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Characterization of the inhibition mechanism of a tissuefactor inhibiting single-chain variable fragment: a combined computational approach

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A Correction to this article was published on 03 May 2020

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Abstract

The interaction of a single-chain variable fragment (scFv) directed against human tissue factor (TF) was predicted using an in silico approach with the aim to establish a most likely mechanism of inhibition. The structure of the TF inhibiting scFv (TFI-scFv) was predicted using homology modeling, and complementarity-determining regions (CDRs) were identified. The CDR was utilized to direct molecular docking between the homology model of TFI-scFv and the crystal structure of the extracellular domains of human tissue factor. The rigid-body docking model was refined by means of molecular dynamic (MD) simulations, and the most prevalent cluster was identified. MD simulations predicted improved interaction between TFI-scFv and TF and propose the formation of stable complex for duration of the 600-ns simulation. Analysis of the refined docking model suggests that the interactions between TFI-scFv would interfere with the allosterical activation of coagulation factor VII (FVII) by TF. This interaction would prevent the formation of the active TF:VIIa complex and in so doing inhibit the initiation phase of blood coagulation as observers during in vitro testing.

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  • 03 May 2020

    One of the co-author’s details (Leon du Preez-lategaan) was printed incorrectly in the above publication. The correct details are provided below.

Abbreviations

CDR:

Complementarity-determining regions

NPT:

Constant number, volume, and pressure

NVT:

Constant number, volume, and temperature

dsFv:

Disulfide-stabilized variable fragments

E. coli :

Escherichia coli

FVII:

Factor VII

FX:

Factor X

Hv:

Heavy chain variable domains

IgG:

Immunoglobulin

Lv:

Light chain variable domains

PME:

Particle mesh Ewald

PBC:

Periodic boundary conditions

pFv:

Permutated variable fragments

ps:

Pico seconds

PSSM:

Position-specific scoring matrix

PDB:

Protein data bank

scFv:

Single-chain variable fragment

TF:

Tissue factor

TF:FVIIa:

Tissue factor: factor VIIa complex

TFI-scFv:

Tissue factor inhibitor scFv

VMD:

Visual molecular dynamics

YASARA:

Yet another scientific artificial reality application

References

  1. Reichert JM (2012) Marketed therapeutic antibodies compendium. MAbs 4:413–415. https://doi.org/10.4161/mabs.19931

    Article  PubMed  PubMed Central  Google Scholar 

  2. Chames P, Van Regenmortel M, Weiss E, Baty D (2009) Therapeutic antibodies: successes, limitations and hopes for the future. Br J Pharmacol 157:220–233. https://doi.org/10.1111/j.1476-5381.2009.00190.x

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Nicolaides NC, Sass PM, Grasso L (2006) Monoclonal antibodies: a morphing landscape for therapeutics. Drug Dev Res 67:781–789. https://doi.org/10.1002/ddr.20149

    Article  CAS  Google Scholar 

  4. Tsumoto K, Shinoki K, Kondo H et al (1998) Highly efficient recovery of functional single-chain Fv fragments from inclusion bodies overexpressed in Escherichia coli by controlled introduction of oxidizing reagent - application to a human single-chain Fv fragment. J Immunol Methods 219:119–129. https://doi.org/10.1016/S0022-1759(98)00127-6

    Article  CAS  PubMed  Google Scholar 

  5. Reiter Y, Brinkmann U, Webber KO et al (1994) Engineering interchain disulfide bonds into conserved framework regions of Fv fragments: improved biochemical characteristics of recombinant immunotoxins containing disulfide-stabilized Fv. Protein Eng Des Sel 7:697–704. https://doi.org/10.1093/protein/7.5.697

    Article  CAS  Google Scholar 

  6. Lawrence LJ, Kortt AA, Iliades P et al (1998) Orientation of antigen binding sites in dimeric and trimeric single chain Fv antibody fragments. FEBS Lett 425:479–484. https://doi.org/10.1016/S0014-5793(98)00292-0

    Article  CAS  PubMed  Google Scholar 

  7. Brinkmann U, Di Carlo A, Vasmatzis G et al (1997) Stabilization of a recombinant Fv fragment by base-loop interconnection and V(H)-V(L) permutation. J Mol Biol 268:107–117. https://doi.org/10.1006/jmbi.1996.0850

    Article  CAS  PubMed  Google Scholar 

  8. Crivianu-Gaita V, Thompson M (2016) Aptamers, antibody scFv, and antibody fab’ fragments: an overview and comparison of three of the most versatile biosensor biorecognition elements. Biosens Bioelectron 85:32–45. https://doi.org/10.1016/j.bios.2016.04.091

    Article  CAS  PubMed  Google Scholar 

  9. Moricoli D, Muller WA, Carbonella DC et al (2014) Blocking monocyte transmigration in in vitro system by a human antibody scFv anti-CD99. Efficient large scale purification from periplasmic inclusion bodies in E. coli expression system. J Immunol Methods 408:35–45. https://doi.org/10.1016/j.jim.2014.04.012

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Witkowski M, Landmesser U, Rauch U (2016) Tissue factor as a link between inflammation and coagulation. Trends Cardiovasc Med 26:297–303. https://doi.org/10.1016/j.tcm.2015.12.001

    Article  CAS  PubMed  Google Scholar 

  11. Breitenstein A, Tanner FC, Lüscher TF (2010) Tissue Factor and Cardiovascular Disease. Circ J 74:3–12. https://doi.org/10.1253/circj.CJ-09-0818

    Article  CAS  PubMed  Google Scholar 

  12. Butenas S, Orfeo T, Brummel-Ziedins KE, Mann KG (2007) Tissue factor in thrombosis and hemorrhage. Surgery 142:2–14. https://doi.org/10.1016/j.surg.2007.06.032

    Article  Google Scholar 

  13. Samad F, Pandey M, Loskutoff DJ (2001) Regulation of tissue factor gene expression in obesity. Blood 98:3353–3358. https://doi.org/10.1182/blood.V98.12.3353

    Article  CAS  PubMed  Google Scholar 

  14. Mackman N (2009) The many faces of tissue factor. J Thromb Haemost 7:136–139. https://doi.org/10.1111/j.1538-7836.2009.03368.x

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Meiring SM, Vermeulen J, Badenhorst PN (2009) Development of an inhibitory antibody fragment to human tissue factor using phage display technology. Drug Dev Res 70:199–205. https://doi.org/10.1002/ddr.20295

    Article  CAS  Google Scholar 

  16. Vermeulen J-G, Burt F, van Heerden E et al (2018) Evaluation of in vitro refolding vs cold shock expression: production of a low yielding single chain variable fragment. Protein Expr Purif. https://doi.org/10.1016/j.pep.2018.06.005

  17. Garet E, Cabado AG, Vieites JM, González-Fernández Á (2010) Rapid isolation of single-chain antibodies by phage display technology directed against one of the most potent marine toxins: Palytoxin. Toxicon 55:1519–1526. https://doi.org/10.1016/j.toxicon.2010.03.005

    Article  CAS  PubMed  Google Scholar 

  18. Yan JP, Ko JH, Qi YP (2004) Generation and characterization of a novel single-chain antibody fragment specific against human fibrin clots from phage display antibody library. Thromb Res 114:205–211. https://doi.org/10.1016/j.thromres.2004.06.013

    Article  CAS  PubMed  Google Scholar 

  19. Proba K, Wörn A, Honegger A, Plückthun A (1998) Antibody scFv fragments without disulfide bonds, made by molecular evolution. J Mol Biol 275:245–253. https://doi.org/10.1006/jmbi.1997.1457

    Article  CAS  PubMed  Google Scholar 

  20. Watkins NA, Ouwehand WH (2000) Introduction to antibody engineering and phage display. Vox Sang 78:72–79 31154

    Article  CAS  Google Scholar 

  21. Arakawa T, Ejima D (2014) Refolding technologies for antibody fragments. Antibodies 3:232–241. https://doi.org/10.3390/antib3020232

    Article  CAS  Google Scholar 

  22. Chen X, Zaro JL, Shen WC (2013) Fusion protein linkers: property, design and functionality. Adv Drug Deliv Rev 65:1357–1369. https://doi.org/10.1016/j.addr.2012.09.039

    Article  CAS  PubMed  Google Scholar 

  23. Wu Q, Wang X, Gu Y et al (2016) Screening and identification of human ZnT8-specific single-chain variable fragment (scFv) from type 1 diabetes phage display library. Sci China Life Sci 59:686–693. https://doi.org/10.1007/s11427-016-5077-7

    Article  CAS  PubMed  Google Scholar 

  24. Ahmad ZA, Yeap SK, Ali AM et al (2012) ScFv antibody: principles and clinical application. Clin Dev Immunol 2012. https://doi.org/10.1155/2012/980250

  25. Sefid F, Rasooli I, Payandeh Z (2016) Homology modeling of a Camelid antibody fragment against a conserved region of Acinetobacter baumannii biofilm associated protein (bap). J Theor Biol 397:43–51. https://doi.org/10.1016/j.jtbi.2016.02.015

    Article  CAS  PubMed  Google Scholar 

  26. Krieger E, Vriend G (2014) YASARA view - molecular graphics for all devices - from smartphones to workstations. Bioinformatics 30:2981–2982. https://doi.org/10.1093/bioinformatics/btu426

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Hooft RWW, Vriend G, Sander C, Abola EE (1996) Errors in protein structures. Nature 381:272–272. https://doi.org/10.1038/381272a0

    Article  CAS  PubMed  Google Scholar 

  28. Hooft RW, Sander C, Scharf M, Vriend G (1996) The PDBFINDER database: a summary of PDB, DSSP and HSSP information with added value. Comput Appl Biosci 12:525–529. https://doi.org/10.1093/bioinformatics/12.6.525

    Article  CAS  PubMed  Google Scholar 

  29. Jones DT (1999) Protein secondary structure prediction based on position-specific scoring matrices. J Mol Biol 292:195–202. https://doi.org/10.1006/jmbi.1999.3091

    Article  CAS  PubMed  Google Scholar 

  30. Harlos K, Martin DMA, O’Brien DP et al (1994) Crystal structure of the extracellular region of human tissue factor. Nature 370:662–666. https://doi.org/10.1038/370662a0

    Article  CAS  PubMed  Google Scholar 

  31. Pierce BG, Wiehe K, Hwang H et al (2014) ZDOCK server: interactive docking prediction of protein-protein complexes and symmetric multimers. Bioinformatics 30:1771–1773. https://doi.org/10.1093/bioinformatics/btu097

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Berendsen HJC, van der Spoel D, van Drunen R (1995) GROMACS: a message-passing parallel molecular dynamics implementation. Comput Phys Commun 91:43–56. https://doi.org/10.1016/0010-4655(95)00042-E

    Article  CAS  Google Scholar 

  33. Davies J, Riechmann L (1996) Affinity improvement of single antibody VH domains: residues in all three hypervariable regions affect antigen binding. Immunotechnology 2:169–179. https://doi.org/10.1016/S1380-2933(96)00045-0

    Article  CAS  PubMed  Google Scholar 

  34. Kabat EA, Wu TT, Perry H et al (1991) Sequences of proteins of immunological Interstfifth edn. US Department of Health and Human Services, Bethesda

    Google Scholar 

  35. Norledge BV, Petrovan RJ, Ruf W, Olson AJ (2003) The tissue factor/factor VIIa/factor Xa complex: a model built by docking and site-directed mutagenesis. Proteins Struct Funct Genet 53:640–648. https://doi.org/10.1002/prot.10445

    Article  CAS  PubMed  Google Scholar 

  36. Turner CE (1998) Molecules in focus paxillin. Int J BioChemiPhysics 30:955–959

    CAS  Google Scholar 

  37. Martin DM, Boys CW, Ruf W (1995) Tissue factor: molecular recognition and cofactor function. FASEB J 9:852–859

    Article  CAS  Google Scholar 

  38. Pike AC, Brzozowski AM, Roberts SM et al (1999) Structure of human factor VIIa and its implications for the triggering of blood coagulation. Proc Natl Acad Sci U S A 96:8925–8930. https://doi.org/10.1073/pnas.96.16.8925

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Butenas S (2012) Tissue factor structure and function. Scientifica (Cairo) 2012:1–15. https://doi.org/10.6064/2012/964862

    Article  CAS  Google Scholar 

  40. Schwede T, Kopp J, Guex N, Peitsch MC (2003) SWISS-MODEL: an automated protein homology-modeling server. Nucleic Acids Res 31:3381–3385. https://doi.org/10.1093/nar/gkg520

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Nayeem A, Sitkoff D, Krystek S (2006) A comparative study of available software for high-accuracy homology modeling: from sequence alignments to structural models. Protein Sci 15:808–824. https://doi.org/10.1110/ps.051892906

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Price II WN, Chen Y, Handelman SK et al (2009) Understanding the physical properties that control protein crystallization by analysis of large-scale experimental data. Nat Biotechnol 27:51–57. https://doi.org/10.1038/nbt.1514

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Kabat EA, Wut T (1991) Identical V regions amino acid sequences and segments of sequences in antibodies of different specificities. Relative contributions of VH and VL genes, minigenes, and complementarity- determining regions to binding of antibody-combining sites. J Immunol 147:1709–1719

    CAS  PubMed  Google Scholar 

  44. Eigenbrot C, Kirchhofer D, Dennis MS et al (2001) The Factor VII Zymogen Structure Reveals Reregistration of β Strands during Activation. Structure 9:627–636. https://doi.org/10.1016/S0969-2126(01)00624-4

  45. Wang W, Singh S, Zeng DL et al (2007) Antibody structure, instability, and formulation. J Pharm Sci 96:1–26. https://doi.org/10.1002/jps.20727

    Article  CAS  PubMed  Google Scholar 

  46. Gu X, Jia X, Feng J et al (2010) Molecular modeling and affinity determination of scFv antibody: proper linker peptide enhances its activity. Ann Biomed Eng 38:537–549. https://doi.org/10.1007/s10439-009-9810-2

    Article  PubMed  Google Scholar 

  47. He K, Zhang X, Wang L et al (2016) Production of a soluble single-chain variable fragment antibody against okadaic acid and exploration of its specific binding. Anal Biochem 503:21–27. https://doi.org/10.1016/j.ab.2015.12.020

    Article  CAS  PubMed  Google Scholar 

  48. Barderas R, Desmet J, Timmerman P et al (2008) Affinity maturation of antibodies assisted by in silico modeling. Proc Natl Acad Sci U S A 105:9029–9034. https://doi.org/10.1073/pnas.0801221105

    Article  PubMed  PubMed Central  Google Scholar 

  49. Yu J, Vavrusa M, Andreani J et al (2016) InterEvDock: a docking server to predict the structure of protein-protein interactions using evolutionary information. Nucleic Acids Res 44:W542–W549. https://doi.org/10.1093/nar/gkw340

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Camacho CJ, Gatchell DW, Kimura SR, Vajda S (2000) Scoring docked conformations generated by rigid-body protein-protein docking. Proteins Struct Funct Genet 40:525–537. https://doi.org/10.1002/1097-0134(20000815)40:3<525::AID-PROT190>3.0.CO;2-F

    Article  CAS  PubMed  Google Scholar 

  51. Halperin I, Ma B, Wolfson H, Nussinov R (2002) Principles of docking: an overview of search algorithms and a guide to scoring functions. Proteins Struct Funct Genet 47:409–443. https://doi.org/10.1002/prot.10115

    Article  CAS  PubMed  Google Scholar 

  52. Salomon-Ferrer R, Case DA, Walker RC (2013) An overview of the Amber biomolecular simulation package. Wiley Interdiscip Rev Comput Mol Sci 3:198–210. https://doi.org/10.1002/wcms.1121

    Article  CAS  Google Scholar 

  53. Ciccotti G, Ferrario M, Schuette C (2014) Molecular dynamics simulation

  54. Thayer KPD, Slaw B, Han I, Quinn T (2015) Structural bioinformatics and molecular dynamics of p53 tumor suppressor protein. FASEB J 29:1 Suppl. http://www.fasebj.org/doi/abs/10.1096/fasebj.29.1_supplement.712.12

  55. Pasi M, Maddocks JH, Lavery R (2015) Analyzing ion distributions around DNA: sequence-dependence of potassium ion distributions from microsecond molecular dynamics. Nucleic Acids Res 43:2412–2423. https://doi.org/10.1093/nar/gkv080

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Satoh M, Saburi H, Tanaka T et al (2015) Multiple binding modes of a small molecule to human Keap1 revealed by X-ray crystallography and molecular dynamics simulation. FEBS Open Bio 5:557–570. https://doi.org/10.1016/j.fob.2015.06.011

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Jeffrey GA, Saenger W (1991) Hydrogen bonding in biological structures. Springer Berlin Heidelberg, Berlin

    Book  Google Scholar 

  58. Carmeliet P, Collen D (1998) Molecules in focus tissue factor. Int J Biochem Cell Biol 30:661–667. https://doi.org/10.1016/S1357-2725(97)00121-0

    Article  CAS  PubMed  Google Scholar 

  59. O’Brien DP, Anderson JS, Martin DM et al (1993) Structural requirements for the interaction between tissue factor and factor VII: characterization of chymotrypsin-derived tissue factor polypeptides. Biochem J 292(Pt 1):7–12

    Article  Google Scholar 

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Acknowledgments

I would like to thank the Department of Haematology and Cell Biology; Department of Virology of the Faculty of Health Sciences; and the Department of Microbial, Biochemical and Food Biotechnology, Faculty of Agricultural Sciences at the University of the Free State as well as High-Performance Computing Cluster at the University of the Free State and the National Health Laboratory Service for their assistance throughout the project.

Funding

This work was funded by the Technology Innovation Agency of South Africa.

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Authors and Affiliations

  1. Department of Microbial, Biochemical and Food Biotechnology, Faculty of Agricultural Sciences, University of the Free State, Bloemfontein, South Africa

    Jan-G Vermeulen, Esta van Heerden & Louis Lategan du Preez

  2. Department of Haematology and Cell Biology, Faculty of Health Sciences, University of the Free State, Bloemfontein, South Africa

    Jan-G Vermeulen & Muriel Meiring

  3. Division of Virology, Faculty of Health Sciences, University of the Free State, Bloemfontein, South Africa

    Felicity Burt

  4. National Health Laboratory Service, Universitas, Bloemfontein, South Africa

    Felicity Burt & Muriel Meiring

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  1. Jan-G Vermeulen
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  2. Felicity Burt
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  3. Esta van Heerden
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  4. Louis Lategan du Preez
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  5. Muriel Meiring
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Contributions

Dr. J Vermeulen: principle researcher and main author

Mr. LL du Preez: experimental design and molecular dynamic analysis

Prof SM Meiring: study leader, experimental design and manuscript editing

Prof. FJ Burt: co-study leader, manuscript editing and infrastructure support

Prof. E van Heerden: co-study leader, experimental design and infrastructure support

Corresponding author

Correspondence to Jan-G Vermeulen.

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The original version of this article was revised: One of the co-author’s details (Leon du Preez-lategaan) was printed incorrectly in the above publication. Leon du Preez-lategaan should be Louis Lategan du Preez.

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Vermeulen, JG., Burt, F., van Heerden, E. et al. Characterization of the inhibition mechanism of a tissuefactor inhibiting single-chain variable fragment: a combined computational approach. J Mol Model 26, 87 (2020). https://doi.org/10.1007/s00894-020-4350-7

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