Nonspecific DNA Binding of cGAS N Terminus Promotes cGAS Activation

Abstract

The cytosolic DNA sensor cyclic GMP-AMP synthase (cGAS) mediates innate immune responses against invading pathogens, or against self-dsDNA, which causes autoimmune disorders. Upon nonspecific binding of cytosolic B-form DNA, cGAS synthesizes the second messenger 2'3'-cGAMP and triggers STING-dependent signaling to produce type I IFNs. The cGAS comprises less-conserved N-terminal residues and highly conserved nucleotidyltransferase/Mab21 domains. The function and structure of the well-conserved domains have been extensively studied, whereas the physiological function of the N-terminal domain of cGAS is largely uncharacterized. In this study we used a single-molecule technique combined with traditional biochemical and cellular assays to demonstrate that binding of nonspecific dsDNA by the N-terminal domain of cGAS promotes its activation. We have observed that the N terminus of human cGAS (cGAS-N160) undergoes secondary structural change upon dsDNA binding in solution. Furthermore, we showed that the cGAS-N160 helps full lengthcGAS to expand the binding range on λDNA and facilitates its binding efficiency to dsDNA compared with cGAS without the 160 N-terminal residues (cGAS-d160). More importantly, cGAS-N160 endows full lengthcGAS relatively higher enzyme activity and stronger activation of STING/IRF3-mediated cytosolic DNA signaling. These findings strongly indicate that the N-terminal domain of cGAS plays an important role in enhancing its function.

Screening and Identification of Novel cGAS Homologues Using a Combination of in Vitro and In Vivo Protein Synthesis

“…Moreover, full-length hscGAS was significantly less active than the truncated version. This finding is contrary to previous studies, which stated an enhancement of enzyme activity due to the highly charged N-terminus of cGAS [15,34]. This might indicate incorrect folding of the full-length enzyme during heterologous gene expression and therefore lowered the total concentration of active enzyme in the activity assay.…”

“…The amino acid sequences can be found in Supplementary Materials 1. Similar alignments for further cGAS homologues emphasize the conservation in these regions likewise [10,15]. In the following, the numbering of amino acids corresponds to that for hscGAS.…”

“…The gene library was extended with the non-truncated full-length variant of human cGAS (fl hscGAS) and a mutant of mMcGAS. The full-length human cGAS was included because it was expected to have a higher catalytic activity compared to truncated human cGAS [15]. The point mutation of mMcGAS causes the exchange of a highly conserved amino acid located in the Zn-binding motif of the enzyme, probably having a major influence on the catalytic activity.…”

“…For the murine and porcine homologue, crystal structures are also available and, in parts, crucial amino acids with relevance for the catalytic function are known. Alignments of amino acid sequences of identified cGAS homologues revealed a low-sequence homology in regions without determined function [10,15]. This high level of variation and the markedly reduced activity of human cGAS in comparison to murine cGAS [11] demonstrate the possibility that other cGAS homologues might be available with enhanced properties or different characteristics and functions.…”

Human cGAS catalytic domain has an additional DNA-binding interface that enhances enzymatic activity and liquid-phase condensation

“…Structure of SRY.h-cGAS CD -DNA Complex. Besides promoting dimerization, the lysine/arginine-rich N-terminal domain of cGAS also has the potential for enhancing cGAS-DNA binding (27) as well as serves as the phosphoinositide-binding domain (28). To mimic FL h-cGAS and stabilize the protein-DNA interaction, we replaced the N-terminal domain of h-cGAS with the sequencespecific HMG box of human SRY (29), resulting in the formation of a hybrid protein, termed SRY.h-cGAS CD , as shown in Fig.…”