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

Abstract

The glycosomal membrane-associated Leishmania donovani protein 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 M sodium citrate tribasic dihydrate, 0.1 M HEPES buffer pH 7.5. The crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 143.98, b = 32.62, c = 95.62 Å, β = 94.68°. Structure determination and characterization are in progress.

Development of Novel Anti-Leishmanials: The Case for Structure-Based Approaches

“…As seen in Table 4, no structures for any of the components of this important pathway from Leishmania are available, though a few trypanosomatid homologs are known [120]. Recently, a crystallization note of the CC domain of LdPEX14 has appeared in the literature [121], and any structural deviations and its critical functional values could be a great opportunity for SBDD [31,104]. The potential value of this pathway for a therapeutic approach thus depends on the structural elucidation of the components.…”