Molecular Dynamics Flexible Fitting Simulations Identify New Models of the Closed State of the Cystic Fibrosis Transmembrane Conductance Regulator Protein

Cystic
fibrosis (CF) is a lethal, genetic disease found in particular
in humans of European origin which is caused by mutations in the cystic
fibrosis transmembrane conductance regulator (CFTR) chloride channel.
The search for CF therapies acting by modulating the impaired function
of mutant CFTR will be greatly advanced by high resolution structures
of CFTR in different states. To date, two medium resolution electron
microscopy (EM) structures of CFTR are available (one of a distant
zebrafish (Danio rerio) CFTR ortholog
and one of human CFTR). The two models are nearly identical to one
another, and both correspond to the inward-facing, nucleotide binding
domains (NBDs) separated, closed state of the channel. In addition,
lower resolution structural data are available for human CFTR in an
alternative conformation which likely features associated NBDs and
thus geometrically resembles the conducting state of the channel.
Multiple homology models of human CFTR in multiple states have been
developed over the years, yet their correspondence to the existing
structural information is unexplored. In this work we use molecular
dynamics flexible fitting (MDFF) simulations to refine two previously
described CFTR models based on the available cryo-EM map of the human protein. This map was recorded in the absence of ATP
and consequently represents closed-state CFTR yet its features likely
correspond to an NBD associated conformation of the protein. Accordingly,
the resulting models feature dimerized NBDs yet with no membrane traversing
pore. Moreover, the open probability of the new models as deduced
from the MDFF trajectories is significantly lower than that deduced
from control MD trajectories initiated from the starting models. We
propose that the new models correspond to a CFTR conformation which
to date was largely unexplored yet is one that is relevant to the
gating cycle of the protein. In particular this conformation may participate
in rapid channel opening and closing through small allosteric movements
controlled by nucleotide binding and dissociation events. Analyzing
the resulting trajectories (and not only the final models as is usually
the case), we demonstrate that the refined models have good stereochemical
properties and are also in favorable agreement with multiple experimental
data. Moreover, despite different starting points, the final models
share many common features. Finally, we propose that the combination
of high resolution cryo-EM maps, which are currently emerging from
multiple laboratories, and MDFF simulations will be of value for the
development of yet more reliable CFTR models as well as for the identification
of binding sites for CFTR modulators.