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Vol. 1, 1237-1241, November 2002     Molecular Cancer Therapeutics
© 2002 American Association for Cancer Research

Molecular Modeling of Mutations in the DNA-binding Domain of the Oncoprotein Qin

Sharmila Banerjee-Basu and Andreas D. Baxevanis1

Genome Technology Branch, National Human Genome Research Institute, NIH, Bethesda, Maryland 20892-4470


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
The retroviral oncogene qin, homologue of mammalian brain factor 1 (FOXG1B), belongs to the family of winged helix transcription factors. Oncogenic transformation by Qin requires sequence-specific DNA binding. Missense mutations in the forkhead domain of Qin modulate its oncogenic transforming ability in chicken embryonic fibroblasts. We used homology model building (threading) techniques to generate atomic structures of wild-type c-Qin and c-Qin mutants, using the solution structure of the forkhead domain of the adipocyte transcription factor as a template (M. J. van Dongen et al., J. Mol. Biol., 296: 351–359, 2000). Energy calculations indicate that the Qin forkhead structure is stabilized primarily by hydrophobic interactions between residues at the helical interface. None of the missense mutations analyzed here were responsible for maintaining the most critical pairwise interactions holding the forkhead domain together. The mutated proteins form the overall structure of the forkhead domain, but the mutations do interfere with DNA binding.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
The qin oncogene was first isolated from the avian sarcoma virus (ASV31) that causes fibrosarcomas in chickens and induces neoplastic transformations in cell culture (1). Qin is the avian orthologue of mammalian BF-12 (also known as FOXG1). BF-1 is a telencephalon-specific gene that is essential for the proliferation of the progenitor cells of the cerebral cortex (2, 3). Targeted disruption of BF-1 is lethal in mice, with severe defects in the development of cerebral cortex and basal ganglia (4). BF-1 mediates the development of telencephalic structures by regulating the rate of neuroepithelial cell proliferation and the timing of neuronal differentiation. The cellular networks involved in proliferation of progenitor cells and asymmetric cell divisions essential for normal development are intimately connected to neoplastic progression.

The viral oncoprotein (v-Qin) and its cellular homologue (c-Qin) belong to the family of forkhead transcription factors (5). Forkhead transcription factors function in key regulatory processes including embryogenesis, tumorigenesis, and maintenance of differentiated cell states (6). Forkhead domains are evolutionarily conserved and exist in a wide range of species from yeast to human. Members of this family share a highly conserved DNA-binding domain of about 100 amino acid residues (the forkhead domain) and regulate expression of the downstream genes by sequence-specific DNA recognition. The DNA-binding domain consists of three major {alpha}-helices that are packed against each other, resting on a small three-stranded anti-parallel ß-sheet from which two loops emerge (7). The highest degree of sequence conservation among forkhead domain-containing proteins is found in the helical regions. DNA binding occurs mainly through the third helix, which interacts with the major groove of the DNA (8, 9). The winged regions of the forkhead domain also participate in DNA recognition. A number of missense mutations introduced in the third helix of c-qin (N189A, H193A, S196A, and N189A/H193A) reduced or completely abolished sequence-specific DNA binding (10). The H193A mutation failed to transform CEFs, whereas other mutations, even with reduced DNA binding, retained oncogenicity for CEFs. However, efficient DNA binding was positively correlated to accelerated transformation in CEFs.

In this article, we present a model of the forkhead domain of Qin generated using standard molecular modeling techniques. The homology within the forkhead family is relatively high, allowing for reliable structure determination. The models were examined with known experimental information and potential interaction surfaces suggested on the basis of conserved exposed residues. This work lays the foundation for future site-directed mutagenesis experiments for mapping biologically relevant residues within the Qin forkhead domain.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Homology Model Building.
Threading experiments were performed by the method of Bryant and Lawrence (11), with detailed derivations and methodology provided therein. Each query sequence was threaded through the atomic coordinates of the solution structure of the forkhead domain of FREAC-11. Three core segments were defined based on the three-dimensional structure. For each possible alignment, individual pairwise residue interactions were determined based on chemical type and distance intervals, lookup tables for which are present in Bryant and Lawrence (11). Using these values, a conformational energy {Delta}GR M, defined as the expected work for substitution of a specific sequence R for a random sequence with the same composition in the context of folding motif M, was then calculated for each alignment. Z-scores (ZR M) and chance occurrence probabilities (ER M) were calculated to compare conformational energies for different alignments. Chance occurrence probabilities give the odds that a random sequence of the same length and amino acid composition would yield a threading energy as low as the query sequence R. Calculations of energies and statistical significance were performed using C and S-PLUS subroutines (11). Critical interactions are defined as those having a pairwise interaction energy <=-1 kcal/mol. All energy scaffold figures were generated using the GRASP software package (12). The MODELER package (13) was also used to build and validate the Qin forkhead domain model, using two experimentally determined structures (FREAC-11 and AFX).

Phylogenetic Analysis.
Phylogenetic trees for the human forkhead family were constructed using algorithms contained within the PHYLIP Phylogeny Inference Package, version 3.5c (14). PROTDIST was used on these sequences to calculate a distance matrix according to the Dayhoff PAM probability model (15), as well as the categories model. The computed distances represent the expected fraction of amino acid substitutions between each pair of sequences. The distance matrix was then used to estimate phylogenies using the NJ method (16). Bootstrapping was carried out using SEQBOOT (1000 replicates for the PAM model of substitution and 100 replicates for the categories model of substitution). To independently confirm the results using alternate methodologies, the data set was also analyzed using the weighted least-squares distance method of Fitch and Margoliash (17) on 100 replicates. All Fitch runs were performed with global rearrangements. CONSENSE was used to compute the consensus tree by the majority-rule method. The final unrooted tree diagram was generated using TREETOOL.3


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
Sequence Comparison.
All available human forkhead domain sequences were compiled from various source databases using the human homologue of Qin, FOXG1B (SWISS-PROT accession number P55315) as the query in a PSI-BLAST (18) search. A final data set of 35 sequences was assembled after elimination of partial, duplicate, or incomplete sequences. A multiple sequence alignment of the complete data set is available online.4 The evolutionary relationships between these 35 human forkhead domains were analyzed using the NJ algorithm from the PHYLIP Phylogeny Inference Package (14). Two different amino acid substitution models (see "Materials and Methods") generated the same tree topology. The resulting unrooted tree showing the relationship of the Qin/FOXG1B forkhead domain to the other members of the forkhead family is shown in Fig. 1 (see supplementary material for bootstrap support values).4 In this phylogenetic analysis, the FOXC subclass (blue) is most closely related to Qin/FOXG1B. The three-dimensional structure of a member the C subclass, FREAC-11, was used to perform threading analysis of the Qin forkhead domain.



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  		Fig. 1. Phylogenetic relationships between the members of the human forkhead family. Unrooted NJ trees were generated using the PHYLIP Phylogeny Inference Package, version 3.5c. TREETOOL, a freestanding tree editor and formatter, was used to generate the tree diagram.

 
The success of homology modeling is critically dependent on a reliable alignment between the target sequences and the templates (19). To date, high-resolution three-dimensional structures of the DNA-binding domain for four different forkhead transcription factors, FREAC-11 (20), AFX (21), HNF3-{gamma} (7), and Genesis (8), have been experimentally determined. Fig. 2 shows the sequence alignment of the DNA-binding domains of Qin and other selected forkhead family members from each of the phylogenetic subclasses (Fig. 1). The sequence conservation is high within the forkhead family, especially within the secondary structural elements (Fig. 2). The multiple sequence alignment also shows that insertions and/or deletions are uncommon in this domain, except in the FOXO subclass (orange) that has an insertion in the loop region before helix 3. The Qin forkhead domain is highly homologous to that of FREAC-11 (70% identity and 83% similarity). The sequence identity of Qin with the other experimentally solved structures of the forkhead domain ranges from 45% (AFX) to 68% (Genesis).



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  		Fig. 2. Multiple sequence alignment of forkhead domains of selected proteins. The sequences shown in single-letter amino acid codes are human FOXG1B (P55315), FOXC1 (Q12948), FOXE1 (O00358), FOXE3 (Q13461), FOXD3 (Q9UJU5), FOXL2 (P58012), YA41 (Q9UPW0), CHE1 (Q9UIE7), FOXP1 (Q9H334), FOXP2 (O15409), FOXP3 (Q9BZS1), HNF3{gamma} (P55318), AFX1 (P98177), and FREAC-11 (Q99958). Amino acid residues showing absolute identity among these proteins are shown in white against a blue background; those positions with conservative substitutions are shown with a yellow background. The numbering scheme at the top of the figure refers to amino acid positions within the FREAC-11 forkhead domain. The missense mutations analyzed in this study (N189A, H193A, and S196A at positions 50, 54, and 57, respectively, in the Qin forkhead domain) are shown as downward arrows. The positions of the three {alpha}-helices defined in the solution structure of human FREAC-11 are schematically represented in the bar below the alignment. ALSCRIPT was used to format the alignment (24).

 
Threading Experiments on the Qin Forkhead Domain.
Molecular models were generated for the Qin forkhead domain using threading algorithms developed by Bryant and Lawrence (11). The three-dimensional structure of the forkhead domain of the adipocyte transcription factor FREAC-11 (20) was used as a template for the threading analysis (see "Materials and Methods"). All possible placements of the core segments of the template structure along the query sequence, given the constraints of sequence length, core segment length, and limits of loop length, were considered. Threading contact energies were corrected for sequence composition bias by random shuffling of the aligned residues to generate composition-corrected threading scores (ZR M). To evaluate the statistical significance of computed threading scores, 100 random permutations of the query sequence were generated, and the alignment optimization procedure was then repeated on these now-shuffled sequences. Based on this collection of scores, the probability (ER M) that the threading score for a query sequence would be observed purely by chance was calculated. A summary of the threading results is shown in Table 1. Considering the wild-type data for Qin and FREAC-11, the conformational energies show only a slight difference (~1 kcal/mol). More importantly, the probability values (ER M) both meet the statistical criteria of being >=0.05 (5% {alpha}-level). As such, it can be concluded that there is a statistically significant match between the sequence of Qin and the structure of FREAC-11.


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  		Table 1 Statistics for optimal threads of Qin and its mutations

 
Because the threading technique searches for the best alignment of sequence to structure, it is possible to represent the results as a "structural alignment" (22, 23). It is important to reinforce that this alignment has a structural, thermodynamic basis as described above; as such, the results would not be the equivalent of what would be learned by doing a simple BLASTP or similar sequence-based search because the theoretical underpinnings of these algorithms are quite different. The structural alignment indicates that there is 70% absolute sequence identity between Qin and FREAC-11 in the DNA-binding region. In addition, there are many positions that are replaced by conservative substitutions within the aligned core segments (Fig. 2, yellow positions). Because of the similarities at the sequence level and at the structural level, the model structure of Qin is deemed to be a reliable one for understanding the significance of point mutations in the forkhead region.

Fig. 3 shows the energy scaffolds for the Qin forkhead domain generated in this threading experiment. The energy scaffolds provide a method for visualizing the important intramolecular interactions taking place within a protein. Here, the winged helical bundle is held together by numerous hydrophobic interactions, represented by the thick, magenta-colored cylinders. Several of the highly conserved, large hydrophobic residues in this protein are involved in maintaining intramolecular interactions within the hydrophobic core and occur primarily between the conserved residues at the helical interfaces. The energy scaffolds indicate that the most favorable hydrophobic interactions observed in the threading model of Qin forkhead domain involve Leu-12 and Ile-13 in helix 1; Ile-30, Phe-33, and Ile-34 in helix 2; Ile-52 and Leu-56 in helix 3; and Tyr-77 and Trp-78 in the last ß-strand (the numbering is according to the three-dimensional structure of FREAC-11). The amino acid residues participating in these critical pairwise interactions are absolutely conserved between Qin and FREAC-11. These data, coupled with the information obtained from the multiple sequence alignments, indicate that these key amino acid residues are essential in maintaining fold stability of the forkhead domains.



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  		Fig. 3. Molecular models of the Qin forkhead domain. A, the {alpha}-carbon backbone of the molecular model of qin is depicted as a curving "worm." Pairwise residue interaction energies between core residues are shown by the width and coloring of the connected {alpha}-carbon positions on the protein backbone. Indicated interactions are limited to those with pairwise interaction energies < -1 kcal/mol. Thick, magenta-colored cylinders indicate the most favorable interactions. Intermediate cylinder thicknesses represent interactions with lower pairwise energies. Scaffolds were generated using the graphics program GRASP. B, the Qin molecular model is rotated 230° on the Y axis. C, a ribbon model showing selected residues that are involved in critical pairwise interactions necessary for maintaining the hydrophobic core of the forkhead domain. The orientation of the model is the same as that in B.

 
Structural Model for the Forkhead Domain of Qin Oncoprotein.
Atomic models for the Qin forkhead domain were generated based on the alignments obtained from threading experiments. The MODELER package was implemented using two experimentally determined structures of the forkhead domain, FREAC-11 (pdb |1D5V) and AFX (pdb|1E17), as the templates. The validation of the models was assessed in terms of divergence from the templates and consensus of the alignments using MODELER target functions (data not shown). The root mean square deviation of the models with the experimentally solved structures of FREAC-11 and AFX, are 1.3 Å and 1.7 Å, respectively (Fig. 4). Detailed examination of the models showed that maximum variability in the Qin forkhead structure is observed in the loop region before the DNA recognition helix 3, as well as the COOH-terminal end of the helix 3. Both of these regions are critical for DNA recognition specificity of the individual forkhead domains (20).



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  		Fig. 4. Superimposition of molecular model of Qin with the template structures FREAC-11 (pdb 1D5V) and AFX (pdb 1E17).

 
Structural Consequences of Qin Oncogenic Mutations.
The Qin forkhead model was used to predict the effect of four missense mutations with altered DNA binding activity (10). All of the missense mutations generated threading scores similar to wild-type Qin (Table 1). None of the mutations significantly altered any critical pairwise interactions or destabilized the forkhead domain of Qin, suggesting that these missense mutations do not significantly alter the structure of the Qin forkhead domain. The missense mutations (N189A, H193A, and S196A at positions 50, 54, and 57, respectively, in the Qin forkhead domain) are all located on the DNA-contacting surface of the forkhead domain (Fig. 5A). The functional properties of a protein reside largely on its surface as it is involved in interactions with other molecular surfaces. It is evident from the molecular model (Fig. 5B) that these missense mutations function by altering the DNA-binding surface of the Qin forkhead domain.



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  		Fig. 5. Molecular models of Qin forkhead mutations. A, ribbon model showing the positions of the residues Asn-189, His-193, and Ser-196 in the third helix of qin. Asn-189, His-193, and Ser-196 correspond to positions 50, 54, and 57, respectively, in the Qin forkhead domain. B, ribbon model of N189A/H193A mutation.

 
The homology model building experiments described in this study have provided additional insight into understanding the structural basis of Qin missense mutations with altered DNA binding activity. These findings provide an important complement to laboratory studies that have examined the effect of such mutations on biochemical properties such as DNA binding and the transactivation of reporter constructs in that observed changes in function can now be explained at a structural level. In addition, the identification of critical residues involved in maintaining the stability of Qin will allow for further study of the biochemical properties of this and other forkhead proteins through site-directed mutagenesis and other similar techniques.


   Footnotes
 
1 To whom requests for reprints should be directed. Phone: (301) 496-8570; Fax: (301) 480-2634; E-mail: andy{at}nhgri.nih.gov Back

2 The abbreviations used are: BF-1, brain factor 1; CEF, chicken embryonic fibroblast; NJ, neighbor-joining. Back

3 http://ftp.sunet.se/pub/Science/Molecular_Biology/unix/treetool/. Back

4 http://genome.nhgri.nih.gov/forkhead/qin. Supplementary data is also available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org). Back

Received 7/24/02; revised 9/13/02; accepted 10/ 1/02.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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