User:Chase Haven/Sandbox 1
From Proteopedia
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==DNA Binding Properties== | ==DNA Binding Properties== | ||
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=====ssDNA Binding Modes===== | =====ssDNA Binding Modes===== | ||
| - | 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> | ||
| - | 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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| - | 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" /> | ||
=====dsDNA Binding Modes===== | =====dsDNA Binding Modes===== | ||
| - | 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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| - | 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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| - | 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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| - | 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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| - | ==Insights from Crystal Structures== | ||
| - | 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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| - | =====His-352===== | ||
| - | 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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| - | 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. | ||
=====Arg-357===== | =====Arg-357===== | ||
| - | 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. | ||
=====N-terminal Domain===== | =====N-terminal Domain===== | ||
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" /> | 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 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" /> | ||
==Evolutionary Conservation== | ==Evolutionary Conservation== | ||
| - | 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, and their presynaptic filaments also show a strong degree of structural homology.<ref name="Liu" /> Yet there are prominent differences worth noting 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> Interestingly, as you consider organisms with increasing genomic complexity, their recombinase enzymes show decreased ATPase activity, much lower turnover rates, and unique dsDNA binding affinities. It has been suggested that this signifies a more prominent role for mediator proteins in higher organisms, enabling them to exhibit tighter regulation of both initiation and progression of homologous recombination.<ref name="Liu" /> This may explain the differences and divergences in evolutionary conservation of sequence and structure, as well as the observed biochemical properties of different recombinases in the RecA family. | ||
==References== | ==References== | ||
<references/> | <references/> | ||
Revision as of 18:13, 22 April 2014
Contents |
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][2]
Fibrinogen, the chief proteinaceous component of a clot, circulates in blood at concentrations genearlly ranging from 2 to 3 mg/mL. 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 performed it releases fibrinopeptides A and B. 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 growth of fibrin fibers and ultimately a fibrin clot network capable of stemming blood loss in vivo.[1][2]
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.[1][2]
DNA Binding Properties
ssDNA Binding Modes
dsDNA Binding Modes
Arg-357
N-terminal Domain
The of Rad51 has been shown to bind DNA, [1][3][4] and is thought to have significant disordered character.[4] 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.[5]
Evolutionary Conservation
References
- ↑ Cite error: Invalid
<ref>tag; no text was provided for refs namedLiu - ↑ 2.0 2.1 2.2 Chi P, Van Komen S, Sehorn MG, Sigurdsson S, Sung P. Roles of ATP binding and ATP hydrolysis in human Rad51 recombinase function. DNA Repair (Amst). 2006 Mar 7;5(3):381-91. Epub 2006 Jan 4. PMID:16388992 doi:10.1016/j.dnarep.2005.11.005
- ↑ Cite error: Invalid
<ref>tag; no text was provided for refs namedChen - ↑ 4.0 4.1 Aihara H, Ito Y, Kurumizaka H, Yokoyama S, Shibata T. The N-terminal domain of the human Rad51 protein binds DNA: structure and a DNA binding surface as revealed by NMR. J Mol Biol. 1999 Jul 9;290(2):495-504. PMID:10390347 doi:10.1006/jmbi.1999.2904
- ↑ Cite error: Invalid
<ref>tag; no text was provided for refs namedConway
