Protein C and the Anticoagulant System Meng Meng Zhu, Townsend Harris High School ‘10 Macaulay Honors Summer Academy 2009 Bioinformatics Class, CCNY
Profile My name is Meng Meng Zhu from Townsend Harris High School. I am interested in biology, mostly environmental biology, engineering, and psychology. When I started this bioinformatics course, I had practically no prior knowledge about protein function and structure nor the technology available to scientists today. Taking this course has taught me how to use Blast, ClustalW, SwissModel, and knowledge about some methods of structure determination for DNA, RNA and proteins such as X-ray crystallography. In addition, the numerous guest speakers invited by Dr. Gosser taught me about research projects and recent advances in the sciences such as research to combat biofilm, application of tissue engineering, and the function of the electron microscope. With this new knowledge, I would like to talk about Protein C, its function in the anticoagulant system and also present 2 images generated using Postscript and POV-Ray.
Biological Application
Background: Protein C is synthesized in human liver and exist in the blood plasma where it plays a crucial role in the anticoagulant system. Activated by protein K and the thrombinthrombomodulin complex, it attaches to endothelial cells on the interior surface of blood vessels to prevent further thrombin formation by inactivating Factor Va and Factor VIII, therefore slowing and stopping the formation of fibrin. Problems arise when one is born with Protein C Deficiency which leads to greater chance of blood clots, strokes and even miscarriage. Protein C also has anti-inflammatory properties.
Fibrin leading to blood clotting http://twistedphysics.typepad.com/cocktail_party_physics/2007/03/index.html
Protein C Activation
http://www.unige.ch/cyberdocuments/theses2003/DimitrovaTirefort Y/images/Diapositive8.jpg
General Information
Name: Protein C First Identified: 1960 Official Names: PC,PROC1,PROC PDB Structures: 1aut,1lqv Location: chromosome: 2 Location: 2q13-q14 Accession: NP_000303 Length : 461 amino acids FASTA sequence: >gi|4506115|ref|NP_000303.1| protein C [Homo sapiens] MWQLTSLLLFVATWGISGTPAPLDSVFSSSERAHQVLRIRKRANSFLEELRHSSLERECIEEICDFEEAK EIFQNVDDTLAFWSKHVDGDQCLVLPLEHPCASLCCGHGTCIDGIGSFSCDCRSGWEGRFCQREVSFLNC SLDNGGCTHYCLEEVGWRRCSCAPGYKLGDDLLQCHPAVKFPCGRPWKRMEKKRSHLKRDTEDQEDQVDP RLIDGKMTRRGDSPWQVVLLDSKKKLACGAVLIHPSWVLTAAHCMDESKKLLVRLGEYDLRRWEKWELDL DIKEVFVHPNYSKSTTDNDIALLHLAQPATLSQTIVPICLPDSGLAERELNQAGQETLVTGWGYHSSREK EAKRNRTFVLNFIKIPVVPHNECSEVMSNMVSENMLCAGILGDRQDACEGDSGGPMVASFHGTWFLVGLV SWGEGCGLLHNYGVYTKVSRYLDWIHGHIRDKEAPQKSWAP
Protein C Activation
PDB Structure y
Structure was determined by X-Ray Diffraction with a resolution of 2.80 Å.
Length: a= 57.06 Å b= 89.60 Å c= 101.23 Å
Angles: α = 90.00 β = 90.00 γ = 90.00
1aut - activated protein c http://www.rcsb.org/pdb/explore/explore.do?structureId=1AUT http://www.ebi.ac.uk/pdbe-srv/view/entry/1aut/secondary.html (structure site)
Sample Conditions
Human Protein C purified and activated with 1 mg of bovine thrombin at 37C in three hours in 0.1M NaCl, 20 MM Tris-HCL, 5mM EDTA pH7.5. Then it was inactivated. Gla domain was removed using chymotrysin.
Crystals gown by hanging drop vapor diffusion method
Crystals (0.05x0.05x0.2 mm formed in around 4 weeks
110-132 -Calcium Binding Site [Yellow] 213-448- Serine Protease Domain [ Red] 24-87- Gla domain (not shown)
Pymol
Domains The Serine Protease Domain interacts with the ThrombinThrombomodulin Complex. This is the enzyme part. The EGF –like domain contains the calcium binding site and allows protein-protein or protein-cell interactions.
The Gla domain allows lipid membrane binding in the presence of Ca2+ ions, which leads to its activation. http://www1.imperial.ac.uk/medicine/about/divisions/is/haemo/coag/
Mutations
E.G. Bovill, J.A. Tomczak, B. Grant, F. Bhashan, etc found that 2 single base substitutions in the DNA from protein C –deficient family members resulted in Gla domain changes that made it hard for Protein C to bind to the Protein C receptors making the Protein less effective.
A-> C resulted in Glu 20 to mutate to Alanine
G-> A resulted in Val34 to mutate to Methionine
The affected individuals had abnormal Protein C levels, 1.5-3.3 that of the norm., in their blood plasma but still had high incidence of clotting. This was the result of the analysis of the Protein C genes of 14 people.
Crystal structure of the Endothelial protein C receptor with phospholipid in the groove in complex with Gla domain of protein C- 1Lqv
Sequence Alignment The sequence alignment of protein C of human, dog, mouse and pig shows much conservation throughout protein demonstrating that much of the same structures are necessary for proper function in a variety of species.
Conclusion
Protein C has anti-coagulant and anti-inflammatory properties that is important to the human body. It is made of 3 main domains: A Trypsin-like Serine Protease domain, A EGF like domain and a Gla domain.
Changes in the nucleotide sequence alters the protein structure and can lead to harmful diseases. Binding to endothelial cells is disrupted when Val34 in the Gla domain is mutated. Future research may find mutations that can help binding and therefore increase and quicken protein c activation.
Identifying the location of mutations that may be the cause of diseases helps chemists and biologists develop treatments.
References
Blood Clotting
Cleaning Industries Research Institute
EG Bovill, JA Tomczak, B Grant, F Bhushan, E Pillemer, IR Rainville, and GL Long. “Protein C Vermont: Symptomatic Type II Protein C Deficiency Associated With Two GLA Domain Mutations” Blood 79: 1456-1465.
EMBL-EBI < http://www.ebi.ac.uk/Tools/es/cgi-bin/clustalw2/result.cgi?tool=clustalw2&jobid=clustalw2-200907200721374997&treendisp=hide&treetype=new&sortby=seqno&color=yes>
National Alliance for Thrombosis & Thrombophilia (NATT)
Oganesyan, Vaheh. “The Crystal Structure of the Endothelial Protein C Receptor and a Bound Phospholipid” JBS Online
ProCMD: Protein C Mutations Database
The National Center for Biotechnological Information (NCBI)
Art of Science and Engineering Project:
Pollock Clot & Molding Corn Meng Meng Zhu, Townsend Harris High School ‘10 Macaulay Honors Summer Academy 2009 Bioinformatics Class, CCNY
Molding Corn This POV-Ray image depicts the microscopic activity that occurs as corn sits on a plate in a room. The growth on the corn is a fungus named Aspergillus niger. It is the cause behind the black mold that infects vegetables and fruits. It is also known as black mold. This fungi is also used in industries to produce citric acid and gluconic acid. The Aspergillus consist of a long stalk like structure or hyphae. The spherical structure at the end contains smaller hypha which produces chains of spores or conidia. In addition to the corn there are white straw-like objects on the plate. These are fibers found commonly on the corn. Finally filling the entire room are magnified air bacterial in green and cyan.
Pollock Clot This Postscript image shows the red blood cells in a blood vessel. Red blood cells deliver oxygen to tissues throughout the body. However, red blood cells can be prevented from doing their job when there is a blood clot. Blood clots or thrombus occurs when blood vessels are injured. An enzyme named thrombin plays a major role in the blood coagulation process. During blood clots, fibrins form which restricts blood flow and stop red blood cells from moving. My picture depicts this process using Pollock style fibrins.
Acknowledgements
Dr. Yuying Gosser and Dr. Peter Brass
Teaching assistants Zi Lu, Olivia PLante, Sam Kim, Noah Lawson
