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Keywords:

  • allosteric;
  • disulfide bond;
  • integrin;
  • tissue factor;
  • von Willebrand factor

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Allosteric −RHStaple disulfides
  5. Tissue factor
  6. von Willebrand factor
  7. Integrin β3
  8. Conclusions
  9. Acknowledgments
  10. Disclosure of Conflict of Interests
  11. References

Summary.  Protein disulfide bonds are covalent links between pairs of Cys residues in the polypeptide chain. Acquisition of disulfide bonds is an important way that proteins have evolved and are continuing to evolve. These bonds serve either a structural or functional role. There are two types of functional disulfide: the catalytic bonds that reside in the active sites of oxidoreductases and the allosteric bonds. Allosteric disulfides are defined as bonds that have evolved to control the manner in which proteins function by breaking or forming in a precise way. The known allosteric bonds have a particular configuration known as the −RHStaple. Several hemostasis proteins contain −RHStaple disulfides and there is increasing evidence that some of these bonds may be involved in the functioning of the protein in which they reside. The best studied of these to date is the −RHStaple disulfide in tissue factor and its role in de-encryption of the cofactor.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Allosteric −RHStaple disulfides
  5. Tissue factor
  6. von Willebrand factor
  7. Integrin β3
  8. Conclusions
  9. Acknowledgments
  10. Disclosure of Conflict of Interests
  11. References

All life forms make proteins that contain disulfide bonds. At least 10% of the proteins made by human cells, for instance, contain one or more of these bonds. Most disulfide bonds assist protein folding by decreasing the entropy of the unfolded form and stabilize the tertiary and quaternary structure. A small proportion of disulfide bonds serve a functional role. There are two types of functional disulfides; the catalytic and allosteric bonds.

The catalytic bonds are found at the active sites of enzymes that mediate thiol/disulfide exchange in other proteins, the oxidoreductases [1]. The allosteric bonds, on the other hand, control the function of the protein in which they reside by mediating a change when they are reduced or oxidized [2,3]. The actions of the two functional disulfides are linked. The redox state of the allosteric disulfides is controlled by catalytic disulfides of the oxidoreductases.

A disulfide bond is made up of the two α-carbons, the two β-carbons of the two sulfur atoms of the Cys residues (inline image). These six atoms define five χ angles, which are the rotation about the bonds linking the atoms. Each χ angle can be either positive or negative, which equates to 20 possible disulfide bond configurations [3]. The three basic types of disulfide are defined by the angle of the central three bonds and are the spirals, hooks or staples. If the χ3 angle is positive, the bond is right-handed and if the angle is negative the bond is left-handed. For example, a disulfide is a minus right handed spiral (−RHSpiral) if the inline image angles are negative, positive, positive, positive and negative, respectively. A tool for analysis of disulfide bonds in X-ray or NMR structures is available at http://www.cancerresearch.unsw.edu.au/CRCWeb.nsf/page/Disulfide+Bond+Analysis.

Allosteric −RHStaple disulfides

  1. Top of page
  2. Abstract
  3. Introduction
  4. Allosteric −RHStaple disulfides
  5. Tissue factor
  6. von Willebrand factor
  7. Integrin β3
  8. Conclusions
  9. Acknowledgments
  10. Disclosure of Conflict of Interests
  11. References

The spirals are typically structural disulfides. With a few exceptions, all the catalytic disulfides are +/−RHHooks, while the known allosteric disulfides are −RHStaples [3]. A defining feature of −RHStaples is the close proximity of the α-carbon atoms of the two Cys residues. The −RHStaple bonds have a mean α-carbon atom distance of 4.3 Å, compared to a mean of 5.6 Å for all disulfides [3]. This is because of the secondary structures that the Cys link. About 60% of these bonds link adjacent strands in the same β-sheet. The strands are often so close in the β-sheet that they need to pucker to accommodate the disulfide bond, which can result in a significant strain on the bond [3,4]. Analysis of −RHStaple disulfides in NMR structures indicates that they often switch to the −LHStaple configuration, so these bonds should also be considered as potential allosteric disulfides [5].

There are currently six examples of functional −RHStaple disulfides that meet the following criteria: there is one or more X-ray structure of the disulfide bond, the consequence of cleavage or formation of the bond for protein function is known and the oxidoreductase that controls the redox state of the disulfide has been determined (with one exception) (Table 1). There are a number of other examples of potential allosteric disulfides but their current characterization does not meet all these criteria [4,6].

Table 1.   −RHStaple disulfides demonstrated to be involved in protein function. The indicated oxidoreductase controls the redox state of the −RHStaple disulfide
OrganismProteinDisulfideActive ConfigurationOxidoreductaseReference
Bacteria (Escherichia coli)PapD207–212OxidizedDsbA31,32
Bacteria (E. coli)Arylsulfate sulfotransferase418–424OxidizedDsbL33,34
Bacteria (E. coli)DsbD103–109ReducedDsbD  Cys461–Cys46435
Plant (Arabidopsis thaliana)FKB13106–111OxidizedChloroplast m-type thioredoxin36
Bacteria (Clostridium botulinum)Botulinum neurotoxins429–453 (type A) 436–445 (type B)Reduced & oxidizedNot known37
HumanCD4130–159ReducedThioredoxin38,39

The redox potentials of four −RHStaple are known and range from −229 mV for the Cys103–Cys109 DsbD bond [7] to −278 mV for the Cys186–Cys209 tissue factor (TF) bond (Liang, H.P.H., Brophy, T.M and Hogg, P.J, unpublished observations). For reference, the catalytic disulfides of protein disulphide isomerase (PDI) and thioredoxin have redox potentials of −175 and −270 mV respectively [1].

In this article, the −RHStaple disulfides in TF, von Willebrand Factor (VWF) and integrin β3 will be discussed. These bonds are the best studied in hemostasis proteins at this time. Other −RHStaple disulfides in hemostasis proteins have been discussed elsewhere [6].

Tissue factor

  1. Top of page
  2. Abstract
  3. Introduction
  4. Allosteric −RHStaple disulfides
  5. Tissue factor
  6. von Willebrand factor
  7. Integrin β3
  8. Conclusions
  9. Acknowledgments
  10. Disclosure of Conflict of Interests
  11. References

TF is a transmembrane glycoprotein that initiates blood coagulation in mammals. The cofactor binds factor VIIa to activate factors X and IX by limited proteolysis. Binary TF-VIIa and ternary TF-VIIa-Xa complexes also signal in inflammation, tumor progression and angiogenesis by cleaving protease activated receptor 2 (PAR2). TF is a member of the class II cytokine superfamily and contains two fibronectin type III domains in the extracellular part, each with a disulfide bond. The N-terminal domain Cys49–Cys57 disulfide is a typical structural bond, while the C-terminal domain Cys186–Cys209 disulfide exists exclusively as a −RHStaple as per the data in all published structures [8] (Fig. 1). The bond straddles the F and G strands of the anti parallel β-sheet and is exposed to solvent.

image

Figure 1.  Structures of the −RHStaple disulfides in TF, the VWC domain and the β-tail domain of integrin β3. The structures are of the C-terminal domain of TF (PDB 2HFT), the VWC domain of crossveinless-2 (PDB 3BK3) (which represents the C domains of VWF) and the β-tail domain of the integrin β3 subunit (PDB 1JV2). Disulfide bonds are in yellow.

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TF can be expressed on the cell-surface in a cryptic configuration (low activity state) that is activated by freeze-thawing of the cells, exposing them to calcium ionophores, or to mercury compounds. Several lines of evidence point to a conformational change in the vicinity of the −RHStaple disulfide upon de-encryption (reviewed in Ref. 6). Moreover, an intact Cys186–Cys209 bond is required for TF coagulant activity [9]. Unpaired Cys thiols exist in cryptic TF that are reduced or lost upon de-encryption and Cys thiols are involved in de-encryption as pretreatment of cells with thiol alkylating compounds blocks TF activation, while dithiol crosslinkers activate TF [8]. The allosteric disulfide is not required for binary TF-VIIa complex signaling [9]. Notably, though, the C209A TF mutant retains signaling activity while the C186A mutant is inactive in signaling [9].

These observations led to the hypothesis that redox change of the −RHStaple disulfide is central to de-encryption of the cofactor [8,9]. Specifically, the bond is thought to be reduced in cryptic TF and de-encryption involves oxidation of the disulfide. It is not clear at this stage what mediates oxidation of the disulfide. PDI [9–11], glutathione [11] and NO [9] have been implicated as redox regulators of the −RHStaple disulfide. PDI is on the surface of platelets [12] and platelet microparticles [13] and PDI ligands markedly impair TF-mediated thrombus formation in mice [10,11]. Studies using the purified oxidoreductase in cell culture systems, though, have yet to establish a role for PDI in TF de-encryption [14,15]. Despite these results, the redox model of TF de-encryption is controversial and other mechanisms have been proposed for de-encryption [16,17].

von Willebrand factor

  1. Top of page
  2. Abstract
  3. Introduction
  4. Allosteric −RHStaple disulfides
  5. Tissue factor
  6. von Willebrand factor
  7. Integrin β3
  8. Conclusions
  9. Acknowledgments
  10. Disclosure of Conflict of Interests
  11. References

VWF is a plasma glycoprotein that mediates adhesion of platelets to exposed subendothelium in the high shear conditions of flowing blood. VWF is synthesized by endothelial cells and megakaryocytes and circulates in the blood as a series of multimers made up of disulfide-linked 500 kDa homodimers. In response to vascular injury and platelet activation, ultra large VWF molecules as big as 20 000 kDa are released from Weibel-Palade bodies in endothelial cells and the α-granules of platelets. Strings or filaments of VWF with lengths of up to 1 mm also occur in Weibel-Palade bodies and in blood [18,19]. The formation of these structures by lateral association of multimers appears to be mediated by a thiol/disulfide exchange process [20].

Notably, nine unpaired Cys thiols in plasma VWF were mapped to the D3 and C domains, with seven of the nine located in the C2 domain and the link between the C2 and C3 domains [21]. More than 1000 proteins contain VWF type C (VWC) domains (see http://smart.embl-heidelberg.de/smart/do_annotation.pl?DOMAIN=VWC), which are 60–80 amino acids in length and defined by 10 conserved Cys residues [22]. In addition, some proteins have been shown to self-associate or interact with other partners via their VWC domains [22]. The Cys3–Cys5 VWC disulfide is a typical −RHStaple bond in both published structures of the VWC domain [23,24] (Fig. 1). The Cys3–Cys5 bond in the C2 domain of VWF is Cys2451–Cys2468, which was shown to be reduced in some molecules of plasma VWF [21]. These observations suggest that the C2 −RHStaple disulfide may be involved in lateral association of VWF molecules.

Integrin β3

  1. Top of page
  2. Abstract
  3. Introduction
  4. Allosteric −RHStaple disulfides
  5. Tissue factor
  6. von Willebrand factor
  7. Integrin β3
  8. Conclusions
  9. Acknowledgments
  10. Disclosure of Conflict of Interests
  11. References

The αIIbβ3 integrin is the platelet fibrinogen receptor. Platelet agonists trigger a conformational change in αIIbβ3 and ligand-binding. Platelet activation also results in a clear increase in cell surface protein thiol groups [25]. In particular, unpaired Cys thiols appear in integrin β3 [26] and the active site disulfide(s) of surface PDI are reduced [25]. There appears to be a functional consequence of these redox changes. There is evidence that thiol/disulfide events are necessary for full activation of αIIbβ3 and that platelet PDI, and perhaps also glutathione and NO, play a role in these events (reviewed in Ref. 27).

The details of the redox change(s) in the β3 subunit are not known although the current information points to a thiol-disulfide rearrangement in the epidermal growth factor (EGF)-like domains (reviewed in Ref. 27). Disruption of disulfide bonds in the 3rd and 4th EGF domains and the β-tail domain by mutagenesis differentially affected αIIbβ3 activation [28]. It is not clear at this point, however, which disulfides have structural or functional roles. Of the 16 defined disulfide bonds in the X-ray structure of the β3 subunit [29] only one is a −RHStaple bond; the Cys663–Cys687 disulfide in the β-tail domain (Fig. 1). Interestingly, ablation of this bond by replacing both Cys with Ala results in a constitutively active αIIbβ3 [30].

Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Allosteric −RHStaple disulfides
  5. Tissue factor
  6. von Willebrand factor
  7. Integrin β3
  8. Conclusions
  9. Acknowledgments
  10. Disclosure of Conflict of Interests
  11. References

Allosteric disulfides defined by the −RHStaple configuration can control the function of the protein in which they reside when they undergo redox change. Several hemostasis proteins contain −RHStaple disulfide bonds. The best studied of these to date are the bonds in TF, VWF and the β3 integrin subunit. Plasmin(ogen), vitronectin, glycoprotein 1bα, thrombomodulin, fibrinogen, tPA, uPA and the uPA receptor also contain −RHStaple disulfides [6]. It may be that some of these bonds contribute to the control of hemostasis.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Allosteric −RHStaple disulfides
  5. Tissue factor
  6. von Willebrand factor
  7. Integrin β3
  8. Conclusions
  9. Acknowledgments
  10. Disclosure of Conflict of Interests
  11. References