Binding of ruthenium(III) anti-tumor drugs to human lactoferrin probed by high resolution X-ray crystallographic structure analyses
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The binding to human lactoferrin of three Ru(III) complexes with anti-tumor activity has been investigated by X-ray crystallography in order to gain insights into how such complexes might be carried during transferrin-mediated delivery to cells. The complexes, HIm[RuIm2Cl4], HInd[RuInd2Cl4] and (HInd)2 [RuIndCl5], where Im = imidazole and Ind = indazole, were diffused into crystals of apo-lactoferrin (apoLf). X-ray diffraction data were collected to 2.6 Å, 2.2 Å and 2.4 Å respectively. The binding sites for the Ru complexes were determined from difference Fouriers, in comparison with native apoLf; the two indazole-apoLf complexes were also refined crystallographically to final R factors of 0.202 (for 8.0 to 2.3 Å data) and 0.192 (for 8.0 to 2.4 Å data) respectively. Two types of binding site were identified, a high-affinity site at His 253 in the open N-lobe iron-binding cleft of apoLf (and by analogy a similar one at His 597 in the C-lobe), and lower-affinity sites at surface-exposed His residues, primarily His 590 and His 654. The exogenous heterocyclic ligands remain bound to Ru, at least at the His 253 site, and modelling suggests that the nature and number of these ligands may determine whether the closed structure that is required for receptor binding could be formed or not. The results also highlight the importance of His residues for binding such complexes and the value of heavy atom binding studies from crystallographic analyses for identifying non-specific binding sites on proteins.
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