Molecular modeling of the heterodimer of human CFTR’s nucleotide-binding domains using a protein–protein docking approach

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Abstract

We have presented a new protein–protein docking approach to model heterodimeric structures based on the conformations of the monomeric units. The conventional modeling method relies on superimposing two monomeric structures onto the crystal structure of a homologous protein dimer. The resulting structure may exhibit severe backbone clashes at the dimeric interface depending on the backbone dissimilarity between the target and template proteins. Our method overcomes the backbone clashing problem and requires no a priori knowledge of the dimeric structure of a homologous protein. Here we used human Cystic Fibrosis Transmembrane conductance Regulator (CFTR), a chloride channel whose dysfunction causes cystic fibrosis, for illustration. The two intracellular nucleotide-binding domains (NBDs) of CFTR control the opening and closing of the channel. Yet, the structure of the CFTR’s NBD1–NBD2 complex has not been experimentally determined. Thus, correct modeling of this heterodimeric structure is valuable for understanding CFTR functions and would have potential applications for drug design for cystic fibrosis treatment. Based on the crystal structure of human CFTR’s NBD1, we constructed a model of the NBD1–NBD2 complex. The constructed model is consistent with the dimeric mode observed in the crystal structures of other ABC transporters. To verify our structural model, an ATP substrate was docked into the nucleotide-binding site. The predicted binding mode shows consistency with related crystallographic findings and CFTR functional studies. Finally, genistein, an agent that enhances CFTR activity, though the mechanism for such enhancement is unclear, was docked to the model. Our predictions agreed with genistein’s bell-shaped dose–response relationship. Potential mutagenesis experiments were proposed for understanding the potentiation mechanism of genistein and for providing insightful information for drug design targeting at CFTR. The method used in this study can be applied to modeling studies of other dimeric protein structures.

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)

  • R. Derand et al.

    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.

    (2002)
  • J. Riordan et al.

    Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA

    Science

    (1989)
  • T.-Y. Chen et al.

    CLC-0 and CFTR: Chloride channels evolved from transporters

    Physiol. Rev.

    (2008)
  • D.C. Gadsby et al.

    The ABC protein turned chloride channel whose failure causes cystic fibrosis

    Nature

    (2006)
  • X. Zou et al.

    ATP hydrolysis-coupled gating of CFTR chloride channels: Structure and function

    Biochemistry

    (2001)
  • F. Becq

    On the discovery and development of CFTR chloride channel activators

    Curr. Pharm. Des.

    (2006)
  • H.A. Lewis et al.

    Structure of nucleotide-binding domain 1 of the cystic fibrosis transmembrane conductance regulator

    EMBO J.

    (2004)
  • P.H. Thibodeau et al.

    Side chain and backbone contributions of Phe508 to CFTR folding

    Nat. Struct. Mol. Biol.

    (2004)
  • I. Callebaut et al.

    Nucleotide binding domains of human cystic fibrosis transmembrane conductance regulator: Detailed sequence analysis and three dimensional modeling of the heterodimer

    Cell. Mol. Life Sci.

    (2004)
  • O. Moran et al.

    Binding site of activators of the cystic fibrosis transmembrane conductance regulator in the nucleotide binding domains

    Cell. Mol. Life Sci.

    (2005)
  • M. Mense et al.

    In vivo phosphorylation of CFTR promotes formation of a nucleotide-binding domain heterodimer

    EMBO J.

    (2006)
  • B. Illek et al.

    cAMP-independent activation of CFTR Cl channels by the tyrosine kinase inhibitor genistein

    Am. J. Physiol.

    (1995)
  • B. Illek et al.

    Alternate stimulation of apical CFTR by genistein in epithelia

    Am. J. Physiol.

    (1996)
  • T.-C. Hwang et al.

    Genistein potentiates wild-type and delta F508-CFTR channel activity

    Am. J. Physiol.

    (1997)
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