ATP-Dependent Signaling in Simulations of a Revised Model of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)

Abstract

Cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the ATP-binding cassette (ABC) transporter superfamily that has uniquely evolved to function as a chloride channel. It binds and hydrolyzes ATP at its nucleotide binding domains to form a pore providing a diffusive pathway within its transmembrane domains. CFTR is the only known protein from the ABC superfamily with channel activity, and its dysfunction causes the disease cystic fibrosis. While much is known about the functional aspects of CFTR, significant gaps remain, such as the structure–function relationship underlying signaling of ATP binding. In the present work, we refined an existing homology model using an intermediate-resolution (9 Å) published cryo-electron microscopy map. The newly derived models have been simulated in equilibrium molecular dynamics simulations for a total of 2.5 μs in multiple ATP-occupancy states. Putative conformational movements connecting ATP binding with pore formation are elucidated and quantified. Additionally, new interdomain interactions between E543, K968, and K1292 have been identified and confirmed experimentally; these interactions may be relevant for signaling ATP binding and hydrolysis to the transmembrane domains and induction of pore opening.

G551D mutation impairs PKA-dependent activation of CFTR channel that can be restored by novel GOF mutations

“…In a recent study of Cryo-EM structure analysis, Fay et al (18a) observed that the dephosphorylated R domain of chicken CFTR possibly interacts with CL1, supporting the idea that the R domain could regulate the interaction between the x-loop and CL1 by limiting the motion of CL1 under the condition of dephosphorylation. It is interesting to note that Strickland et al (38) recently reported that charge interaction between E543 in the x-loop of NBD1 and K968 in CL3 promotes channel phosphorylation by showing fast time course of activation, supporting the idea that interaction between the x-loop and CLs is important for signal transmission generated by ATP-dependent NBD dimerization and also implying a functional asymmetry between the two x-loops and their counterpart of CLs. Taken together, our present data provide new evidence that phosphorylation regulates CFTR channel activity independently of ATP-dependent NBD dimerization and support the idea that the R domain has divided labors that allosterically promote CFTR channel activity by targeting different gating elements (46), including interaction between the x-loop and CLs that is involved in conformational change requisite for CFTR channel opening.…”

The molecular evolution of function in the CFTR chloride channel

“…In addition, several homology models of CFTR were developed using structures of related ABC transporters as a template. These studies contributed to the understanding of the molecular interface encompassing the most common CF-causing mutation (ΔF508; Mornon et al, 2008 ; Serohijos et al, 2008 ), as well as several details relating to the conformational transitions underlying CFTR gating ( Corradi et al, 2015 ; Dalton et al, 2012 ; Furukawa-Hagiya et al, 2013 ; Mornon et al, 2015 ; Mornon et al, 2009 ; Rahman et al, 2013 ; Strickland et al, 2019 ). However, the disparity between the wide variety of substrates of nonchannel ABC transporters and the chloride channel function of CFTR resulted in intrinsically limited confidence in these homology models, at least with respect to the TMDs.…”

“…9 A depicts a simplified model of these sites, wherein each ABS is shown to consist of the so-termed Walker A, Walker B, and H loop regions from one NBD and the ABC signature and D loops from the other NBD. ATP binding to an ABS promotes NBD dimerization, which “powers” active transport by driving conformational changes in the TMDs ( Rahman et al, 2013 ; Strickland et al, 2019 ); in ABC exporters, this flips the TMD conformation from inward to outward facing ( Rees et al, 2009 ). ATP hydrolysis at these sites leads to dissociation of the NBD dimer, which allows the readoption of the inward-facing conformation to bind new intracellular substrates, although there is significant disagreement regarding the degree of dissociation undergone at the NBDs to accomplish this ( George and Jones, 2012 ; Hohl et al, 2014 ; Puljung, 2015 ; Zoghbi et al, 2012 ).…”

“…Second, ATP binds to two sites at the interface of the cytoplasmic NBDs, which promotes a stable NBD dimer ( Mense et al, 2006 ; Vergani et al, 2005 ). Finally, the wave of conformational changes associated with ATP-induced dimerization of the NBDs is transmitted to the pore domain, resulting in pore opening ( Rahman et al, 2013 ; Simhaev et al, 2017 ; Sorum et al, 2015 ; Strickland et al, 2019 ). In related ABC exporters, ATP-dependent dimerization of the NBDs drives an overall transition from inward- to outward-facing conformation of the TMDs; this function was coopted by CFTR to drive ATP-induced channel opening ( Fig.…”

“…At the level of individual residues, there is high conservation with transporters among amino acids in CFTR that are proposed to stabilize the inward-facing (closed) conformation in the absence of ATP ( Wang et al, 2010 ; Wei et al, 2014 ; Wei et al, 2016 ), suggesting conservation of motifs integral to energetic signaling ( Wang et al, 2014b ; Wei et al, 2014 ; Wei et al, 2016 ). The close proximity of intracellular loops 2 and 4 (ICL2 and ICL4, respectively; Doshi et al, 2013 ; Wang et al, 2014b ), constriction of the intracellular vestibule ( Bai et al, 2011 ), and dilation of the extracellular vestibule, relative to the closed state, are all associated with channel opening ( Beck et al, 2008 ; Infield et al, 2016 ; Norimatsu et al, 2012b ; Rahman et al, 2013 ; Strickland et al, 2019 ). The CFTR pore opens in stages, requiring the sequential breaking and forming of intraprotein residue–residue interactions ( Cui et al, 2013 , 2014 ; Rahman et al, 2013 ), resulting in two subconductance states in addition to the full-conductance state ( Gunderson and Kopito, 1995 ; Zhang et al, 2005a ; Zhang et al, 2005b ; Fig.…”