1. Molecular dynamics simulations reveal structural instability of human trypsin inhibitor upon D50E and Y54H mutations Surasak Chunsrivirot Biostatistics and Informatics Laboratory National Center for Genetic Engineering and Biotechnology (BIOTEC) PACCON 2013 24 January 2013

2. Outline Pancreatitis Trypsin and trypsin inhibitor Methods Results

3. Pancreatitis Inflammation of the pancreas Characterized by – Repeated attacks of abdominal pain – Irreversible morphological changes – Impairment of both exocrine and endocrine functions It can be – Acute: beginning suddenly and lasting a few days – Chronic: occurring over many years Chen, J. and Ferec, C. (2009). Chronic Pancreatitis: Genetics and Pathogenesis. Annu. Rev. Genomics. Hum. Genet. 10:63-87. http://uvahealth.com/services/digestive-health/conditions-and-treatments/11634

4. Pancreatitis It occurs when pancreatic enzymes (especially trypsin) are prematurely activated in the pancreas instead of the small intestine – Resulting in digestion of the pancreas itself Chen, J. and Ferec, C. (2009). Chronic Pancreatitis: Genetics and Pathogenesis. Annu. Rev. Genomics. Hum. Genet. 10:63-87. http://www.medindia.net/patients/patientinfo/pancreatitis.htm

5. Trypsin A pancreatic digestive enzyme stored as an inactive precursor named trypsinogen Strictly controlled under normal conditions to prevent autodigestion of the pancreas In some circumstances, excessive activation of trypsinogen to trypsin leads to – Activation of other zymogens – Autodigestion of the pancreas – Pancreatitis that can be acute or chronic

6. Human trypsin inhibitor Serine protease inhibitor Kazal type 1 (SPINK1)

7. Human trypsin inhibitor Synthesized in acinar cells of the pancreas Inactivate trypsin activity if trypsinogen is accidentally converted to trypsin in acinar cells Mutations in the SPINK1 gene were shown to be associated with patients with pancreatitis by various studies Examples of these mutations are N34S, D50E, Y54H, R65Q, R67C, and P55S

8. Physiological role of trypsin and trypsin inhibitor (SPINK1) 8 Chen, J. and Ferec, C. (2009). Chronic Pancreatitis: Genetics and Pathogenesis. Annu. Rev. Genomics. Hum. Genet. 10:63-87.

9. Asp50 Tyr54 Human trypsin inhibitor Recent studies by Király et al. – identified intracellular folding defects as A common mechanism that reduces SPINK1 secretion A possible novel mechanism of SPINK1 deficiency associated with chronic pancreatitis. – Found that D50E and Y54H mutations Caused complete loss or marked reduction of SPINK1 secretion Did not change trypsin inhibitory activity. Király O, Wartmann T, Sahin-Tóth M (2007) Missense mutations in pancreatic secretory trypsin inhibitor (spink1) cause intracellular retention and degradation. Gut 56:1433

10. Human trypsin inhibitor – Proposed that D50E and Y54H mutations may cause Mutation induced misfolding  Intracellular retention  Degradation of SPINK1. – SPINK1 misfolding is most likely caused by Elimination of the conserved hydrogen bond between Asp50 and Tyr54. – Proposed that pancreatitis caused by these mutations may join a group of “protein folding disease” Asp50 Tyr54

11. Objective Investigate the effects of D50E and Y54H mutations on SPINK1 dynamics and conformations at 300 K

12. Methods D50E model Asp50 Glu50

13. Methods For Y54H model, His can be protonated at δ or ε positions at neutral pH  2 models of SPINK1 were created Y54H(δ) Y54H(ε) Hid54(δ) Hie54(ε) Tyr54

14. Methods AMBER 10 package and AMBER FF03 force- field parameters were used for all minimization and simulations. Three independent simulations were performed for each system (50 ns).

15. RMSD values of D50E and Y54H mutants are higher than those of the wild type. Suggests that changes of the conformation of the mutants may be more than that of the wild type

16. RMSF values of the mutants are higher than those of the wild type on average Especially residues 67-69: top of the helix H and the loop connecting to it. May suggest the decreased stability of the mutants, as compared to the wild type

17. DSSP shows the disappearance of the top of helix H (especially at residue 67) of the mutants. WTD50E Y54H(δ)Y54H(ε)

18. Partial unwinding of helix H and distortion of the loop on top of helix H of the mutants FrontBack

19. Partial unwinding of helix H and distortion of the loop on top of helix H of the mutants WT after minimization After 50 ns simulations WTD50E Y54H(δ)Y54H(ε)

20. Partial unwinding of helix H and distortion of the loop on top of helix H of the mutants WT after minimization WT D50E Y54H(δ)Y54H(ε) After 50 ns simulations

21. Partial unwinding of helix H and distortion of the loop above helix H is caused by the loss of the hydrogen bond between Asn64 and Gln68. WT D50E Y54H(δ)Y54H(ε)

22. DSSP shows the disappearance of the top of helix H (especially at residue 67) of the mutants. WTD50E Y54H(δ)Y54H(ε)

23. Y54H(δ) The hydrogen bond between Asn64 and Gln68 is maintained by the intricate hydrogen bond networks formed by Tyr54, Asp50, Arg67, Glu63, Thr69 and Asn64 WT

24. Conclusions The structures of the D50E and Y54H mutants were less stable than those of the wild type These mutations caused – Partial unwinding of the top of helices H. – The distortions of the loops on top of the helices. The results from molecular dynamics support the results and hypothesis of Király et al.

25. Acknowledgements Wanwimon Mokmak (BIOTEC) Dr. Sissades Tongsima, Dr. Anunchai Assawamakin (BIOTEC) Assistant Professor Kiattawee Choowongkomon (Kasetsart University) Chumpol Ngamphiw, Pongsakorn Wangkumhang, Supasak Kulawonganunchai (BIOTEC) Funding – the Office of the Higher Education Commission and Mahidol University under the National Research Universities Initiative – the Thailand Research Fund (TRF) – the “Research Chair Grant” National Science and Technology Development Agency – the Higher Education Research Promotion and National Research University Project of Thailand, the Office of the Higher Education Commission

26. Thank you