Hidetoshi Kono

Sequence-dependent DNA deformability studied using molecular dynamics simulations

Proteins recognize specific DNA sequences not only through direct contact between amino acids and bases, but also indirectly based on the sequence-dependent conformation and deformability of the DNA (indirect readout). We used molecular dynamics simulations to analyze the sequence-dependent DNA conformations of all 136 possible tetrameric sequences sandwiched between CGCG sequences. The deformability of dimeric steps obtained by the simulations is consistent with that by the crystal structures. The simulation results further showed that the conformation and deformability of the tetramers can highly depend on the flanking base pairs. The conformations of xATx tetramers show the most rigidity and are not affected by the flanking base pairs and the xYRx show by contrast the greatest flexibility and change their conformations depending on the base pairs at both ends, suggesting tetramers with the same central dimer can show different deformabilities. These results suggest that analysis of dimeric steps alone may overlook some conformational features of DNA and provide insight into the mechanism of indirect readout during protein–DNA recognition. Moreover, the sequence dependence of DNA conformation and deformability may be used to estimate the contribution of indirect readout to the specificity of protein–DNA recognition as well as nucleosome positioning and large-scale behavior of nucleic acids.

Sequence analysis of the gliding protein Gli349 in Mycoplasma mobile

The motile mechanism of Mycoplasma mobile remains unknown but is believed to differ from any previously identified mechanism in bacteria. Gli349 of M. mobile is known to be responsible for both adhesion to glass surfaces and mobility. We therefore carried out sequence analyses of Gli349 and its homolog MYPU2110 from M. pulmonis to decipher their structures. We found that the motif “YxxxxxGF” appears 11 times in Gli349 and 16 times in MYPU2110. Further analysis of the sequences revealed that Gli349 contains 18 repeats of about 100 amino acid residues each, and MYPU2110 contains 22. No sequence homologous to any of the repeats was found in the NCBI RefSeq non-redundant sequence database, and no compatible fold structure was found among known protein structures, suggesting that the repeat found in Gli349 and MYPU2110 is novel and takes a new fold structure. Proteolysis of Gli349 using chymotrypsin revealed that cleavage positions were often located between the repeats, implying that regions connecting repeats are unstructured, flexible and exposed to the solvent. Assuming that each repeat folds into a structural domain, we constructed a model of Gli349 that fits well the shape and size of images obtained with electron microscopy.

Sequence Dependencies of DNA Deformability and Hydration in the Minor Groove

DNA deformability and hydration are both sequence-dependent and are essential in specific DNA sequence recognition by proteins. However, the relationship between the two is not well understood. Here, systematic molecular dynamics simulations of 136 DNA sequences that differ from each other in their central tetramer revealed that sequence dependence of hydration is clearly correlated with that of deformability. We show that this correlation can be illustrated by four typical cases. Most rigid basepair steps are highly likely to form an ordered hydration pattern composed of one water molecule forming a bridge between the bases of distinct strands, but a few exceptions favor another ordered hydration composed of two water molecules forming such a bridge. Steps with medium deformability can display both of these hydration patterns with frequent transition. Highly flexible steps do not have any stable hydration pattern. A detailed picture of this correlation demonstrates that motions of hydration water molecules and DNA bases are tightly coupled with each other at the atomic level. These results contribute to our understanding of the entropic contribution from water molecules in protein or drug binding and could be applied for the purpose of predicting binding sites.

A generalized conformational energy function of DNA derived from molecular dynamics simulations

Proteins recognize DNA sequences by two different mechanisms. The first is direct readout, in which recognition is mediated by direct interactions between the protein and the DNA bases. The second is indirect readout, which is caused by the dependence of conformation and the deformability of the DNA structure on the sequence. Various energy functions have been proposed to evaluate the contribution of indirect readout to the free-energy changes in complex formations. We developed a new generalized energy function to estimate the dependence of the deformability of DNA on the sequence. This function was derived from molecular dynamics simulations previously conducted on B-DNA dodecamers, each of which had one possible tetramer sequence embedded at its center. By taking the logarithm of the probability distribution function (PDF) for the base-step parameters of the central base-pair step of the tetramer, its ability to distinguish the native sequence from random ones was superior to that with the previous method that approximated the energy function in harmonic form. From a comparison of the energy profiles calculated with these two methods, we found that the harmonic approximation caused significant errors in the conformational energies of the tetramers that adopted multiple stable conformations.

Rational design of DNA sequence-specific zinc fingers.

We developed a rational scheme for designing DNA binding proteins. The scheme was applied for a zinc finger protein and the designed sequences were experimentally characterized with high DNA sequence specificity. Starting with the backbone of a known finger structure, we initially calculated amino acid sequences compatible with the expected structure and the secondary structures of the designed fingers were then experimentally confirmed. The DNA‐binding function was added to the designed finger by reconsidering a section of the amino acid sequence and computationally selecting amino acids to have the lowest protein–DNA interaction energy for the target DNA sequences. Among the designed proteins, one had a gap between the lowest and second lowest protein–DNA interaction energies that was sufficient to give DNA sequence‐specificity.

EVALUATION OF DNA INTRAMOLECULAR INTERACTIONS FOR NUCLEOSOME POSITIONING IN YEAST

We calculated intramolecular interaction energies of DNA by threading DNA sequences around crystal structures of nucleosomes. The strength of the intramolecular energy oscillations at frequency approximately 10 bps for dinucleotides was in agreement with previous nucleosome models. The intramolecular energy calculated along yeast genome positively correlated with nucleosome positioning experimentally measured.