CRITERIO RECURSOS HUMANOS

The structure and function of the fibrin(ogen) αC region has been subject to much debate over the past 40 years. (13, 14, 26, 141-145) Originally, Doolittle proposed that these structures were largely unstructured and acted as “free swimming appendages.” (4, 142) This view was supported by earlier circular dichroism studies indicating that the α- C plasmin degradation product was a random coil and by amino acid sequence comparisons revealing a similarity between the αC region and other unstructured proteins. (146, 147) However, this work was contradicted by some calorimetric and EM studies which indicated that part of the α-C region contained compact structures. (143, 148-150) In 1983, Erickson and Fowler showed EM images indicating that the fibrinogen α-C region has some globular structure and is connected to the central region of the molecule.(148) This globular structure was dubbed the αC domain. This work has

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been followed up with further EM studies of fibrinogen, fibrin, and various fragments, which show a structured portion of the αC domain in a high percentage of the molecules imaged. (150, 151) Recent NMR studies have indicated that recombinant truncated variants of both bovine and human fibrinogen αC-domains form beta sheet structure. (14, 152, 153) However, a debate still exists as to whether an ordered structure is present in the fibrin(ogen) αC region due to the lack of an electron density in crystal structures.(13)

EM images of fibrinogen, indicate that the αC domains interact with each other intramolecularly and are positioned near the central region of the molecule.(151) It has now been established that αC domains of fibrin molecules also interact intermoleculary through non-covalent interactions. (73, 152, 154) These interactions are concentration and pH dependent, and a recent laser tweezers study indicates some interactions between αC-domains that could withstand forces of up to 50pN at pH 7.4.(73, 152, 154)

Fibrin αC domains also interact via FXIIIa cross-linking to form networks termed α-polymers. FXIIIa is another blood protein that acts as a transglutaminase, catalyzing the formation of a covalent bond between the free amine group of a Lysine (Lys) and the gamma-carboxamid group of a glutamine (Gln). Each αC domain contains 23 potential Lys donor sites and 6 Gln sites, however not all are equally used in cross-linking. Sobel et al. showed that the primary Lys sites are Lys556 and Lys580, and Cottrell and co- workers indicated that the primary Gln sites are α328 and α366, indicating that some sites are more accessible to FXIIIa than other.(30, 155) The mechanism for this selection does not seem well understood.

To address these issues, we have undertaken a study of the αC region structure using discrete molecular dynamics modeling. We began by searching for a homology

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model of the αC region using I-TASSER.(156, 157) I-TASSER is a web-based protein structure prediction tool, which is able to find homologs and even remote homologs (with relatively low sequence similarity). Using these identified homolog and remote homolog structures, a structural model of the query sequence was built. The model was then energy minimized and thermally unfolded to define the equilibrium structure at various temperatures. The resulting structure, which was not constrained by any of the previous, EM, calorimetry, or NMR data, is yet remarkably consistent with the aforementioned data. The model consists of an unstructured αC connector region, terminating in a β-helix structure which contains the di-sulfide bond. Beyond the β-helix is a second unstructured region, containing the most prevalent Lys donors for cross- linking. This structure is consistent with the binding and cross-linking requirements of the αC region and also suggests a possible mechanism by which FXIIIa can select residues for ligation.

4.2.2 A computationally Identified β helix structure of the fibrin αC region

A series of homology models were generated for amino acids 196-610 of the fibrin αC domain using the online modeling resource I-TASSER.(140) One model of particular interest showed a β-helix structure for the αC region and within the structure Cys442 and Cys472 were within 1nm of each other indicating a potential for di-sulfide bond formation. Following homology modeling, the structure was relaxed using 400 steps. The di-sulfide bond was formed by adding constraint potentials between the Cys442 and Cys472,(158) and the system was allowed to relax for 10ns of DMD simulations at 276K. The system was then allowed to evolve for an additional 10ns of DMD simulations at 300K after di-sulfide formation.(159) Figure 4.1 shows the resulting

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structure after equilibration at 300K along with the homology derived structure. The equilibrium resultant structure displays several remarkable structural features.

In Figure 4.1, the αC region has been separated by color to show several distinct regions. Amino acids 196-240 are seen folding back on each other, however in simulations of the whole fibrinogen molecule where α196 was attached to the 4th coil, the bonding was not present. The repeat region, amino acids 264-391, displays a random coil arrangement, with no discernable fold, in agreement with previous studies.(145) A novel structure is seen between amino acids 392 and 520, where a partial β-helix structure is formed. The helix measures approximately 2nm in diameter, and appears destabilized on the side of the di-sulfide bond. The αC domain then terminates in a second random coil region containing the most active transglutaminase sites, Lys556 and Lys580, which account for 50% of all FXIIIa-catalyzed cross-links.

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Figure 4.1 Structure of the fibrin αC region: A) The homology derived structure. B) The equilibriated structure at 300K. The blue region consists of amino acids 196-220, which are not in the human fibrinogen crystal structure implying an inherent flexibility (12), but have not historically been considered part of the αC region. (13) The orange region consists of amino acids 221-391, typically called the αC connector region. (160) Within this region, amino acids 264-391 contain the series of 10, non-identical (13 amino acid each), repeats. The green region, consisting of amino acids 392-610 is traditionally called the αC domain. The Cys442-Cys472 di-sulfide bond is highlighted in yellow, while all the potential cross-linking sights are highlighted in red (Lysines) and teal (Glutamine). All amino acids noted above are represented in atomic sphere mode, while the structure as a whole is represented in cartoon mode to highlight the beta sheet structures. Lys580 and Lys556 contribute to 50% of αC cross-linking.

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4.2.3 Thermal unfolding of the αC region indicates the relative stability of the β