Sulphate removal induces a major conformational change in Leishmania mexicana pyruvate kinase in the crystalline state.
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 Aalpha6' helix and the inward hinge movement of the B domain are coupled with a significant widening (4 A) 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.
Tulloch
Lindsay B
LB
Institute of Structural and Molecular Biology, The University of Edinburgh, Michael Swann Building, The King's Buildings, Edinburgh, UK.
Morgan
Hugh P
HP
Hannaert
Véronique
V
Michels
Paul A M
PA
Fothergill-Gilmore
Linda A
LA
Walkinshaw
Malcolm D
MD
D000255:Adenosine Triphosphate / Q000737:chemistry; D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D018360:Crystallography, X-Ray; D004789:Enzyme Activation; D006801:Humans; D007894:Leishmania mexicana / Q000201:enzymology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D020836:Protein Structure, Quaternary; D011770:Pyruvate Kinase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013431:Sulfates / Q000737:chemistry*
D013431:Sulfates; D000255:Adenosine Triphosphate; D011770:Pyruvate Kinase