Biochemical characterization and NMR study of the region E748-A785 of the human protein MRP6/ABCC6.
Multidrug-resistance-associated protein 6 (MRP6/ABCC6) is a protein belonging to the ABC transporter family which couple ATP hydrolysis with the transport of molecules across biological membranes. MRP6 topology presents three transmembrane domains and two nucleotide-binding domains (NBDs). The protein is structurally and functionally poorly characterized. Mutations in ABCC6 gene cause Pseudoxanthoma elasticum, a recessive genetic disorder affecting the elastic tissues. Most mutations have been found in NBDs that are critical for ATP binding and hydrolysis. With the aim to better characterize MRP6, we have performed a preliminary study on the fragment E748-A785 of MRP6-NBD1, with the wild type sequence and the R765Q mutation found in PXE affected patients. CD and NMR spectroscopy show the presence of helical structures in both peptides. Fluorescence experiments demonstrate that peptides bind ATP. The NMR structure of the mutated peptide is compared with the corresponding region of the MRP6-NBD1 modeled structure using as a template the X-ray structure of MRP1-NBD1. The finding that both wild type and mutated peptide present the same structure and similar affinity for ATP suggests that the onset of PXE symptoms is a consequence of the different type of interactions involving residue 765 R/Q inside the protein.
Department of Chemistry, University of Basilicata, Potenza, via Nazario Sauro 85, 85100, Italy. maria.castiglione@unibas.it
Angela
A
Ostuni
Rocchina
R
Miglionico
Faustino
F
Bisaccia
Maria Antonietta
MA
Castiglione Morelli
D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D002942:Circular Dichroism; D006801:Humans; D008969:Molecular Sequence Data; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000737:chemistry / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D013050:Spectrometry, Fluorescence
D000255:Adenosine Triphosphate; D027425:ATP-Binding Cassette, Sub-Family C Proteins; C102029:ABCC6 protein, human