Functional and structural studies of the hydrophilic acylated surface proteins from Leishmania donovani

• Main Author: McKenna, Sophie
• Other Authors: Brzozowski, Andrzej Marek ; Wilkinson, Tony
• Published: University of York 2015
• Subjects: 540
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.680615

Protozoan parasites of the genus Leishmania cause a diverse range of tropical diseases referred to as Leishmaniasis that lack effective treatment or licensed vaccines. The hydrophilic acylated surface proteins (HASPs) are present in all human infective Leishmania species, are highly immunogenic and their expression is stage-regulated during human infection. HASPs share highly conserved N- and C-terminal domains, but a subset, the HASPBs, have a divergent central domain containing extensive hydrophilic amino acid repeats that vary in number and composition, both within and between Leishmania species. HASPs associate with the membrane via an N-terminal dual acylation motif that is myristoylated and palmitoylated. The focus of this project was the HASPs from Leishmania donovani. Extensive crystallisation screening of the proteins in combination with an array of directed crystallisation strategies did not produce crystals. Biochemical and biophysical characterisation supported bioinformatic predicted intrinsic disorder. NMR analysis of HASPA was performed, including resonance assignment of 94% of the backbone nuclei. This required implementation of unlabelling protocols to resolve problems associated with spectral congestion and low sequence complexity. HN, N, Cα and Cβ chemical shift analysis revealed that HASPA does not contain elements of structural propensity. Myristoylation of the HASPs is catalysed by N-Myristoyltransferase (NMT). It was shown for the first time that recombinant NMT is able to catalyse recombinant HASP myristoylation in vitro. A fluorescence based assay, where a fluorescent CPM-CoA adduct is formed, was implemented to establish the kinetic parameters for this reaction. HASPA myristoylation was also monitored by real-time NMR spectroscopy, which revealed that the residues proximal to the N-terminus experienced the most pronounced changes in chemical shift or resonance intensity. A 2.45 Å resolution crystal structure of Leishmania major NMT in complex with 2-oxopentadecyl-CoA was elucidated. The data presented here, particularly IDP classification, will contribute to deciphering the functional role of HASPs.