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Highly Conserved Arg Residue of ERFNIN Motif of Pro-Domain is Important for pH-Induced Zymogen Activation Process in Cysteine Cathepsins K and L

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Abstract

Pro-domain of a cysteine cathepsin contains a highly conserved Ex2Rx2Fx2Nx3Ix3N (ERFNIN) motif. The zymogen structure of cathepsins revealed that the Arg(R) residue of the motif is a central residue of a salt-bridge/H-bond network, stabilizing the scaffold of the pro-domain. Importance of the arginine is also demonstrated in studies where a single mutation (Arg → Trp) in human lysosomal cathepsin K (hCTSK) is linked to a bone-related genetic disorder “Pycnodysostosis”. In the present study, we have characterized in vitro Arg → Trp mutant of hCTSK and the same mutant of hCTSL. The R → W mutant of hCTSK revealed that this mutation leads to an unstable zymogen that is spontaneously activated and auto-proteolytically degraded rapidly. In contrast, the same mutant of hCTSL is sufficiently stable and has proteolytic activity almost like its wild-type counterpart; however it shows an altered zymogen activation condition in terms of pH, temperature and time. Far and near UV circular dichroism and intrinsic tryptophan fluorescence experiments have revealed that the mutation has minimal effect on structure of the protease hCTSL. Molecular modeling studies shows that the mutated Trp31 in hCTSL forms an aromatic cluster with Tyr23 and Trp30 leading to a local stabilization of pro-domain and supplements the loss of salt-bridge interaction mediated by Arg31 in wild-type. In hCTSK-R31W mutant, due to presence of a non-aromatic Ser30 residue such interaction is not possible and may be responsible for local instability. These differences may cause detrimental effects of R31W mutation on the regulation of hCTSK auto-activation process compared to altered activation process in hCTSL.

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Abbreviations

hCTSL:

Human cathepsin L

hCTSK:

Human cathepsin K

SDS:

Sodium dodecylsulfate

PEG:

Polyethylene glycol

MW:

Molecular weight

CD:

Circular dichroism

WT:

Wild type

RT:

Room temperature

IPTG:

Isopropyl-β-d-thiogalactopyranoside

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Acknowledgements

This study was supported by institutional grant (MSACR project) and funding from DBT, Govt. of India (BT/PR13895/BRB/10/789/2010 dt. 15-06-2011).

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Author notes

  1. Pulakesh Aich

    Present address: Center for Self-Assembly and Complexity, Institute for Basic Science, Pohang University of Science and Technology, Pohang, Republic of Korea

Authors and Affiliations

  1. Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, 1/AF Bidhannagar, Kolkata, 700 064, India

    Pulakesh Aich & Sampa Biswas

  2. Homi Bhaba National Institute, Anushaktinagar, Mumbai, 400 094, India

    Sampa Biswas

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  1. Pulakesh Aich
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Correspondence to Sampa Biswas.

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Aich, P., Biswas, S. Highly Conserved Arg Residue of ERFNIN Motif of Pro-Domain is Important for pH-Induced Zymogen Activation Process in Cysteine Cathepsins K and L. Cell Biochem Biophys 76, 219–229 (2018). https://doi.org/10.1007/s12013-017-0838-x

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