3F6U

Crystal structure of human Activated Protein C (APC) complexed with PPACK

Summary for 3F6U

• Entry DOI: 10.2210/pdb3f6u/pdb
• Related PRD ID: PRD_000020
• Descriptor: Vitamin K-dependent protein C heavy chain, Vitamin K-dependent protein C light chain, D-phenylalanyl-N-[(2S,3S)-6-{[amino(iminio)methyl]amino}-1-chloro-2-hydroxyhexan-3-yl]-L-prolinamide, ... (6 entities in total)
• Functional Keywords: blood coagulation, serine protease, hydrolase-hydrolase inhibitor complex, hydrolase/hydrolase inhibitor
• Biological source: Homo sapiens; More
• Total number of polymer chains: 2
• Total formula weight: 38285.32

Authors

Schmidt, A.E.,Padmanabhan, K.,Underwood, M.C.,Bode, W.,Mather, T.,Bajaj, S.P. (deposition date: 2008-11-06, release date: 2008-11-25, Last modification date: 2024-10-16)

Primary citation

Schmidt, A.E.,Padmanabhan, K.,Underwood, M.C.,Bode, W.,Mather, T.,Bajaj, S.P.
Thermodynamic linkage between the S1 site, the Na+ site, and the Ca2+ site in the protease domain of human activated protein C (APC).
J.Biol.Chem., 277:28987-28995, 2002

PubMed Abstract: The serine protease domain of activated protein C (APC) contains a Na+ and a Ca2+ site. However, the number and identity of the APC residues that coordinate to Na+ is not precisely known. Further, the functional link between the Na+ and the Ca2+ site is insufficiently defined, and their linkage to the substrate S1 site has not been studied. Here, we systematically investigate the functional significance of these two cation sites and their thermodynamic links to the S1 site. Kinetic data reveal that Na+ binds to the substrate-occupied APC with K(d) values of approximately 24 mm in the absence and approximately 6 mm in the presence of Ca2+. Sodium-occupied APC has approximately 100-fold increased catalytic efficiency ( approximately 4-fold decrease in K(m) and approximately 25-fold increase in k(cat)) in hydrolyzing S-2288 (H-d-Ile-Pro-Arg-p-nitroanilide) and Ca2+ further increases this k(cat) slightly ( approximately 1.2-fold). Ca2+ binds to the protease domain of APC with K(d) values of approximately 438 microm in the absence and approximately 105 microm in the presence of Na+. Ca2+ binding to the protease domain of APC does not affect K(m) but increases the k(cat) approximately 10-fold, and Na+ further increases this k(cat) approximately 3-fold and decreases the K(m) value approximately 3.7-fold. In agreement with the K(m) data, sodium-occupied APC has approximately 4-fold increased affinity in binding to p-aminobenzamidine (S1 probe). Crystallographically, the Ca2+ site in APC is similar to that in trypsin, and the Na+ site is similar to that in factor Xa but not thrombin. Collectively, the Na+ site is thermodynamically linked to the S1 site as well as to the protease domain Ca2+ site, whereas the Ca2+ site is only linked to the Na+ site. The significance of these findings is that under physiologic conditions, most of the APC will exist in Na2+-APC-Ca2+ form, which has 110-fold increased proteolytic activity.

PubMed: 12029084
DOI: 10.1074/jbc.M201892200

Experimental method

X-RAY DIFFRACTION (2.8 Å)