# Structural insights into RNase H catalytic mechanism from room-temperature X-ray and neutron crystallography of apo- and RNA/DNA hybrid-bound enzyme

## TL;DR

This study uses advanced imaging techniques to reveal how RNase H enzymes cut RNA strands, which is important for developing new drugs and gene therapies.

## Contribution

The study provides structural insights into the RNase H catalytic mechanism using room-temperature neutron and X-ray crystallography.

## Key findings

- Neutron structures show E109 in the DEDD motif changes protonation states, suggesting its role in RNA cleavage.
- RNA/DNA hybrids bind to BhRNase H1 to mimic product and Michaelis complexes depending on metal site occupancy.
- Protonation of the leaving O3’ group of RNA is proposed to occur via the side chain of E109.

## Abstract

RNase H enzymes are sequence-nonspecific endonucleases that cleave RNA strands in RNA/DNA hybrid duplexes, an enzymatic process essential in DNA replication and repair in both prokaryotes and eukaryotes. Also, RNase H activity of the reverse transcriptase in human immunodeficiency viruses (HIV-1 and HIV-2) is indispensable for the viral replication cycle. RNase H enzymes play an central role in the development of gene therapies and are targets for novel antivirals. It is therefore of great importance to gain a detailed understanding of the RNase H catalytic mechanism to improve drug design. We utilized Bacillus halodurans RNase H1 (BhRNase H1) to shed light on its function and catalytic mechanism. Room-temperature neutron crystallography of the wild-type and inactive D132N mutant enzymes revealed that E109, belonging to the catalytic DEDD motif, can change its protonation state, allowing us to propose its role in the protonation of the leaving O3′ hydroxyl group of RNA. X-ray crystallography has demonstrated the ability of the RNA/DNA duplex to slide along the protein surface upon metal ion binding at site MA, transforming a product mimic into a Michaelis-like complex, which confirms an essential role of the MA metal ion in catalysis.

Image 1

•Neutron structures of apo-wild-type and apo-D132N mutant BhRNase H1 map protonation states in the enzyme active site.•E109 from the catalytic DEDD motif changes its protonation state between apo-wild-type and apo-D132N structures.•RNA/DNA hybrids bind to BhRNase H1 to mimic product and Michaelis complexes depending on the metal site occupancy.•Protonation of the leaving O3’ group of cleaved RNA is proposed to occur from O2’ through the side chain of E109.

Neutron structures of apo-wild-type and apo-D132N mutant BhRNase H1 map protonation states in the enzyme active site.

E109 from the catalytic DEDD motif changes its protonation state between apo-wild-type and apo-D132N structures.

RNA/DNA hybrids bind to BhRNase H1 to mimic product and Michaelis complexes depending on the metal site occupancy.

Protonation of the leaving O3’ group of cleaved RNA is proposed to occur from O2’ through the side chain of E109.

X-ray and neutron structures of BhRNase H1 in the apo-form and in complex with RNA/DNA hybrids are described.