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Molecular dynamics simulations reveal structural instability of human trypsin inhibitor upon D50E and Y54H mutations

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Abstract

Serine protease inhibitor Kazal type 1 (SPINK1) plays an important role in protecting the pancreas against premature trypsinogen activation that causes pancreatitis. Various mutations in the SPINK1 gene were shown to be associated with patients with pancreatitis. Recent transfection studies identified intracellular folding defects, probably caused by mutation induced misfolding of D50E and Y54H mutations, as a common mechanism that reduces SPINK1 secretion and as a possible novel mechanism of SPINK1 deficiency associated with chronic pancreatitis. Using molecular dynamics, we investigated the effects of D50E and Y54H mutations on SPINK1 dynamics and conformation at 300 K. We found that the structures of D50E and Y54H mutants were less stable than and were distorted from those of the wild type, as indicated by the RMSD plots, RMSF plots and DSSP series. Specifically, unwinding of the top of helices (the main secondary structures) and the distortion of the loops above the helices were observed. It may be possible that this distorted protein structure may be recognized as “non-native” by members of the chaperone family; it may be further retained and targeted for degradation, leading to SPINK1 secretion reduction and subsequently pancreatitis in patients as Király et al. (Gut 56:1433, 2007) proposed.

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Acknowledgments

The authors would like to thank the National Center for Genetic Engineering and Biotechnology (BIOTEC) and the National Science and Technology Development Agency (NSTDA) for the use of high performance computer clusters. The National Nanotechnology Center (NANOTEC) for the use of Discovery Studio. Mr. Chumpol Ngamphiw for his technical supports on the clusters. Mr. Pongsakorn Wangkumhang and Mr. Supasak Kulawonganunchai for helpful technical discussions. This work was supported by the Office of the Higher Education Commission and Mahidol University under the National Research Universities Initiative, the Thailand Research Fund (TRF) under Project no. RSA5480026, the “Research Chair Grant” National Science and Technology Development Agency, and the Higher Education Research Promotion and National Research University Project of Thailand, the Office of the Higher Education Commission.

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Authors and Affiliations

  1. National Center for Genetic Engineering and Biotechnology, 113 Thailand Science Park, Phahonyothin Road, Khlong Nueng, Khlong Luang, Pathum Thani, 12120, Thailand

    Wanwimon Mokmak, Surasak Chunsrivirot, Anunchai Assawamakin & Sissades Tongsima

  2. Interdisciplinary Graduate Program in Genetic Engineering, Graduate School, Kasetsart University, Chatuchak, Bangkok, 10900, Thailand

    Wanwimon Mokmak & Kiattawee Choowongkomon

  3. Center for Advanced Studies in Tropical Natural Resources, National Research University-Kasetsart University, Kasetsart University, Chatuchak, Bangkok, 10900, Thailand

    Kiattawee Choowongkomon

  4. Department of Biochemistry, Faculty of Sciences, Kasetsart University, Chatuchak, Bangkok, 10900, Thailand

    Kiattawee Choowongkomon

Authors
  1. Wanwimon Mokmak
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  2. Surasak Chunsrivirot
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  3. Anunchai Assawamakin
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  4. Kiattawee Choowongkomon
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Corresponding authors

Correspondence to Kiattawee Choowongkomon or Sissades Tongsima.

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Wanwimon Mokmak and Surasak Chunsrivirot contributed equally to this work

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Fig. S1

RMSD of the models of wild type, D50E, Y54H(δ) and Y54H(ε) mutants of the second and third runs (excluding the flexible N-terminals). (a) wild type 2, (b) wild type 3, (c) D50E 2, (d) D50E 3, (e) Y54H(δ) 2, (f) Y54H(δ) 3, (g) Y54H(ε) 2, (h) Y54H(ε) 3. (JPEG 112 kb)

High resolution image

(TIFF 366 kb)

Fig. S2

RMS fluctuation of the models of wild type, D50E, Y54H(δ) and Y54H(ε) mutants of the second and third runs. (a) wild type 2, (b) wild type 3, (c) D50E 2, (d) D50E 3, (e) Y54H(δ) 2, (f) Y54H(δ) 3, (g) Y54H(ε) 2, (h) Y54H(ε) 3. (JPEG 74 kb)

High resolution image

(TIFF 198 kb)

Fig. S3

Secondary structure as a function of simulation time (as determine by DSSP) of the second and third runs. The arrows indicate the points where helices H start to unwind. (a) wild type 2 (b) wild type 3, (c) D50E 2 (d) D50E 3, (e) Y54H(δ) 2, (f) Y54H(δ) 3, (g) Y54H(ε) 2, (h) Y54H(ε) 3. (JPEG 304 kb)

High resolution image

(TIFF 727 kb)

Fig. S4

Front views of the structures of the wild type and mutants after 50 ns simulations of the second run. Important residues are shown in licorice. Unwinding of the top of the main helices H is circled in red. Important hydrogen bonds are shown as green dashed lines. (a) wild type, (b) D50E, (c) Y54H(δ), (d) Y54H(ε). (JPEG 97 kb)

High resolution image

(TIFF 734 kb)

Fig. S5

Back views of the structures of the wild type and mutants after 50 ns simulations of the second run. Important residues are shown in licorice. Important hydrogen bonds are shown as green dashed lines. The absences of hydrogen bonds between the backbone carbonyl oxygens of Asn64 and the backbone amino hydrogens of Gln68 are circled in red. (a) wild type, (b) D50E, (c) Y54H(δ), (d) Y54H(ε). (JPEG 90 kb)

High resolution image

(TIFF 724 kb)

Fig. S6

Front views of the structures of the wild type and mutants after 50 ns simulations of the third run. Important residues are shown in licorice. Unwinding of the top of the main helices H is circled in red. Important hydrogen bonds are shown as green dashed lines. (a) wild type, (b) D50E, (c) Y54H(δ), (d) Y54H(ε). (JPEG 97 kb)

High resolution image

(TIFF 746 kb)

Fig. S7

Back views of the structures of the wild type and mutants after 50 ns simulations of the third run. Important residues are shown in licorice. Important hydrogen bonds are shown as green dashed lines. The absences of hydrogen bonds between the backbone carbonyl oxygens of Asn64 and the backbone amino hydrogens of Gln68 are circled in red. (a) wild type, (b) D50E, (c) Y54H(δ), (d) Y54H(ε). (JPEG 92 kb)

High resolution image

(TIFF 762 kb)

Fig. S8

Hydrogen bond distance profiles of the second and third runs between backbone oxygen of the carbonyl group of Asn64 and backbone hydrogen of the amino group of Gln68. (a) wild type 2, (b) wild type 3, (c) D50E 2, (d) D50E 3, (e) Y54H(δ) 2, (f) Y54H(δ) 3, (g) Y54H(ε) 2, (h) Y54H(ε) 3. (JPEG 99 kb)

High resolution image

(TIFF 309 kb)

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Mokmak, W., Chunsrivirot, S., Assawamakin, A. et al. Molecular dynamics simulations reveal structural instability of human trypsin inhibitor upon D50E and Y54H mutations. J Mol Model 19, 521–528 (2013). https://doi.org/10.1007/s00894-012-1565-2

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