BioSysBio:abstracts/2007/Evgeny Zatulovskiy

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Docking Study on Mammalian CTR1 Copper Importer Motifs

Author(s): Evgeny Zatulovskiy, Sergey Samsonov
Affiliations: Saint-Petersburg State Polytechnical University, Research Institute for Experimental Medicine, Saint-Petersburg, Russia
Contact:email: ZatulovskyE@mail.ru
Keywords: 'copper metabolism' 'CTR1' 'Cu(I)-importer' 'docking molecules'

Background/Introduction

Copper is an essential trace element that is required for a number of vitally important functions in all types of cells in the majority of living organisms [1]. In mammals cuproenzymes (copper-containing proteins) take part in such processes as oxidative phosphorylation (cytochrome c oxidase), free radicals detoxication (superoxide dismutase), connective tissue formation (lysyl oxidase), regulation of central nervous system functions (dopamine beta-monoxygenase, peptidyl alfa-amidating monooxygenase), iron transmembrane transport (ceruloplasmin, gefestin), blood coagulation (blood coagulation factor VIII) [2]. Copper deficiency in cell leads to defitiency of these enzymes. At the same time free copper ions are extremely toxic for all types of biomolecules due to reactive oxygene species formation caused by copper ions reversible redox changes Cu(I) <--> Cu(II). Safety copper obtention to cells, its distribution to cuproenzymes formation regions and its excretion are provided by a special copper metabolic system. To the present time some genes of this system are cloned and main principles of its work are studied. Copper matabolism systems of all eukariots are similar, they include integral membrane and soluble cytosolic proteins, coded by orthological genes, structures and functions of which are highly conservative. Tranportation of copper ions to the sites of cuproenzymes formation is provided by proteins of this system unidirectionally and consecutively within direct protein-protein interactions [3]. Copper transition through cell membrane is the main stage of its metabolism. High affinity Cu(I) transporter from CTR1 family (encoded by SLC31A1) is the main candidate for the role of mammalian transmembrane Cu(I)-importer. All proteins of this family exist as homotrimers. They contain extracellular N-terminal and cytosolic C-terminal copper binding sites, separated by transmembrane cuprophylic channel [4]. Existing experimental data evidences that CTR1 plays a central role in copper ions transportation. It is a reason of high interest to studies on CTR1 structure and functions. Despite some success in theese studies many aspects of CTR1 functionality is unclear yet. In particular, it is unknown, what is the donor of copper to N-terminal domain of CTR1, that is localized on cell surface. It could be ceruloplasmin (CP) - a copper-transporting protein, which is the main donor of copper for nonhepatic cells and which holds more than 95% of all extracellular copper [5]. Intracellular acceptor of copper from CTR1 is also not identified yet. It was shown that there is own Cu(I)-chaperone for each cuproenzyme. But it is unknown, if all of these chaperones can accept copper from CTR1 or not. Menkes ATPase is one of such chaperones. And it is interesting to clear up if copper-binding domain of this ATPase can interact with C-terminal domain of CTR1. Finding answers to described questions is very important for general understanding of copper thansport mechanism in cells.

Results

The calculations showed that the first N-terminal copper binding motif of CTR1 (MDHSHHMGMSYMD) is favoured to interact with the site on CP surface that consists of such residues of CP (PDB ID: 1KCW) as: Asp233, Glu236, Tyr241, Glu272, Asn323, Glu593, Lys600, Asn632, Glu633 Val903, Asp929, Glu931, Ile934, Lys938, Glu971, and also with labile Cu atom bound by CP molecule (Cu62 in 1KCW PDB structure) and with residues, which are directly close to this labile copper atom: Glu935, His940, Asp1025. The second N-terminal copper binding motif of CTR1 (QPSHHHPT) is mostly favoured to interact with the site on CP molecule surface that is close to the site of the first motif binding. This second site includes Gln235, Glu272, Arg239, Val273,Glu633, Asn907, Glu908, Lys928, Asp929, Ile934, Lys938, Glu971 residues of CP. Also the second motif contacts with the same labile Cu atom bound by CP molecule as the first one and with residues, which directly contact with this labile copper atom: Glu935, Asp1025. The third N-terminal copper binding motif of CTR1 (SMMMMPMT) is favoured to interact with two different sites on CP molecule. The first of these sites is nearly same as the site for binding with the first and second N-terminal copper-binding motifs. This site includes Asp230, Gln235, Asn271, Glu272, Ser745, Asn746, Ala747, Phe748, Phe947, Thr1024, His1028 residues of CP and also the same labile Cu atom (Cu62 in 1KCW PDB structure) and residues, which directly contact with this labile copper atom: Glu935, Asp1025. The second possible site consists of such residues of CP as: His32, Tyr36, Asn323, Lys326, Glu593, Gln596, Lys600, Glu633, Lys928, Asp929, Glu931, Ile934, and also includes another labile Cu atom bound by CP molecule (Cu42 in 1KCW PDB structure) and residues, which directly contact with this labile copper atom: Glu597 and Asp684. As the control for docking specificity we used albumin, another copper binding protein. No copper containing sites in CP were found to be favourable for the interaction with albumin. That means the possibility of the CP role as the Cu donor for CTR1 N-terminus. The docking of CTR1 C-terminal copper binding monomeric, dimeric and trimeric HCH motifs with reduced 4th Menkes ATPase metal binding also revealed the sites of favoured interactions. So monomeric HCH copper-bound motif is favoured to interact with Met12, Thr13, Cys14, Leu38, Ser41 residues of reduced metal-binding domain 4 of Menkes ATPase (PDB ID: 1AW0). In this situation Cu(I) could be coordinated by Met12 and Cys14 residues of metal-binding domain, that agrees with NMR-analisys data (PDB ID: 2AW0). Dimeric HCH copper-bound motifs are favoured to interact with some different sites on the reduced 4th Menkes ATPase metal binding domain surface. The best energies correspond to interactions with: 1. Arg35, Val36, Ser37, Asn42, Thr44; 2. Gly23, Val24, Lys27; 3. Cys17, Ser20, Met64, Phe66; 4. Lys27, Lys28, Pro29; 5. Asp10, Gly11, Met12, Thr13, Asp67. Trimeric HCH copper-bound motifs are also favoured to interact with several sites on the Menkes ATPase metal binding domain surface. For trimeric motifs the best energies correspond to interactions with: 1. Gln19, Glu22, Arg35; 2. Gln19, Gly23; 3. Lys28, Pro29; 3. Asn15, Val18, Leu38; 4. Ser37, Leu38, Ala39, Asn40; 5. Cys17, Met64, Phe66; 6. Thr2, Lys32, Ser33; Glu46; 7. Lys28, Ala60, Asp63. Thus, most possible sites of reduced 4th Menkes ATPase metal binding domain for interaction with C-terminal copper-bound HCH motifs of CTR1 are: 1) Met12, Thr13, Cys14; 2) Arg35, Thr44; 3) Cys17, Met64, Phe66; 4) Lys27, Lys28, Pro29. These results agree with experimental data and confirm the posibility of CTR1 C-termial motifs to transport copper to Cu-binding domain of Menkes ATPase. They show that copper from CTR1 can be brought to CXXC motif of Cu(I) chaperone (Cys14-X-X-Cys17 motif of 4th Menkes ATPase metal binding domain - PDB ID: 1AW0).

Images/Tables

Model of interaction between domain 4 of Menkes ATPase (PDB ID: 1AW0) and monomeric C-terminal HCH motif of CTR1 File:I:\SS\interaction model.jpg NMR structure of metal binding domain 4 of Menkes ATPase (PDB ID: 2AW0) File:I:\SS\NMR structure.jpg

Materials/Methods

Hex 4.5 docking software was used in our study for the protein molecules docking calculations. Hex is an interactive molecular graphics program for calculating and displaying feasible docking modes of pairs of protein and DNA molecules. Hex can also calculate small-ligand/protein docking (provided the ligand is rigid). The main thing which distinguishes Hex from other macromolecular docking programs and molecular graphics packages is its use of spherical polar Fourier correlations to accelerate the docking and superposition calculations. In Hex's docking calculations, each molecule is modelled using 3D parametric functions which are used to encode both surface shape and electrostatic charge and potential distributions. By writing an expression for the overlap of pairs of parametric functions, one can derive an expression for a docking score as a function of the six degrees of freedom in a rigid body docking search. With suitable scaling factors, this docking score can be interpreted as an interaction energy, which we seek to minimise. Use of spherical polar Fourier correlations to accelerate the docking calculations and heteroatoms parametrization implemented into the sofware are the advantages of Hex software. The main disadvantages consist in that model of rigid ligand is used and the solvent is not accounted for in docking calculations. In our study we used reduced extracellular N-terminal copper-binding motifs of CTR1 as ligands for docking with ceruloplasmin molecule (PDB structure: 1KCW). Also we used monomeric, dimeric and trimeric C-terminal copper-binding motifs of CTR1 for calculation of docking with reduced 4th Menkes ATPase metal binding domain (PDB structure: 1AW0). We used docking of random oligopeptides with albumine, ceruloplasmin, reduced 4th Menkes ATPase metal binding domain and also docking albumine with ceruloplasmin as the control for docking specificity. Conformations of used motifs and oligopeptides were obtained in geometry optimization by AMBER force field package with point charges on atoms obtained in CNDO semi-empirical method. We used these methods implemented in HyperChem 7.0 software.

Conclusion

The calculations showed that all the reduced CTR1 extracellular N-terminal copper binding motifs are strongly favoured to interact with labile Cu bound with CP molecule. As the control for docking specificity we used albumin, another copper binding protein. No copper containing sites in CP were found to be favourable for the interaction with albumin. That means the possibility of the CP role as the Cu donor for CTR1 N-terminus. The docking of C-terminal copper binding monomeric, dimeric and trimeric HCH motifs with reduced metal binding domain 4 of Menkes ATPase revealed the sites of favoured interactions that coincide with Cu-binding site CXXC in Menkes ATPase. The results allow us to conclude that CTR1 is able to accept Cu from extracellular transporter, CP, and transport it further into the cell to Cu binding domain of Menkes ATPase. That agrees with other in vivo and in vitro experiments suggesting CTR1 role as membrane copper importer.

References

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