Book of Abstracts: Albany 2009
June 16-20 2009
© Adenine Press (2008)
Minor Groove Shape and Electrostatics Provide a Molecular Origin for Protein-DNA Specificity
The molecular basis for protein-DNA recognition and its specificity is still widely unknown. Complexes of proteins from various families bound to DNA have been solved by means of X-ray crystallography and NMR spectroscopy. However, the molecular mechanisms through which proteins specifically recognize their DNA binding sites are only partially understood. Direct readout through specific contacts between amino acids and bases dominates recognition within the DNA major groove. Different base pairs account for specific patterns of hydrogen bond donors and acceptors in the major groove with thymine additionally offering a methyl group for hydrophobic contacts. Direct readout in the minor groove is limited because there is no differentiation in terms of the location of hydrogen bond donors or acceptors between A-T and T-A or between G-C and C-G base pairs. Indirect readout accounts for the recognition of the overall shape of a DNA binding site by proteins. Overall shape is a function of base sequence and comprises global deformation effects such as DNA bending .
In a recent study of the Hox family of transcription factors, we have identified a third mode of protein-DNA recognition that involves recognition of minor groove shape [2, 3]. Hox proteins bind DNA by making nearly identical major groove contacts via the recognition helices of their homeodomains. In vivo specificity, however, depends on extended and unstructured regions that link Hox homeodomains to their cofactors. Crystal structures were determined for one of the eight Drosophila Hox proteins, Scr, bound to its specific DNA sequence (fkh250) and a consensus Hox site (fkh250con*). The structures of these two Hox-Exd-DNA ternary complexes only differ by an Arg3/His-12 pair that inserts into a narrow region of the fkh250 minor groove whereas these residues are disordered when presented with the fkh250con* sequence. For both the fkh250 and fkh250con* sequences, minor groove width and the magnitude of the negative electrostatic potential are strongly correlated. All-atom Monte Carlo simulations of the free DNA binding sites predict that the DNA conformation being recognized is an intrinsic property of the base sequence, and thus, already prevalent in unbound DNA rather than induced by protein binding . Our results on Hox-DNA recognition indicate that the intrinsically narrow minor groove of fkh250 induces an enhanced negative electrostatic potential, which in turn attracts the positively charged Arg/His pair.
In current studies we ask if the local shape recognition that we found for Hox proteins is of a more general nature . Electrostatics calculations along with MC structure predictions of DNA binding sites indicate that several protein families employ this readout mechanism. Homeodomains, as an example of such a family, often bind to A-tracts, which are rigid AT-rich DNA regions of three or more consecutive ApT or ApA (TpT) base pair steps. Narrow minor grooves are a common structural feature of A-tracts. TpA steps break A-tract structure since they act as flexible hinges due to unfavorable stacking interactions. Our studies on Hox proteins have proven that the location of a TpA step is key for the intrinsic structure of a binding site. Strikingly, our data shows a correlation of A-tract sequence and structure with electrostatic potential in the DNA minor groove as a result of shape-induced electrostatic focusing. Our observation of the causal relationship between minor groove structure and enhanced negative electrostatic potentials reveals the biological function of A-tract motifs. In addition, our results suggest recognition of local DNA shape as a novel readout mechanism crucial for proteins that bind DNA with narrow minor groove regions.
References and Footnotes