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The NBD1 Domain of the Cystic Fibrosis Transmembrane Conductance Regulator Image made using pymol & PDB 1QH3 from Lewis, EMBO,23,2,283-293,2004 By Eladio Abreu Cystic Fibrosis Cystic Fibrosis (CB) is a lethal autosomal recessive genetic disease Symptoms: – mucus build up – debilitating chronic pulmonary inflammation and blockage – excess loss of salts in sweat – reduced pancreatic enzyme release Reduced life span Source: Defective CFTR Dysfunctional chloride ion transport in epithelial cells is responsible for the disease A defect in the cystic fibrosis transmembrane conductance regulator (CFTR) protein leads to the obvious transport problem CFTR The CFTR was first identified in 1989 Sequence analysis predicted that it was comprised of: – 2 MSDs – 2 NBDs – 1 R-region Characteristics consistent with members of the ABC super family of membrane transporters. MSD1 MSD2 Thought to satisfy binding interactions L of ATP bound to N S ATP NBD2 G G Q H NBD1 G NBD2 ATP H S L C Signature R Sequence ATP Model for The Cystic Fibrosis Trans-Membrane Regulator, and Position of NBD1 Within it NBD1 Image made using pymol,powerpoint & PDB 1QH3 from Lewis, EMBO,23,2,283-293,2004 NBD1 and its Significance In most ABC transporters ATP hydrolysis cycles between the 2 NBDs lead to confirmational changes in the MSDs and subsiquent channel gating. NBDs have several structural motifs that aid in ATP binding and hydrolysis. All of the mutations that lead to CF are in NBD1 of the CFTR Tertiary Structure and Subdomain Composition of mNBD1 F1-type ATP binding core c ABC ά-subdomain β-subdomain Unique to mNBD1 N mNBD1 Image made using pymol, powerpoint & PDB 1QH3 from Lewis, EMBO,23,2,283-293,2004 ATP Binds between helices Hl, H1B, and H1C Walker motif A (GSTGSGKTS) extends from the N- terminal end of H1. – ATP directly binds:Gly463, Ser466, Gly461, Ser462, and Lys464 of walker motif A. – also binds with the nucleotide indirectly by coordinating Mg+2 . Mg+2 is coordinated by Thr465 of Walker Motif A Walker motif B (LYLLDSPFG) which extends from the S7 β-strand of the F1-type ATP binding core provides an ASP residue which aids in Mg+2 coordination Glu493 from an adjacent Q-loop contributes toward Mg+2 coordination. Phe430 of helix H1C makes an edge to face interaction with the adenine of ATP. Position and Mode of ATP Binding in mNBD1 Image made using pymol & PDB 1QH3 from Lewis, EMBO,23,2,283-293,2004 The image above identifies the H- bonding, ionic and Vander Vaals interactions responsible for binding ATP to NBD1 General position of ATP binding site mNBD1 Binds ATP but does not hydrolize it Structural characteristics Structural Differences in shared with other ABC NBDs mNBD1 that eliminate implicated in ATP binding hydrolytic activity ATP Binding core – Walker motifs A and B – Catalytic charged residues in – Q and H loops these motifs replaced by inactive serine residues. ATP only has face to edge ATP stacks against several interaction with a PHE residue as aromatic residues when bound result to a different torsional angle CF mutations are not in positions involved in ATP hydrolysis ATP binding site Image made using pymol & PDB 1QH3 from Lewis, EMBO,23,2,283-293,2004 Positions of the common CB causative mutations are highlighted in cyan, the most common (occurring in 90% of CB patients) is the deletion of Phe508 (highlighted in green) Conclusions NBD1 binds ATP but does not hydrolyze it. CFTR channel gating is dependent on ATP binding at NBD1 but not its hydrolysis. The NBDS come together to form a nucleotide sandwich, below the MSDs (as seen in slide number 5).
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