ABSTRACT
We have carried out molecular dynamics simulation of the lambda OL1 DNA operator on the free and the protein-bound forms. Our results lead us to conclude that the binding of the repressor actually makes the N-7 atom of Gua8' more solvent exposed, thereby enhancing its reactivity to chemical methylation. This increase in solvent accessibility surface area occurs simultaneously with the formation of hydrogen bonds between Lys-4 of the nonconsensus flexible N-terminal arm and Gua6' of the nonconsensus half-site operator DNA. Calculations of protein--DNA interaction energies reveal that among the residues of the arm, Lys-4 contributes the most favorably to the interaction energies. This result is consistent with mutagenesis studies that established that lysine at position 4 is absolutely required for tight binding. We find that the nonconsensus arm and the nonconsensus monomer interacts less favorably with DNA than do their respective counterparts of the consensus monomer. Moreover, the six-residue flexible arm accounts for at least half the total protein--DNA interactions energy. These results are in agreement with previous experimental studies. In accord with the diffuse electron density map observed in crystallographic studies of the nonconsensus flexible arm, we find that our model built for this region is more flexible and exhibits more conformations than its consensus counterpart. The simulation also reveals that DNA bending observed near the outer edge of the operator site is an intrinsic sequence-dependent property. By contrast, the DNA-bending features observed toward the center of the operator are induced by the protein. On the whole, stepwise protein-induced bending is more pronounced in the consensus half-site operator. We also find that the unusually large helical twist (49 degrees ) observed in the protein-bound form near the center of the operator results from the binding of the protein at a base step with some propensity for high twists.
Subject(s)
DNA, Viral/chemistry , Bacteriophage lambda/chemistry , Bacteriophage lambda/genetics , Base Sequence , Binding Sites , Biopolymers/chemistry , Biopolymers/genetics , Crystallography, X-Ray , DNA Methylation , DNA, Viral/genetics , DNA-Binding Proteins/chemistry , Hydrogen Bonding , Models, Biological , Nucleic Acid Conformation , Operator Regions, Genetic , Protein Binding , Repressor Proteins/chemistry , Thermodynamics , Viral Proteins/chemistry , Viral Regulatory and Accessory ProteinsABSTRACT
We have carried out molecular dynamics simulation of the N-terminal domain of the lambda repressor protein in a surrounding environment including explicit waters and ions. We observe two apparent dynamics substates in the nanosecond protein simulation, the transition occurring around 500 ps. The existence of these two apparent substates results from a high flexibility of the arm in each monomer, a relative flexibility of both arms with respect to each other, and a relative displacement of the recognition helices from 30 to 40 A of interhelical distance. Many amino acid residues, including those involved in DNA recognition, undergo a simultaneous transition in their side-chain conformations, consistent with the relationship between side-chain conformation and secondary structural elements, as observed in protein crystal structures. This result suggests plausible conformational changes experienced by the protein upon DNA binding. On the whole, the non-consensus monomer appears to be more flexible than its consensus counterpart.
Subject(s)
Repressor Proteins/chemistry , Amino Acid Sequence , Computer Simulation , DNA-Binding Proteins/chemistry , Models, Molecular , Monte Carlo Method , Peptide Fragments/chemistry , Protein Conformation , Protein Structure, Secondary , Static Electricity , Time Factors , Viral Proteins , Viral Regulatory and Accessory ProteinsABSTRACT
A molecular dynamics simulation of the operator binding domain of the lambda repressor protein has been carried out. The protein was embedded in explicit waters, Na(+) and CL(-) ions. The Amber 4.1 computer package and the Cornell et al. Force field were used for energy-minimization and molecular dynamics simulation. We find that the atoms distributions in the environment of waters and Na(+) ions are in excellent agreement with those derived from the analysis of water molecules in crystal structures and ion-binding proteins. We also find that, on the whole, both distributions are similar to each other.
Subject(s)
Carbon/chemistry , Models, Molecular , Nitrogen/chemistry , Oxygen/chemistry , Water/chemistry , Computer Simulation , Crystallography, X-Ray , DNA-Binding Proteins/chemistry , Macromolecular Substances , Monte Carlo Method , Protein Structure, Tertiary , Repressor Proteins/chemistry , Structure-Activity Relationship , Thermodynamics , Viral Proteins , Viral Regulatory and Accessory ProteinsABSTRACT
Binding of the N-terminal domain of the lambda repressor to DNA is coupled to dimerization. Hydrophobic interactions between helix-5 and helix-5' drive the packing at the dimer interface. We have carried out computations of the conformational energy of packing of the fifth helices (and of the helix-4-loop-helix-5 portions) of variants of the lambda repressor operator binding domain, using an ECEPP/3-based packing algorithm. Here, we report the results for 26 mutants chosen among those that hve been characterized experimentally. We find that the relative orientation of the fifth helices for active mutants is very similar to the wild-type. The fifth helices of the inactive mutants have a significantly different relative orientation. This result illustrates that a unique specific orientation pattern of helix-5 relative to helix-5' is required for dimerization-coupled DNA binding activity. This finding is further supported by computational studies of the whole N-terminal domain of ten variants that showed that the active mutants, including the wild-type protein, have similar values of the number of contacts between the two monomers in the dimer, involving two amino acid residues of the fifth helices (positions 84 and 87 in each monomer). A decrease in the number of such contacts abolishes DNA-binding activity. Furthermore, all active mutants have their "DNA-recognition helices", numbers 3 and 3' positioned so that they can fit in the DNA operator like those of the wild-type protein, while some inactive mutants exhibit a substantial change in the relative orientation of their recognition helices.
Subject(s)
Computer Simulation , DNA-Binding Proteins , Models, Molecular , Mutation , Repressor Proteins/chemistry , Algorithms , DNA/metabolism , Helix-Loop-Helix Motifs , Operator Regions, Genetic , Protein Binding , Protein Conformation , Protein Structure, Secondary , Repressor Proteins/genetics , Repressor Proteins/metabolism , Viral Proteins , Viral Regulatory and Accessory ProteinsABSTRACT
Conformational energy computations have been carried out on the N-acetyl-N'-methylamide of 5-hydroxytryptophan (5OH-Trp) using ECEPP/3. As observed with tryptophan (Trp), the most preferred conformation about the C alpha-C beta bond of the side chain is g+ or t. This preference is reduced to only the t conformational state when 5-hydroxyTrp is in the middle of a right-handed poly(L-alanine) alpha-helix. A similar result has been obtained with Trp [Piela et al. (1987), Biopolymers 1987, 1273-1286]. These results suggest that replacement of Trp by its analog 5-hydroxyTrp may be tolerated in an alpha-helix. To test this hypothesis, we have replaced Trp by 5OH-Trp in the fifth helices of two functionally active mutants of the N-terminal domain of the bacteriophage lambda repressor. Computations on the packing of these helices have shown that no significant structural changes results from the replacement of Trp by 5OH-Trp. The DNA-binding activity of these mutants, as assessed indirectly through geometrical parameters, is also unaltered.
