Sulphate Removal Induces a Major Conformational Change in Leishmania mexicana Pyruvate Kinase in the Crystalline State Lindsay B. Tulloch 1 , Hugh P. Morgan 1 , Véronique Hannaert 2 , Paul A. M. Michels 2 , Linda A. Fothergill-Gilmore 1 and Malcolm D. Walkinshaw 1 ⁎ 1 Structural Biochemistry Group, Institute of Structural and Molecular Biology, The University of Edinburgh, Michael Swann Building, The King's Buildings, Mayfield Road, Edinburgh EH9 3JR, UK 2 Research Unit for Tropical Diseases, de Duve Institute and Laboratory of Biochemistry, Université catholique de Louvain, Avenue Hippocrate 74, B-1200 Brussels, Belgium Received 11 August 2008; accepted 14 August 2008 Available online 23 August 2008 We report X-ray structures of pyruvate kinase from Leishmania mexicana (LmPYK) that are trapped in different conformations. These, together with the previously reported structure of LmPYK in its inactive (T-state) conformation, allow comparisons of three different conformers of the same species of pyruvate kinase (PYK). Four new site point mutants showing the effects of side-chain alteration at subunit interfaces are also enzymatically characterised. The LmPYK tetramer crystals grown with ammonium sulphate as precipitant adopt an active-like conformation, with sulphate ions at the active and effector sites. The sulphates occupy positions similar to those of the phosphates of ligands bound to active (R-state) and constitutively active (nonallosteric) PYKs from several species, and provide insight into the structural roles of the phosphates of the substrates and effectors. Crystal soaking in sulphate-free buffers was found to induce major conformational changes in the tetramer. In particular, the unwinding of the Aα6′ helix and the inward hinge movement of the B domain are coupled with a significant widening (4 Å) of the tetramer caused by lateral movement of the C domains. The two new LmPYK structures and the activity studies of site point mutations described in this article are consistent with a developing picture of allosteric activity in which localised changes in protein flexibility govern the distribution of conformer families adopted by the tetramer in its active and inactive states. © 2008 Elsevier Ltd. All rights reserved. Edited by M. Guss Keywords: conformational transitions; Leishmania mexicana; pyruvate kinase; X-ray crystallography Introduction Pyruvate kinase (PYK) catalyses the final reaction of glycolysis in which phosphoenolpyruvate (PEP) and ADP are converted into pyruvate and ATP, respectively. The enzyme is a homotetramer (Fig. 1a) composed of identical monomers of 50–60 kDa, depending on species. In mammals, the M1 (adult) isoenzyme is normally unregulated, whilst the M2 (embryonic and tumour), R (erythrocyte), and L (liver) isoenzymes are allosterically activated by fructose 1,6-bisphosphate (F-1,6-BP) and other effec- tors. Many PYKs from other species are also activated by F-1,6-BP, but trypanosomatid (Trypano- soma and Leishmania) PYKs are allosterically acti- vated primarily by fructose 2,6-bisphosphate (F-2,6- *Corresponding author. E-mail address: m.walkinshaw@ed.ac.uk. Present address: L. B. Tulloch, Division of Biological Chemistry and Molecular Microbiology, School of Life Sciences, The Wellcome Trust Biocentre, The University of Dundee, Dow Street, Dundee DD1 5EH, UK. Abbreviations used: LmPYK, pyruvate kinase from Leishmania mexicana; PYK, pyruvate kinase; PEP, phosphoenolpyruvate; F-1,6-BP, fructose 1,6- bisphosphate; F-2,6-BP, fructose 2,6-bisphosphate; FBP, fructose bisphosphate; rM1PYK, rabbit M1 PYK; LmPYK Glu451Trp, Glu451Trp mutant of LmPYK; AS, ammonium sulphate; PG, phosphoglycollate; PDB, Protein Data Bank; TEA, triethanolamine. doi:10.1016/j.jmb.2008.08.037 J. Mol. Biol. (2008) 383, 615–626 Available online at www.sciencedirect.com 0022-2836/$ - see front matter © 2008 Elsevier Ltd. All rights reserved.