Exosite

Exosites are an alternative binding site on an enzyme that can trigger changes in the protein. They have multiple uses for an enzyme, such as substrate-recognition, inhibitor-binding, cofactor-binding, and others, depending on the physiological needs. An enzyme having this alternative binding region can help the positioning and recognition of substrates. They can also hold substrates in place to perform reactions at the active site. Exosites are commonly seen in thrombin since they help improve the specificity and efficiency of blood clotting reactions and immune responses. == Structure ==

residue, this makes the exosite II be more flexible which as seen in the research is important to escort the elastin to the catalytic site for hydrolysis. Exosites can also be produced from the spatial clustering of catalytic residues of similar properties in Pl molecules. These are stabilized by bacterial cofactors. They can also temporarily occupy plasmin and alter its specificity; but not permanent structural pockets like in thrombin. == Function ==

For protease exosites, there are three functions: substrate recognition, inhibitor binding, or cofactor binding. Exosites work in thrombin to recognize substrates and inhibitors. To initialize these functions, exosites flank the active site and promote binding of substrates, cofactors, and inhibitors, stabilizing enzyme-ligand complexes. This process regulates thrombin activity through allosteric effects. In the binding of substrates, exosites help position the substrate onto the catalyst, which increases specificity and efficiency. The binding of cofactors at exosites improves enzyme function by enhancing the interactions with substrates. In the binding of inhibitors, exosites create docking regions that accelerate the binding of inhibitors to thrombin or other enzymes. Exosites use the active site for inhibitor binding and cofactor binding, but for substrate recognition, exosites impact protease function without active site binding. The active site only recognizes small peptide sequences. This limits specificity, since some proteases need their target protein to unfold in order to cleave substrates. When interacting with extended regions of substrates, it allows for broader specificity and for exosites to bind to different kinds of substrates. Thrombin is an example of a protease that has a broad specificity, and exosite binding to its substrates allows it to overcome the limitations of its active sites. In thrombin, antithrombin, and other inhibitors inhibit blood coagulation, so the ability to cleave substrates allows these inhibitors to be blocked. However, antithrombin has evolved to inhibit coagulation factors that could not be blocked by binding to substrates. Figure 3 below shows exosite I and exosite II and their locations on thrombin. Exosite I primarily is a fibrinogen-binding site, and exosite II is a heparin-binding site. Studies have shown that exosites are not just rigid binding regions, but they are highly flexible surfaces that can adapt to different ligands. Exosite I and II also communicate with each other. There is long-range inter-exosite communication that creates dynamic transmissions of the structural information from one exosite to another. This exosite communication helps thrombin interact with multiple partners simultaneously during coagulation. These partners that thrombin communicates with include substrates, inhibitors, and ligands, as mentioned above. These ligands include proteins, peptides, and nucleic acid aptamers. The aptamers bind to specific targets, and they are single-stranded DNA or RNA molecules. Aptamers can bind to thrombin. Two examples of an aptamer are TBA (a DNA aptamer) and Toggle-25t (an RNA aptamer). TBA binds to thrombin via exosite I, and Toggle-25t binds to thrombin via exosite II. Studies on aptamers found that binding at exosite I or II can affect thrombin activity without directly blocking the active site of thrombin. This shows that exosites can fine-tune enzyme function through indirect mechanisms. In regard to docking regions on exosites, an example of this is the prothrombin docking to various exosites. This process involves two steps and leads to thrombin formation. The first step involves the docking of prothrombin FXa-Va membrane on exosites. FXa (factor XA) is an enzyme, and FVa (factor Va) is a cofactor that binds to the catalytic site of FXa. The docking of prothrombin forms a complex. On this complex, the active site of FXa is accessible to substrates and inhibitors. The FXa-Va membrane complex is also known as the prothrombinase complex. When this happens, the FXa catalytic site gets ready for prothrombin cleavage due to a conformational change. The second step is this conformational change, which results in prothrombin activation, and the prothrombin competes with other substrates to bind to the same exosite on prothrombinase. When prothrombinase is catalyzed by prothrombin, thrombin is produced. Figure 4 below shows how thrombin is produced: the green dashed line represents exosite involvement in generating FXa, ultimately leading to the formation of the prothrombinase complex and production of thrombin. The involvement of exosite in blood coagulation is described in the exosite application section. == Uses/Applications ==

Cathepsin V One example of exosites applications is in Cathepsin V. Cathepsin V is an elastase involved in extracellular matrix degradation; this cathepsin is very effective. It was found that the structurally homologous cathepsin L, which shares a 78% amino acid sequence, has only a minimal proteolatic activity toward insoluble elastin. This study then suggests there are distinct domains between both of them. It was found that there are two exosites that contribute to cathepsin V elastolytic activity effectiveness. These exosites are located in the surface loop regions. In the study the replacement of exosite I or II with analogous residues from cathepsin L led to 75% loss in elastolytic activity for exosite I and 43% loss for exosite II, when both exosites were replaced it led to a non-elastase variant, similar to cathepsin L. It was found that the hydrophobicity of both exosites increase when going from the appropriate sequences present in cathepsin L to cathepsin V, indicating that hydrophobicity of both exosites is a critical matter for the elastolytic activity that cathepsin V. Plasminogen Plasminogen activation is largely regulated and driven by exosites rather than catalytic sites alone. Plasminogen possesses a closed conformation that limits access to its activation site, making activation into plasmin extremely hard without additional interactions. Plasminogen has kringle domains that are classic exosite-binding regions that allow it to bind to cofactors. These cofactors promote confirmation changes that enhance activation. Exosites are crucial as they act as the main binding site for these cofactors distinct from the active site and induce the conformational change. The binded cofactors enhance substrate affinity and specificity by stabilizing the form of plasminogen and catalyzing the conversion to plasmin. == References ==