Article https://doi.org/10.1038/s41467-024-483 65-3
The structural basis for 2′−5′/3′−5′-cGAMP
synthesis by cGAS
Shuai Wu
1
,SandraB.Gabelli
1,2,3,5
& Jungsan Sohn
1,2,4
cGAS activates innate immune responses against cytosolic double-stranded
DNA. Here, by determining crystal structures of cGAS at various reaction
stages, we report a unifying catalytic mechanism. apo-cGAS assumes an array
of inactive conformations and binds NTPs nonproductively. Dimerization-
coupled double-stranded DNA-binding then affixes the active site into a rigid
lock for productive metal•substrate binding. A web-like network of pro-
tein•NTP, intra-NTP, and inter-NTP interactions ensures the stepwise synthesis
of 2′−5′/3′−5′-linked cGAMP while discriminating against noncognate NTPs and
off-pathway intermediates. One divalent metal is sufficient for productive
substrate binding, and capturing the second divalent metal is tightly coupled
to nucleotide and linkage specificities, a process which manganese is preferred
over magnesium by 100-fold. Additionally, we elucidate how mouse cGAS
achieves more stringent NTP and linkage specificities than human cGAS.
Together, our results reveal that an adaptable, yet precise lock-and-key-like
mechanism underpins cGAS catalysis.
Cyclic GMP-AMP (cGAMP) synthase (cGAS) is essential for the host
defense against cytosolic double-stranded (ds)DNA arising from var-
ious maladies (e.g., pathogen invasion, ionizing irradiation, and gen-
otoxic chemicals)
1–3
. Upon directly binding to and dimerizing on
dsDNA, cGAS cyclizes ATP and GTP into 2′−5′/3′−5′-linked cGAMP, a
unique metazoan second messenger for initiating type-I interferon
(IFN-I)-mediated inflammatory responses (Fig. 1A)
1,4–7
.cGASiscentral
to antitumor immunity, host defense against an array of pathogens,
and regulating autoimmunity
2,3,8
.
It is increasingly appreciated that cGAS-like enzymes have an
ancestral origin and cGAMPs are widely employed as second messen-
gers for both prokaryotic and eukaryotic innate immune pathways
9
.
However, only metazoan enzymes generate the 2′−5′/3′−5′-linkage
combination
4,5,9,10
, and currently, the mechanisms by which cGAS
specifically generates this uniquely linked cyclic dinucleotide remain
poorlyunderstood. Persisting questions include: how cGAS specifically
recognizes ATP and GTP at each substrate binding pocket and coor-
dinates its signature 2′−5′ linkage formation; how the same substrate
binding sites then switch their nucleotide specificity and precisely
position the GTP-AMP (pppGpA) intermediate for the second 3′−5′-
linkage formation while barring the cyclization of other off-pathway
dinucleotides; how dsDNA and dimerization regulate these processes,
how it utilizes different divalent metals; and finally, how active site
reactivity and promiscuity are regulated across different mammalian
species. Here, we resolve these numerous fundamental mechanistic
questions in innate immunology and present a unifying catalytic
mechanism of cGAS.
We find that apo-cGAS assumes an array of inactive conforma-
tions and binds ATP/GTP nonproductively without involving divalent
metals, which is then affixed into the catalytically competent con-
formation by dimerization-coupled dsDNA binding. We also delineate
how web-like network of protein•NTP, intra-NTP, and inter-NTP inter-
actions underpin the substrate-dependent linkage specificity. One
Mg
2+
is sufficient for productive substrate binding and Mn
2+
is pre-
ferred as the second catalytic metal without involving an inverted
intermediate. In the cyclization step, the adenosine of the pppGpA
intermediate binds ~30° rotated compared to the guanine of the GTP
substrate to precisely position the 3′-OH for the second linkage
Received: 31 May 2023
Accepted: 26 April 2024
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1
Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
2
Department of Oncology, Johns
Hopkins University School of Medicine, Baltimore, MD, USA.
3
Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
4
Division of Rheumatology, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
5
Present address: Discovery Chemistry, Merck Laboratories,
West Point, PA, USA.
e-mail: jsohn@jhmi.edu
Nature Communications | (2024) 15:4012 1
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