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Structural and biochemical studies of TREX1 inhibition by metals. Identification of a new active histidine conserved in DEDDh exonucleases
Author(s) -
Brucet Marina,
QuerolAudí Jordi,
Bertlik Kamila,
Lloberas Jorge,
Fita Ignacio,
Celada Antonio
Publication year - 2008
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.036426.108
Subject(s) - exonuclease , active site , histidine , chemistry , klenow fragment , divalent , enzyme , stereochemistry , biochemistry , binding site , biophysics , biology , polymerase , organic chemistry
TREX1 is the major exonuclease in mammalian cells, exhibiting the highest level of activity with a 3′→5′ activity. This exonuclease is responsible in humans for Aicardi‐Goutières syndrome and for an autosomal dominant retinal vasculopathy with cerebral leukodystrophy. In addition, this enzyme is associated with systemic lupus erythematosus. TREX1 belongs to the exonuclease DEDDh family, whose members display low levels of sequence identity, while possessing a common fold and active site organization. For these exonucleases, a catalytic mechanism has been proposed that involves two divalent metal ions bound to the DEDD motif. Here we studied the interaction of TREX1 with the monovalent cations lithium and sodium. We demonstrate that these metals inhibit the exonucleolytic activity of TREX1, as measured by the classical gel method, as well as by a new technique developed for monitoring the real‐time exonuclease reaction. The X‐ray structures of the enzyme in complex with these two cations and with a nucleotide, a product of the exonuclease reaction, were determined at 2.1 Å and 2.3 Å, respectively. A comparison with the structures of the active complexes (in the presence of magnesium or manganese) explains that the inhibition mechanism is caused by the noncatalytic metals competing with distinct affinities for the two metal‐binding sites and inducing subtle rearrangements in active centers. Our analysis also reveals that a histidine residue (His124), highly conserved in the DEDDh family, is involved in the activity of TREX1, as confirmed by mutational studies. Our results shed further light on the mechanism of activity of the DEDEh family of exonucleases.