Abstract
Ribonuclease H1 (RNase H) enzymes are well-conserved endonucleases that are present in all domains of life and are particularly important in the life cycle of retroviruses as domains within reverse transcriptase. Despite extensive study, especially of the E. coli homolog, the interaction of the highly negatively charged active site with catalytically required magnesium ions remains poorly understood. In this work, we describe molecular dynamics simulations of the E. coli homolog in complex with magnesium ions, as well as simulations of other homologs in their apo states. Collectively, these results suggest that the active site is highly rigid in the apo state of all homologs studied and is conformationally preorganized to favor the binding of a magnesium ion. Notably, representatives of bacterial, eukaryotic, and retroviral RNases H all exhibit similar active-site rigidity, suggesting that this dynamic feature is only subtly modulated by amino acid sequence and is primarily imposed by the distinctive RNase H protein fold.
Highlights
Ribonuclease H1 (RNase H) proteins are well-conserved endonucleases that are found in all domains of life and cleave the RNA strand of an RNA-DNA duplex substrate
Given that ions in this position are not observed in the substrate-bound structures of RNase H homologs (Figure 2D), and that the B-factor of the Mg2+ ion in the 1RDD structure is much higher than those of the surrounding residues (Figure 4B), it is likely that this position does not reflect the most stable conformation of the protein-ion complex in solution
In this work we aimed to use molecular dynamics simulations to understand the dynamic behavior of the RNase H family in complex with catalytically required Mg2+ ions
Summary
Ribonuclease H1 (RNase H) proteins are well-conserved endonucleases that are found in all domains of life and cleave the RNA strand of an RNA-DNA duplex substrate. The RNase H active site canonically consists of a highly conserved DED(D) motif (Figure 1), three to four carboxylate-containing residues collectively participating in the binding of catalytically required divalent cations, Mg2+ under physiological conditions. This active-site sequence motif and requirement for Mg2+ is widely shared with other nucleases, suggesting a common catalytic mechanism[1]. The pH optimum for the RNase H reaction in vitro is approximately 7.5–8.54, a value at which all active-site residues should be deprotonated[3] and accessible for ion binding
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