).…”

“…21 Our recent biochemical and biophysical work demonstrated that calcium binding to the CDGI EF hand domain leads to release of autoinhibition and induction of nucleotide exchange activity. 22 The C1 domains comprise a family of ~60 members, and contain two β-sheets and an α-helix that promote zinc coordination via two sets of three cysteines and one histidine. 23 C1 domains were originally characterized as diacylglycerol (DAG) binding motifs in protein kinase C (PKC) proteins where they facilitate activation by translocation of the enzyme to the membrane and by release of autoinhibitory interactions.…”

“…The Cdc25 domain contains a helical hairpin that removes GDP when Ras or Rap binds to the catalytic pocket, thereby facilitating exchange of GDP for GTP to promote GTPase activation . Our recent biochemical and biophysical work demonstrated that calcium binding to the CDGI EF hand domain leads to release of autoinhibition and induction of nucleotide exchange activity . The C1 domains comprise a family of ~60 members, and contain two β‐sheets and an α‐helix that promote zinc coordination via two sets of three cysteines and one histidine .…”