Molecular modeling of the heterodimer of human CFTR’s nucleotide-binding domains using a protein–protein docking approach
Introduction
Many proteins form dimers as functional units. One such example is the Cystic Fibrosis Transmembrane conductance Regulator (CFTR). CFTR is a chloride channel in the apical membrane of epithelial cells that is regulated by cAMP-dependent protein kinase (PKA) and ATP [1]. The 1480-amino acid protein belongs to the superfamily of ATP-binding cassette (ABC) transporters. CFTR consists of two transmembrane domains (TMDs), two intracellular nucleotide-binding domains (NBD1 and NBD2) and one unique intracellular regulatory (R) domain. It has been proposed that the ATP-driven dimerization of NBD1 and NBD2 leads to the opening of the channel, and the subsequent hydrolysis of ATP at NDB2 results in dimer dissociation which is responsible for the closing of the channel (for review, see Refs. [2], [3], [4]).
Mutations in the CFTR gene cause cystic fibrosis (CF), the most common fatal, inherited disease in Caucasian populations [5]. CFTR is therefore an important therapeutic target for CF treatment. Despite significant advances in synthesizing novel compounds that can modulate the activity of CFTR, there is still no cure for CF (see [6], [7], [8] for review). The three-dimensional structure of CFTR is highly desirable for functional studies and therapeutic design regarding CFTR.
A recent breakthrough in the CF field is the determination of the crystal structures of mouse and human CFTR’s NBD1 [9], [10], [11]. These structures allow computational modeling of the unknown structure of the NBD1–NBD2 heterodimer of human CFTR.
A few efforts have recently been devoted to constructing the human CFTR’s NBD1–NBD2 structures [12], [13], [14] by utilizing the crystal structures of the NBD homodimers of other members in the ABC transporter superfamily such as MJ0796 [15]. Despite providing new insights into CFTR’s structural features, these modeled structures contain limitations, such as the low levels of sequence identity in regions between human CFTR and other ABC transporters that will directly affect the accuracy of those structures modeled by homology [12]. Another limitation is that the superimposed NBD dimer can yield severe backbone clashes at the NBD1–NBD2 interface which need to be removed by deleting some protein segments [13]. These maneuvers could potentially introduce some artifacts into the modeled dimeric structure. Lastly, this type of overlying methods requires the dimeric structure of a homologous protein, restricting their application to more general cases.
In the present work, we proposed to use a new protein–protein docking approach to construct the human CFTR’s NBD1–NBD2 heterodimeric complex based on the crystal structure of human CFTR’s NBD1 and a modeled structure of human CFTR’s NBD2 built by using NBD1 as a mold. The method requires no a priori knowledge of any dimeric structure of homologous proteins. It also overcomes the potential severe backbone clashing problem in the conventional overlying method. The constructed NBD1–NBD2 model was consistent with the experimentally observed NBD dimers of ABC transporters (see [16] for review). To further validate our model, we docked ATP molecules to the CFTR’s NBD1–NBD2 dimer, and reproduced the observed ATP binding mode in the NBD1 monomer. Lastly, we used the modeled NBD heterodimer to investigate the interaction between genistein and human CFTR’s NBD dimer by molecular docking. Genistein is an isoflavonoid found in soybeans and soy products that has a prominent effect in potentiating CFTR channel activity [17], [18], [19], [20], [21], [22], [23], [24]. It is regarded as the gold standard for drug screening programs spearheaded by the Cystic Fibrosis Foundation. However, the molecular mechanism of genistein’s activation remains unclear. Based on our computational results, the putative binding sites of genistein on CFTR were identified, showing consistency with experimental findings.
Section snippets
Model human NBD2 structure by homology modeling
The structure of the human CFTR’s NBD2 was modeled using the crystal structure of the human CFTR’s NBD1 as a template (PDB code: 1XMI) [11]. The sequence alignment, giving a sequence identity of 24.8% and a similarity of 43.6% between NBD1 and NBD2, is shown in Fig. 1. Before modeling, the bound ATP was removed from the template. Based on the crystal structure of NBD1, the MODELLER program [25] was used to generate the three-dimensional structure of NBD2. Finally, AMBER force field minimization
The modeled structure of human CFTR’s NBD1–NBD2 dimer
The top human NBD1–NBD2 dimer structure predicted by our protein–protein approach is shown in Fig. 2. A notable feature of the predicted structure is the head-to-tail conformation in which the Walker A and B motifs of one NBD and the signature motif of the other NBD form the ATP-binding sites (see Fig. 1 for the definition of the motifs). The same unique dimeric mode is observed in the recently solved crystal structures of the other ABC transporters (e.g., [40], [41], [42], [43], [44], [15],
Conclusion
In the present study, we have used NBD1–NBD2 heterodimer of human CFTR as an example to present a protein–protein docking approach for modeling dimeric structures based on the monomeric structures without a priori structural knowledge about homologous protein dimers. It also avoids the problem of severe backbone clashes at the interface, another potential disadvantage of the conventional overlying method.
The dimeric mode of our constructed human CFTR’s NBD1–NBD2 structure is consistent with the
Acknowledgements
Support to XZ from OpenEye Scientific Software Inc. (Santa Fe, NM) and Tripos, Inc. (St. Louis, MO) is gratefully acknowledged. Zou is supported by the Cystic Fibrosis Foundation grant ZOU07I0, NIH grant DK61529, and the Research Board Award of the University of Missouri RB-07-32. Hwang is supported by NIH R01DK55835 and NIH R01HL53445. The support from Cystic Fibrosis Foundation Therapeutics, Inc. CLARKE06XXO grant is gratefully acknowledged. The work is also supported by Federal Earmark NASA
References (49)
- et al.
Molecular mechanisms of the CFTR chloride channel dysfunction in cystic fibrosis
Cell
(1993) - et al.
Molecular targeting of CFTR as a therapeutic approach to cystic fibrosis
Trends Pharmacol. Sci.
(2007) - et al.
Molecular pharmacology of the CFTR Cl− channel
Trends Pharmacol. Sci.
(1999) - et al.
Impact of the delta-F508 mutation in first nucleotide-binding domain of human cystic fibrosis transmembrane conductance regulator on domain folding and structure
J. Biol. Chem.
(2005) - et al.
ATP binding to the motor domain from an ABC transporter drives formation of a nucleotide sandwich dimer
Mol. Cell
(2002) - et al.
Structure and mechanism of ABC transporter proteins
Curr. Opin. Struct. Biol.
(2007) - et al.
A geometric approach to macromolecule-ligand interactions
J. Mol. Biol.
(1982) - et al.
A tweezers-like motion of the ATP-binding cassette dimer in an ABC transport cycle
Mol. Cell
(2003) - et al.
Nucleotide occlusion in the human cystic fibrosis transmembrane conductance regulator: Different patterns in the two nucleotide binding domains
J. Biol. Chem.
(1999) - et al.
The first nucleotide binding domain of cystic fibrosis transmembrane conductance regulator is a site of stable nucleotide interaction, whereas the second is a site of rapid turnover
J. Biol. Chem.
(2002)
The cystic fibrosis mutation G551D alters the non-Michaelis-Menten behavior of the cystic fibrosis transmembrane conductance regulator (CFTR) channel and abolishes the inhibitory genistein binding site
J. Biol. Chem.
Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA
Science
CLC-0 and CFTR: Chloride channels evolved from transporters
Physiol. Rev.
The ABC protein turned chloride channel whose failure causes cystic fibrosis
Nature
ATP hydrolysis-coupled gating of CFTR chloride channels: Structure and function
Biochemistry
On the discovery and development of CFTR chloride channel activators
Curr. Pharm. Des.
Structure of nucleotide-binding domain 1 of the cystic fibrosis transmembrane conductance regulator
EMBO J.
Side chain and backbone contributions of Phe508 to CFTR folding
Nat. Struct. Mol. Biol.
Nucleotide binding domains of human cystic fibrosis transmembrane conductance regulator: Detailed sequence analysis and three dimensional modeling of the heterodimer
Cell. Mol. Life Sci.
Binding site of activators of the cystic fibrosis transmembrane conductance regulator in the nucleotide binding domains
Cell. Mol. Life Sci.
In vivo phosphorylation of CFTR promotes formation of a nucleotide-binding domain heterodimer
EMBO J.
cAMP-independent activation of CFTR Cl channels by the tyrosine kinase inhibitor genistein
Am. J. Physiol.
Alternate stimulation of apical CFTR by genistein in epithelia
Am. J. Physiol.
Genistein potentiates wild-type and delta F508-CFTR channel activity
Am. J. Physiol.
Cited by (30)
Targeting different binding sites in the CFTR structures allows to synergistically potentiate channel activity
2020, European Journal of Medicinal ChemistryCitation Excerpt :On another hand, SBCs can be considered as tools to understand the mechanisms at play in CFTR (co-potentiation), in particular relative to our theoretical data, which suggested potential binding sites at the level of ATP-binding sites, with a noticeable evolution of the protein conformation to adapt such small molecules and possibly to allow allosteric regulation of the CFTR channel activity. The relevance of such potentiator binding sites at the NBDs interface has already been supported by other docking experiments based on 3D models [72,73], but remains to be supported at the experimental level. Of note, the free energy binding values calculated here are lower for SBC219 than for SBC040 in the two potential binding sites, a result consistent with (i) the higher efficiency observed for SBC219 and (ii) with the additional contacts predicted to exist between SBC219 and the intracellular loops (at least at the level of the canonical ATP-binding site).
The implications of CFTR structural studies for cystic fibrosis drug development
2017, Current Opinion in PharmacologyCitation Excerpt :Even though limitations of in silico approaches must be emphasized, especially at the level of the resolution needed for drug design, such a strategy is supported by the relevance of the targeted sites, similarities between different models and experimental data. The first in silico docking experiments were done on models of the CFTR NBD1:NBD2 heterodimer for predicting potentiator binding sites [38,39]. Later, based on models of the MSDs:NBDs architecture, a lot of effort has been put into the search for the VX-809-binding site, assuming that this one would be present on a folded 3D structure, and not on short-lived intermediate states, which are inaccessible through current structural studies.
Exploring the potential of global protein-protein docking: an overview and critical assessment of current programs for automatic ab initio docking
2015, Drug Discovery TodayCitation Excerpt :Targeting protein–protein interactions for drug design by designing small-molecule modulators has received a growing interest in pharmaceutical science [1–5], in which determination of the complex structure between interacting proteins is valuable [6–12].
Search strategies and evaluation in protein-protein docking: Principles, advances and challenges
2014, Drug Discovery TodayMolecular modelling approaches for cystic fibrosis transmembrane conductance regulator studies
2014, International Journal of Biochemistry and Cell BiologyCitation Excerpt :This effect was overcome in studies of Callebaut et al. (2004). An entirely different concept based on the protein-protein docking approach instead of homology modelling has been used by Huang at al. to propose a new model of NBD1–NBD2 interactions (Huang et al., 2009). The rationale was to overcome the backbone clashing problem that resulted from the superposition methods used in previous homology modelling.
Do main location within the cystic fibrosis transmembrane conductance regulator protein investigated by electron microscopy and gold labelling
2011, Biochimica et Biophysica Acta - BiomembranesCitation Excerpt :These observations might also suggest a way in which PDZ-binding proteins such as NHERF might influence the activity of CFTR in vivo [52,59]. The data presented here suggest that current homology models for CFTR [24,25,60,61] based on other ATP-binding cassette proteins appear to be reasonable, at least at a resolution sufficient to resolve the major core domains of the protein. An earlier study of negatively stained CFTR dimers, however, was not consistent with such homology models, and core domains were not resolved [14].