The coiled-coil domain of glycosomal membrane-associatedLeishmania donovaniPEX14: cloning, overexpression, purification and preliminary crystallographic analysis

Abstract

The glycosomal membrane-associatedLeishmania donovaniprotein PEX14, which plays a crucial role in protein import from the cytosol to the glycosomal matrix, consists of three domains: an N-terminal domain where the signalling molecule binds, a transmembrane domain and an 84-residue coiled-coil domain (CC) that is responsible for oligomerization. CCs are versatile domains that participate in a variety of functions including supramolecular assembly, cellular signalling and transport. Recombinant PEX14 CC was cloned, overexpressed, affinity-purified with in-column thrombin cleavage and further purified by size-exclusion chromatography. Crystals that diffracted to 1.98 Å resolution were obtained from a condition consisting of 1.4 Msodium citrate tribasic dihydrate, 0.1 MHEPES buffer pH 7.5. The crystals belonged to the monoclinic space groupC2, with unit-cell parametersa = 143.98,b= 32.62,c= 95.62 Å, β = 94.68°. Structure determination and characterization are in progress.