Evolutionary conservation of the allosteric activation of factor VIIa by tissue factor in lamprey: comment
2018; Elsevier BV; Volume: 16; Issue: 7 Linguagem: Inglês
10.1111/jth.14142
ISSN1538-7933
AutoresJesper J. Madsen, Egon Persson, Ole H. Olsen,
Tópico(s)Hemophilia Treatment and Research
ResumoWe read with great interest the article on allostery in a protease from the blood coagulation cascade by Beeler et al. 1.Beeler D.L. Aird W.C. Grant M.A. Evolutionary conservation of the allosteric activation of factor VIIa by tissue factor in lamprey.J Thromb Haemost. 2018; 16: 734-48Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar in a recent issue of the Journal of Thrombosis and Haemostasis. Herein, the authors conclude that the allosteric activation of coagulation factor VIIa (FVIIa) by tissue factor (TF) in lampreys appears to be indistinguishable from, and conserved as compared with, that in humans, meaning that lamprey FVIIa (l‐FVIIa) is assumed to be catalytically incompetent unless allosterically activated by lamprey TF (l‐TF). The lamprey, an ancient jawless eel‐like vertebrate, is a fascinating creature, and study of its coagulation system can certainly lead to insights into molecular mechanisms of coagulation and evolutionary adaptations over hundreds of millions of years (reviewed in 2.Davidson C.J. Tuddenham E.G. McVey J.H. 450 Million years of hemostasis.J Thromb Haemost. 2003; 1: 1487-94Crossref PubMed Scopus (130) Google Scholar). Nevertheless, the conclusion presented by the authors is, in our opinion, fairly surprising or even improbable. In this Letter, we address the subject of possible allostery in l‐FVIIa, and demonstrate that a diametrically opposite hypothesis arises organically from mechanistic discoveries in human FVIIa (h‐FVIIa) biochemistry based on our own investigations and those of others. Readers are left to form their own opinion, but we hope that our perspective sheds some light on the need for allosteric activation in FVIIa (or lack thereof in carefully engineered variants) and provides the impetus for future studies in this important area of research. The remainder of this Letter is organized as follows. We briefly review central concepts in FVIIa biochemistry with a focus on the human protease and the relevant variants subject to allosteric modulation. We go on to discuss similarities between these variants and l‐FVIIa from a combined sequence and three‐dimensional structure point of view. Finally, we provide a critical comparison with the work of Beeler et al., and end with concluding remarks. It is known that the coagulation system of the lamprey is simplified, containing fewer components and completely lacking the intrinsic pathway (FIX and FVIII are missing), as is also emphasized by Beeler et al. This, perhaps coincidentally, makes the lamprey coagulation system somewhat similar to that of human hemophilia A/B patients, who have genetically inherited deficiencies in the intrinsic pathway. This raises the question of how exactly blood coagulation is properly controlled in the lamprey, which does not appear to have symptoms of hemophilia such as a prolonged bleeding time. The coagulation system of the lamprey has previously been the subject of investigations (e.g. within the field of genome bioinformatics) 3.Doolittle R.F. Jiang Y. Nand J. Genomic evidence for a simpler clotting scheme in jawless vertebrates.J Mol Evol. 2008; 66: 185-96Crossref PubMed Scopus (40) Google Scholar, 4.Kimura A. Ikeo K. Nonaka M. Evolutionary origin of the vertebrate blood complement and coagulation systems inferred from liver EST analysis of lamprey.Dev Comp Immunol. 2009; 33: 77-87Crossref PubMed Scopus (43) Google Scholar, 5.Doolittle R.F. Bioinformatic characterization of genes and proteins involved in blood clotting in lampreys.J Mol Evol. 2015; 81: 121-30Crossref Scopus (9) Google Scholar; however, none of the published articles that we found mention l‐FVIIa allostery explicitly. A wealth of biochemical information regarding h‐FVIIa has matured the understanding of subtle mechanisms that contribute to activity and TF‐induced allostery (reviewed in [6]), and enabled rational engineering of variants with enhanced activity independent of stimulation by endogenous TF 7.Persson E. Kjalke M. Olsen O.H. Rational design of coagulation factor VIIa variants with substantially increased intrinsic activity.Proc Natl Acad Sci USA. 2001; 98: 13583-8Crossref PubMed Scopus (114) Google Scholar, 8.Nielsen A.L. Sorensen A.B. Holmberg H.L. Gandhi P.S. Karlsson J. Buchardt J. Lamberth K. Kjelgaard‐Hansen M. Ley C.D. Sørensen B.B. Ruf W. Olsen O.H. Østergaard H. Engineering of a membrane‐triggered activity switch in coagulation factor VIIa.Proc Natl Acad Sci USA. 2017; 114: 12454-9Crossref PubMed Scopus (6) Google Scholar. Let us at this point emphasize that h‐FVIIa can be distinguished from l‐FVIIa by means of multiple sequence alignment (MSA) analysis of their respective heavy chains, which, together with biochemical evidence and structural modeling, will serve as premises for our argument. Figure S3 of Beeler et al. shows MSAs of FVII from human, mouse, cow, chicken, frog, zebrafish, and lamprey, to which we shall refer. The structural model of l‐FVIIa is shown in Fig. 1A (with close‐ups in B–D). Observation 1: In h‐FVIIa, the M306D{c164} mutation bidirectionally short‐circuits TF‐induced allostery 9.Persson E. Nielsen L.S. Olsen O.H. Substitution of aspartic acid for methionine‐306 in factor VIIa abolishes the allosteric linkage between the active site and the binding interface with tissue factor.Biochemistry. 2001; 40: 3251-6Crossref PubMed Scopus (51) Google Scholar. l‐FVIIa has aspartic acid in the position corresponding to M306{c164} of h‐FVIIa, as also noted by Beeler et al., but we interpret this observation differently. Curiously, the majority of mammals have methionine in that position, whereas cold‐blooded animals have a charged residue or proline, suggesting a specific role for this residue. Beeler et al. speculate that a corresponding mutation (to another residue, i.e. not aspartic acid) in the same position of l‐FVIIa will abrogate the allosteric connection. We suggest a much more direct interpretation, whereby the allosteric 'on/off switch' is innately set to off in l‐FVIIa, as corroborated by comparison with h‐FVIIa‐M306D{c164}. Observation 2: The triple mutant V158D{c21}/E296V{c154}/M298Q{c156} of h‐FVIIa (h‐FVIIaDVQ) appears to mediate the structural transition that is also inducible by TF, largely eliminating the need for TF 7.Persson E. Kjalke M. Olsen O.H. Rational design of coagulation factor VIIa variants with substantially increased intrinsic activity.Proc Natl Acad Sci USA. 2001; 98: 13583-8Crossref PubMed Scopus (114) Google Scholar, 10.Persson E. Olsen O.H. Bjørn S.E. Ezban M. Vatreptacog alfa from conception to clinical proof of concept.Semin Thromb Hemost. 2012; 38: 274-81Crossref PubMed Scopus (12) Google Scholar, 11.Lentz S.R. Ehrenforth S. Abdul Karim F. Matsushita T. Weldingh K.N. Windyga J. Mahlangu J.N. Weltermann A. de Paula E. Cerqueira M. Zupancic‐Salek S. Katsarou O. Economou M. Nemes L. Boda Z. Santagostino E. Tagariello G. Hanabusa H. Fukutake K. Taki M. et al.Recombinant factor VIIa analog in the management of hemophilia with inhibitors: results from a multicenter, randomized, controlled trial of vatreptacog alfa.J Thromb Haemost. 2014; 12: 1244-53Crossref PubMed Scopus (57) Google Scholar, 12.Petrovan R.J. Ruf W. Residue Met156 contributes to the labile enzyme conformation of coagulation factor VIIa.J Biol Chem. 2001; 276: 6616-20Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar. DVQ is a canonical motif present in thrombin, which has considerable constitutive activity. In the corresponding positions, we see the presence of matching motifs in frogs, zebrafish, and lampreys (MLQ, ELR, and EIQ, respectively), which are all cold‐blooded. Of these, the lamprey's EIQ motif (Fig. 1B) is the closest chemical match to the DVQ motif, and, probably, is similar enough to have an analogous stabilizing effect by means of neatly coordinating a water molecule that strengthens the connection between the N‐terminus and the surface of the body of the protease domain 7.Persson E. Kjalke M. Olsen O.H. Rational design of coagulation factor VIIa variants with substantially increased intrinsic activity.Proc Natl Acad Sci USA. 2001; 98: 13583-8Crossref PubMed Scopus (114) Google Scholar. We note that the h‐FVIIa‐M298Q{c156} single mutant, i.e. even without the DV part of DVQ, has enhanced amidolytic and proteolytic activity, by three‐fold and nine‐fold, respectively 12.Petrovan R.J. Ruf W. Residue Met156 contributes to the labile enzyme conformation of coagulation factor VIIa.J Biol Chem. 2001; 276: 6616-20Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar. Observation 3: The L305V{c163} mutation in h‐FVIIa enhances amidolytic and proteolytic activity by three‐fold to four‐fold, and contributes synergistically to enhanced activity in double mutants of h‐FVIIa 7.Persson E. Kjalke M. Olsen O.H. Rational design of coagulation factor VIIa variants with substantially increased intrinsic activity.Proc Natl Acad Sci USA. 2001; 98: 13583-8Crossref PubMed Scopus (114) Google Scholar, 13.Persson E. Bak H. Olsen O.H. Substitution of valine for leucine 305 in factor VIIa increases the intrinsic enzymatic activity.J Biol Chem. 2001; 276: 29195-9Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar. l‐FVIIa has valine in the position corresponding to L305{c163} of h‐FVIIa (Fig. 1C). This could mean that l‐FVIIa, by analogy, has enhanced intrinsic activity as compared with h‐FVIIa and relies less on l‐TF. Observation 4: Shortening of the 170‐loop by grafting the trypsin loop into h‐FVIIa seems to affect the structural integrity of the TF‐binding helix and enhances activity 14.Soejima K. Mizuguchi J. Yuguchi M. Nakagaki T. Higashi S. Iwanaga S. Factor VIIa modified in the 170 loop shows enhanced catalytic activity but does not change the zymogen‐like property.J Biol Chem. 2001; 276: 17229-35Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 15.Soejima K. Yuguchi M. Mizuguchi J. Tomokiyo K. Nakashima T. Nakagaki T. Iwanaga S. The 99 and 170 loop‐modified factor VIIa mutants show enhanced catalytic activity without tissue factor.J Biol Chem. 2002; 277: 49027-35Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 16.Sorensen A.B. Madsen J.J. Svensson L.A. Pedersen A.A. Østergaard H. Overgaard M.T. Olsen O.H. Gandhi P.S. Molecular basis of enhanced activity in factor VIIa‐trypsin variants conveys insights into tissue factor‐mediated allosteric regulation of factor VIIa activity.J Biol Chem. 2016; 291: 4671-83Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar. We and others have shown that shortening of the 170‐loop (Fig. 1D) confers increased activity to the protease and diminishes the contribution of allosteric activation by TF. Again, it is interesting to note that mammals have a 170‐loop of the same length as in h‐FVIIa, whereas cold‐blooded vertebrates have a loop truncated by four or five residues. Observation 5: The combination of sequence elements (mutations and grafts) that modulate protease activity and TF‐induced allostery, in particular those mentioned in Observations 1–4 above, generally produces expected additive effects, albeit with some possibility of synergy 7.Persson E. Kjalke M. Olsen O.H. Rational design of coagulation factor VIIa variants with substantially increased intrinsic activity.Proc Natl Acad Sci USA. 2001; 98: 13583-8Crossref PubMed Scopus (114) Google Scholar, 9.Persson E. Nielsen L.S. Olsen O.H. Substitution of aspartic acid for methionine‐306 in factor VIIa abolishes the allosteric linkage between the active site and the binding interface with tissue factor.Biochemistry. 2001; 40: 3251-6Crossref PubMed Scopus (51) Google Scholar, 13.Persson E. Bak H. Olsen O.H. Substitution of valine for leucine 305 in factor VIIa increases the intrinsic enzymatic activity.J Biol Chem. 2001; 276: 29195-9Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 15.Soejima K. Yuguchi M. Mizuguchi J. Tomokiyo K. Nakashima T. Nakagaki T. Iwanaga S. The 99 and 170 loop‐modified factor VIIa mutants show enhanced catalytic activity without tissue factor.J Biol Chem. 2002; 277: 49027-35Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 16.Sorensen A.B. Madsen J.J. Svensson L.A. Pedersen A.A. Østergaard H. Overgaard M.T. Olsen O.H. Gandhi P.S. Molecular basis of enhanced activity in factor VIIa‐trypsin variants conveys insights into tissue factor‐mediated allosteric regulation of factor VIIa activity.J Biol Chem. 2016; 291: 4671-83Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar. This combination heuristic is supported by biochemical evidence whose finer nuances are addressed in the cited articles. The similarities between the well‐studied h‐FVIIa variants discussed above and l‐FVIIa are nothing short of striking. The emerging and very tempting hypothesis is that, because l‐FVIIa carries the equivalents of D306{c164}, V305{c163}, a DVQ‐like motif, and a short 170‐loop, it is, at least to a considerable degree, constitutively active and does not rely much on allosteric activation by l‐TF (see the summary graphic in Fig. 1E). How, then, is this compatible with the reported findings of Beeler et al.? Setting aside for a moment technical concerns about the modeling and simulation procedures, the conclusion drawn by Beeler et al. relies on three key assumptions: (i) that the structures of individual domains of the lamprey complex can be reliably modeled; (ii) that the l‐FVIIa–l‐TF complex forms in vitro and in vivo to give the precise interface contacts consistent with the homology model; and (iii) that the in silico methodology employed is sufficiently sensitive. Crucially, we are still missing the essential control experiments in the establishment of the computational protocol used by Beeler et al. (and others 17.Sethi A. Eargle J. Black A.A. Luthey‐Schulten Z. Dynamical networks in tRNA: protein complexes.Proc Natl Acad Sci USA. 2009; 106: 6620-5Crossref PubMed Scopus (530) Google Scholar, 18.Gasper P.M. Fuglestad B. Komives E.A. Markwick P.R.L. McCammon J.A. Allosteric networks in thrombin distinguish procoagulant vs. anticoagulant activities.Proc Natl Acad Sci USA. 2012; 109: 21216-22Crossref PubMed Scopus (119) Google Scholar, 19.Fuglestad B. Gasper P.M. McCammon J.A. Markwick P.R.L. Komives E.A. Correlated motions and residual frustration in thrombin.J Phys Chem B. 2013; 117: 12857-63Crossref PubMed Scopus (37) Google Scholar, including us 20.Madsen J.J. Persson E. Olsen O.H. Tissue factor activates allosteric networks in factor VIIa through structural and dynamic changes.J Thromb Haemost. 2015; 13: 262-7Crossref PubMed Scopus (17) Google Scholar), for the specific purpose that they pursue, namely the comparison between allosteric activation in h‐FVIIa and that in a minimally perturbed variant that is constitutively active in the absence of h‐TF, such as h‐FVIIaDVQ. If the protocol fails to correctly identify the substantial differences in the allosteric activation between the two variants, and, vice versa, the similarity between allosteric activation in h‐FVIIa–h‐TF and h‐FVIIaDVQ, the results must ultimately be deemed inconclusive, meaning that the protocol requires improvement (e.g. by extending and improving trajectory sampling significantly, considering multiple independent configurational ensembles, or limiting the use to reliable and relevant starting structures). For these reasons, the apparent conservation of the allosteric activation between the homology model of l‐FVIIa–l‐TF and the empirical human complex claimed by Beeler et al. could be a red herring. The comparative functional clotting results presented by Beeler et al. (their Fig. 1A) shows species specificity of the molecular interactions, and do not prove that there is allosteric activation of l‐FVIIa by l‐TF. It is likely that the detected effect of lamprey thromboplastin on lamprey plasma clotting is a mere consequence of localization of l‐FVIIa to membrane‐bound l‐TF. Hence, this observation is not necessarily indicative of conservation of TF‐dependent allosteric activation of FVIIa between lampreys and humans. The considerations provided in this Letter support the hypothesis that FVIIa in lampreys (and possibly in other cold‐blooded vertebrates) is, to some degree, constitutively more active than the human counterpart and has less need for allosteric activation by TF. The lack of the intrinsic coagulation pathway in these cold‐blooded vertebrates appears to necessitate compensatory adaptations in the extrinsic pathway, materializing as enhanced free FVIIa activity and diminished TF‐induced allostery. To put this another way, the evolutionary emergence of an intrinsic pathway demands more regulatory complexity. If this were the case, a provisional role for TF in these organisms could be to provide exosites for spatiotemporal colocalization and alignment of FVIIa with its substrate(s), but not allosteric activation of the enzyme. We will be looking forward with anticipation to additional evidence that can help to settle the apparent contradiction regarding the matter of allostery in l‐FVIIa. J. J. Madsen designed the research, performed the experiments, analyzed results, and wrote the manuscript. E. Persson and O. H. Olsen designed the research, analyzed results, and revised the manuscript. The authors state that they have no conflict of interest. J. J. Madsen gratefully acknowledges financial support from the Carlsberg Foundation in the form of a postdoctoral fellowship (grants CF15‐0552, CF16‐0639, and CF17‐0783) and the research framework provided by the Research Computing Center at the University of Chicago.Carlsberg FoundationCF15‐0552CF16‐0639CF17‐0783University of Chicago
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