Microscopic insight to specificity of metal ion cofactor in DNA cleavage by restriction endonuclease EcoRV

Abstract

Restriction endonucleases protect bacterial cells against bacteriophage infection by cleaving the incoming foreign DNA into fragments. In presence of Mg 2+ ions, EcoRV is able to cleave the DNA but not in presence of Ca 2+ , although the protein binds to DNA in presence of both metal ions. We make an attempt to understand this difference using conformational thermodynamics. We calculate the changes in conformational free energy and entropy of conformational degrees of freedom, like DNA base pair steps and dihedral angles of protein residues in Mg 2+ (A)-EcoRV-DNA complex compared to Ca 2+ (S)-EcoRV-DNA complex using all-atom molecular dynamics (MD) trajectories of the complexes. We find that despite conformational stability and order in both complexes, the individual degrees of freedom behave differently in the presence of two different metal ions. The base pairs in cleavage region are highly disordered in Ca 2+ (S)-EcoRV-DNA compared to Mg 2+ (A)-EcoRV-DNA. One of the acidic residues ASP90, coordinating to the metal ion in the vicinity of the cleavage site, is conformationally destabilized and disordered, while basic residue LYS92 gets conformational stability and order in Ca 2+ (S) bound complex than in Mg 2+ (A) bound complex. The enhanced fluctuations hinder placement of the metal ion in the vicinity of the scissile phosphate of DNA. Similar loss of conformational stability and order in the cleavage region is observed by the replacement of the metal ion. Considering the placement of the metal ion near scissile phosphate as requirement for cleavage action, our results suggest that the changes in conformational stability and order of the base pair steps and the protein residues lead to cofactor sensitivity of the enzyme. Our method based on fluctuations of microscopic conformational variables can be applied to understand enzyme activities in other protein-DNA systems.

In-silico studies on hydration in EcoRI-cognate DNA complex

“…Figure 1 The complexes of metal ions, such as Mg 2+ and Ca 2+ loaded EcoRI-DNA are not available. To model these complexes, we follow the similar protocol as explained in our earlier publication [15]. We model Mg 2+ -EcoRI-DNA complex by inserting one Mg 2+ ion in the vicinity of each active site of EcoRI-DNA.…”

“…Several experimental and theoretical studies suggest that Mg 2+ plays pivotal roles: It might aid in the binding and positioning of DNA, as well as partially neutralizing the extra negative charge in the transition state and protonate the leaving group after cleavage by providing a water molecule in its coordination sphere [1]. Our recent molecular dynamics (MD) simulation studies show that replacement of Mg 2+ ions by Ca 2+ ions in RE-DNA complex makes the cleavage region unstable and disordered due to enhanced uctuations of the catalytically active residues and the cleavage base pairs of the DNA [2][3][4][5][6]. The other divalent metal ions such as Mn 2+ and Co 2+ also make the cleavage probability signi cantly lower [6,7].…”

Fluctuation-Dominated Ligand Binding in Molten Globule Protein

“…Our objective is to understand the microscopic fluctuations in the MG state upon binding to the ligand. We consider in particular the bovine aLA, namely, a model BAMLET complex, constructed with the help of conformational thermodynamics data − from a constant pH MD (CpHMD) trajectory. We carry out MD simulations along with umbrella sampling (US) to understand the stability of BAMLET within the model.…”

Conformational stability and order of Hoogsteen base pair induced by protein binding

“…The changes in conformational stability and order in bio-molecular systems are quantified in terms of conformational free energy and entropy changes derived from the fluctuations of microscopic conformational variables [12][13][14] . Negative changes in conformational free energy and entropy indicate structural stability and ordering, respectively, in a given structure with respect to a reference structure, while positive changes indicate conformational destabilization and disorder respectively.…”

“…Negative changes in conformational free energy and entropy indicate structural stability and ordering, respectively, in a given structure with respect to a reference structure, while positive changes indicate conformational destabilization and disorder respectively. Although originally developed for proteins, the conformational thermodynamics calculations are extended to DNA-protein systems as well 12 . The fluctuations of microscopic conformational variables can be captured via long molecular dynamics (MD) simulation from the changes in fluctuations of microscopic conformational variables of the DNA duplex.…”