Figure 6. Distinct roles of the two ATP sites in promoting CFTR channel gating.(A) Homology models (Corradi et al., 2015) of closed (left) and open (right) conformations of phosphorylated CFTR gating in ATP, based on the structures of inward-facing Tm287-288 (left) and outward-occluded McjD (right); target positions are highlighted in spacefill. Labels identify the native residues (left) and the Φ values of each position (right). For both conformations full structures (top) and NBDs with coupling helices only (bottom) are shown. (B) Current traces showing a single pore opening event in the absence of ATP in a patch containing hundreds of WT CFTR channels (top trace), and gating of a single K1250R CFTR channel in 5 mM ATP (bottom trace). Green and red bars identify closed interburst and open burst events, respectively. Cartoons below the traces illustrate the mechanism of gating of WT CFTR in the absence (top) and of K1250R CFTR in the presence (bottom) of ATP. Color coding as in Figure 1B, the upper ATP binding site represents non-catalytic site 1, the lower site represents catalytic site 2. Red semi-circles around ATP (yellow circles) represent tight bonding of the nucleotide with the opposing NBD surface. (C) Standard Gibbs energy profiles of a CFTR channel in the absence (orange profile) and presence (black profile) of bound ATP during progress from the closed state (left) through the transition state (center) to the open state (right). Vertical arrows illustrate the heights of the energetic barriers for the forward (opening) and backward (closing) steps under both conditions.Figure 6—figure supplement 1. Location of aspartate 1370, mutated in our background construct.Structures of NBD1 (blue) and NBD2 (green) of human dephosphorylated CFTR in a closed ATP-free state (PDBID: 5UAK) viewed top-down from the direction of the membrane. ATP sites 1 and 2 are highlighted by dotted ellipses, target positions in both sites are shown in spacefill. Aspartate 1370 (salmon spacefill) is located between the two ATP sites, at the boundary between the core and α-helical subdomains of NBD2, and interacts with Q-loop glutamine 1291 (yellow spacefill).
Image published in: Sorum B et al. (2017)
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