Created by: Dory DeWeese

         Blood Coagulation Factor VIIa (PDB: 1FAK), molecular weight 50,000 Da and isoelectric point 4.8-5.1, when complexed with Tissue Factor, TF (PDB: 1AHW) is a biologically active enzyme involved in the extrinsic blood coagulation pathway (1,2,3). The complexed form of VIIa and TF is an enzyme and its cofactor respectively, and the complex is the necessary first line of cellular response to tissue injury or vascular damage. Factor VIIa is a member of the serine protease family, implying that its main enzymatic functionality involves the cleavage of specific peptide bonds. Specifically, VIIa, when in complex with TF, cleaves a peptide bond in factors X and IX. This converts both factors to their active protease forms, IXa and Xa. Structural necessities for the VIIa molecule functionality include four factors: critical residues and hydrogen bonding affecting the catalytic domain, residues involved in the interaction and binding of the VIIa molecule to TF and the presence of a calcium ion ligand. As determined by the PSI-Blast and Dali Server bioinformatics searches, VIIa has many similar proteins in both primary and tertiary structures, but the most intriguing of these is Nerve Growth Factor, or NGF, (PDB: ISGF) found in Mus musculus (4,5,6,7). 

          Blood Coagulation Factor, VII, is activated when in complex with Tissue Factor, TF, and together they nececitate a structure with four subunits. Both are the primary components in the induction pathway of blood coagulation. The pathway of blood coagulation leads to the formation of blood clots, which are vital for preventing internal bleeding and blood loss. The complex activates factors IX and X, stimulating a cascade that leads to the generation of a cross-linked fibrin clot, or blood clot. The VII zymogen, or inactive form, termed VII, is in constant circulation in the blood stream. TF resides on the outsides of blood vessels and is normally not present in the blood stream (1). However, upon vessel injury or damage, the tissue factors become exposed to the circulating blood and the inactivated Blood Coagulation Factor VII. VII binds to TF, and VII is activated to VIIa through the action of a series of proteases (factor IIa, Xa, IXa and XIIa). The VIIa-TFcomplex catalyzes the conversion of factor X and IX into their active proteases, factor Xa and IXa, which convert prothrombin to thrombin. Thrombin stimulates the transformation of fibrinogen to fibrin, which is converted by factor XIIIa to a cross-linked fibrin clot (8) (Figure 1). 

          Activated forms of VII are often administered as a first line of treatment for hemophilic patients, as well as to those suffering from uncontrolled blood loss.  This indicates that not only is factor VIIa the first step in the extrinsic blood coagulation pathway, it is the necessary first step, and blood coagulation cannot take place without its activated presence. VIIa is the current focus of much research into generating drugs for the treatment and control of all varieties of uncontrolled bleeding (9). Conversely, overactive VIIa-TF complexes can also be the cause of extreme blood clotting, leading to heart attacks and strokes. This fact has encouraged scientists to begin developing molecular VIIa inhibitors (1). 

          VIIa is a single chain protein, with four seperate protein domains– the N-terminal γ-carboxyglutamate-rich domain (GLA), two epidermal growth factor domains (EGF1 and EGF2) and a serine protease (catalytic) domain (1). In its zymogen form, the VII molecule's secondary structure includes four regions of alpha helices and five regions of beta pleated sheets. Upon activation, one helix increases in length by 16 residues (7). This differs from the secondary structure of the VIIa molecule, as this activated molecule is in complex with TF, so the full activated protein contains the secondary structure of both the VII molecule and the TF molecule. 

           Activation converting VII to VIIa occurs by specific peptide bond cleavage, or proteolytic cleavage, of the Leu-153 - Arg-154 peptide bond (1). This bond defines the linker region between the ERG2 and serine protease domain. Cleavage of this bond results in the formation of a light chain and a heavy chain, held together by a disulfide bridge between Cys-135 - Cys-262. This gives VIIa a more flexible and extended coverage across the TF molecule, which acts as a scaffold-like support for the VIIa molecules (1). 

           The functionality of the active VIIa enzyme is dependent on the presence of a calcium ion ligand (7). VIIa possesses a high-affinity binding site for calcium ions, which is located in the catalytic domain. The catalytic domain is a slightly distorted octahedral arrangement of residues around the Ca+ atom, which is also hydrated by two water molecules. This forms an extended hydrogen-bonding network, which significantly stabilizes the catalytic region, providing necessary enhancement of VIIa’s catalytic properties (7). 

           The K_d , or dissociation constant, of the TF-VIIa complex is approximately 1.5 micromolar in the absence of the calcium ion, and  0.4 picomolar in its presence (1). This implies that the presence of the calcium ion conveys a conformational change on the binding of the protein complex that occurs in the presence of calcium, allowing the protein complex to enzymatically function at a higher capacity. Additionally, the exceptionally large difference in the K_d values implies that the ion’s presence must have a cumulative effect, involving the calcium-mediated folding of the catalytic domain (1). 

            In terms of functionally important residues, Phe-328 is the most important functional residue in the VIIa molecule (1). If mutated, the molecule shows a twofold decrease in its ability to bind to TF, as well as not being able to activate either X or IX. A mutation of Phe-328 induces new hydrogen bond formation between Tyr-377and Asp-338 in neighboring regions of the VIIa molecule, changing the conformation of the VIIa protein and critically reducing VIIa’s specificity for both X and IX factors (1). 

           The cofactor TF defines the cascade that leads to the activation of factor X and IX (3). TF is a cell surface glycoprotein with three domains that function as a high-affinity receptor for the Blood Coagulation Factor VII. The molecule consists of a soluble extracellular component (N terminal, 1-219), a single alpha helix transmembrane component (220-242) and another soluble region, containing the C terminal, residing in the cytoplasm (243-262). In order for VII to bind to TF, TF must be both exposed to the blood stream and be activated (3). This activation stems from an increase in the levels of calcium in the blood stream due to vascular injury. The calcium induces an increase in phosphatidylserine exposure on the outer leaflet of the cell membrane lipid bilayer, resulting in a loss of bilayer asymmetry and the induction of TF. A disulfide linkage between 186-Cys and 209-Cys mediates this induction (3). 

           The result of the binding of VIIa to TF is a two chain, multi-domain enzyme bridged by a disulfide bond (1). The complex is composed of a light chain N-terminal gamma-carboxyglutamic acid rich domain, followed by two epidermal growth factors (EGF1 and EGF2), and a heavy chain trypsin-like catalytic domain. Domain 1 of the TF molecule (TF1) contributes 73% of the binding surface area between the TF and VIIa molecule. This implies a presence of a higher concentration of polar molecules in this domain, which interact favorably with the charged molecules present in the VIIa molecule.  Specifically, the Asp-44 residue of TF1 forms a hydrogen bond with Arg-277 of the VIIa molecule. This bond is significantly involved in the enhancement and regulation of the VIIa catalytic domain, emphasizing TF’s role as a cofactor to VIIa’s functionality (1). The N terminal of TF1, residues 39-45, interact with TF1 residues Phe-275-Phe-278, forming a shallow hydrophobic pocket. This loop undergoes a significant shift upon binding with the VIIa molecule (7).

           Bioinformatics searches are a necessary and vital tool used when exploring the structure and function of proteins. Different assays allow scientists to explore similarities and differences in primary, secondary and tertiary structures across known proteins. Two such searches are PSI-Blast and the Dali Server. PSI-Blast allows scientists to explore proteins that have similar sequences, or primary structures, to their protein of interest. The search generates a list of proteins with similar sequences with corresponding E values – those values closer to 0 indicate a higher conservation of sequence, as the E value rises with increasing gaps between the comparison sequence and the protein query input sequence. Values below 0.05 are considered significant (4). Dali Server searches for proteins that have similar protein folding, or tertiary structures, to the protein of interest. This search also generates a Z score for each protein, which indicates the similarity in tertiary structure – a higher Z score indicates a higher similarity in structure, where above a score of 2 is significant. This Z score is generated through comparisons of intramolecular or van der Waals distances, using the sum-of-pairs method (5).   

          These searches can be useful when answering questions regarding necessary conserved sequences for proteins of similar function, or necessary conserved folding for similar function, regardless of sequence. They can also explore proteins that have similar sequence and/or structure, but do not follow in function, another interesting case. One example of such a protein is Nerve Growth Factor (PDB: ISGF), or NGF, which has a Z score of 32.4 (5). Nerve Growth Factor is responsible for the growth and maintenance of certain populations of neurons in both the central and peripheral nervous systems in Mus musculus, or house mice (6). This protein was chosen because it had a high level of similarity to VIIa in term of tertiary structure but differs in the role the protein takes in the cell and in the species it is present in. 

          It is clear from inspection of VIIa’s and NGF’s overall functionality that there is not much similarity between the role these two proteins take in their respective cells. However, upon closer inspection into how both these proteins fulfill their cellular objective, it is clear that there is a large area of overlap – both proteins are considered part of the serine proteinase family. All of NGF’s domains acts as a serine proteases, which contributes to the similarity in tertiary structure of the two proteins (6). However, the VIIa protein is also a multi-domain protein, where only one of its domain acts a a serine protease. Though the role in their respective cells may be completely different, both proteins’ serine protease functionality is intergral to their functionality, and this functionality is aided by their similarity in tertiary structure. 

          One fundamental difference between these two proteins is that NGF does not need a cofactor be functionally active. However, after VIIa is activated and bound to the TF molecule, it extends its conformation over the entirety of the TF molecule, which acts as a scaffold like support for the VIIa molecule. Much like this, the gamma-NFG subunits make extensive interactions with the central beta-NGF dimer, where the central dimer makes a scaffold like support for the gamma-NGF subunits. Additionally, there is considerable similarities between the function of metal ions in the functionality of both of these proteins. Where the VIIa protein functionality hinges on the presence of a calcium ion in its binding pocket, the NGF protein utilizes a zinc ion. Both of these binding pockets occur in similar locations in the molecule. Also in terms of functionality, the three β-hairpin loops of the NGF molecule are critical for its functionality, wherein these loops in the VIIa molecule hold no significance. In terms of similarity in critical residues and therefore primary structure, there is no overlap because of the lack of similarity in the primary sequence between these two molecules. In conclusion, both proteins contain at least one serine protease domain, which characterize their functionality, but not their role in the cell. The similarity in tertiary structure is coincidental in that the spatial locations of all the domains arises not because of similarity in role in the cell.