Probing the Interaction of Cisplatin with the Human Copper Chaperone Atox1 by Solution and In-Cell NMR Spectroscopy

Among anticancer therapeutics, platinum-based drugs have a prominent role. They carry out their antitumor activity by forming stable adducts with DNA, thus interfering with replication and transcription processes. Cellular uptake of these drugs is tightly connected to copper transport. The major Cu(I) influx transporter Ctr1 has been found to mediate transport of cisplatin and its analogues. Evidence also suggests that ATP7A and ATP7B mediate cisplatin sequestration and efflux from cells, thus influencing drug resistance. The copper-chaperone Atox1, which normally binds Cu(I) via two cysteines and delivers the metal to ATP7A/B, has also been reported to interact with cisplatin in in vitro experiments. In the present investigation we apply a combined approach, using solution and in-cell NMR spectroscopy methods, to probe intracellular drug delivery and interaction of cisplatin with Atox1. The intracellular environment provides itself the suitable conditions for the preservation of the protein in its active form. Initially a {Pt(NH₃)₂}-Atox1 adduct is formed. At longer reaction time we observed protein dimerization and loss of the ammines. Such a process is reminiscent of the copper-promoted formation of Atox1 dimers which have been proposed to be able to cross the nuclear membrane and act as a transcription factor. We also show that overexpression of Atox1 in <i>E. coli</i> reduces the amount of DNA platination and, consequently, the degree of cell filamentation

Dynamic Nuclear Polarization of Sedimented Solutes

Using the 480 kDa iron-storage protein complex, apoferritin (ApoF), as an example, we demonstrate that sizable dynamic nuclear polarization (DNP) enhancements can be obtained on sedimented protein samples. In sedimented solute DNP (SedDNP), the biradical polarizing agent is co-sedimented with the protein, but in the absence of a glass-forming agent. We observe DNP enhancement factors ε > 40 at a magnetic field of 5 T and temperatures below 90 K, indicating that the protein sediment state is “glassy” and suitable to disperse the biradical polarizing agent upon freezing. In contrast, frozen aqueous solutions of ApoF yield ε ≈ 2. Results of SedDNP are compared to those obtained from samples prepared using the traditional glass-forming agent glycerol. Collectively, these and results from previous investigations suggest that the sedimented state can be functionally described as a “microcrystalline glass” and in addition provide a new approach for preparation of samples for DNP experiments

Structural Insights into the Ferroxidase Site of Ferritins from Higher Eukaryotes

The first step of iron biomineralization mediated by ferritin is the oxidation at the ferroxidase active site of two ferrous ions to a diferric oxo/hydroxo species. Metal-loaded ferritin crystals obtained by soaking crystals of frog ferritin in FeSO₄ and CuSO₄ solutions followed by flash freezing provided X-ray crystal structures of the tripositive iron and bipositive copper adducts at 2.7 and 2.8 Å resolution, respectively. At variance with the already available structures, the crystal form used in this study contains 24 independent subunits in the asymmetric unit permitting comparison between them. For the first time, the diferric species at the ferroxidase site is identified in ferritins from higher eukaryotes. Anomalous difference Fourier maps for crystals (iron crystal 1) obtained after long soaking times in FeSO₄ solution invariantly showed diferric species with a Fe–Fe average distance of 3.1 ± 0.1 Å, strongly indicative of the presence of a μ-oxo/hydroxo bridge between the irons; protein ligands for each iron ion (Fe1 and Fe2) were also unequivocally identified and found to be the same in all subunits. For copper bound ferritin, dicopper­(II) centers are also observed. While copper at site 1 is essentially in the same position and has the same coordination environment as Fe1, copper at site 2 is displaced toward His54, now acting as a ligand; this results in an increased intermetal distance (4.3 ± 0.4 Å). His54 coordination and longer metal–metal distances might represent peculiar features of divalent cations at the ferroxidase site. This oxidation-dependent structural information may provide key features for the mechanistic pathway in ferritins from higher eukaryotes that drive uptake of bivalent cation and release of ferric products at the catalytic site. This mechanism is supported by the X-ray picture obtained after only 1 min of soaking in FeSO₄ solutions (iron crystal 2) which reasonably contain the metal at different oxidation states. Here two different di-iron species are trapped in the active site, with intermetal distances corresponding to those of the ferric dimer in crystal 1 and of the dicopper centers and corresponding rearrangement of the His54 side chain

Structural Basis for Matrix Metalloproteinase 1-Catalyzed Collagenolysis

The proteolysis of collagen triple-helical structure (collagenolysis) is a poorly understood yet critical physiological process. Presently, matrix metalloproteinase 1 (MMP-1) and collagen triple-helical peptide models have been utilized to characterize the events and calculate the energetics of collagenolysis via NMR spectroscopic analysis of 12 enzyme–substrate complexes. The triple-helix is bound initially by the MMP-1 hemopexin-like (HPX) domain via a four amino acid stretch (analogous to type I collagen residues 782–785). The triple-helix is then presented to the MMP-1 catalytic (CAT) domain in a distinct orientation. The HPX and CAT domains are rotated with respect to one another compared with the X-ray “closed” conformation of MMP-1. Back-rotation of the CAT and HPX domains to the X-ray closed conformation releases one chain out of the triple-helix, and this chain is properly positioned in the CAT domain active site for subsequent hydrolysis. The aforementioned steps provide a detailed, experimentally derived, and energetically favorable collagenolytic mechanism, as well as significant insight into the roles of distinct domains in extracellular protease function

Interaction of Cisplatin with Human Superoxide Dismutase

<i>cis</i>-Diamminedichloroplatinum­(II) (cisplatin) is able to interact with human superoxide dismutase (hSOD1) in the disulfide oxidized apo form with a dissociation constant of 37 ± 3 μM through binding cysteine 111 (Cys111) located at the edge of the subunit interface. It also binds to Cu₂–Zn₂ and Zn₂–Zn₂ forms of hSOD1. Cisplatin inhibits aggregation of demetalated oxidized hSOD1, and it is further able to dissolve and monomerize oxidized hSOD1 oligomers <i>in vitro</i> and <i>in cell</i>, thus indicating its potential as a leading compound for amyotrophic lateral sclerosis