Defects in DNA degradation revealed in crystal structures of TREX1 exonuclease mutations linked to autoimmune disease

Abstract

Mutations within the human TREX1 3' exonuclease are associated with Aicardi-Goutières Syndrome (AGS) and familial chilblain lupus (FCL). Both AGS and FCL are autoimmune diseases that result in increased levels of interferon alpha and circulating antibodies to DNA. TREX1 is a member of the endoplasmic reticulum (ER)-associated SET complex and participates in granzyme A-mediated cell death to degrade nicked genomic DNA. The loss of TREX1 activity may result in the accumulation of double-stranded DNA (dsDNA) degradation intermediates that trigger autoimmune activation. The X-ray crystal structures of the TREX1 wt apoprotein, the dominant D200H, D200N and D18N homodimer mutants derived from AGS and FCL patients, as well as the recessive V201D homodimer mutant have been determined. The structures of the D200H and D200N mutant proteins reveal the enzyme has lost coordination of one of the active site metals, and the catalytic histidine (H195) is trapped in a conformation pointing away from the active site. The TREX1 D18N and V201D mutants are able to bind both metals in the active site, but with inter-metal distances that are larger than optimal for catalysis. Additionally, all of the mutant structures reveal a reduced mobility in the catalytic histidine, providing further explanation for the loss of catalytic activity. The structures of the mutant TREX1 proteins provide insight into the dysfunction relating to human disease. Additionally, the TREX1 apoprotein structure together with the previously determined wild type substrate and product structures allow us to propose a distinct mechanism for the TREX1 exonuclease.

The Arg-62 Residues of the TREX1 Exonuclease Act Across the Dimer Interface Contributing to Catalysis in the Opposing Protomers

“…Biochemical and structural studies of the TREX1 and TREX2 enzymes have highlighted the remarkable stability and unique dimeric structures of these 3Ј-exonucleases (26,29,30,32,36,38). The results presented here provide evidence that the TREX1 Arg-62 residues act across the dimer interface in the active sites of the opposing protomers.…”

“…In addition to Arg-114, the TREX1 structure reveals residues positioned at the dimer interface such as Arg-62 that have not yet been identified as positions of disease-causing mutations but might contribute to enzyme activity through inter-protomer coordination (26,32). The studies here test the hypothesis that the conserved TREX1 Arg-62 residues extend across the dimer interface and contribute to catalysis in the active site of the opposing protomer specifically through DNA binding.…”

“…This across the dimer interface interaction of Arg-62 suggests a role for this residue in the catalytic function of the opposing protomer and provides a possible biological rationale for the dimeric structure of TREX1. The Arg-62 is highly conserved in all species of TREX enzymes and is nearly identically positioned in all the TREX1 structures (26,32,36).…”

“…The backbone hydrogen bonds between the outermost ␤3-strands of each TREX1 protomer result in the formation of an extended, central antiparallel ␤-sheet that stretches the length of the dimer. 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). The highly reinforced TREX1 interface generates a kinetically stable dimer that does not dissociate and re-equilibrate at measurable rates (29).…”

“…7, compare D, lanes 8 -12, with D, lanes 3-7). Our structural studies of the TREX1 D200N/D200N enzyme indicate that altered side chain conformations and the absence of a divalent metal at position A in the active site could result in repositioning of Glu-20 and Asn-200 sufficiently to generate unique electrostatic interactions (data not shown) (32). The altered TREX1 D200N active site configuration could stabilize the orientation of Ala-21 and Leu-24 sufficiently to preserve the substrate DNA-binding site conformation.…”