Skip to main content
SpringerLink
Log in

Conformational dynamics of threonine 195 and the S1 subsite in functional trypsin variants

  • Original Paper
  • Published:
Journal of Molecular Modeling Aims and scope Submit manuscript

Abstract

Replacing the catalytic serine in trypsin with threonine (S195T variant) leads to a nearly complete loss of catalytic activity, which can be partially restored by eliminating the C42-C58 disulfide bond. The 0.69 μs of combined explicit solvent molecular dynamics (MD) simulations revealed continuous rearrangement of T195 with different conformational preferences between five trypsin variants tested. Among three conformational families observed for the T195 residue, one showed the T195 hydroxyl in a conformation analogous to that of the serine residue in wild-type trypsin, positioning the hydroxyl oxygen atom for attack on the carbonyl carbon of the peptide substrate. MD simulations demonstrated that this conformation was more populated for the C42A/C58V/S195T and C42A/C58A/S195T triple variants than for the catalytically inactive S195T variant and correlated with restored enzymatic activities for triple variants. In addition, observation of the increased motion of the S214-G219 segment in the S195T substituted variants suggested an existence of open and closed conformations for the substrate binding pocket. The closed conformation precludes access to the S1 binding site and could further reduce enzymatic activities for triple variants. Double variants with intact serine residues (C42A/C58A/S195 and C42A/C58V/S195) also showed interchange between closed and open conformations for the S214-G219 segment, but to a lesser extent than the triple variants. The increased conformational flexibility of the S1 subsite, which was not observed for the wild-type, correlated with reduced enzymatic activities and suggested a possible mode of substrate regulation for the trypsin variants tested.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

Abbreviations

MD:

Molecular dynamics

WT:

Wild-type trypsin

S195T:

C42/C58/S195T variant

AA:

C42A/C58A/S195 variant

AV:

C42A/C58V/S195 variant

AAT:

C42A/C58A/S195T variant

AVT:

C42A/C58V/S195T variant

PDB:

Protein data bank

RMSD:

Root mean square deviations

References

  1. Page MJ, Di Cera E (2008) Serine peptidases: classification, structure and function. Cell Mol Life Sci 65(7–8):1220–1236. doi:10.1007/s00018-008-7565-9

    Article  CAS  Google Scholar 

  2. Ross J, Jiang H, Kanost MR, Wang Y (2003) Serine proteases and their homologs in the Drosophila melanogaster genome: an initial analysis of sequence conservation and phylogenetic relationships. Gene 304:117–131

    Article  CAS  Google Scholar 

  3. Puente XS, Sanchez LM, Overall CM, Lopez-Otin C (2003) Human and mouse proteases: a comparative genomic approach. Nat Rev Genet 4(7):544–558. doi:10.1038/nrg1111

    Article  CAS  Google Scholar 

  4. Puente XS, Sanchez LM, Gutierrez-Fernandez A, Velasco G, Lopez-Otin C (2005) A genomic view of the complexity of mammalian proteolytic systems. Biochem Soc Trans 33(Pt 2):331–334

    CAS  Google Scholar 

  5. Ding X, Rasmussen BF, Petsko GA, Ringe D (1994) Direct structural observation of an acyl-enzyme intermediate in the hydrolysis of an ester substrate by elastase. Biochemistry 33(31):9285–9293

    Article  CAS  Google Scholar 

  6. Blanchard H, James MN (1994) A crystallographic re-investigation into the structure of Streptomyces griseus proteinase A reveals an acyl-enzyme intermediate. J Mol Biol 241(4):574–587

    Article  CAS  Google Scholar 

  7. Katona G, Wilmouth RC, Wright PA, Berglund GI, Hajdu J, Neutze R, Schofield CJ (2002) X-ray structure of a serine protease acyl-enzyme complex at 0.95-A resolution. J Biol Chem 277(24):21962–21970. doi:10.1074/jbc.M200676200

    Article  CAS  Google Scholar 

  8. Katona G, Berglund GI, Hajdu J, Graf L, Szilagyi L (2002) Crystal structure reveals basis for the inhibitor resistance of human brain trypsin. J Mol Biol 315(5):1209–1218. doi:10.1006/jmbi.2001.5305

    Article  CAS  Google Scholar 

  9. Radisky ES, Lee JM, Lu CJ, Koshland DE Jr (2006) Insights into the serine protease mechanism from atomic resolution structures of trypsin reaction intermediates. Proc Natl Acad Sci USA 103(18):6835–6840

    Article  CAS  Google Scholar 

  10. Brady K, Wei AZ, Ringe D, Abeles RH (1990) Structure of chymotrypsin-trifluoromethyl ketone inhibitor complexes: comparison of slowly and rapidly equilibrating inhibitors. Biochemistry 29(33):7600–7607

    Article  CAS  Google Scholar 

  11. McGrath ME, Vasquez JR, Craik CS, Yang AS, Honig B, Fletterick RJ (1992) Perturbing the polar environment of Asp102 in trypsin: consequences of replacing conserved Ser214. Biochemistry 31(12):3059–3064

    Article  CAS  Google Scholar 

  12. Neidhart D, Wei Y, Cassidy C, Lin J, Cleland WW, Frey PA (2001) Correlation of low-barrier hydrogen bonding and oxyanion binding in transition state analogue complexes of chymotrypsin. Biochemistry 40(8):2439–2447

    Article  CAS  Google Scholar 

  13. Hedstrom L (2002) Serine protease mechanism and specificity. Chem Rev 102(12):4501–4523. doi:10.1021/Cr000033x

    Article  CAS  Google Scholar 

  14. Perona JJ, Tsu CA, Craik CS, Fletterick RJ (1993) Crystal structures of rat anionic trypsin complexed with the protein inhibitors APPI and BPTI. J Mol Biol 230(3):919–933

    Article  CAS  Google Scholar 

  15. Frey PA, Whitt SA, Tobin JB (1994) A low-barrier hydrogen bond in the catalytic triad of serine proteases. Science 264(5167):1927–1930

    Article  CAS  Google Scholar 

  16. Warshel A, Papazyan A (1996) Energy considerations show that low-barrier hydrogen bonds do not offer a catalytic advantage over ordinary hydrogen bonds. Proc Natl Acad Sci USA 93(24):13665–13670

    Article  CAS  Google Scholar 

  17. Wahlgren WY, Pal G, Kardos J, Porrogi P, Szenthe B, Patthy A, Graf L, Katona G (2011) The catalytic aspartate is protonated in the Michaelis complex formed between trypsin and an in vitro evolved substrate-like inhibitor: a refined mechanism of serine protease action. J Biol Chem 286(5):3587–3596

    Article  CAS  Google Scholar 

  18. Perona JJ, Craik CS (1997) Evolutionary divergence of substrate specificity within the chymotrypsin-like serine protease fold. J Biol Chem 272(48):29987–29990

    Article  CAS  Google Scholar 

  19. Helland R, Otlewski J, Sundheim O, Dadlez M, Smalas AO (1999) The crystal structures of the complexes between bovine beta-trypsin and ten P1 variants of BPTI. J Mol Biol 287(5):923–942

    Article  CAS  Google Scholar 

  20. Helland R, Berglund GI, Otlewski J, Apostoluk W, Andersen OA, Willassen NP, Smalas AO (1999) High-resolution structures of three new trypsin-squash-inhibitor complexes: a detailed comparison with other trypsins and their complexes. Acta Crystallogr D: Biol Crystallogr 55(Pt 1):139–148. doi:10.1107/S090744499801052X

    Article  CAS  Google Scholar 

  21. Zakharova E, Horvath MP, Goldenberg DP (2009) Structure of a serine protease poised to resynthesize a peptide bond. Proc Natl Acad Sci U S A 106(27):11034–11039

    Article  CAS  Google Scholar 

  22. Ascenzi P, Bocedi A, Bolognesi M, Spallarossa A, Coletta M, De Cristofaro R, Menegatti E (2003) The bovine basic pancreatic trypsin inhibitor (Kunitz inhibitor): a milestone protein. Curr Protein Pept Sci 4(3):231–251

    Article  CAS  Google Scholar 

  23. Katz BA, Finer-Moore J, Mortezaei R, Rich DH, Stroud RM (1995) Episelection: novel Ki approximately nanomolar inhibitors of serine proteases selected by binding or chemistry on an enzyme surface. Biochemistry 34(26):8264–8280

    Article  CAS  Google Scholar 

  24. Perona JJ, Craik CS (1995) Structural basis of substrate specificity in the serine proteases. Protein Sci 4(3):337–360. doi:10.1002/pro.5560040301

    Article  CAS  Google Scholar 

  25. Liu SQ, Meng ZH, Fu YX, Zhang KQ (2010) Insights derived from molecular dynamics simulation into the molecular motions of serine protease proteinase K. J Mol Model 16(1):17–28. doi:10.1007/s00894-009-0518-x

    Article  Google Scholar 

  26. Rawlings ND, Tolle DP, Barrett AJ (2004) MEROPS: the peptidase database. Nucleic Acids Res 32:D160–D164. doi:10.1093/nar/gkh071

    Article  CAS  Google Scholar 

  27. Baird TT Jr, Wright WD, Craik CS (2006) Conversion of trypsin to a functional threonine protease. Protein Sci 15(6):1229–1238

    Article  CAS  Google Scholar 

  28. Lee EH, Hsin J, Sotomayor M, Comellas G, Schulten K (2009) Discovery through the computational microscope. Structure 17(10):1295–1306

    Article  CAS  Google Scholar 

  29. Karplus M, McCammon JA (2002) Molecular dynamics simulations of biomolecules. Nat Struct Biol 9(9):646–652. doi:10.1038/nsb0902-646

    Article  CAS  Google Scholar 

  30. Case DA, Darden TA, Cheatham TEI, Simmerling CL, Wang J, Duke RE, Luo R, Walker RC, Zhang W, Merz KM, Roberts B, Wang B, Hayik S, Roitberg A, Seabra G, Kolossváry I, Wong KF, Paesani F, Vanicek J, Liu J, Wu X, Brozell SR, Steinbrecher T, Gohlke H, Cai Q, Ye X, Wang J, Hsieh M-J, Cui G, Roe DR, Mathews DH, Seetin MG, Sagui C, Babin V, Luchko T, Gusarov S, Kovalenko A, Kollman PA (2010) AMBER 11. University of California, San Francisco

    Google Scholar 

  31. McGrath ME, Haymore BL, Summers NL, Craik CS, Fletterick RJ (1993) Structure of an engineered, metal-actuated switch in trypsin. Biochemistry 32(8):1914–1919

    Article  CAS  Google Scholar 

  32. Jorgensen WL, Chandrasekhar J, Madura JD, Impey RW, Klein ML (1983) Comparison of simple potential functions for simulating liquid water. J Chem Phys 79(2):926–935

    Article  CAS  Google Scholar 

  33. Darden T, York D, Pedersen L (1993) Particle Mesh Ewald - an N.Log(N) method for Ewald Sums in large systems. J Chem Phys 98(12):10089–10092

    Article  CAS  Google Scholar 

  34. Steinbrecher T, Mobley DL, Case DA (2007) Nonlinear scaling schemes for Lennard-Jones interactions in free energy calculations. J Chem Phys 127(21):214108. doi:10.1063/1.2799191

    Article  Google Scholar 

  35. Berendsen HJC, Postma JPM, Vangunsteren WF, Dinola A, Haak JR (1984) Molecular-dynamics with coupling to an external bath. J Chem Phys 81(8):3684–3690

    Article  CAS  Google Scholar 

  36. Case DA, Cheatham TE, Darden T, Gohlke H, Luo R, Merz KM, Onufriev A, Simmerling C, Wang B, Woods RJ (2005) The Amber biomolecular simulation programs. J Comput Chem 26(16):1668–1688. doi:10.1002/Jcc.20290

    Article  CAS  Google Scholar 

  37. Adcock SA, McCammon JA (2006) Molecular dynamics: survey of methods for simulating the activity of proteins. Chem Rev 106(5):1589–1615. doi:10.1021/Cr040426m

    Article  CAS  Google Scholar 

  38. Cerutti DS, Freddolino PL, Duke RE, Case DA (2010) Simulations of a protein crystal with a high resolution x-ray structure: evaluation of force fields and water models. J Phys Chem B 114(40):12811–12824. doi:10.1021/Jp105813j

    Article  CAS  Google Scholar 

  39. Guliaev AB, Hang B, Singer B (2004) Structural insights by molecular dynamics simulations into specificity of the major human AP endonuclease toward the benzene-derived DNA adduct, pBQ-C. Nucleic Acids Res 32(9):2844–2852. doi:10.1093/Nar/Gkh594

    Article  CAS  Google Scholar 

  40. Rodriguez B, Yang YN, Guliaev AB, Chenna A, Hang B (2010) Benzene-derived N-2-(4-hydroxyphenyl)-deoxyguanosine adduct: UvrABC incision and its conformation in DNA. Toxicol Lett 193(1):26–32. doi:10.1016/j.toxlet.2009.12.005

    Article  CAS  Google Scholar 

  41. Beck DAC, Daggett V (2004) Methods for molecular dynamics simulations of protein folding/unfolding in solution. Methods 34(1):112–120. doi:10.1016/j.ymeth.2004.03.008

    Article  CAS  Google Scholar 

  42. Humphrey W, Dalke A, Schulten K (1996) VMD: visual molecular dynamics. J Mol Graph 14(1):33–38

    Article  CAS  Google Scholar 

  43. Rastelli G, Del Rio A, Degliesposti G, Sgobba M (2010) Fast and accurate predictions of binding free energies using MM-PBSA and MM-GBSA. J Comput Chem 31(4):797–810. doi:10.1002/Jcc.21372

    CAS  Google Scholar 

  44. Onufriev A, Case DA, Bashford D (2002) Effective born radii in the generalized born approximation: the importance of being perfect. J Comput Chem 23(14):1297–1304. doi:10.1002/Jcc.10126

    Article  CAS  Google Scholar 

  45. Frishman D, Argos P (1995) Knowledge-based protein secondary structure assignment. Proteins 23(4):566–579

    Article  CAS  Google Scholar 

  46. Perona JJ, Tsu CA, McGrath ME, Craik CS, Fletterick RJ (1993) Relocating a negative charge in the binding pocket of trypsin. J Mol Biol 230(3):934–949. doi:10.1006/jmbi.1993.1211

    Article  CAS  Google Scholar 

  47. Ishida T, Kato S (2004) Role of Asp102 in the catalytic relay system of serine proteases: a theoretical study. J Am Chem Soc 126(22):7111–7118. doi:10.1021/ja030405u

    Article  CAS  Google Scholar 

  48. Krem MM, Prasad S, Di Cera E (2002) Ser(214) is crucial for substrate binding to serine proteases. J Biol Chem 277(43):40260–40264. doi:10.1074/jbc.M206173200

    Article  CAS  Google Scholar 

  49. Spraggon G, Hornsby M, Shipway A, Tully DC, Bursulaya B, Danahay H, Harris JL, Lesley SA (2009) Active site conformational changes of prostasin provide a new mechanism of protease regulation by divalent cations. Protein Sci 18(5):1081–1094. doi:10.1002/pro.118

    Article  CAS  Google Scholar 

  50. Marquardt U, Zettl F, Huber R, Bode W, Sommerhoff C (2002) The crystal structure of human alpha1-tryptase reveals a blocked substrate-binding region. J Mol Biol 321(3):491–502

    Article  CAS  Google Scholar 

  51. Rohr KB, Selwood T, Marquardt U, Huber R, Schechter NM, Bode W, Than ME (2006) X-ray structures of free and leupeptin-complexed human alphaI-tryptase mutants: indication for an alpha– > beta-tryptase transition. J Mol Biol 357(1):195–209

    Article  CAS  Google Scholar 

  52. Bah A, Carrell CJ, Chen Z, Gandhi PS, Di Cera E (2009) Stabilization of the E* form turns thrombin into an anticoagulant. J Biol Chem 284(30):20034–20040

    Article  CAS  Google Scholar 

  53. Gandhi PS, Chen Z, Mathews FS, Di Cera E (2008) Structural identification of the pathway of long-range communication in an allosteric enzyme. Proc Natl Acad Sci USA 105(6):1832–1837

    Article  CAS  Google Scholar 

  54. Niu W, Chen Z, Gandhi PS, Vogt AD, Pozzi N, Pelc LA, Zapata F, Di Cera E (2011) Crystallographic and kinetic evidence of allostery in a trypsin-like protease. Biochemistry 50(29):6301–6307. doi:10.1021/bi200878c

    Article  CAS  Google Scholar 

  55. Shia S, Stamos J, Kirchhofer D, Fan B, Wu J, Corpuz RT, Santell L, Lazarus RA, Eigenbrot C (2005) Conformational lability in serine protease active sites: structures of hepatocyte growth factor activator (HGFA) alone and with the inhibitory domain from HGFA inhibitor-1B. J Mol Biol 346(5):1335–1349

    Article  CAS  Google Scholar 

  56. Forneris F, Ricklin D, Wu J, Tzekou A, Wallace RS, Lambris JD, Gros P (2010) Structures of C3b in complex with factors B and D give insight into complement convertase formation. Science 330(6012):1816–1820

    Article  CAS  Google Scholar 

  57. Carvalho AL, Sanz L, Barettino D, Romero A, Calvete JJ, Romao MJ (2002) Crystal structure of a prostate kallikrein isolated from stallion seminal plasma: a homologue of human PSA. J Mol Biol 322(2):325–337

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by the National Institutes of Health grant 5SC2GM095448 (to Anton Guliaev), Center for Computing for Life Sciences (http://cs.sfsu.edu/ccls/) Mini-grant, San Francisco State University (to Anton Guliaev) and National Science Foundation Award MCB0643988 (to Teaster T. Baird Jr.).

Author information

Authors and Affiliations

  1. Department of Chemistry and Biochemistry, San Francisco State University, San Francisco, CA, 94132, USA

    Trevor Gokey, Teaster T. Baird Jr. & Anton B. Guliaev

Authors
  1. Trevor Gokey
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Teaster T. Baird Jr.
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Anton B. Guliaev
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Anton B. Guliaev.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Gokey, T., Baird, T.T. & Guliaev, A.B. Conformational dynamics of threonine 195 and the S1 subsite in functional trypsin variants. J Mol Model 18, 4941–4954 (2012). https://doi.org/10.1007/s00894-012-1541-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00894-012-1541-x

Keywords

Search

Navigation