ABSTRACT
Induced fit effects in the complex of a DNA decamer with two even-skipped transcriptional repressor homeodomain molecules were investigated by means of molecular dynamics simulations. Dynamics of these effects are found to be in the time scale from pico- to nanoseconds. First steps are made by the fast-moving DNA backbone phosphates, which upon binding change their B(I)/B(II) substate distribution. Further rearrangements in the DNA double helix induced upon complexation, like bending of the helix axis, changes of the minor groove width, and of different helical parameters, are slower and occur within a few nanoseconds. The flexibility of the DNA, especially of its backbone, seems thereby to play an important role for specific DNA ligand recognition.
Subject(s)
Bacterial Proteins , DNA/genetics , Drosophila Proteins/genetics , Homeodomain Proteins/genetics , Repressor Proteins/genetics , Transcription Factors , Animals , Base Sequence , Binding Sites , Computer Simulation , Crystallography, X-Ray , DNA/chemistry , Models, Molecular , Molecular Conformation , Oligodeoxyribonucleotides/chemistry , Protein Conformation , Protein Structure, Secondary , Sensitivity and SpecificityABSTRACT
A nonoriented hydrated film of poly(dG-dC) with ?20 water molecules per nucleotide (called B* by Loprete and Hartman (Biochem. 32, 4077-4082 (1993)) was studied by Fourier transform infrared (FT-IR) spectroscopy either as equilibrated sample between 290 and 270 K or, after quenching into the glassy state, as nonequilibrated film isothermally at 200 and 220 K. IR spectral changes on isothermal relaxation at 200 and 220 K, caused by interconversion of two conformer substates, are revealed by difference spectra. Comparison with difference curves obtained in the same manner from two classical B-DNA forms, namely the d(CGCGAATTCGCG)(2) dodecamer and polymeric NaDNA from salmon testes, revealed that the spectral changes on B(I)-to-B(II) interconversion in the classical B-DNA forms are very similar to those in the B*-form, and that the spectroscopic differences between the B(I) and B(II) features from classical B-DNA and those from the modified B*-form are minor. Nonexponential kinetics of the B(I)-->B(II) transition in the B*-form of poly(dG-dC) at 200 K showed that the structural relaxation time is about three times of that in the classical B-DNA forms (approximately equal to 30 versus approximately equal to 10 min at 200 K). The unexpected reversal of conformer substates interconversion (that is B(II)-->B(I) transition on cooling from 290 K and B(I)-->B(II) transition on isothermal relaxation at 200 K) observed for classical B-DNA occurs also in the modified B*-form. We therefore conclude that restructuring of hydration shells rules the low-temperature dynamics of the B*-form via its two conformer substates in the same manner reported for classical B-DNA by Pichler et al. (J. Phys. Chem. B 106, 3263-3274 (2002)).
Subject(s)
DNA/chemistry , Polydeoxyribonucleotides/chemistry , Animals , Kinetics , Male , Nucleic Acid Conformation , Salmon , Spectroscopy, Fourier Transform Infrared , TemperatureABSTRACT
We investigated the PvuII endonuclease with its cognate DNA by means of molecular dynamics simulations. Comparing the complexed DNA with a reference simulation of free DNA, we saw structural changes at the scissile phosphodiester bond. At this GpC step, the enzyme induces the highest twist and axial rise, inclination is increased and the minor groove widened. The distance between the scissile phosphate group and the phosphate group of the following thymine base is shortened significantly, indicating a substrate-assisted catalysis. A feasible reason for this vicinity is the catalytically important amino acid residue lysine 70, which bridges the free oxygen atoms of the successive phosphate groups. Due to this geometry, a compact reaction pocket is formed where a water molecule can be held, thus bringing the reaction partners for hydrolysis into contact. The O1-P-O2 angle of the scissile nucleotide is decreased, probably due to a complexation of the negative oxygen atoms through protein and solvent contacts.
Subject(s)
DNA/chemistry , DNA/metabolism , Deoxyribonucleases, Type II Site-Specific/chemistry , Deoxyribonucleases, Type II Site-Specific/metabolism , Phosphates/chemistry , Binding Sites , Calcium/metabolism , Catalytic Domain , Computer Simulation , Crystallography, X-Ray , Magnesium/chemistry , Magnesium/metabolism , Models, Molecular , Nucleic Acid Conformation , Structural Homology, Protein , WaterABSTRACT
Although the trp-repressor-operator complex is one of the best studied transcriptional controlling systems, some questions regarding the specific recognition of the operator by the repressor remain. We performed a 2.35 ns long molecular dynamics simulation to clarify the influence of the two B-DNA backbone conformational substates B(I) and B(II) on complexation. The trp-repressor-operator is an ideal biological system for this study because experimental results have already figured out that the interaction between the internucleotide phosphates and the protein is essential for the formation of the high affinity complex. Our simulation supports these results, but more important it shows a strong correlation between the B(I)/B(II) phosphate substate and the number of interactions with this phosphate. In particular the B(I) <==> B(II) transitions occur synchronous to hydrogen bond breaking or formation. To the best of our knowledge, this was observed for the first time. Thus, we conclude that the sequence specific B(I)/B(II) behavior contributes via indirect readout to sequence specific recognition. These results have implication for the design of transcription-controlling drugs in view of the recently published influence of minor groove binders on the B(I)/B(II) pattern. The simulation also agrees with crystallographically observed hydration sites. This is consistent with experimental results and indicates the correctness of the model used.
