User:Oluwapeluwa Sangoseni/Sandbox 1
6P9U, Crystal Structure of Human Thrombin Mutant W215A
OverviewThrombin is one of the many essential molecules that our body produces and uses to consistently maintain homeostasis. It is a serine protease that acts as both a procoagulant and an anticoagulant, and is essential for blood clot formation, among other functions, such as causing inflammation, repairing tissue, and forming new blood vessels [1].As a procoagulant, in order to promote blood clotting, thrombin cleaves fibrinogen, a soluble protein that can be found in blood plasma, to produce fibrin. Fibrin is an insoluble protein that can be used, with the addition of aggregated platelets (which are also activated by thrombin), to form blood clots by creating meshes that stop the flow of blood. This is important to prevent the loss of too much blood in the event of an injury that ruptures or damages blood vessels. Thrombin can also induce the coagulation pathway to produce more thrombin by activation of factor XI, and cofactors V and VIII [1]. As said before, it also acts as a regulatory anticoagulant by binding to thrombomodulin. Thrombomodulin is a receptor glycoprotein found on the surface of the membranes of endothelial cells, and when thrombin binds to it, it activates the protein C pathway to start a process known as fibrinolysis, which breaks down fibrin and destroys blood clots [2].This is required to prevent excessive blood clotting, which would be problematic as required blood flow may be impeded. Thrombin also regulates fibrinolysis by activating carboxypeptidase B2, also known as thrombin activatable fibrinolysis inhibitor. With this wide array of necessary functions, thrombin is a very important enzyme for maintenance of our bodies. Function and StructureThrombin alone does not cleave fibrinogen, however, and the process is far more complex than one would initially be led to believe. Several molecules also participate in the process, including 6P9U. Most research and data for this molecule has only been conducted and compiled very recently, as the protein data was only deposited in the PDB in mid-late 2019, so, compared to other well-known molecules, there is still quite a bit of work that can be done to learn more about it [3]. 6P9U is the crystallized structure of the human thrombin mutant, W215A. Data about this molecule’s structure was compiled using X-ray diffraction, at a resolution of 3.3 Å. It is found in humans, and features an 8-carbon chain structure. It has a cyclic C2 symmetry, and is a hetero 4 mer with the stoichiometry equation A2B2, meaning it has 2 alpha sub units, and 2 beta sub units. In terms of its macromolecular content, it has a total structural weight of 141170.61 Daltons, and features an atom count of 9004. It has a residue count of 1216, with two unique protein chains. These two unique chains consist of two different prothrombin or coagulation factor II macromolecules, with different sequence lengths (31 and 273). Both are expressed by the gene F2. The macromolecule also contains two ligands that bind to it: a Zinc ion (Zn2+), and n-acetyl-D-glucosamine (C8H15NO6). 6P9U/W215A serves as a residue for a hydrolase, facilitating hydrolysis for thrombin, in which a molecule of water is used to break bonds between atoms or molecules. This allows for the required peptide bonds to be cleavedto enable the conversion of fibrinogen to fibrin. 6P9U/W215A’s functionality and uses extend to a wider variety of possibilities than just cleaving peptide bonds, however. W215, along with E217 and E192, are residues that work together to regulate thrombin’s activity. Specifically, W215 is responsible for maintaining the allosteric equilibrium of thrombin’s closed and open conformations, which would maintain thrombin’s catalytic activity, bycontrolling the rate of transition between the two forms, if need be [4]. It does this by use of hydrophobic interactions with the benzene ring portion of thrombin’s F227 residue. This keeps the enzyme’s active site open, which causes the rate at which the open conformation closes to decrease. This is because thrombin’s zymogen, which is the inactive, immature form of the enzyme, is the more common form of the enzyme when it is in its closed conformation. By opening, the zymogen matures along with it, being converted into the active and mature form. It was discovered that W215A functions similarly to W215, as if you were to replace W215 with W215A, the rate of transition between the two conformations would still be maintained, almost as if it were still W215. If W215 or W215A were to be removed completely, or its hydrophobic interaction with F227 is otherwise disrupted, then the rate at which thrombin’s conformation closes would be increased, and thrombin’s activity would be decreased, as the equilibrium between the closed and open conformations are responsible for the enzyme’s activity [4]. RelevanceThese observations have several beneficial implications. Using the knowledge of these mechanisms, enzymes could possibly be created using W215 and/or W215A that maintains the open and closed conformation equilibrium in such a way that thrombin’s activity increases and remains boosted. This could be used to promote blood clot formation in patients that have difficulty with it, like hemophiliacs, or patients that have been seriously injured [5]. On the same token, an enzyme or some other molecule could be developed that has the effect of disrupting the hydrophobic interaction between W215 and F227. Ultimately, this would decrease thrombin’s activity,preventing or regulating the various processes that thrombin is part of, such as the formation of fibrin and the activation of fibrinolysis.These are some of the many ways that W215A can interact with thrombin and other molecules to elicit different reactions or changes in our body. In fact, this is not all theoretical; W215A, along with E217A, has been shown to have the potential to be used in treatments for various conditions and ailments [6]. For example, in an effort to find a suitable medication better than tissue plasminogen activatorfor lessening or alleviating the catastrophic results of an ischemic stroke, various treatments, including WE (the combined form of W215A and E217A) have been tested on mice that were induced to have an ischemic stroke [6]. An ischemic stroke occurs when an artery that allows blood to flow to and from the brain has been blocked for some reason. As blood carries oxygen and important nutrients to the brain, and carries waste and carbon dioxide away from the brain, having such an important avenue shut down results in brain cells dying and the brain potentially shutting off [7]. Tissue plasminogen activator, also known as tPa, is the current treatment that is used to treat ischemic strokes, and, at the time of writing this paper, it is the only FDA-approved treatment as well [6]. It acts as an anticoagulant by successfully catalyzing the fibrinolysis process to clear the clots obstructing the flow of blood to the brain, but it can also cause hemorrhaging in the brain. Recombinant activated protein C, also known as APC, has also been tested as an anticoagulant for ischemic stroke clots. It functions by inhibiting the activation of the coagulation factors V and VIII [6]. Unlike tPa, APC works to prevent damage to vascular and neuronal structure, and it also keeps the blood brain barrier safe.However, as APC activates protein C and its pathway, continued administration can have the unintended side effect ofnegatively affecting the bodies’ ability to stop the flow of blood too severely [6]. When WE was tested, however, scientists found something interesting. When used to treat an ischemic stroke, it was found to catalyze the fibrinolysis process and clear blood clots with efficiency similar to that of tPa. Use of WE was also found to naturally generate APC, which means that use of WE also confers the patient with APC’s ability to maintain vascular and neuronal structure, while preventing the blood brain barrier from being damaged. Since it is now naturally produced, treatment no longer runs the risk of impairing the patient’s ability to stop the flow of blood too severely [8]. These benefits show that WE may have a bright future as an anticoagulant for treatment, and not just for ischemic strokes. W215A and 6P9U are evidently potentially very important molecules. However, as said before, these molecules in particular only have relatively new data collected, research conducted, and papers written. Their functions are plentiful, and their structure and mechanisms are complex. As such, the current field has likely not even scratched the surface of all that these molecules can be used for. With more testing and research done, for example, the previously mentioned WE could be used to severely lessen the effects of ischemic strokes, and beyond. The medical field, and science in general would benefit greatly from more focused study on these molecules.
| ||||||||||||
