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===Rad51===
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__NOTOC__
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<Structure load='1szp' size='400' color='white' frame='true' align='left' caption='S. cerevisiae Rad51, pdb:1SZP' />
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===Fibrinogen===
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==Homologous Recombination==
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The process of [http://en.wikipedia.org/wiki/Homologous_recombination homologous recombination]is essential for genomic stability through the high-fidelity repair of DNA double stranded breaks. The recombination event is orchestrated by a family of enzymes called recombinases, which assemble into presynaptic filaments on single stranded DNA. The recombinases then perform a homology search, initiate strand invasion, and ultimately resolve the double stranded break. Humans and yeast share the evolutionarily related recombinase Rad51, a homolog of the well-documented prokaryotic [http://en.wikipedia.org/wiki/RecA RecA]protein.<ref name="Liu">PMID: 21599536</ref>
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''S. cerevisiae'' Rad51 is a 43 kDa protein that shows a remarkable degree of conservation with its human homolog. They share a core ATPase domain as well as an additional N-terminal domain (which is not observed in the bacterial RecA protein), although they lack a C-terminal extension characteristic of RecA. The overall secondary structure of a Rad51 <scene name='User:Chase_Haven/Sandbox_1/Rad51_monomer/1'>monomer</scene> is comprised of 16% beta sheets, with 17 helices totaling up to 38% helical character. Coordination of the DNA binding properties of recombinase protomers directs their assembly into the functional unit of homologous recombination, the presynaptic filament, through nucleotide binding and hydrolysis as well as interactions with recombination mediator and auxiliary proteins.<ref name="Liu" /><ref name="San">PMID: 16388992</ref>
 
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==DNA Binding Properties==
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==Biological Role==
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As Rad51 comprises the paradigm of eukaryotic DNA strand exchange, extensive work has gone into characterizing its very unique DNA binding properties. It has been shown to bind ssDNA and dsDNA, each with two distinct modes.
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The process of haemostasis is crucial to stemming blood loss following vascular injury. It involves a complex balance of pro- and anti-coagulant activities by a multitude of enzymes and cofactors that ultimately lead to a fibrin clot followed by attenuation of the coagulation response to restore normal blood flow. In short, the serine protease thrombin can be thought of as the star player: thrombin cleaves fibrinogen to fibrin (forming the clot) and also activates a trans-glutaminase (Factor XIII) which will create cross-links in the clot to enhance it's tensile strength. Furthermore, upon the formation of the clot thrombin also activates protein C as part of a negative feedback loop, which in turn degrades various cofactors and ultimately shuts down the coagulation response.<ref>PMID:16500122</ref>
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=====ssDNA Binding Modes=====
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Fibrinogen, the chief proteinaceous component of a clot, is a 340 kDa multidomain glycoprotein consisting of <scene name='49/493720/Murica_fbgn/1'>three pairs of peptide chains</scene> termed <scene name='49/493720/Alpha_chains_green/4'>alpha</scene>, <scene name='49/493720/Beta_chains_green/1'>beta</scene> and <scene name='49/493720/Gamma_chains_green/1'>gamma</scene>. It circulates in blood at concentrations generally ranging from 2 to 3 mg/mL.<ref>PMID:19296670</ref> Vascular injury exposes tissue factor-bearing subendothelial cells to flowing blood, triggering a coagulation response in order to generate thrombin. This serine protease has a unique specificity for cleavage sites on fibrinogen alpha and beta chains, and when cleaved fibrinopeptides A and B are released. This results in the exposure of polymerization sites known as "knobs", which are complementary to "holes" on other fibrinogen molecules. This interaction is non-covalent but strong enough to support the spontaneous growth of fibrin fibers and ultimately a fibrin clot network capable of stemming blood loss in vivo.<ref>PMID:20888119</ref>
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At pH 7.5 Rad51 monomers assemble onto ssDNA in the presence of ATP and Mg2+ ions. This occurs with an apparent stoichiometric ratio of 1 protein molecule to 4 nucleotides. This binding capability is lost in the absence of ATP, which contrasts with RecA’s ability to retain ssDNA binding capabilities without ATP. Similarly, pre-incubation of Rad51 with TRIS acetate prevents ssDNA binding if no ATP is present (due to the sequestering of Mg2+). Addition of ATP before this incubation prevents this inactivation. However, addition of ATP subsequent to incubation with TRIS acetate is unable to rescue any DNA binding ability. This signifies the necessity of the nucleotide cofactor with regards to its protective role in preserving Rad51’s ssDNA binding capabilities and preventing inactivation, which is believed to occur due to protein aggregation..<ref name="Zaitseva">PMID: 9915828 </ref>
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The aforementioned binding characteristics are observed over a pH range of 6.8-8.5. However, at a pH lower than 6.8, the binding of ATP and presence of Mg2+ is no longer required. Interestingly, at these acidic pH values presynaptic filament assembly occurs in a different modality. Instead of the 4 nucleotides per Rad51 monomer, as observed at pH 7.5 with ATP and Mg2+, this observed binding mode occurs with a stoichiometry of either 6 or 7 nucleotides per monomer at pH 6.8 or 9 nucleotides per monomer at pH 6.2.<ref name="Zaitseva" />
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Fibrin clots are highly heterogeneous; numerous factors play a role in determining fiber diameter, types of branch points, and number of branching fibers per unit area. Some of these factors are well understood, such as the effects of higher thrombin or fibrinogen concentrations. Others are less understood, such as calcium and metal ion levels, circulating lipid content, coagulation or fibrinolytic protein levels, some studies have even shown smoking and diabetes may change the structural layout or integrity of fibrin clots.<ref>PMID:22759629</ref>
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The stability of these protein-DNA interactions can be evaluated by their resistance to disruption by addition of NaCl. The salt titration midpoint for the modes observed at pH 6.8 and 6.2 are 60 mM and 110 mM NaCl, respectively. Conversely, the salt titration midpoint for the binding mode observed at pH 7.5 in the presence of ATP and Mg2+ is 550 mM NaCl. This demonstrates that decreasing pH stabilizes the Rad51-ssDNA interaction when no nucleotide cofactor or Mg2+ is present, but also further conveyed the importance of ATP to proper filament assembly at pH values closer to physiological levels. Thus Rad51 exhibits two distinct and non-inter-convertible binding modes for ssDNA: a highly stable ATP-dependent one, and a weaker, ATP-independent manner.<ref name="Zaitseva" />
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<StructureSection load='3ghg' size='400' side='right' caption='Human Fibrinogen (PDB entry [[3ghg]])' scene=''>
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=====dsDNA Binding Modes=====
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==Basic Crystallographic Features==
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The binding of Rad51 to dsDNA has been shown to be Mg2+ dependent; even with the presence of ATP, binding to dsDNA is quite poor at concentrations less than 3 mM. Increasing this concentration leads to improved binding, with saturating conditions at approximately 10 mM Mg2+. The binding stoichiometry of this mode is similar to that observed for ATP/ Mg2+ dependent ssDNA binding: approximately 1 Rad51 monomer per 4 or 5 nucleotides. This binding mode occurs over a wide range of pH values. This contrasts with the bacterial RecA protein, which binds to dsDNA so slowly under neutral or basic conditions that it is virtually negligible. Rad51 is therefore less subject to pH-induced inhibition of filament assembly, while the nucleation step for RecA remains a rate-limiting factor that is much more sensitive to pH changes.<ref name="Zaitseva" />
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The heterogeneity of fibrin clots make them virtually impossible to crystallize, and fibrinogen itself has intrinsic properties that enabled it to elude crystallization efforts until 2009. Some of the interesting aspects of this crystal are discussed below.
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In the absence of both ATP and Mg2+ dsDNA binding by Rad51 becomes pH dependent and displays a different stoichiometry. The formation of Rad51-dsDNA complexes under these conditions is not detected at pH 7.5, but is observed under more acidic conditions with protomer to nucleotide ratio of 1:6. This diverges further from the properties of RecA, whose dsDNA binding ability is greatly enhanced in acidic conditions even with a low concentration of Mg2+.<ref name="Zaitseva" />
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=====Coiled-Coil Domains=====
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The coiled-coil domains separate the two outer D domains from the central globular E domain. These coils are mostly helical, with a notable exception being the “out-loop” in gamma chains residues <scene name='49/493720/G70-76/1'>70-76</scene>. Certain areas also have runs of polar amino acids, coinciding with a pronounced bending at this location. Three notable runs include Alpha: <scene name='49/493720/A99-107_ball_and_stick/2'>99-107</scene>, Beta: <scene name='49/493720/B126-131_ball_and_stick/1'>126-131</scene>, and Gamma: <scene name='49/493720/G69-77_ball_and_stick/1'>69-77</scene>. These non-helical regions share structural similarity and conservation with that of previously-crystalized chicken fibrinogen, even though they have strikingly different amino acid sequences.<ref>PMID:19296670</ref>
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The formation of the presynaptic filament on ssDNA is a crucial step in homologous recombination, which makes the dsDNA binding properties of Rad51 appear deleterious to its primary function. The low rate of ATP hydrolysis by Rad51 likely plays a role in the formation of dsDNA-nucleoprotein filaments, where the ATPase activity experiences up to a 10-fold reduction. This reduction corresponds to a much more stable interaction, as ATP hydrolysis has been shown to promote filament disassembly and redistribution.<ref name="Liu" /><ref name="Zaitseva" />
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It has been proposed that the coiled coils are the source of (at least in part) fibrinogen's inherent "floppiness". The non-helical stretches may act as flexible hinges interspersed with the more rigid helical components. This also fits with data regarding the compressible and stretchable nature of fibrin gels. Also fitting to this "hinge hypothesis", are two known mutations that cause defective fibrin formation occur in the polar (nonhelical) regions.<ref>PMID:19296670</ref>
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Furthermore, filament assembly by Rad51 has been shown to absolutely require RPA. RPA is a ssDNA binding protein that prevents the formation of secondary structures. It is believed that without RPA, Rad51 binds to and stabilizes secondary structures formed within ssDNA due to its dsDNA binding capabilities, and thus hinders the process of homologous recombination. This illustrates the limitations of the eukaryotic Rad51 recombinases in catalyzing a recombination event. However, the high affinity of RPA for ssDNA poses a rate-limiting barrier for Rad51 filament assembly. RPA must be displaced by Rad51, which does not occur to an appreciable level without the aid of mediator proteins such as Rad52, BRCA2, or Rad51 paralogs.<ref name="Liu" /><ref name="Beernick">PMID: 9548953</ref>
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=====Indiscernible Regions=====
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The elusive alpha-C termini regions were unable to be resolved. This was actually expected, as these regions are thought to be intrinsically disordered.<ref>PMID:19296670</ref> A study in 2006 used the PONDR VL-XT algorithm (Predictor Of Natural Disordered Regions)and found that of the 866 residues of fibrinogen, 397 or 46% were predicted to be disordered. The longest stretch being 129 consecutive disordered residues. Large portions of these figures correspond with the alpha-C terminal regions. Interestingly, these regions have been shown to be involved in binding numerous proteins including those involved in coagulation (FXIII, other fibrin molecules) fibrinolysis (plasminogen, tissue-plasminogen activator), and others.<ref>PMID:16939197</ref> The intrinsic disorder could be beneficial for fibrinogen by allowing a broad binding diversity, while also stabilizing the protein in a variety of trauma-induced scenarios such as hypothermia and acidosis. The results of this algorithm also coincide with, up until the past decade, the distinct lack of available structural data concerning the different domains of fibrinogen, and likely contribute to the difficulty of crystalization attempts.
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As it pertains to this crystal structure, it is also possible that these portions of the molecule were proteolytically removed sometime during purification, as has been observed in the literature. N-terminal segments of the alpha and B chains (source of fibrinopeptides) in the central globular region were also unresolved, but are known to be mobile.<ref>PMID:19296670</ref>
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==Insights from Crystal Structures==
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=====Cation-Pi Interactions=====
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Despite extensive biochemical characterization of Rad51, our understanding of its role in homologous recombination has been limited by a lack of structural data regarding the presynaptic filament. The recent solving of crystal structures for yeast Rad51 nuceloprotein filaments has provided numerous insights into the nature of the <scene name='User:Chase_Haven/Sandbox_1/Important_residues1/1'>interactions</scene> that mediate filament assembly as well as nucleotide binding and hydrolysis.
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When ionic interactions are evaluated in the gaseous phase, they are incredibly strong. However, in aqueous media the water molecules negates some of the advantage of an electrostatic interaction due to the energy expense required to shed the solvation shell surrounding each of the charged molecules in the bridge. Conversely, cation-pi interactions do not have such an extensive penalty to pay and can thus be highly favorable. In fibgrinogen there are an estimated 94 energetically significant cation-pi interactions, providing a considerable amount of stability to the molecule. The strongest interaction occurs between <scene name='49/493720/Cation_pi/1'>Lys380 and Trp253</scene> of the gamma chain, providing an interaction energy of -7.24 kcal/mol. Of those that are energetically significant, 13 cation-pi interactions are between arginine and phenylalanine, 27 between arginine and tryptophan, and only 7 between arginine and tyrosine. There are also 12 between lysine and phenylalanine, 15 for lysine to tyrosine, and 20 between lysine and tryptophan.<ref>PMID:10449714</ref>
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=====His-352=====
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=====Disulfide Bonds=====
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Notably, the nucleotide binding pocket of one protomer is in direct contact with the ATPase domain of the adjacent monomer.<ref name="Conway">PMID: 15235592</ref> H352 is a highly conserved residue among in Rad51 homologs and certain other recombinases, and was found in close proximity to the phosphate binding loop within the ATPase domain of Rad51. It protrudes out from an α-helix and hovers directly over the ATPase domain of the subsequent protomer. This finding has implicated its involvement in protein-protein interactions that may mediate filament assembly. The equivalent residue in RecA is F217, which is known to participate in allosteric communication in RecA nucleoprotein filaments.<ref name="Conway" /><ref name="Chen">PMID: 20371520 </ref>
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Each fibrinogen has <scene name='49/493720/Disulfide_bonds/1'>29 disulfide</scene> bonds and no free thiol groups. All of these are considered structural; they hold the six peptide chains together and are crucial for proper folding and assembly.<ref>PMID:8276866</ref> There are three obvious clusters of disulfide bonds in the molecule, located at the N-terminal region, C-terminal region and in an intermediate location. In the central E domain containing the N-termini of the chains, a series of five symmetrical disulfide bridges secure it's globular structure. In the alpha chain two reside at Aalpha28 and Aalpha36, in the Bbeta chain at position 65, and two more in the gamma chain at positions 8 and 9. Since the two at the gamma chain are reciprocal, they orient the chains in an antiparallel manner. Other non-symmetrical inter-chain disulfides in the E domain form what has been referred to as a disulfide "ring."<ref>PMID:19296670</ref><ref>PMID:8276866</ref>
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The critical importance of H352 residue has been demonstrated by several studies which showed that a H352A mutation produces a marked decrease in ssDNA binding ability.<ref name="Conway" /><ref name="Chen" /><ref name="Arabela">PMID: 19066203 </ref> In contrast, a H352Y mutation resulted in a constitutive ssDNA binder, but this mutant was unable to catalyze strand exchange in vitro due to a defect in nucleotide exchange and an inability to displace RPA.<ref name="Chen" /> These mutational studies further demonstrated the importance of inter-subunit communication via ATP hydrolysis and exchange, and demonstrated the crucial role residue H352 plays in this in these interactions.
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==The interaction of Fibrinogen with Thrombin==
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<scene name='49/493720/Fbgn_and_iia/1'>Thrombin surface with FPA</scene>
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=====N-terminal Domain=====
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Proteolytic cleavage of fibrinopeptide A, the N-terminal sixteen amino acid residues of fibrinogen alpha chains, initiates fibrin assembly and the formation of a clot. This reaction is performed by thrombin, a trypsin-like serine protease. Unlike trypsin however, thrombin shows a remarkable specificity for fibrinogen; a highly unique quality that is in contrast with the very broad substrate specificity of other canonical serine proteases. Of the 376 arginine or lysines located in fibrinogen, thrombin only cleaves four (corresponding with the two each of fibrinopeptide A and B).<ref>PMID:6052745</ref> A crystal structure depicting a ternary complex of thrombin with synthetic analogs of fibrinopeptide A and hirudin’s C-terminal domain was produced in attempt to elucidate the interactions necessary for the enzyme’s specificity.<ref>PMID:1587268</ref>
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The <scene name='User:Chase_Haven/Sandbox_1/N-terminal_domain/1'>N-terminal domain </scene> of Rad51 has been shown to bind DNA, <ref name="Liu" /><ref name="Chen" /><ref name="Aihara">PMID: 10390347</ref> and is thought to have significant disordered character.<ref name="Aihara" /> It contains a conserved glycine residue at position 103, although this is not shared by the ''Drosophila melanogaster'' Rad51 protein. Mutation of this residue to glutamate results in a greatly reduced ability to bind both ssDNA as well as dsDNA. This defect then leads to a significant reduction in ATPase activity. G103 lies at the surface of the N-terminal domain facing the core ATPase site, yet crystal structures show that G103 is removed from the NTP binding site, so there is no direct interaction that could explain the loss of ATPase activity.<ref name="Conway" />
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In particular, G103 is adjacent to three residues which form a positively charged patch: R260, H302 and K305. Molecular modeling studies show that these residues are close enough to potentially form charge interactions when glycine is mutated to glutamate at position 103. It is believed that the G103E mutant is thus able to statically interact with these positively charged residues, which would greatly restrict the flexibility of the N-terminal domain and freeze Rad51 in an inactive state that can no longer bind DNA or ATP. In this respect the flexibility of glycine preserves the overall plasticity of the N-terminal domain, thus allowing formation of a proper filament.<ref name="Zhang">PMID: 15908697 </ref>
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The fibrinopeptide A analog showed little secondary structure, although the sections that did demonstrate some important features about the interaction of thrombin with fibrinogen. A short helical sequence of residues 7-10 show a hydrogen bond between the carboxylate oxygen of Asp7 to the amide nitrogen of Ala10. This alternative hydrogen bonding pattern is not characteristic of alpha helices, and is thought to help give rise to the chain reversal occurring after Ala10, which is further stabilized by main-chain hydrogen bonds in the brief helical region, the subsequent amino acids 11 and 12.<ref>PMID:1587268</ref>
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The crystal structures also revealed contacts between highly conserved residues Y112 and Y253. Y112 is found in the N-terminal domain, and stacks with the Y112 residue of the adjacent protomer. Y253 is found in the ATPase domain, further explicating the link between nucleation and the Rad51 ATPase cycle.<ref name="Conway" />
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This folding pattern facilitates the the formation of a <scene name='49/493720/Basic_hydrophobic_patch1/2'>hydrophobic patch</scene> consisting of the side chains of Phe8, Leu9 and Val15. Analysis of this regions shows a considerable number of contacts occur as this hydrophobic face inserts into the apolar binding site on thrombin. There are however, 3 hydrogen bonds that form a canonical beta-ladder, which are believed to orient substrates into the binding site of the protease. In this case, the antiparallel beta ladder occurs between Gly14 of FPA and Gly216 of thrombin. Other polar contacts include a number of ionic interactions between <scene name='49/493720/Glu11_arg173_intrxn/1'>Arg173 of thrombin and Glu11.</scene> These are believed to also facilitate proper substrate orientation into the active site.<ref>PMID:1587268</ref>
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R357 is another highly conserved residue that resides at this protomer interface. It forms an ion pair with a conserved glutamate residue, E182, which packs it against that α-helix containing H352. Mutation of R357 has been shown to greatly impair Rad51’s ability to catalyze strand exchange and its ATPase activity. However, both of these activities are not fully eliminated. This demonstrates the importance of R357 not necessarily as a catalytically relevant ATPase residue, such as that seen in trans-acting arginine fingers, but as an ATP sensor.<ref name="Arabela" /> In this respect R357 determines the state of nucleotide binding, and may translate that signal to allow for ATP-dependent polymerization via conformational changes into an active presynaptic filament.
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Sequence comparisons of fibrinopeptide A with different species show conservation of the basic features of their interactions with thrombin. Residues 8 and 9, which form part of the hydrophobic patch, are well conserved and only found to be replaced with other hydrophobic residues. Residue ten (site of chain reversal) makes no contacts with the enzyme and therefore can accomadate several alternative amino acids. The highly conserved glycine at position 12 illustrates the importance of it’s rotational flexibility. In order for the peptide to fold back into the active site following this residue, the peptide has to adopt dihedral angles that no other amino acids are capable of.<ref>PMID:2521950</ref>
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==Evolutionary Conservation==
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Looking at the binding of the C-terminal <scene name='49/493720/Basic_hirudin_binding/1'>hirudin</scene> analog, we see an extended chain with a short 3-10 helix. It’s interaction with thrombin shows numerous ionic contacts with residues 54-65 of hirudin to the anion-binding site of the protease, which is flanked by two hydrophobic faces. Despite the large number of ionic amino acids in this region there are relatively few hydrogen or ionic bonds; rather binding may be mediated more by their complementary structure. The sequence of the fibrinogen alpha chain from residues 35-43 is believed to contain these necessary features, as kinetic studies show that this region is important for optimum enzymatic efficiency. Therefore, it is hypothesized that there are numerous interactions of fibrinogen with thrombin over a rather large area of both molecules, enabling for the incredible specificity of the protease to its substrate.<ref>PMID:1587268</ref>
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Rad51 belongs to the RecA family of recombinases, which all share distinct regions of conservation. Rad51 and RecA contain a highly conserved core ATPase domain, yet there are prominent differences as well. RecA contains a C-terminal domain which Rad51 lacks, while on the other hand Rad51 contains a N-terminal domain which RecA does not posess. Various studies have demonstrated functional similarities between these two domains, most notably that they both bind DNA.<ref name="Aihara" /><ref name="Aihara2">PMID: 9398528</ref> It has also been suggested that both contain regions of disorder; approximately 25 residues in the C-terminus of RecA have not been visualized in crystal structures,and neither have 15 N-terminal residues in human Rad51.<ref name="Aihara" /> Despite these similarities, they domains do not share any significant degree of sequence or structural homology.<ref name="Vitold">PMID: 16765891</ref>
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</StructureSection>
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==Sources of Flexibility==
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The function of fibrinogen requires an intrinsic level of flexibility; this protein has to form a dense yet resilient network that can withstand shear forces within the vasculature. A clot must be able to close gaps caused by injury, and for that reason the clot components must be capable of assuming a variety of shapes. Several studies, many utilizing atomic force microscopy, have demonstrated a remarkable ability of fibrinogen and fibrin fibers to stretch and bend to an extreme extent: under some conditions, nearly an additional 30% of it’s resting state length.<ref>PMID:20888119</ref> A lot of work has been undertaken in order to better understand the origin this remarkable ability in the molecule and the clots that it makes up.
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=====D-D Interface=====
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One group analyzed their crystal structure of a fibrin D-Dimer that had been crosslinked by factor XIII. The junction between these two globular regions was not found to be tight; there were no salt bridges and minimal hydrogen bonding. In fact, there appeared to be a layer of solvent between them.<ref>PMID:9333233</ref> This makes sense for the formation of a dynamic fibrin network; as too strong of an end to end interaction would make the clot too rigid. Also interesting about this particular crystal structure was the location of the gamma-gamma cross-link between the D regions: they weren’t visible. Density could not attribute these to a single location, meaning that the covalent bond between E396 and Q398 was highly mobile. This mobility was also observed by another group, who noted that there were several possible orientations in D-Dimer crystals. Taken altogether this provides evidence for the existence of weak interactions at the D-D interface that can slide around and “play,” potentially lending flexibility to the overall network.<ref>PMID:9333233</ref><ref>PMID:20888119</ref>
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=====A:a Interaction=====
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The knob-hole interaction responsible for fibrin polymerization is non-covalent, but incredibly strong. In fact, it has been shown that regions of the protein actually unfold due to stress before this interaction is broken. A group found that when force was applied to small fibrin polymers, a stepwise unfolding of the gamma nodule occurred with increasing force prior to rupture of the knob-hole interaction. This means that the interaction is sufficiently strong and will persist as the fiber experiences localized unfolding that accounts for fiber stretching. The in-vivo role of this may not be significant however, as FXIII cross-links gamma chains diametrically opposite of the knob-hole interactions. The strength of this covalent bond (despite being mobile) would likely circumvent the utility of stretching due to gamma-nodule unfolding.<ref>PMID:20888119</ref>
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=====Alpha-c Regions=====
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As previously mentioned, many studies have observed an inherent flexibility of fibrin fibers as well as individual molecules themselves, characteristic of other protein fibers such as elastin and spider silk. Some researchers have hypothesized that these mechanical properties may stem from the phenomena of straightening of unstructured peptide sequences. Fibrin contains two regions per molecule that possess such a significant amount of intrinsic disorder: the elusive alpha-c regions. It is already accepted that these regions play a key role in determining some of the mechanical properties of fibrin clots through work with recombinant protein lacking these regions, but more recent studies are pointing to specific portions of these regions and relating them to the extensibility of the fibers themselves.<ref>PMID:20888119</ref><ref>PMID:16091450</ref>
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In particular, the length of tandem repeats in the alpha-c regions varies from species to species: low in chicken fibrinogen, which has no repeats, to high in lampreys which have 20 repeats of 18 residues.<ref>PMID:8497848</ref><ref>PMID:16288455</ref><ref>PMID:2611265</ref> Interestingly, the amino acid content of the repeats seen in lamprey (as well as human and others) are reminiscent of the sequences seen in previously mentioned protein fibers like elastin.<ref>PMID:11911780</ref>
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Further supporting a potential role for these tandem repeats was a study that looked at fibrin extensibility from a variety of species and found that the length of the repeating sequence correlated well. Chicken fibrinogen (no repeats) had an extensibility of 47%, mouse fibrinogen (5 repeats of ~13 residues each) could extend 187%, and that of human (10 repeats of ~13 residues each) could be extended 217%. This significant finding clearly supports the hypothesis that the alpha-c regions provide a structural basis for fiber flexibility, but it is limited in it’s interpretative value. For example, there are other significant differences in sequence and structure in the different species of fibrinogen, including even the terminal region of the alpha-c chain, not just the tandem repeats.<ref>PMID:18761721</ref><ref>PMID:8497848</ref>
==References==
==References==
<references/>
<references/>

Current revision

Fibrinogen

Biological Role

The process of haemostasis is crucial to stemming blood loss following vascular injury. It involves a complex balance of pro- and anti-coagulant activities by a multitude of enzymes and cofactors that ultimately lead to a fibrin clot followed by attenuation of the coagulation response to restore normal blood flow. In short, the serine protease thrombin can be thought of as the star player: thrombin cleaves fibrinogen to fibrin (forming the clot) and also activates a trans-glutaminase (Factor XIII) which will create cross-links in the clot to enhance it's tensile strength. Furthermore, upon the formation of the clot thrombin also activates protein C as part of a negative feedback loop, which in turn degrades various cofactors and ultimately shuts down the coagulation response.[1]

Fibrinogen, the chief proteinaceous component of a clot, is a 340 kDa multidomain glycoprotein consisting of termed , and . It circulates in blood at concentrations generally ranging from 2 to 3 mg/mL.[2] Vascular injury exposes tissue factor-bearing subendothelial cells to flowing blood, triggering a coagulation response in order to generate thrombin. This serine protease has a unique specificity for cleavage sites on fibrinogen alpha and beta chains, and when cleaved fibrinopeptides A and B are released. This results in the exposure of polymerization sites known as "knobs", which are complementary to "holes" on other fibrinogen molecules. This interaction is non-covalent but strong enough to support the spontaneous growth of fibrin fibers and ultimately a fibrin clot network capable of stemming blood loss in vivo.[3]

Fibrin clots are highly heterogeneous; numerous factors play a role in determining fiber diameter, types of branch points, and number of branching fibers per unit area. Some of these factors are well understood, such as the effects of higher thrombin or fibrinogen concentrations. Others are less understood, such as calcium and metal ion levels, circulating lipid content, coagulation or fibrinolytic protein levels, some studies have even shown smoking and diabetes may change the structural layout or integrity of fibrin clots.[4]

Human Fibrinogen (PDB entry 3ghg)

Drag the structure with the mouse to rotate

Sources of Flexibility

The function of fibrinogen requires an intrinsic level of flexibility; this protein has to form a dense yet resilient network that can withstand shear forces within the vasculature. A clot must be able to close gaps caused by injury, and for that reason the clot components must be capable of assuming a variety of shapes. Several studies, many utilizing atomic force microscopy, have demonstrated a remarkable ability of fibrinogen and fibrin fibers to stretch and bend to an extreme extent: under some conditions, nearly an additional 30% of it’s resting state length.[20] A lot of work has been undertaken in order to better understand the origin this remarkable ability in the molecule and the clots that it makes up.

D-D Interface

One group analyzed their crystal structure of a fibrin D-Dimer that had been crosslinked by factor XIII. The junction between these two globular regions was not found to be tight; there were no salt bridges and minimal hydrogen bonding. In fact, there appeared to be a layer of solvent between them.[21] This makes sense for the formation of a dynamic fibrin network; as too strong of an end to end interaction would make the clot too rigid. Also interesting about this particular crystal structure was the location of the gamma-gamma cross-link between the D regions: they weren’t visible. Density could not attribute these to a single location, meaning that the covalent bond between E396 and Q398 was highly mobile. This mobility was also observed by another group, who noted that there were several possible orientations in D-Dimer crystals. Taken altogether this provides evidence for the existence of weak interactions at the D-D interface that can slide around and “play,” potentially lending flexibility to the overall network.[22][23]

A:a Interaction

The knob-hole interaction responsible for fibrin polymerization is non-covalent, but incredibly strong. In fact, it has been shown that regions of the protein actually unfold due to stress before this interaction is broken. A group found that when force was applied to small fibrin polymers, a stepwise unfolding of the gamma nodule occurred with increasing force prior to rupture of the knob-hole interaction. This means that the interaction is sufficiently strong and will persist as the fiber experiences localized unfolding that accounts for fiber stretching. The in-vivo role of this may not be significant however, as FXIII cross-links gamma chains diametrically opposite of the knob-hole interactions. The strength of this covalent bond (despite being mobile) would likely circumvent the utility of stretching due to gamma-nodule unfolding.[24]

Alpha-c Regions

As previously mentioned, many studies have observed an inherent flexibility of fibrin fibers as well as individual molecules themselves, characteristic of other protein fibers such as elastin and spider silk. Some researchers have hypothesized that these mechanical properties may stem from the phenomena of straightening of unstructured peptide sequences. Fibrin contains two regions per molecule that possess such a significant amount of intrinsic disorder: the elusive alpha-c regions. It is already accepted that these regions play a key role in determining some of the mechanical properties of fibrin clots through work with recombinant protein lacking these regions, but more recent studies are pointing to specific portions of these regions and relating them to the extensibility of the fibers themselves.[25][26]

In particular, the length of tandem repeats in the alpha-c regions varies from species to species: low in chicken fibrinogen, which has no repeats, to high in lampreys which have 20 repeats of 18 residues.[27][28][29] Interestingly, the amino acid content of the repeats seen in lamprey (as well as human and others) are reminiscent of the sequences seen in previously mentioned protein fibers like elastin.[30]

Further supporting a potential role for these tandem repeats was a study that looked at fibrin extensibility from a variety of species and found that the length of the repeating sequence correlated well. Chicken fibrinogen (no repeats) had an extensibility of 47%, mouse fibrinogen (5 repeats of ~13 residues each) could extend 187%, and that of human (10 repeats of ~13 residues each) could be extended 217%. This significant finding clearly supports the hypothesis that the alpha-c regions provide a structural basis for fiber flexibility, but it is limited in it’s interpretative value. For example, there are other significant differences in sequence and structure in the different species of fibrinogen, including even the terminal region of the alpha-c chain, not just the tandem repeats.[31][32]

References

  1. ↑ Mann KG, Brummel-Ziedins K, Orfeo T, Butenas S. Models of blood coagulation. Blood Cells Mol Dis. 2006 Mar-Apr;36(2):108-17. Epub 2006 Feb 23. PMID:16500122 doi:http://dx.doi.org/10.1016/j.bcmd.2005.12.034
  2. ↑ Kollman JM, Pandi L, Sawaya MR, Riley M, Doolittle RF. Crystal structure of human fibrinogen. Biochemistry. 2009 May 12;48(18):3877-86. PMID:19296670 doi:10.1021/bi802205g
  3. ↑ Falvo MR, Gorkun OV, Lord ST. The molecular origins of the mechanical properties of fibrin. Biophys Chem. 2010 Nov;152(1-3):15-20. doi: 10.1016/j.bpc.2010.08.009. PMID:20888119 doi:http://dx.doi.org/10.1016/j.bpc.2010.08.009
  4. ↑ Wolberg AS. Determinants of fibrin formation, structure, and function. Curr Opin Hematol. 2012 Sep;19(5):349-56. doi: 10.1097/MOH.0b013e32835673c2. PMID:22759629 doi:http://dx.doi.org/10.1097/MOH.0b013e32835673c2
  5. ↑ Kollman JM, Pandi L, Sawaya MR, Riley M, Doolittle RF. Crystal structure of human fibrinogen. Biochemistry. 2009 May 12;48(18):3877-86. PMID:19296670 doi:10.1021/bi802205g
  6. ↑ Kollman JM, Pandi L, Sawaya MR, Riley M, Doolittle RF. Crystal structure of human fibrinogen. Biochemistry. 2009 May 12;48(18):3877-86. PMID:19296670 doi:10.1021/bi802205g
  7. ↑ Kollman JM, Pandi L, Sawaya MR, Riley M, Doolittle RF. Crystal structure of human fibrinogen. Biochemistry. 2009 May 12;48(18):3877-86. PMID:19296670 doi:10.1021/bi802205g
  8. ↑ Cheng Y, LeGall T, Oldfield CJ, Dunker AK, Uversky VN. Abundance of intrinsic disorder in protein associated with cardiovascular disease. Biochemistry. 2006 Sep 5;45(35):10448-60. PMID:16939197 doi:http://dx.doi.org/10.1021/bi060981d
  9. ↑ Kollman JM, Pandi L, Sawaya MR, Riley M, Doolittle RF. Crystal structure of human fibrinogen. Biochemistry. 2009 May 12;48(18):3877-86. PMID:19296670 doi:10.1021/bi802205g
  10. ↑ Gallivan JP, Dougherty DA. Cation-pi interactions in structural biology. Proc Natl Acad Sci U S A. 1999 Aug 17;96(17):9459-64. PMID:10449714
  11. ↑ Zhang JZ, Redman CM. Role of interchain disulfide bonds on the assembly and secretion of human fibrinogen. J Biol Chem. 1994 Jan 7;269(1):652-8. PMID:8276866
  12. ↑ Kollman JM, Pandi L, Sawaya MR, Riley M, Doolittle RF. Crystal structure of human fibrinogen. Biochemistry. 2009 May 12;48(18):3877-86. PMID:19296670 doi:10.1021/bi802205g
  13. ↑ Zhang JZ, Redman CM. Role of interchain disulfide bonds on the assembly and secretion of human fibrinogen. J Biol Chem. 1994 Jan 7;269(1):652-8. PMID:8276866
  14. ↑ Blomback B, Blomback M, Hessel B, Iwanaga S. Structure of N-terminal fragments of fibrinogen and specificity of thrombin. Nature. 1967 Sep 30;215(5109):1445-8. PMID:6052745
  15. ↑ Stubbs MT, Oschkinat H, Mayr I, Huber R, Angliker H, Stone SR, Bode W. The interaction of thrombin with fibrinogen. A structural basis for its specificity. Eur J Biochem. 1992 May 15;206(1):187-95. PMID:1587268
  16. ↑ Stubbs MT, Oschkinat H, Mayr I, Huber R, Angliker H, Stone SR, Bode W. The interaction of thrombin with fibrinogen. A structural basis for its specificity. Eur J Biochem. 1992 May 15;206(1):187-95. PMID:1587268
  17. ↑ Stubbs MT, Oschkinat H, Mayr I, Huber R, Angliker H, Stone SR, Bode W. The interaction of thrombin with fibrinogen. A structural basis for its specificity. Eur J Biochem. 1992 May 15;206(1):187-95. PMID:1587268
  18. ↑ Mosesson MW, Siebenlist KR, Amrani DL, DiOrio JP. Identification of covalently linked trimeric and tetrameric D domains in crosslinked fibrin. Proc Natl Acad Sci U S A. 1989 Feb;86(4):1113-7. PMID:2521950
  19. ↑ Stubbs MT, Oschkinat H, Mayr I, Huber R, Angliker H, Stone SR, Bode W. The interaction of thrombin with fibrinogen. A structural basis for its specificity. Eur J Biochem. 1992 May 15;206(1):187-95. PMID:1587268
  20. ↑ Falvo MR, Gorkun OV, Lord ST. The molecular origins of the mechanical properties of fibrin. Biophys Chem. 2010 Nov;152(1-3):15-20. doi: 10.1016/j.bpc.2010.08.009. PMID:20888119 doi:http://dx.doi.org/10.1016/j.bpc.2010.08.009
  21. ↑ Spraggon G, Everse SJ, Doolittle RF. Crystal structures of fragment D from human fibrinogen and its crosslinked counterpart from fibrin. Nature. 1997 Oct 2;389(6650):455-62. PMID:9333233 doi:http://dx.doi.org/10.1038/38947
  22. ↑ Spraggon G, Everse SJ, Doolittle RF. Crystal structures of fragment D from human fibrinogen and its crosslinked counterpart from fibrin. Nature. 1997 Oct 2;389(6650):455-62. PMID:9333233 doi:http://dx.doi.org/10.1038/38947
  23. ↑ Falvo MR, Gorkun OV, Lord ST. The molecular origins of the mechanical properties of fibrin. Biophys Chem. 2010 Nov;152(1-3):15-20. doi: 10.1016/j.bpc.2010.08.009. PMID:20888119 doi:http://dx.doi.org/10.1016/j.bpc.2010.08.009
  24. ↑ Falvo MR, Gorkun OV, Lord ST. The molecular origins of the mechanical properties of fibrin. Biophys Chem. 2010 Nov;152(1-3):15-20. doi: 10.1016/j.bpc.2010.08.009. PMID:20888119 doi:http://dx.doi.org/10.1016/j.bpc.2010.08.009
  25. ↑ Falvo MR, Gorkun OV, Lord ST. The molecular origins of the mechanical properties of fibrin. Biophys Chem. 2010 Nov;152(1-3):15-20. doi: 10.1016/j.bpc.2010.08.009. PMID:20888119 doi:http://dx.doi.org/10.1016/j.bpc.2010.08.009
  26. ↑ Collet JP, Moen JL, Veklich YI, Gorkun OV, Lord ST, Montalescot G, Weisel JW. The alphaC domains of fibrinogen affect the structure of the fibrin clot, its physical properties, and its susceptibility to fibrinolysis. Blood. 2005 Dec 1;106(12):3824-30. Epub 2005 Aug 9. PMID:16091450 doi:http://dx.doi.org/10.1182/blood-2005-05-2150
  27. ↑ Murakawa M, Okamura T, Kamura T, Shibuya T, Harada M, Niho Y. Diversity of primary structures of the carboxy-terminal regions of mammalian fibrinogen A alpha-chains. Characterization of the partial nucleotide and deduced amino acid sequences in five mammalian species; rhesus monkey, pig, dog, mouse and Syrian hamster. Thromb Haemost. 1993 Apr 1;69(4):351-60. PMID:8497848
  28. ↑ Doolittle RF, Kollman JM. Natively unfolded regions of the vertebrate fibrinogen molecule. Proteins. 2006 May 1;63(2):391-7. PMID:16288455 doi:http://dx.doi.org/10.1002/prot.20758
  29. ↑ Wang YZ, Patterson J, Gray JE, Yu C, Cottrell BA, Shimizu A, Graham D, Riley M, Doolittle RF. Complete sequence of the lamprey fibrinogen alpha chain. Biochemistry. 1989 Dec 12;28(25):9801-6. PMID:2611265
  30. ↑ Tatham AS, Shewry PR. Comparative structures and properties of elastic proteins. Philos Trans R Soc Lond B Biol Sci. 2002 Feb 28;357(1418):229-34. PMID:11911780 doi:http://dx.doi.org/10.1098/rstb.2001.1031
  31. ↑ Falvo MR, Millard D, O'Brien ET 3rd, Superfine R, Lord ST. Length of tandem repeats in fibrin's alphaC region correlates with fiber extensibility. J Thromb Haemost. 2008 Nov;6(11):1991-3. doi: 10.1111/j.1538-7836.2008.03147.x., Epub 2008 Aug 28. PMID:18761721 doi:http://dx.doi.org/10.1111/j.1538-7836.2008.03147.x
  32. ↑ Murakawa M, Okamura T, Kamura T, Shibuya T, Harada M, Niho Y. Diversity of primary structures of the carboxy-terminal regions of mammalian fibrinogen A alpha-chains. Characterization of the partial nucleotide and deduced amino acid sequences in five mammalian species; rhesus monkey, pig, dog, mouse and Syrian hamster. Thromb Haemost. 1993 Apr 1;69(4):351-60. PMID:8497848

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