As a component of the innate immune response, cyclic GMP–AMP synthase (cGAS) synthesizes the second messenger cGAMP to stimulate an interferon response, which in turn provides defense against pathogens, DNA damage and tumorigenesis. After allosteric binding of cGAS to double-stranded DNA, activation of the enzyme occurs via disruption of the N-terminal α-helix, ordering of an active site loop, and movement of the core nucleotidyltransferase (NTase) domain. With the aim of generating a therapeutically useful cGAS variant, Dowling et al. used a protein-design approach to stabilize the active conformation of the enzyme in the absence of its DNA ligand. In the first design stage, the authors used Rosetta to calculate the energies of single amino acid substitutions and score potential mutations for their ability to stabilize the active conformation and/or destabilize the inactive one. In a second design stage, the authors used bioinformatics to identify distantly related proteins whose structures align well with the active conformation of cGAS (particularly the NTase domain), and multiple sequence alignments to identify positions to target for mutagenesis. The combination of mutations identified in both rounds of design resulted in several constitutively active variants of cGAS, with the activity level of one (CA-cGAS-50) being comparable to that of the wild-type cGAS in vitro. Structural analysis of CA-cGAS-50 confirmed that its structure was consistent with the active cGAS confirmation in the absence of ligand — notably, the disrupted N-terminal α-helix and positioning of the NTase domain. Importantly, expression of CA-cGAS-50 was effective at stimulating tumor regression in mice via activation of immunity in host cells. Molecules such as CA-cGAS-50 demonstrate the power of computational design for the development of therapeutically useful protein-based technologies.
Original reference: Nat. Struct. Mol. Biol. 30, 72–80 (2023)
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