Allosteric Bonds Research Articles

Targeting allosteric disulphide bonds in cancer

Protein action in nature is generally controlled by the amount of protein produced and by chemical modification of the protein, and both are often perturbed in cancer. The amino acid side chains and the peptide and disulphide bonds that bind the polypeptide backbone can be post-translationally modified. Post-translational cleavage or the formation of disulphide bonds are now being identified in cancer-related proteins and it is timely to consider how these allosteric bonds could be targeted for new therapies.

Post-Translational Control of Protein Function by Disulfide Bond Cleavage

Protein action in nature is largely controlled by the level of expression and by post-translational modifications. Post-translational modifications result in a proteome that is at least two orders of magnitude more diverse than the genome. There are three basic types of post-translational modifications: covalent modification of an amino acid side chain, hydrolytic cleavage or isomerization of a peptide bond, and reductive cleavage of a disulfide bond. This review addresses the modification of disulfide bonds. Protein disulfide bonds perform either a structural or a functional role, and there are two types of functional disulfide: the catalytic and allosteric bonds. The allosteric disulfide bonds control the function of the mature protein in which they reside by triggering a change when they are cleaved. The change can be in ligand binding, substrate hydrolysis, proteolysis, or oligomer formation. The allosteric disulfides are cleaved by oxidoreductases or by thiol/disulfide exchange, and the configurations of the disulfides and the secondary structures that they link share some recurring features. How these bonds are being identified using bioinformatics and experimental screens and what the future holds for this field of research are also discussed.

Tissue Factor De-encryption, Thrombus Formation, and Thiol-disulfide Exchange

Tissue factor (TF) by forming a complex with factor VIIa (FVIIa) initiates blood coagulation. It was traditionally believed that the separation of FVIIa in circulation from subendothelial TF was the main control that was preventing spontaneous initiation of thrombosis and that circulating cells and endothelium did not express TF protein at rest in healthy individuals. However, TF has been detected in healthy human plasma and animal models of thrombosis, which indicate that TF in circulation can contribute to thrombin generation and fibrin formation after an activation event. Circulating TF-and indeed, most of the TF on the cell surface-is "encrypted" or coagulation inactive. The de-encryption step involves exposure of phosphatidylserine (PS), but PS exposure alone is insufficient for full TF activity. Allosteric disulfide bonds control protein function by mediating conformal change through the formation and breaking of disulfide bonds. TF contains a typical surface exposed allosteric bond in the membrane proximal fibronectin type III domain. Thiol-disulfide exchange involving this disulfide is implicated in TF activation with the formation of the disulfide bond corresponding with the active conformation of TF and free thiol or thiol-modified forms corresponding with encryption. Although the exact mechanism by which TF de-encryption occurs remains a subject of debate, thiol blockade and inhibition of oxidoreductases show an important role for thiol-disulfide reactions in platelet-independent pathways of coagulation in vitro and in vivo. In particular, redox active extracellular protein disulfide isomerase is involved in the earliest stages of thrombus initiation and has proven to be a potential target for antithrombotic drug development.

Allosteric Cysteine Oxidation Does Not Play a Role in Tissue Factor Decryption

Abstract 1174Tissue factor (TF) has two disulfides in the extracellular domain of the protein. The integrity of one cysteine pair, Cys186–Cys209, has been hypothesized to be essential for an allosteric “decryption” phenomenon regulating TF function, a subject of a lengthy debate. The encryption/decryption hypothesis asserts that cell-surface expressed TF, devoid of procoagulant activity, becomes converted to an active form when Cys186 and Cys209 are oxidized. Unlike a catalytic disulfide bond, which enzymatically mediates thiol-disulfide interchanges in substrate proteins, the hypothesized allosteric bond changes protein structure. The subsequent change in TF conformation is hypothesized to influence the intermolecular interactions between TF, a protease component of the extrinsic factor (F)Xase (FVIIa) and the substrate (FX), leading to enhanced dynamics of FX activation. The conclusions in support of this hypothesis are based on indirect evidence obtained with reagents with oxidizing/reducing properties and mutated recombinant TF.In our study, we determined the status of disulfides in both the recombinant TF1-263 (rTF1-263) and natural placental TF (pTF) by mass spectrometry. We analyzed TF peptides generated by proteolytic trypsin digestion in the non-reduced (NR) and reduced (R) (using10 mM dithiothreitol) TF without alkylation (NA) or with alkylation (A) (using 50 mM iodoacetamide). We found that all four cysteines of the extracellular domain of TF are oxidized in the native TF proteins. There was no evidence for peptides with reduced Cys49 and Cys57 (mass of 1996 Da) in the NR/NA samples of both pTF and rTF1-263. We did, however, observe peptides with mass of 1994 Da corresponding to a peptide with oxidized Cys49 and Cys57. In the R/A samples we identified a peptide (mass of 2110 Da) corresponding to reduced and alkylated peptide containing Cys49 and Cys57. Mass spectrometry analysis of peptides containing Cys186 and Cys209, two cysteines with close proximity to the membrane, revealed that these two residues are also oxidized. We did not observe peptides containing reduced Cys186 (mass of 3060 Da) or Cys209 (mass of 1409 Da) in the NR/NA samples of both pTF and rTF1-263, indicating that they are oxidized. In the R/A samples we identified peptides with mass 3116 Da and 1466 Da corresponding to reduced and alkylated Cys186 and Cys209, respectively. To further confirm that all four cysteines are oxidized, we treated TF with iodoacetamide without prior reduction. We did not detect peptides with alkylated cysteines in the NR/A sample in both pTF and rTF1-263. The mass spectrometry profile was the same as that of the NR/NA sample. The lack of incorporation of iodoacetamide into the NR sample indicates that all four cysteines are quantitatively involved in disulfide bridges.In functional assays, TF with reduced or non-reduced cysteines, with or without alkylation, showed similar activity of the TF/FVIIa complex in either membrane-independent synthetic substrate hydrolysis or membrane-dependent FX activation. The affinity of pTF for FVIIa was not altered by the reduction and alkylation (Kdapp 1.8±0.13 nM NR/NA, 1.8±0.19 nM R/NA, 1.7±0.16 nM R/A). Similarly, the maximum rate of synthetic substrate hydrolysis by the TF/FVIIa complex (FIU/sec) was not significantly affected by the treatment (Vmax 2662 FIU/sec NR/NA, 2434 FIU/sec R/NA and 3038 FIU/sec R/A). In FX activation, reduction and alkylation had little effect on the affinity of the pTF/FVIIa complex for FX (Km 0.9±0.2 μM NR/NA, 1.1±0.1 μM R/NA and 1.1±.2 μM R/A). Furthermore, the treatment only slightly altered the catalytic efficiency of the complex (kcat 7.4±0.06 sec−1 NR/NA, 7.2±0.03 sec−1 R/NA and 6.3±1.04 sec−1 R/A sam). In conclusion, our data indicate that the status of disulfides in the extracellular domain of TF does not contribute to the regulation of FVIIa catalytic function. Disclosures:Mann:Merck, Daiichi Sankyo, Baxter, GTI: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau; NIH, DOD, Baxter: Research Funding; corn trypsin inhibitor: Patents & Royalties; Haematologic Technologies: Chairman of the Board, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees.

Control of Mature Protein Function by Allosteric Disulfide Bonds

Protein disulfide bonds are the links between the sulfur atoms of two cysteine amino acids. All the known life forms appear to make this bond. Most disulfide bonds perform a structural role by stabilizing the tertiary and quaternary structures. Some perform a functional role and can be characterized as either catalytic or allosteric disulfides. Catalytic disulfides/dithiols transfer electrons between proteins, whereas the allosteric bonds control the function of the protein in which they reside when they undergo redox change. There are currently five clear examples of allosteric disulfide bonds and a number of potential allosteric disulfides at various stages of characterization. The features of these bonds and how they control the activity of the respective proteins are discussed. A common aspect of the allosteric disulfides identified to date is that they all link β-strands or β-loops.

Analysis of disulfide bonds in protein structures.

Disulfide bonds are covalent links between the sulfur atoms of two cysteine residues in the polypeptide chain. To assist with identification of possible functional disulfide bonds in proteins, we have generated a simple interface to obtain relevant information about disulfide bonds in crystal or solution structures. This includes secondary structural information, solvent accessibility values and different geometric measures. Most disulfide bonds are structural motifs that assist protein folding and stabilize the mature form. Some disulfide bonds serve a functional role and have been classified as either catalytic or allosteric bonds. The catalytic bonds are found in the active sites of oxidoreductases, proteins that mediate thiol/disulfide exchange in other proteins. The allosteric bonds are defined as bonds that control the function of the protein in which they reside by mediating a change when they break (reduce) or form (oxidize). The redox state of the allosteric disulfides are controlled by the catalytic disulfides. A disulfide bond is defined by six atoms of the two cysteine residues (Cα–Cβ–Sγ–Sγ′–Cβ′–Cα’) and five chi (χ) angles, which are the rotation about the bonds linking the atoms. There are 20 possible disulfide bond configurations based on the sign of each chi angle. For example, a disulfide is a positive left handed spiral (+LHSpiral) if the χ1, χ2, χ3, χ2′ and χ1′ angles are positive, positive, negative, positive and negative, respectively. The functional disulfides have been associated with particular conformations. The catalytic disulfides are nearly always ±RHHooks because they occur in a common secondary structure. The allosteric disulfides will have different conformations, although most allosteric bonds identified so far have a –RHStaple configuration and link β‐strands or β‐loops. Specific structural information from PDB structures is generally difficult to extract without expert knowledge in the scripting of 3D structure viewing and manipulation programs such as PyMol, Swiss‐PDB and RasMol. In particular, there are often multiple disulfide bonds within a single structure making it a formidable task in extracting geometric information. The issue is exacerbated for NMR structures where it is often useful to analyze the disulfide conformations within each model separately. We have developed a simple interface for researchers interested in obtaining information about disulfide bonds within any PDB structure: http://www.cancerresearch.unsw.edu.au/CRCWeb.nsf/page/Disulfide+Bond+Analysis. The software, Disulfide Bond Analysis, can be accessed through any modern web browser. It is able to accept any file that complies with the PDB format (.pdb or.ent). Alternatively, a user may input a list of PDB IDs that are already present in the RSCB protein data bank. Each PDB file is analyzed using DSSP to generate secondary structure characteristics and solvent accessibility values of the disulfide bond cysteines. Subsequently, the program computes the chi angles of the disulfide bond, calculates the dihedral strain energy and assigns the conformation. The distance between the α‐carbon atoms of the disulfide cysteines is also computed as a value < 5 Å is predictive of allosteric –RHStaple bonds. All the information is then compiled and a summary of the results are immediately displayed on the webpage, or the complete analysis can be downloaded in Microsoft® Excel format (.xls). A tutorial describing the use of Disulfide Bond Analysis along with a detailed description of the output can be found at the website. Disulfide Bond Analysis provides a simple web interface for researchers to obtain disulfide bond characteristics from PDB structures. The analysis can be completed in seconds and without detailed knowledge of the PDB file format. The program can be used to identify potential functional disulfides in proteins based on the known features of these bonds. The authors state that they have no conflict of interest. The authors thank B. Schmidt, L. Ho and K. Hu for their contributions to early versions of this program. This study was supported by a grant from the National Health and Medical Research Council of Australia.

Contribution of allosteric disulfide bonds to regulation of hemostasis.

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.

Evidence for Activation of Tissue Factor by an Allosteric Disulfide Bond

Tissue Factor (TF) is the mammalian plasma membrane cofactor responsible for initiation of blood coagulation. Binding of blood coagulation factor VIIa to TF activates the serine proteinase zymogens factors IX and X by limited proteolysis leading to the formation of a thrombin and fibrin meshwork that stabilizes the thrombus. TF on the plasma membrane of cells resides mostly in a cryptic configuration, which rapidly transforms into an active configuration in response to certain stimuli. The extracellular part of TF consists of two fibronectin type III domains. The disulfide bond in the membrane proximal domain (Cys186-Cys209) is atypical for domains of this type in that it links adjacent strands in the same beta sheet, what we have called an allosteric bond. Ablation of the allosteric disulfide by mutating both cysteine residues severely impairs procoagulant activity. The thiol-alkylating agents N-ethylmaleimide and methyl methanethiolsulfonate block TF activation by ionomycin, while the thiol-oxidizing agent HgCl2 and dithiol cross-linkers promote activation. TF activation could not be explained by exposure of phosphatidylserine on the outer leaflet of the plasma membrane. Cryptic TF contained unpaired cysteine thiols that were depleted upon activation, and de-encryption was associated with a change in the conformation of the membrane-proximal domain. These findings imply that the Cys186-Cys209 disulfide bond is reduced in the cryptic form of TF and that activation involves formation of the disulfide. It is likely that formation of this disulfide bond changes the conformation of the domain that facilitates productive binding of factors IX and X.

Allosteric Disulfide Bonds

Disulfide bonds have been generally considered to be either structural or catalytic. Structural bonds stabilize a protein, while catalytic bonds mediate thiol-disulfide interchange reactions in substrate proteins. There is emerging evidence for a third type of disulfide bond that can control protein function by triggering a conformational change when it breaks and/or forms. These bonds can be thought of as allosteric disulfides. To better define the properties of allosteric disulfides, we have analyzed the geometry and dihedral strain of 6874 unique disulfide bonds in 2776 X-ray structures. A total of 20 types of disulfide bonds were identified in the dataset based on the sign of the five chi angles that make up the bond. The known allosteric disulfides were all contained in 1 of the 20 groups, the -RHStaple bonds. This bond group has a high mean potential energy and narrow energy distribution, which is consistent with a functional role. We suggest that the -RHStaple configuration is a hallmark of allosteric disulfides. About 1 in 15 of all structurally determined disulfides is a potential allosteric bond.