Substrate specificity and the effect of calcium on Trypanosomabrucei metacaspase 2

Machado, M.K.M., Marcondes, M.F., Juliano, M.A., McLuskey, K., Mottram, J.C. , Moss, C.X., Juliano, L. and Oliveira, V. (2013) Substrate specificity and the effect of calcium on Trypanosomabrucei metacaspase 2. FEBS Journal, 280(11), pp. 2608-2621. (doi: 10.1111/febs.12248)

Abstract

Metacaspases are cysteine peptidases found only in yeast, plants and lower eukaryotes, including the protozoa. To investigate the extended substrate specificity and effects of Ca²⁺ on the activation of these enzymes, detailed kinetic, biochemical and structural analyses were carried out on metacaspase 2 from Trypanosoma brucei (TbMCA2). These results reveal that TbMCA2 has an unambiguous preference for basic amino acids at the P₁ position of peptide substrates and that this is most probably a result of hydrogen bonding from the P₁ residue to Asp95 and Asp211 in TbMCA2. In addition, TbMCA2 also has a preference for charged residues at the P₂ and P₃positions and for small residues at the prime side of a peptide substrate. Studies into the effects of Ca²⁺ on the enzyme revealed the presence of two Ca²⁺ binding sites and a reversible structural modification of the enzyme upon Ca²⁺ binding. In addition, the concentration of Ca²⁺ used for activation of TbMCA2 was found to produce a differential effect on the activity of TbMCA2, but only when a series of peptides that differed in P₂ were examined, suggesting that Ca²⁺activation of TbMCA2 has a structural effect on the enzyme in the vicinity of the S2 binding pocket. Collectively, these data give new insights into the substrate specificity and Ca²⁺ activation of TbMCA2. This provides important functional details and leads to a better understanding of metacaspases, which are known to play an important role in trypanosomes and make attractive drug targets due to their absence in humans.

• Item Type: Articles
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: McLuskey, Dr Karen and Moss, Dr Catherine and Mottram, Professor Jeremy
• Authors: Machado, M.K.M., Marcondes, M.F., Juliano, M.A., McLuskey, K., Mottram, J.C., Moss, C.X., Juliano, L., and Oliveira, V.
• College/School: College of Medical Veterinary and Life Sciences > School of Infection & Immunity
• Journal Name: FEBS Journal
• Publisher: Wiley-Blackwell Publishing
• ISSN: 1742-464X
• ISSN (Online): 1742-4658
• Copyright Holders: Copyright © 2013 The Authors
• First Published: First published in FEBS Journal 280(11):2608-2621
• Publisher Policy: Reproduced in accordance with the copyright policy of the publisher

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Project Code	Award No	Project Name	Principal Investigator	Funder's Name	Funder Ref	Lead Dept
53634	1	Structure and activity of trypanosomatid metacaspases	Jeremy Mottram	Wellcome Trust (WELLCOME)	091790/Z/10/Z	III - PARASITOLOGY
45414	1	Analysing the roles of petidases in Leishmania infectivity and pathogenicity	Jeremy Mottram	Medical Research Council (MRC)	G0700127	III - PARASITOLOGY