Structure-based domain assignment in Leishmania infantum EndoG: characterization of a pH-dependent regulatory switch and a C-terminal extension that largely dictates DNA substrate preferences

Abstract

Mitochondrial endonuclease G from Leishmania infantum (LiEndoG) participates in the degradation of double-stranded DNA (dsDNA) during parasite cell death and is catalytically inactive at a pH of 8.0 or above. The presence, in the primary sequence, of an acidic amino acid-rich insertion exclusive to trypanosomatids and its spatial position in a homology-built model of LiEndoG led us to postulate that this peptide stretch might act as a pH sensor for self-inhibition. We found that a LiEndoG variant lacking residues 145–180 is indeed far more active than its wild-type counterpart at pH values >7.0. In addition, we discovered that (i) LiEndoG exists as a homodimer, (ii) replacement of Ser211 in the active-site SRGH motif with the canonical aspartate from the DRGH motif of other nucleases leads to a catalytically deficient enzyme, (iii) the activity of the S211D variant can be restored upon the concomitant replacement of Ala247 with Arg and (iv) a C-terminal extension is responsible for the observed preferential cleavage of single-stranded DNA (ssDNA) and ssDNA–dsDNA junctions. Taken together, our results support the view that LiEndoG is a multidomain molecular machine whose nuclease activity can be subtly modulated or even abrogated through architectural changes brought about by environmental conditions and interaction with other binding partners.

Characterization of an atypical, thermostable, organic solvent- and acid-tolerant 2′-deoxyribosyltransferase from Chroococcidiopsis thermalis

“…PyMOL was used for structure visualization, molecular editing, and figure preparation (DeLano 2013). The AMBER force field (Case et al 2015) was used for progressive energy refinement in explicit solvent (Salomon-Ferrer et al 2013) following a previously described protocol (Oliva et al 2017).…”

Characterization of Vibrio cholerae’s Extracellular Nuclease Xds

“…Real time activity assays elucidated that Xds D787A is heavily impaired for DNA degradation and Xds H837A shows no nuclease activity. The H837 might have a crucial role to act as the general base in activation of the water molecule for the nucleophilic in-line attack on the phosphorous atom during the nuclease reaction as suggested by other studies (Bueren-Calabuig et al, 2011; Oliva et al, 2017). Mechanisms of substrate binding as well as the catalytic process focusing on the predicted active site residues have to be further investigated but we herein confirm that both AA play an important role in the DNase activity of the protein.…”

Cell death in Leishmania

“…Under normal conditions, the enzyme is localized in the mitochondrion of promastigotes as well as of axenic amastigotes [11, 31, 61], where it appears to be essential for parasite survival [62]. In the mitochondrion, where the pH is relatively high, a small amino acid sequence inhibits the nuclease activity [53]. Different apoptotic stimuli, such as H 2 O 2 or edelfosine, induce EndoG translocation from the mitochondrion to the nucleus where it forms a complex, on the one hand, with the Flap endonuclease-1 FEN-1 and, on the other, with the nuclease TatD [11].…”

“…This results in DNA degradation through endonuclease and exonuclease activities, notably on single-stranded DNA [11, 31, 61, 62]. As a consequence, despite unique properties of Leishmania EndoG compared to other EndoG [53], this enzyme is a well-characterized cell death effector in the parasite.…”