University of Central Florida Undergraduate Research Journal - Computational Analysis of Broad Complex Zinc-Finger Transcription Factors
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Computational Analysis of Broad Complex
Zinc-Finger Transcription Factors

By: Barbara Mascareno-Shaw
Mentor: Dr. Thomas Selby


Figure 1It is known that prokaryotic and eukaryotic cells contain genetic material in the form of double stranded deoxyribonucleic acid (DNA).  Use of this DNA is controlled by transcription events that control the production of RNA molecules that are eventually translated into proteins with specific functions.  These transcription events are often used to control developmental stages of the cell through the production of specific signaling proteins.  Transcription factors are used to control the production of specific RNA molecules through their sequence specific association with DNA. 

One class of transcription factors is known as zinc-finger DNA binding proteins.  This type of protein contains a characteristic three-dimensional (3D) structural motif, composed of 2 cysteine and 2 histidine residues bound to a zinc ion.  These proteins have been well characterized by Peter Wright and coworkers at The Scripps Research Institute using NMR spectroscopy to analyze a synthetic form of the Xenopus protein called Xfin (Lee, Gippert, Soman, Case, & Wright,, 1989).  These studies provided important information about the 3D structure of the proteins as well as the dynamic properties of the DNA interaction with zinc-finger proteins.  These studies confirmed that the zinc atom is an essential component of the protein structure since, in the absence of zinc, the protein did not fold properly in aqueous solution (Lee et al., 1989).  The protein fold can be described as having a hairpin structure in the first 10 amino acids.  This hairpin helix contains two conserved histidine residues, and two cysteine residues in the b-strand making up a 2H, 2C zinc-finger binding motif (Lee et al., 1989) (a structural example is shown in Figure 1).  Another important feature of zinc-finger proteins is that the zinc atoms are found in the interior of the protein.  Although, the zinc ions do not interact with the DNA, they are essential for the stability of the protein and assist in maintaining the hairpin fold.  This structural stability enables the appropriate amino acids to interact with the DNA and provides the basis for sequence specific DNA interaction as well as thermodynamic stability for the DNA-protein complex.  A recent study with fruit flies has provided new sequences of zinc-finger DNA proteins that appear to have amino acid substitutions (hereafter referred to as mutants) that influence the morphology of fruit flies.  This study conducted in the Dept. of Biology at the University of Central Florida, led by L. von Kalm, has investigated 4 new sequences of zinc-finger proteins at the genetic level, known as Z1, Z2, Z3, and Z4.  Each of these proteins has a zinc-finger DNA motif. 

The Z1, Z2, Z3, and Z4 proteins are part of the Broad or Broad-Complex (BR-C) locus of the fruit fly(Bayer, Zhou, Zhou, Riddiford, & von Kalm, 2003).  The Z1 protein is genetically linked to the rbp (reduced bristles on palp) function, Z2 to the br (broad) function, and Z3 to the 2Bc function.  The complete contribution of the Z4 protein to the morphology of the fruit flyhas not been determined.  The BR-C locus encompasses a complex of zinc-finger proteins that are associated with the function of rbp, the br, the 2Bc, and a non-pupariating (np) that does not complement any of the genetic functions (Bayer, von Kalm & Fristrom, 1997; Bayer et al., 2003).  Studies have demonstrated that the hormone ecdysone regulates BR-C (von Kalm, Crossgrove, von Seggern, Guild, & Beckendorf, 1994; Hodgetts, Clark, O'Keefe, Schouls, Crossgrove, Guild & von Kalm, 1995).  This study showed that the Z2 protein has br genetic function and regulates expression of DOPA decarboxylase in the epidermis.  In the same manner, the Z1 protein has the rbp genetic function that also regulates the Sgs-4 gene.  The Sgs-4 gene is associated with the rbp and 2Bc genetic functions.  The activation of the Sgs-4 gene is based upon the accumulation of the BR-C complex in tissues of fruit flies and is directly involved in gene expression of transcription factors (Bayer et al., 1997). 

Figure 2Bayer et al investigated the genetic function of the Z1-Z4 proteins as complements for certain mutations in the BR-C complex.  The study demonstrated that the Z1 protein had rbp function and Z4 had partial rbp function.  Moreover, the Z3 protein had a full 2Bc genetic function but Z2 had only partial function.  This study also emphasizes that the genetic functions of these proteins are not specific, but can actually have more than one function within the complex (Bayer et al., 1997).  Mutations in these zinc-finger proteins established the genetic relationship of the BR-C complex to the overall morphology of the fruit fly.

The amino acid sequences (Z1-Z4) were determined through DNA sequencing, using genetic material from the fruit flies and aligned to compare their sequence to other organisms.  Two crystal structures were available that had high sequence homology to the sequences of the Z1-Z4 proteins.  The first structure is named Tramtrack two zinc-finger DNA protein (also from the fruit fly), which is similar in sequence to the Z1 and Z3 sequences.  The crystal structure of this protein (Figure 1), which was solved as a dimer at 2.8 Å resolution (Fairall, Schwabe, Chapman, Finch, & Rhodes, 1993), demonstrates that the protein binds to the major groove of DNA where the zinc atoms are within the hydrophobic region of the helices.  The second crystal structure is known as Zif268 zinc-finger DNA complex and was solved at 1.6 Å (Elrod-Erickson, Benson & Pabo, 1998) as seen in Figure 2, This protein is most similar in sequence to Z2 and Z4.  This type of zinc-finger protein has a slightly different motif and 3 zinc atoms bound within three helices.  The helices still recognize the major groove of DNA as observed with the Z1 and Z3, but the protein has an overall fold that is slightly different.  Although the function of these zinc-finger proteins is well characterized, the effect of the amino acid changes relative to the structural stability and DNA interaction energy has not been investigated. 

The research in this report entails the collaboration between the Selby and von Kalm labs to use computational analysis to construct homology models of Z1, Z2, Z3, and Z4 zinc-finger proteins.  These homology models are constructed using the known crystal structures and the amino acid sequences of the mutant zinc-finger proteins.  The computational analysis is based on molecular mechanics (MM3) calculations to determine the relative interaction energies between the experimentally determined crystal structure, the homology model (without mutations), and the mutants that have the specific amino acid changes. 

Molecular mechanics is a theoretical approach used to measure the different vibrational, rotational, and transitional energies around specific chemical bonds.  Molecular mechanics initially disregards the electronic distribution around atoms and determines the energy with respect to the arrangement of atoms in space (Leach, 1996).  Therefore, molecular mechanics is mainly based on the stretching, bending, and rotational movement of atoms (Leach, 1996; NIH, 2002).  Other interactions involved in molecular mechanics are Van der Waals, electrostatic, and steric repulsions for atoms that are 2 or more bonds apart (NIH, 2002). hese values are determined following the final energy minimization of all atoms.  The purpose of this research is to study the structures of the Z1, Z2, Z3, and Z4 proteins based on molecular mechanics analysis to determine energetic differences between wild type and mutants.  This study provides information about the role of each amino acid change with respect to the DNA interaction energy and helps explain how the changes in the morphology of the fruit fly are altered at the molecular level. 

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