Structural studies of several clinically important oncology drugs in complex with human serum albumin.

BACKGROUND: Serum albumin is a major pharmacokinetic effector of drugs. To gain further insight into albumin binding chemistry, the crystal structures of six oncology agents were determined in complex with human serum albumin at resolutions of 2.8 to 2.0Å: camptothecin, 9-amino-camptothecin, etoposide, teniposide, bicalutamide and idarubicin. METHODS: Protein crystal growth and low temperature X-ray crystallography RESULTS: These large, complex drugs are all bound within the subdomain IB binding region which can be described as a hydrophobic groove formed by α-helices h7, h8 and h9 covered by the extended polypeptide L1. L1 creates a binding cavity with two access sites, one between loop L1 and α-helices h7 and h8 (distal site: IBd) and the other between L1 and α-helix h9 (proximal site: IBp). Camptothecin (2.4Å) and 9 amino camptothecin (2.0Å) are clearly bound as the open lactone form (IBp). Idarubicin (2.8Å) binds in a DNA like dimer complex via an intermolecular π stacking arrangement in IBd. Bicalutamide (2.4Å) is bound in a folded intramolecular π stacking arrangement between two aromatic rings in IBd similar to idarubicin. Teniposide (2.7Å) and etoposide (2.7Å), despite small chemical differences, are bound in two distinctly different sites at or near IB. Teniposide is internalized via primarily hydrophobic interactions and spans through both openings (IBp-d). Etoposide is bound between the exterior of IB and IIA and exhibits an extensive hydrogen bonding network. CONCLUSIONS: Subdomain IB is a major binding site for complex heterocyclic molecules. GENERAL SIGNIFICANCE: The structures have important implications for drug design and development. This article is part of a Special Issue entitled Serum Albumin.

Structure of human ferritin L chain.

Ferritin is the major iron-storage protein present in all cells. It generally contains 24 subunits, with different ratios of heavy chain (H) to light chain (L), in the shape of a hollow sphere hosting up to 4500 ferric Fe atoms inside. H-rich ferritins catalyse the oxidation of iron(II), while L-rich ferritins promote the nucleation and storage of iron(III). Several X-ray structures have been determined, including those of L-chain ferritins from horse spleen (HoSF), recombinant L-chain ferritins from horse (HoLF), mouse (MoLF) and bullfrog (BfLF) as well as recombinant human H-chain ferritin (HuHF). Here, structures have been determined of two crystal forms of recombinant human L-chain ferritin (HuLF) obtained from native and perdeuterated proteins. The structures show a cluster of acidic residues at the ferrihydrite nucleation site and at the iron channel along the threefold axis. An ordered Cd2+ structure is observed within the iron channel, offering further insight into the route and mechanism of iron transport into the capsid. The loop between helices D and E, which is disordered in many other L-chain structures, is clearly visible in these two structures. The crystals generated from perdeuterated HuLF will be used for neutron diffraction studies.

Convergent-beam method in macromolecular crystallography.

A data-collection method for macromolecular crystals using convergent sources is described here. Because of the unique characteristics of the diffraction patterns, a software package CBMPRO has been developed specifically for processing data images collected with the convergent beam method (CBM). The resulting data sets from crystals with two different sets of unit-cell parameters are presented and compared. There is good agreement between data sets from the same type of crystals under slightly different experimental conditions and data sets collected and processed with CBM also agree well with those from conventional oscillation methods, marking an important step to establishing CBM as a viable alternate data-collection method for macromolecular crystals.

The atomic structure of human methemalbumin at 1.9 A.

The high resolution structure of hemalbumin was determined by single crystal X-ray diffraction to a resolution of 1.9 A. The structure revealed the protoporphyrin IX bound to a single site within a hydrophobic cavity in subdomain IB, one of the principal binding sites for long chain fatty acid. The iron is penta coordinated with the fifth ligand comprised of the hydroxyl oxygen of Tyr-161 (phenolic oxygen to heme plane distance: 2.73 A) in an otherwise completely hydrophobic pocket. The heme propionic acid residues form salt bridges with His-142 and Lys-190, which together with a series of hydrophobic interactions, enclose and secure the heme within the IB helical motif. A detailed discussion of the structure together with its implications for the development of potential blood substitutes is presented.