Structural and functional characterization of human coagulation factor XIII

Abstract

The formation of a fibrin clot in blood plasma is a two-step event which involves formation of a “primary clot” comprising of fibrin polymers by from fibrin monomers under the action of thrombin and subsequently the development of this primary clot into a stronger, insoluble, network structure that plugs the wound and thus prevents bleeding. The second step of this process is mediated by coagulation Factor XIII (FXIII), a pro-transglutaminase circulating in the plasma that covalently crosslinks the aforementioned “primary clot” (within itself and to fibrinolytic inhibitors) thereby preventing premature fibrinolysis of the “primary clot” under the action of fibrinolytic enzymes leading to fatal bleeding eventualities. FXIII, circulates in plasma in the form of non-covalently associated hetero-tetrameric FXIIIA2B2 complex comprising of the catalytic dimeric subunits A (FXIIIA2) combined with the protective/regulatory dimeric subunit B (FXIIIB2. The catalytic FXIIIA2 subunit belongs to a class of enzyme called Transglutaminase (TG; protein-glutamine:amine γ-glutamyltransferase, EC 2.3.2.13), and is responsible for the formation of ε(γ-glutamyl)lysyl crosslinks between the two polypeptide chains. The FXIIIB2 subunit is a protective partner towards the FXIIIA2 subunit dimer in the heterotetramer but more recently regulatory roles for this subunit have also come to light. The zymogenic FXIIIA2B2 complex is activated in the plasma by combination of proteolytic cleavage (thrombin) of an N-terminal region of the FXIIIA2 subunit called the activation peptide followed by binding of Calcium ions to three Ca binding sites on the FXIIIIA2 subunit that result in conformational changes that dissociate the FXIIIB2 subunit and open up the FXIIIA molecule to an open activated FXIIIA form (FXIIIAa). The current thesis picks up from aspects of this hetero-tetrameric complex that are not known i.e. about the individual subunits or the complete complex itself. Then it proceeds in a stepwise manner unravelling these aspects using lab investigations driven by in silico driven hypothesis´s. Naturally, therefore the start of thesis involves primarily in silico cheminformatic work that delves into activation path of the FXIIIA2 subunit and the major structural (like the N-terminal activation peptide of the FXIIIA2 subunit) and physiological partners contributing to it (i.e. cationic ligands like Calcium and partner FXIIIB2). This work revealed some major insights into these aspects which were a) the importance of the activation peptide in the dimeric stability of the FXIIIA2 subunit b) the importance of cross-talk within the Ca binding sites of FXIIIA2 subunit for its activation c) the regulatory role of the FXIIIB2 in accelerating the activation of the FXIIIA2 subunit d) plausible after-events in life cycle of FXIIIA2 subunit post-activation and finally e) the dynamics of assembly and dissociation of the heterotetrameric FXIIIA2B2 complex . However, since most of these insights were at a hypothetical level, the next natural step was to verify them on the bench. While some of the insights from these early investigations were substantiated by bench work done from other groups (like the importance of activation peptide), a majority of the other investigations and their follow ups form the core of this thesis. Therefore subsequent to this early investigation, this thesis delves into a) characterizing the role of Calcium binding sites on activation of FXIIIA2 subunit b) adopt a combined disulfide-bond mutating approach combined with experiment guided model generation to shed some light on the structure and functional aspects of the FXIIIB2 subunit, about which not much is known so far c) run preliminary investigations into possibilities of pleiotropic roles for the FXIIIB2 and finally d) the thesis concludes by presenting a structural all-atom model of the FXIIIA2B2 complex combined with a look into thermodynamic patterns emerging from the assembly and dissociation of this complex. <br /> The characterization of the three major Calcium binding sites in the FXIIIA2 subunit involved a series of in silico exercises probing the relative conservation, cross-talk and interaction with other ions in the physiological system which is combined with mutating the binding sites themselves to corroborate the effect they would have on the activation of this subunit. With this work this thesis clearly drives home the point that a) there is an antagonistic equilibrium between the first and the second-third Calcium binding sites at play that regulates the speed and rate of FXIIIA2 activation b) the thermodynamics underlying FXIIIA2 activation upon Calcium favors the formation of a monomeric and not dimeric activated FXIIIA (FXIIIAa) species and c) the presence of ions regardless of whether they actually co-ordinate with the FXIIIA2 subunit or not can influence the activation status of FXIIIA2 subunit by altering its surface electrostatic properties. Moving onto the structural functional aspects of the FXIIIB2 subunit, this thesis provides the first structural model of the FXIIIB molecule as it presents itself bound to the FXIIIA2 subunit. Using Mass spectrometry based chemical cross-linking data from FXIIIA2B2 complex purified from a plasma based FXIII concentrate, FibrogaminP putative distance restraints between intra-residue contact for the FXIIIB molecule were ascertained. These distance restraints aided by a combination of homology/threading modeling drove the domain assembly and final all atom structure of a monomeric FXIIIB molecule. The structural perspective was combined with mutagenesis work that ablated 20 disulfide bonds that are a consistent feature of the FXIIIB2 subunit. They form the structural framework of the FXIIIB2 molecule by stabilizing the 10 repetitive sushi domains that the whole molecule is comprised of. By investigating the functional aspects of these disulfide mutated variants combined with the structural perspective from above, this thesis was able to define structural functional correlations for this subunit in a manner not touched upon so far. This thesis also investigated the possibility of the FXIIIB2 subunit having pleiotropic roles in the complement system (because of its high homology to some proteins from the complement system) by using a host of mixing as well as pull down assays. However, the thesis clearly determined that physiologically the FXIIIB2 has no role in the complement system. Finally, the thesis takes a detailed structural and functional look at the FXIIIA2B2 complex itself. Once again, the Mass spectrometry based chemical cross-linking data from FXIIIA2B2 complex is used. However, this time instead of intra-residue, inter-residue contact data from the crosslinking is used to define the putative interface residues between the FXIIIA2 and FXIIIB2 subunits in the hetero-tetrameric FXIIIA2B2 complex. This information is then used to drive flexible docking of the FXIIIB molecule model onto the crystal structure of the FXIIIA2 to finally generate the first all atom model for the hetero-tetrameric FXIIIA2B2 complex. This model is also compared by surface fitting exercises to surface scans/ Atomic force microscopy images obtained by contact driven Atomic force microscopy performed on the purified FXIIIA2B2 complex. Therefore, this thesis for the first time provides a structural perspective of entire complex. In addition, an intensive investigation into the association and dissociation of this complex was conducted on an Isothermal titration calorimetry(ITC) platform that yielded a) the first Kd (dissociation constants) values for the FXIIIA2 and FXIIIB2 subunit established in a non-labelled/non-immobilized setting b) the co-operative mode of association followed by these two subunits. Therefore, to sum up, this thesis begins by asking fundamental questions about the complex and its particular subunits and ends by presenting major insights into the structural and functional aspects of both the complex and its subunits. <br /> To conclude, this thesis presents a) Structure-functional basis of FXIII complex activation, and roles of its individual subunits; b) A combinatorial approach for dissection of structure-function aspects of complex derived from plasma, here Factor XIII; c) Potential druggable sites for the generation of new anti-coagulants targeting either FXIII-A calcium binding sites, FXIII-B sushi domains, and most importantly FXIII complex interfaces, which may lead to development of new FXIII inhibitors, or more regulated forms of FXIII, which is a major contributor towards maintaining balance between thrombosis and bleeding.