Structures of adenosine kinase from Trypanosoma brucei brucei

Abstract

Trypanosoma brucei is a single-cellular parasite of the genus Kinetoplastida and is the causative agent of African sleeping sickness in humans. Adenosine kinase is a key enzyme in the purine-salvage pathway, phosphorylating adenosine to AMP, and also activates cytotoxic analogues such as cordycepin and Ara-A by their phosphorylation. The structures of T. brucei brucei adenosine kinase (TbAK) in its unliganded open conformation and complexed with adenosine and ADP in the closed conformation are both reported to 2.6 Å resolution. The structures give insight into the binding mode of the substrates and the conformational change induced upon substrate binding. This information can be used to guide the improvement of cytotoxic substrate analogues as potential antitrypanosomal drugs.

Keywords: Trypanosoma brucei brucei; adenosine kinase; ligand complex.

Figure 1

Diffraction pattern from the apo crystal of TbAK. The figure shows a close-up of one of the images used for structure solution, with the rings annotating the resolution created using the adxv software (Arvai, 2012 ▶). The crystal is clearly split, with more than one lattice evident in the image. While the data appear to extend beyond 2.6 Å resolution on this image, the diffraction was anisotropic and the integration was restricted to 2.6 Å resolution overall.

Figure 2

The structure of TbAK. The views are all of the A chain, but there is essentially no difference between the four molecules in the asymmetric unit. (a) The unbiased maximum-likelihood difference map calculated with REFMAC5 output coefficients contoured at 3σ before the ligands were added to the model. The positive contour is in blue and the negative contour is in red. The protein chain is shown as a semi-transparent ribbon. (b) Worm representations of the fold of the open apo form (coral core subunit and red lid) superposed on the closed form (ice blue). The structures were superposed using SSM (Krissinel & Henrick, 2004 ▶) based on the core subunit. The adenosine and ADP ligands are shown as spheres. (c) Surface view of the A chain, in which the two adenines can be seen to be buried in deep pockets. (d) Superposition of the anion-hole region of the TbAK–adenosine–ADP complex (ice blue; the protein was co-crystallized with AMPPNP, which is presumed to have hydrolysed) reported here with those of TbAK–AP5A (PDB entry 3otx, coral; Kuettel et al., 2011 ▶) and human AK in complex with two adenosines (PDB entry 1bx4, lemon; Mathews et al., 1998 ▶). The structures were superposed using SSM based on the C^α atoms in chain A of both. The ligands are shown as cylinders, with the C and P atoms coloured according to chain for clarity. (e) Superposition of TbAK–adenosine–ADP with TbAK–AP5A (PDB entry 3otx). The ribbon shows the TbAK–adenosine–ADP trace. The ligands are shown as cylinders coloured as in (d). This figure was made with CCP4mg (McNicholas et al., 2011 ▶).