Study of the nucleotide-binding domain 1 of the human transporter protein MRP6.
Multidrug-resistance-associated protein 6 (MRP6/ABCC6) is a protein belonging to the ABC transporter family. Proteins in this family share many characteristic structural features, including two membrane-spanning domains and two nucleotide-binding domains (NBD1 and NBD2), that function cooperatively but not equally bind and hydrolyze ATP. The MRP6 protein is structurally and functionally poorly characterized. Previously, we showed, by NMR spectroscopy, that a fragment of MRP6-NBD1 presents helical structure and fluorescence experiments demonstrated that peptide binds ATP. These data suggested that the study on selected regions could be a valid approach to define the structure of MRP6 . In the present study, to better characterize MRP6-NBD1, we report data of CD spectroscopy, nucleotide binding and ATP hydrolysis on two different polypeptides, one corresponding to the full-length NBD1 domain (residues from Asp-627 to Leu-851) and the other is a shorter polypeptide (residues from Arg-648 to Thr-805) without some key residues. We report that both polypeptides are highly structured in aqueous buffer and in 20% trifluoroethanol showing considerable helical and β-structure content. The ATP hydrolysis activity is exhibited only by the full-length NBD1 domain. Comparison between our findings and the structurally well characterized MRP1-NBD1 supports the role of H-loop for the ATP hydrolysis and of A-loop in stabilizing the ATP binding.
Ostuni
Angela
A
Department of Chemistry, University of Basilicata, Potenza, viale Ateneo Lucano 10, 85100, Italy. angela.ostuni@unibas.it
Miglionico
Rocchina
R
Castiglione Morelli
Maria Antonietta
MA
Bisaccia
Faustino
F
D000251:Adenosine Triphosphatases / Q000737:chemistry; D000595:Amino Acid Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D005453:Fluorescence; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000096:biosynthesis / Q000737:chemistry*; D009711:Nucleotides / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry
D000251:Adenosine Triphosphatases; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D009711:Nucleotides; D011994:Recombinant Proteins; C102029:ABCC6 protein, human