Human CGAS-DNA complex and encoded protein
The present invention is based, in part, on the discovery of the human-specific regulatory control of cGAS and the structure of the active human cGAS-DNA complex, as well as compositions comprising the modified hcGAS polypeptide, hcGAS-DNA complex, hcGAS-DNA-ATP complex, and methods of screening for modulators of the structure, expression, and/or activity of such polypeptides and complexes.
1. An isolated polypeptide that binds double-stranded DNA and catalyzes production of 2′-5′/3′-5′ cyclic GMP-AMP (2′3′ cGAMP), wherein the polypeptide comprises an amino acid sequence having at least 90% identity to the human cyclic GMP-AMP synthase (hcGAS) amino acid sequence of SEQ ID NO: 1 and further comprises amino acid substitutions at positions corresponding to K187 and L195 of SEQ ID NO: 1.
2. The isolated polypeptide of claim 1 , wherein the polypeptide comprises an amino acid sequence having at least 95% identity to the human cGAS (hcGAS) amino acid sequence of SEQ ID NO: 1 and further comprises amino acid substitutions at positions corresponding to K187 and L195 of SEQ ID NO: 1.
3. The isolated polypeptide of claim 1 , wherein the polypeptide comprises an amino acid sequence having the amino acid sequence of SEQ ID NO: 1 and further comprises amino acid substitutions at positions corresponding to K187 and L195 of SEQ ID NO: 1.
4. The isolated polypeptide of claim 1 , wherein the residue corresponding to K187 is substituted with a residue selected from the group consisting of a) a residue that makes direct contact with the DNA phosphate backbone; b) a polar residue selected from the group consisting of tyrosine, serine, threonine, asparagine, glutamine and cysteine; and c) an asparagine residue, and wherein the residue corresponding to L195 is substituted with a residue selected from the group consisting of i) a residue that increases the overall positive charge of the A-site DNA-binding surface; ii) a basic residue selected from the group consisting of lysine, arginine and histidine; and iii) an arginine residue.
5. The isolated polypeptide of claim 2 , wherein the residue corresponding to K187 is substituted with a residue selected from the group consisting of a) a residue that makes direct contact with the DNA phosphate backbone; b) a polar residue selected from the group consisting of tyrosine, serine, threonine, asparagine, glutamine and cysteine; and c) an asparagine residue, and wherein the residue corresponding to L195 is substituted with a residue selected from the group consisting of i) a residue that increases the overall positive charge of the A-site DNA-binding surface; ii) a basic residue selected from the group consisting of lysine, arginine and histidine; and iii) an arginine residue.
6. The isolated polypeptide of claim 3 , wherein the residue corresponding to K187 is substituted with a residue selected from the group consisting of a) a residue that makes direct contact with the DNA phosphate backbone; b) a polar residue selected from the group consisting of tyrosine, serine, threonine, asparagine, glutamine and cysteine; and c) an asparagine residue, and wherein the residue corresponding to L195 is substituted with a residue selected from the group consisting of i) a residue that increases the overall positive charge of the A-site DNA-binding surface; ii) a basic residue selected from the group consisting of lysine, arginine and histidine; and iii) an arginine residue.
7. The isolated polypeptide of claim 1 , wherein the isolated polypeptide further comprises amino acid substitutions at positions corresponding to S328, K350 and/or L354 of SEQ ID NO: 1.
8. The isolated polypeptide of claim 1 , wherein the isolated polypeptide has one or more biological properties selected from the group consisting of:
i) increased 2′3′ cGAMP synthesis compared to hcGAS having the amino acid sequence of SEQ ID NO: 1, optionally wherein the 2′3′ cGAMP synthesis is increased by at least 5-fold;
ii) increased repression of Vibrio cholera chemotaxis compared to hcGAS having the amino acid sequence of SEQ ID NO: 1;
iii) similar enzyme kinetics to mouse cGAS having the amino acid sequence of SEQ ID NO: 5;
iv) recognizing DNA shorter than 45 bp;
v) stabilized active enzyme conformation;
vi) stabilized interactions with DNA; and
vii) increased minimal cGAS-DNA complex formation compared to hcGAS having the amino acid sequence of SEQ ID NO: 1.
9. The isolated polypeptide of claim 1 , further comprising a heterologous polypeptide, optionally wherein the heterologous polypeptide is selected from the group consisting of a signal peptide, a peptide tag, a dimerization domain, an oligomerization domain, and an antibody.
10. A pharmaceutical composition comprising:
an isolated polypeptide that binds double-stranded DNA and catalyzes production of 2′-5′/3′-5′ cyclic GMP-AMP (2′3′ cGAMP), wherein the polypeptide comprises an amino acid sequence having at least 90% identity to the human cyclic GMP-AMP synthase (hcGAS) amino acid sequence of SEQ ID NO: 1 and further comprises amino acid substitutions at positions corresponding to K187 and L195 of SEQ ID NO: 1; and
a pharmaceutically acceptable excipient, diluent, or carrier.
11. An isolated polypeptide that binds double-stranded DNA and catalyzes production of 2′-5′/3′-5′ cyclic GMP-AMP (2′3′ cGAMP), wherein the polypeptide comprises an amino acid sequence having at least 90% identity to the human cyclic GMP-AMP synthase (hcGAS) amino acid sequence of SEQ ID NO: 2 and further comprises amino acid substitutions at positions corresponding to K31 and L39 of SEQ ID NO: 2.
12. The isolated polypeptide of claim 11 , wherein the polypeptide comprises an amino acid sequence having at least 95% identity to the human cGAS (hcGAS) amino acid sequence of SEQ ID NO: 2 and further comprises amino acid substitutions at positions corresponding to K31 and L39 of SEQ ID NO: 2.
13. The isolated polypeptide of claim 11 , wherein the polypeptide comprises an amino acid sequence having the amino acid sequence of SEQ ID NO: 2 and further comprises amino acid substitutions at positions corresponding to K31 and L39 of SEQ ID NO: 2.
14. The isolated polypeptide of claim 11 , wherein the residue corresponding to K31 is substituted with a residue selected from the group consisting of a) a residue that makes direct contact with the DNA phosphate backbone; b) a polar residue selected from the group consisting of tyrosine, serine, threonine, asparagine, glutamine and cysteine; and c) an asparagine residue, and wherein the residue corresponding to L39 is substituted with a residue selected from the group consisting of i) a residue that increases the overall positive charge of the A-site DNA-binding surface; ii) a basic residue selected from the group consisting of lysine, arginine and histidine; and iii) an arginine residue.
15. The isolated polypeptide of claim 12 , wherein the residue corresponding to K31 is substituted with a residue selected from the group consisting of a) a residue that makes direct contact with the DNA phosphate backbone; b) a polar residue selected from the group consisting of tyrosine, serine, threonine, asparagine, glutamine and cysteine; and c) an asparagine residue, and wherein the residue corresponding to L39 is substituted with a residue selected from the group consisting of i) a residue that increases the overall positive charge of the A-site DNA-binding surface; ii) a basic residue selected from the group consisting of lysine, arginine and histidine; and iii) an arginine residue.
16. The isolated polypeptide of claim 13 , wherein the residue corresponding to K31 is substituted with a residue selected from the group consisting of a) a residue that makes direct contact with the DNA phosphate backbone; b) a polar residue selected from the group consisting of tyrosine, serine, threonine, asparagine, glutamine and cysteine; and c) an asparagine residue, and wherein the residue corresponding to L39 is substituted with a residue selected from the group consisting of i) a residue that increases the overall positive charge of the A-site DNA-binding surface; ii) a basic residue selected from the group consisting of lysine, arginine and histidine; and iii) an arginine residue.
17. The isolated polypeptide of claim 11 , wherein the isolated polypeptide further comprises amino acid substitutions at positions corresponding to S172, K194 and/or L198 of SEQ ID NO: 2.
18. The isolated polypeptide of claim 11 , wherein the isolated polypeptide has one or more biological properties selected from the group consisting of:
i) increased 2′3′ cGAMP synthesis compared to hcGAS having the amino acid sequence of SEQ ID NO: 1, optionally wherein the 2′3′ cGAMP synthesis is increased by at least 5-fold;
ii) increased repression of Vibrio cholera chemotaxis compared to hcGAS having the amino acid sequence of SEQ ID NO: 1;
iii) similar enzyme kinetics to mouse cGAS having the amino acid sequence of SEQ ID NO:
5;
iv) recognizing DNA shorter than 45 bp;
v) stabilized active enzyme conformation;
vi) stabilized interactions with DNA; and
vii) increased minimal cGAS-DNA complex formation compared to hcGAS having the amino acid sequence of SEQ ID NO: 1.
19. The isolated polypeptide of claim 11 , wherein the isolated polypeptide further comprises a heterologous polypeptide, optionally wherein the heterologous polypeptide is selected from the group consisting of a signal peptide, a peptide tag, a dimerization domain, an oligomerization domain, and an antibody.
20. A pharmaceutical composition comprising:
an isolated polypeptide that binds double-stranded DNA and catalyzes production of 2′-5′/3′-5′ cyclic GMP-AMP (2′3′ cGAMP), wherein the polypeptide comprises an amino acid sequence having at least 90% identity to the human cyclic GMP-AMP synthase (hcGAS) amino acid sequence of SEQ ID NO: 2 and further comprises amino acid substitutions at positions corresponding to K31 and L39 of SEQ ID NO: 2; and
a pharmaceutically acceptable excipient, diluent, or carrier.