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from Saccharomyces cerevisiae - My Favorite Protein
| What is Gal4? Gal4 is a transcription factor found in yeast and involved in galctose and melibiose metabolism (1). Gal4 binds DNA in a sequence specific manner recognizing 17 base pair sequence (1). It uses a conformation known as a zinc finger-binding motif, one of multiple motifs identified in DNA binding proteins (2). The complete protein has been sequenced consists of 881 amino acids with many distinct functional domains: a DNA binding domain, a dimerization domain, and three acidic activation domains. However to be functional, the Gal4 protein must dimerize with an identical subunit (1). Why does it have to dimerize? Hong et al., reported the structure of the first 100 residues of the Gal4 homodimer bound to a 20 base pair region of DNA and discussed the effects of dimerization on Gal4 function (Figure 1) (1). They found that once dimerized, the affinity for DNA was increased. |
| To Bind, or Not To Bind…
Hong et al. completed a number biochemical studies (both theoretical and bench-top) on the function of Gal4 as a monomer and as a dimer. First they verified previous experiments that showed the protein without the dimerization domain (and hence functioning as a monomer) has a lower affinity for DNA than the homodimer has (1). As stated previously, it was known that the dimerization was necessary for function. This sheds some light on the topic: without dimerization the protein has a lower affinity for DNA. To further understand how the dimerization domain functions, they created various mutant versions of the Gal4 protein to see how this would affect DNA binding (1). First they made single alanine substituations within the dimerization interface (where the protein subunits interact), many of which are highly conserved among fungal Gal4 homologs (1). The fact that they are conserved implies that they are most likely important for function and if changed in a non-conservative manner will affect its function. Therefor upon affecting how the subunits interact, it was thought that there should also be a change in the ability to bind DNA. The single amino acid mutations showed very little affect on Gal4’s ability to bind DNA, however double and triple mutations reduced binding affinity of Gal4 for DNA (1). This meant that one mutation within the interface was not sufficient to reduce binding by much, however with multiple they were able to reduce the functioning of Gal4. With too many mutations on the other hand, they found that the protein could not be purified, implying that it had disrupted structure too much to be stable upon trying to purify the protein (1). In the end, this set of experiments supported that the interactions between both subunits was necessary to facilitate the binding of DNA (1). Further analysis showed that a Gal4 dimer was more stable than a monomer using melting temperature experiments (1). Dimerization is therefore important for correct function of Gal4. |
| Figure 1. Image of Gal4 (residues 1-100) bound to 20 bp DNA. Picture courtesy of PDB |
| How Does Binding DNA Affect Gal4 Structure? When comparing to previous structural analysis, Hong et al., saw that a nuclear magnetic resonance (NMR) structure of Gal4 had a slightly elongated conformation when not bound to DNA (1). NMR structures are measured in solution, making them usually a better representation of the protein structure in vivo, as it is an aqueous environment. It looks at locations of usually hydrogens and carbons in a protein to determine its structure. On the other hand X-ray crystallography (the technique used by Hong et al.) uses a crystallized structure of the protein. This gives exact coordinates of each atom (except hydrogens which do not have a large enough electric field to scatter X-rays) in the structure. Given that each uses a different method, it is possible that the protein takes slightly different conformations in each. To determine if the protein did in fact change shape upon binding DNA, they performed a series of sedimentation velocity experiments (1). The data suggests that there may be slight difference when bound and unbound, however it was not as exaggerated as was seen comparing the NMR structure to the X-ray crystallography structure obtained by Hong et al. (1). Perhaps there is slight change upon binding of DNA, however it doesn’t seem to be significant. The conditions of each experiment are likely the cause of this. However Hong et al. argue that their conformation is more representative of the actual complex that forms (1). Conclusions The structure of the Gal4 homodimer is crucial in its function as a transcription factor (1). Hong et al. show that the dimerized Gal4 has a higher affinity to DNA than does a monomer (1). Further, upon binding only small changes in conformation are most likely seen upon binding DNA, not as expected based on comparing the bound (identified via X-ray crystallography and unbound via NMR) (1). References 1. Hong M, Fitzgerald M, Harper S, Lueo C, Speicher D, Marmorstein R. Structural basis for dimerization in DNA recognition by Gal4. Structure 2008; 16: 1019-1026. 2. Marmorstein R, Carey M, Ptashne M, Harrison S. DNA recognition by GAL4: structure of a protein-DNA complex. Nature 1992; 256; 408-414. |
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