Abstract
TREX1 is a 3'-deoxyribonuclease that degrades single- and double-stranded DNA (ssDNA and dsDNA) to prevent inappropriate nucleic acid-mediated immune activation. More than 40 different disease-causing TREX1 mutations have been identified exhibiting dominant and recessive genetic phenotypes in a spectrum of autoimmune disorders. Mutations in TREX1 at positions Asp-18 and Asp-200 to His and Asn exhibit dominant autoimmune phenotypes associated with the clinical disorders familial chilblain lupus and Aicardi-Goutières syndrome. Our previous biochemical studies showed that the TREX1 dominant autoimmune disease phenotype depends upon an intact DNA-binding process coupled with dysfunctional active site chemistry. Studies here show that the TREX1 Arg-62 residues extend across the dimer interface into the active site of the opposing protomer to coordinate substrate DNA and to affect catalysis in the opposing protomer. The TREX1(R62A/R62A) homodimer exhibits ∼50-fold reduced ssDNA and dsDNA degradation activities relative to TREX1(WT). The TREX1 D18H, D18N, D200H, and D200N dominant mutant enzymes were prepared as compound heterodimers with the TREX1 R62A substitution in the opposing protomer. The TREX1(D18H/R62A), TREX1(D18N/R62A), TREX1(D200H/R62A), and TREX1(D200N/R62A) compound heterodimers exhibit higher levels of ss- and dsDNA degradation activities than the homodimers demonstrating the requirement for TREX1 Arg-62 residues to provide necessary structural elements for full catalytic activity in the opposing TREX1 protomer. This concept is further supported by the loss of dominant negative effects in the TREX1 D18H, D18N, D200H, and D200N compound heterodimers. These data provide compelling evidence for the required TREX1 dimeric structure for full catalytic function.
Highlights
The structure of TREX1 exonuclease identifies key residues positioned at the stable dimer interface
The DNA degradation activities and DNA binding measured in the TREX1 R62A-containing mutants demonstrate that Arg-62 from one TREX1 protomer contributes to DNA degradation in the opposing protomer. These results show that the mechanism of the TREX1 Arg-62 “across the dimer interface” contribution is through DNA binding in the opposing protomer
The dimeric structure is further stabilized through multiple side chain-side chain, side chain backbone, and water-bridged interactions across the dimer interface and from a hydrophobic face generated between the protomers [26, 32]
Summary
The structure of TREX1 exonuclease identifies key residues positioned at the stable dimer interface. The TREX1 disease alleles include missense mutations, insertions, duplications, and frame shifts that locate to positions throughout the 314-amino acid-coding gene These findings have established a causal relationship between TREX1 genetic variants and multiple mechanisms of TREX1 enzyme dysfunction that might explain the spectrum of human autoimmune disorders with overlapping clinical symptoms related to TREX1 DNA degradation and immune activation. The TREX1 Arg-114 residue is ϳ15 Å away from the active site and forms side chain-backbone interactions across the dimer interface, contributing to the catalytic function of the opposing protomer and helping to explain the immune dysfunction phenotype in the R114H heterozygous individual [28, 29]. The stable dimeric structure contributes to the FCL and AGS-dominant disease phenotypes in the TREX1 Asp-18 and Asp-200 active site mutations through the actions of a catalytically deficient protomer that retains DNA binding proficiency and blocks access to the DNA by TREX1WT enzyme [28, 30]. These results show that the mechanism of the TREX1 Arg-62 “across the dimer interface” contribution is through DNA binding in the opposing protomer
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