title: Structural and functional studies of γ-carboxyglutamic acid domains of factor VIIa and activated Protein C: role of magnesium at physiological calcium.
abstract: Crystal structures of factor (F) VIIa/soluble tissue factor (TF), obtained under high Mg(2+) (50mM Mg(2+)/5mM Ca(2+)), have three of seven Ca(2+) sites in the γ-carboxyglutamic acid (Gla) domain replaced by Mg(2+) at positions 1, 4, and 7. We now report structures under low Mg(2+) (2.5mM Mg(2+)/5mM Ca(2+)) as well as under high Ca(2+) (5mM Mg(2+)/45 mM Ca(2+)). Under low Mg(2+), four Ca(2+) and three Mg(2+) occupy the same positions as in high-Mg(2+) structures. Conversely, under low Mg(2+), reexamination of the structure of Gla domain of activated Protein C (APC) complexed with soluble endothelial Protein C receptor (sEPCR) has position 4 occupied by Ca(2+) and positions 1 and 7 by Mg(2+). Nonetheless, in direct binding experiments, Mg(2+) replaced three Ca(2+) sites in the unliganded Protein C or APC. Further, the high-Ca(2+) condition was necessary to replace Mg4 in the FVIIa/soluble TF structure. In biological studies, Mg(2+) enhanced phospholipid binding to FVIIa and APC at physiological Ca(2+). Additionally, Mg(2+) potentiated phospholipid-dependent activations of FIX and FX by FVIIa/TF and inactivation of activated factor V by APC. Since APC and FVIIa bind to sEPCR involving similar interactions, we conclude that under the low-Mg(2+) condition, sEPCR binding to APC-Gla (or FVIIa-Gla) replaces Mg4 by Ca4 with an attendant conformational change in the Gla domain ω-loop. Moreover, since phospholipid and sEPCR bind to FVIIa or APC via the ω-loop, we predict that phospholipid binding also induces the functional Ca4 conformation in this loop. Cumulatively, the data illustrate that Mg(2+) and Ca(2+) act in concert to promote coagulation and anticoagulation.
authors: UCLA/Orthopaedic Hospital Department of Orthopaedic Surgery, University of California, Los Angeles, CA 90095, USA.
authors: Kanagasabai
authors: K
authors: Vadivel
authors: UCLA/Orthopaedic Hospital Department of Orthopaedic Surgery, University of California, Los Angeles, CA 90095, USA.
authors: Sayeh
authors: S
authors: Agah
authors: UCLA/Orthopaedic Hospital Department of Orthopaedic Surgery, University of California, Los Angeles, CA 90095, USA.
authors: Amanda S
authors: AS
authors: Messer
authors: UCLA-DOE Institute for Genomics and Proteomics, Los Angeles, CA 90095, USA.
authors: Duilio
authors: D
authors: Cascio
authors: Division of Pulmonology and Critical Care, Department of Medicine, University of California, Los Angeles, CA 90095, USA.
authors: Madhu S
authors: MS
authors: Bajaj
authors: Department of Hematology, The Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA.
authors: Sriram
authors: S
authors: Krishnaswamy
authors: Oklahoma Medical Research Foundation, Howard Hughes Medical Institute, Oklahoma City, OK 73104, USA.
authors: Charles T
authors: CT
authors: Esmon
authors: Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824, USA.
authors: Kaillathe
authors: K
authors: Padmanabhan
authors: UCLA/Orthopaedic Hospital Department of Orthopaedic Surgery, University of California, Los Angeles, CA 90095, USA; Molecular Biology Institute, University of California, Los Angeles, CA 90095, USA. Electronic address: pbajaj@mednet.ucla.edu.
authors: S Paul
authors: SP
authors: Bajaj
mesh_terms: D015055:1-Carboxyglutamic Acid / Q000737:chemistry*; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D018360:Crystallography, X-Ray; D015942:Factor VIIa / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D008274:Magnesium / Q000378:metabolism*; D008958:Models, Molecular; D010743:Phospholipids / Q000378:metabolism; D011485:Protein Binding; D011486:Protein C / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation
chemical_list: D010743:Phospholipids; D011486:Protein C; D015055:1-Carboxyglutamic Acid; D015942:Factor VIIa; D008274:Magnesium; D002118:Calcium