Structure
Previews The Nature’s Clever Trick for Making Cyclic Dinucleotide Pengfei Fang1 and Min Guo1,* 1Department of Cancer Biology, The Scripps Research Institute, Scripps Florida, 130 Scripps Way, Jupiter, FL 33458, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.str.2015.04.011
Ever since their initial discovery few years ago, cyclic dinucleotides (cDNs), their biosynthesis, and their biological function have been in focus of intense research efforts. In this issue, Kato et al. (2015) present strong evidence that the key enzyme in cDN biosynthesis, DncV, is poised on a tipping point such that, given a nudge, the enzyme, can link the nucleotides into a distinct cyclic loop, leading to a specific innate immune response. Cyclic dinucleotides (cDNs) are important secondary messages in bacteria. Three cDNs have been discovered so far: cyclic di-GMP (cDG), cyclic di-AMP (cDA), and the hybrid cyclic AMP-GMP (cGAMP) (Commichau et al., 2015; Davies et al., 2012; Romling et al., 2013). The cellular cDN levels regulate many bacterial processes via a number of mechanisms. For example, cDG regulates microbial virulence, motility, and biofilm formation (Romling et al., 2013), and cDA is involved in multiple functions, such as DNA repair, cell-wall synthesis, potassium homeostasis, and virulence (Commichau et al., 2015). Interestingly, both a lack of and high-level accumulation of cDA can be harmful for the growth of bacteria. Firmicutes such as B. subtilis are unable to live without cDA, making cDA the only known essential second messenger (Mehne et al., 2013). The new type of cDN, cGAMP, was recently identified as a signaling molecule in V. cholerae that is involved in virulence (Davies et al., 2012). This hybrid molecule is synthesized by the enzyme dinucleotide cyclase (DncV), orthologs of which are predicted to exist in many other bacterial species, suggesting the important signaling roles of cGAMP. DncV is particularly interesting for its ability to synthesize not only the hybrid cGAMP, but also non-hybrid cDG and cDA (Davies et al., 2012). Among the exciting achievements of the intense research that followed the discovery of the cDNs were the isolation of the enzyme cyclic GMP-AMP synthase or cGAS in mammals and its ability to synthesize a cDN ligand that activated the stimulator of interferon genes (STING) (Sun et al., 2013; Wu et al., 2013). Further
characterization of cGAS showed that the enzyme produces a cDN that has a rare 20 -50 phosphodiester linkage between GMP and AMP followed by a 30 -50 return linkage from AMP to GMP. This is different from all characterized cDNs, including cAMP-GMP synthesized by DncV, which is a 30 -50 , 30 -50 isomer (Ablasser et al., 2013; Davies et al., 2012). Given the similarity in the nucleotidyl transferase (NT) fold/domain of DncV and cGAS and the fact that both enzymes make secondary messenger molecules, it seems likely that cGAS and DncV constitute a novel evolutionarily conserved group that evolved to detect cellular stimulus. Although how cGAS could specifically synthesize the 20 -50 , 30 -50 cGAMP is known from recent work (Civril et al., 2013), how the bacteria DncV exclusively make 30 -50 , 30 -50 cDNs (cGAMP, cDA, and cDG) remained unclear. In this work, Kato et al. (2015) solved five high-resolution crystal structures of V. cholerae and E. coli DncV in the apo form and in complex with substrates, providing the molecular basis for the bacteria-specific mechanism of cDN production. Like cGAS, DncV contains two nucleotide-binding pockets for the acceptor and donor, respectively. In both acceptor and donor pockets, DncV binds the substrate GTP or ATP mostly through the base moiety, with fewer interactions with the ribose and phosphate moieties. The substrates are held in a way that the 30 -OH group of the acceptor nucleotide is proximal to the a-phosphate group of the donor nucleotide and the catalytic Asp193 residue (Figure 1A). DncV can recognize both GTP and ATP by either acceptor or donor pocket, giving DncV
the ability to synthesize all three cDNs (cDG, cDA, and cGAMP). The donor pocket recognizes GTP and ATP in similar manners, but with more interaction with GTP than ATP, suggesting a higher affinity for GTP, and thereby DncV favors GTP as the donor nucleotide. In contrast, the acceptor pocket recognized GTP and ATP in distinctive manners. In the acceptor pocket, the 30 -OH group of ATP is closer than that of GTP (3.6 A˚ versus 4.4 A˚) and is in a better geometry, which explains why DncV favors ATP as the acceptor nucleotide (Figure 1A). Therefore, the predominant production of cGAMP by DncV in the presence of both GTP and ATP is an outcome of different binding affinity and reactive orientation of the substrates in the two pockets. The next question is how DncV makes only 30 -50 , 30 -50 phosphodiester bond. In this work, Kato et al. discovered that, no matter whether GTP or ATP is held in the acceptor pocket, the distance from the 30 -OH group of acceptor to the a-phosphate group of the donor is closer than that from the 20 -OH group (in GTP case, that is 0.6 A˚ closer). Therefore, DncV catalyzes 30 -50 phosphodiester formation in the first step of the reaction. Distinctively, the cGAS catalyzes 20 -50 linkage in the first step reaction. By structural comparison, Kato et al. showed that two different acceptor-binding residues are responsive for this distinct linkage specificity. In DncV, the shorter Ile257 residue allows deeper binding of the adenosine moiety, leaving 30 -OH group at the proximal position for the reaction, whereas the corresponding residue in porcine cGAS is a longer Arg353 residue, which pushes the
Structure 23, May 5, 2015 ª2015 Elsevier Ltd All rights reserved 801
Structure
Previews
Figure 1. A Tipping Point for Making Discrete Cyclic Dinucleotides
(A) The acceptor pocket of DncV recognizes GTP and ATP in distinctive manners. Kato et al. (2015) show that the 30 -OH group of ATP is bound in a better geometry than that of GTP in acceptor pocket. In contrast, the donor pocket recognizes GTP and ATP in similarly, but with more interaction with GTP than ATP, suggesting a higher affinity for GTP in the donor pocket. (B) In DncV, the shorter acceptor binding residue Ile257 allows deeper binding of the adenosine moiety, leaving the 30 -OH group at the proximal position for reaction with phosphate from the donor GTP, whereas the corresponding residue in porcine cGAS is a longer Arg353 residue, which pushes the acceptor ATP molecule more outside of the pocket, making instead the 20 -OH more accessible for the reaction. (C) With strict linkage specificity, the bacterial DncV and mammalian cGAS produce two different cGAMP isomers, each as an important secondary message in the producing organisms.
acceptor ATP molecule a little outside of the pocket, making only 20 -OH accessible for the reaction (Figure 1B). In the end, Kato et al. went on to explain why DncV can’t be switched to cGAS-like synthase by an Ile257Arg mutation. They suggested that due to the different structural contexts of the DncV and cGAS acceptor pockets, the side chain of the mutated Arg257 is not fixed in the position close to the acceptor nucleotide as in the cGAS and thus failed to affect the orientation of the acceptor nucleotide. On the other hand, the corresponding Arg376Ile mutation of human cGAS did regain the 30 -50 cGAMP formation activity (Kranzusch et al., 2014). Several questions remain for DncV. The crystal structure of human cGAS revealed a positively charged cleft adjacent to the enzymatic scaffold. The positively charged cleft specifically engages dsDNA and induces structural rearrangement and dimerization of cGAS, which are important for the activation of cGAS (Kranzusch et al., 2013). Intriguingly, despite retaining the basic cleft, DncV does not require dsDNA binding to stimulate enzymatic ac-
tivity (Davies et al., 2012; Kranzusch et al., 2014). The possible role of the basic cleft in DncV remains unknown. Additionally, why DncV has evolved to make the 30 -50 , 30 -50 cGAMP and, in particular, whether it has downstream receptor(s) other than riboswitches (Nelson et al., 2015) remain a mystery. Nonetheless, the thorough structural analysis of DncV and deep understanding of its catalytic mechanism pave the way for the rational design of new antibacterial drugs targeting DncV, which could affect a broad spectrum of pathogenic bacteria.
ACKNOWLEDGMENTS M.G. is supported by NIH grants R01GM100136, R01GM106134, and R01CA178315.
Commichau, F.M., Dickmanns, A., Gundlach, J., Ficner, R., and Stulke, J. (2015). Mol. Microbiol. Published online April 13, 2015. http://dx.doi.org/ 10.1111/mmi.13026. Davies, B.W., Bogard, R.W., Young, T.S., and Mekalanos, J.J. (2012). Cell 149, 358–370. Kato, K., Ishii, R., Hirano, S., Ishitani, R., and Nureki, O. (2015). Structure 23, this issue, 843–850. Kranzusch, P.J., Lee, A.S., Berger, J.M., and Doudna, J.A. (2013). Cell Rep. 3, 1362–1368. Kranzusch, P.J., Lee, A.S., Wilson, S.C., Solovykh, M.S., Vance, R.E., Berger, J.M., and Doudna, J.A. (2014). Cell 158, 1011–1021. Mehne, F.M., Gunka, K., Eilers, H., Herzberg, C., Kaever, V., and Stu¨lke, J. (2013). J. Biol. Chem. 288, 2004–2017. Nelson, J.W., Sudarsan, N., Phillips, G.E., Stav, S., Lunse, C.E., McCown, P.J., and Breaker, R.R. (2015). Proc. Natl. Acad. Sci. USA. Published online April 6, 2015. http://dx.doi.org/10.1073/pnas. 1419264112.
REFERENCES Ablasser, A., Goldeck, M., Cavlar, T., Deimling, T., Witte, G., Ro¨hl, I., Hopfner, K.P., Ludwig, J., and Hornung, V. (2013). Nature 498, 380–384. Civril, F., Deimling, T., de Oliveira Mann, C.C., Ablasser, A., Moldt, M., Witte, G., Hornung, V., and Hopfner, K.P. (2013). Nature 498, 332–337.
802 Structure 23, May 5, 2015 ª2015 Elsevier Ltd All rights reserved
Romling, U., Galperin, M.Y., and Gomelsky, M. (2013). Microbiol. Mol. Biol. Rev. 77, 1–52. Sun, L., Wu, J., Du, F., Chen, X., and Chen, Z.J. (2013). Science 339, 786–791. Wu, J., Sun, L., Chen, X., Du, F., Shi, H., Chen, C., and Chen, Z.J. (2013). Science 339, 826–830.
Previews The Nature’s Clever Trick for Making Cyclic Dinucleotide Pengfei Fang1 and Min Guo1,* 1Department of Cancer Biology, The Scripps Research Institute, Scripps Florida, 130 Scripps Way, Jupiter, FL 33458, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.str.2015.04.011
Ever since their initial discovery few years ago, cyclic dinucleotides (cDNs), their biosynthesis, and their biological function have been in focus of intense research efforts. In this issue, Kato et al. (2015) present strong evidence that the key enzyme in cDN biosynthesis, DncV, is poised on a tipping point such that, given a nudge, the enzyme, can link the nucleotides into a distinct cyclic loop, leading to a specific innate immune response. Cyclic dinucleotides (cDNs) are important secondary messages in bacteria. Three cDNs have been discovered so far: cyclic di-GMP (cDG), cyclic di-AMP (cDA), and the hybrid cyclic AMP-GMP (cGAMP) (Commichau et al., 2015; Davies et al., 2012; Romling et al., 2013). The cellular cDN levels regulate many bacterial processes via a number of mechanisms. For example, cDG regulates microbial virulence, motility, and biofilm formation (Romling et al., 2013), and cDA is involved in multiple functions, such as DNA repair, cell-wall synthesis, potassium homeostasis, and virulence (Commichau et al., 2015). Interestingly, both a lack of and high-level accumulation of cDA can be harmful for the growth of bacteria. Firmicutes such as B. subtilis are unable to live without cDA, making cDA the only known essential second messenger (Mehne et al., 2013). The new type of cDN, cGAMP, was recently identified as a signaling molecule in V. cholerae that is involved in virulence (Davies et al., 2012). This hybrid molecule is synthesized by the enzyme dinucleotide cyclase (DncV), orthologs of which are predicted to exist in many other bacterial species, suggesting the important signaling roles of cGAMP. DncV is particularly interesting for its ability to synthesize not only the hybrid cGAMP, but also non-hybrid cDG and cDA (Davies et al., 2012). Among the exciting achievements of the intense research that followed the discovery of the cDNs were the isolation of the enzyme cyclic GMP-AMP synthase or cGAS in mammals and its ability to synthesize a cDN ligand that activated the stimulator of interferon genes (STING) (Sun et al., 2013; Wu et al., 2013). Further
characterization of cGAS showed that the enzyme produces a cDN that has a rare 20 -50 phosphodiester linkage between GMP and AMP followed by a 30 -50 return linkage from AMP to GMP. This is different from all characterized cDNs, including cAMP-GMP synthesized by DncV, which is a 30 -50 , 30 -50 isomer (Ablasser et al., 2013; Davies et al., 2012). Given the similarity in the nucleotidyl transferase (NT) fold/domain of DncV and cGAS and the fact that both enzymes make secondary messenger molecules, it seems likely that cGAS and DncV constitute a novel evolutionarily conserved group that evolved to detect cellular stimulus. Although how cGAS could specifically synthesize the 20 -50 , 30 -50 cGAMP is known from recent work (Civril et al., 2013), how the bacteria DncV exclusively make 30 -50 , 30 -50 cDNs (cGAMP, cDA, and cDG) remained unclear. In this work, Kato et al. (2015) solved five high-resolution crystal structures of V. cholerae and E. coli DncV in the apo form and in complex with substrates, providing the molecular basis for the bacteria-specific mechanism of cDN production. Like cGAS, DncV contains two nucleotide-binding pockets for the acceptor and donor, respectively. In both acceptor and donor pockets, DncV binds the substrate GTP or ATP mostly through the base moiety, with fewer interactions with the ribose and phosphate moieties. The substrates are held in a way that the 30 -OH group of the acceptor nucleotide is proximal to the a-phosphate group of the donor nucleotide and the catalytic Asp193 residue (Figure 1A). DncV can recognize both GTP and ATP by either acceptor or donor pocket, giving DncV
the ability to synthesize all three cDNs (cDG, cDA, and cGAMP). The donor pocket recognizes GTP and ATP in similar manners, but with more interaction with GTP than ATP, suggesting a higher affinity for GTP, and thereby DncV favors GTP as the donor nucleotide. In contrast, the acceptor pocket recognized GTP and ATP in distinctive manners. In the acceptor pocket, the 30 -OH group of ATP is closer than that of GTP (3.6 A˚ versus 4.4 A˚) and is in a better geometry, which explains why DncV favors ATP as the acceptor nucleotide (Figure 1A). Therefore, the predominant production of cGAMP by DncV in the presence of both GTP and ATP is an outcome of different binding affinity and reactive orientation of the substrates in the two pockets. The next question is how DncV makes only 30 -50 , 30 -50 phosphodiester bond. In this work, Kato et al. discovered that, no matter whether GTP or ATP is held in the acceptor pocket, the distance from the 30 -OH group of acceptor to the a-phosphate group of the donor is closer than that from the 20 -OH group (in GTP case, that is 0.6 A˚ closer). Therefore, DncV catalyzes 30 -50 phosphodiester formation in the first step of the reaction. Distinctively, the cGAS catalyzes 20 -50 linkage in the first step reaction. By structural comparison, Kato et al. showed that two different acceptor-binding residues are responsive for this distinct linkage specificity. In DncV, the shorter Ile257 residue allows deeper binding of the adenosine moiety, leaving 30 -OH group at the proximal position for the reaction, whereas the corresponding residue in porcine cGAS is a longer Arg353 residue, which pushes the
Structure 23, May 5, 2015 ª2015 Elsevier Ltd All rights reserved 801
Structure
Previews
Figure 1. A Tipping Point for Making Discrete Cyclic Dinucleotides
(A) The acceptor pocket of DncV recognizes GTP and ATP in distinctive manners. Kato et al. (2015) show that the 30 -OH group of ATP is bound in a better geometry than that of GTP in acceptor pocket. In contrast, the donor pocket recognizes GTP and ATP in similarly, but with more interaction with GTP than ATP, suggesting a higher affinity for GTP in the donor pocket. (B) In DncV, the shorter acceptor binding residue Ile257 allows deeper binding of the adenosine moiety, leaving the 30 -OH group at the proximal position for reaction with phosphate from the donor GTP, whereas the corresponding residue in porcine cGAS is a longer Arg353 residue, which pushes the acceptor ATP molecule more outside of the pocket, making instead the 20 -OH more accessible for the reaction. (C) With strict linkage specificity, the bacterial DncV and mammalian cGAS produce two different cGAMP isomers, each as an important secondary message in the producing organisms.
acceptor ATP molecule a little outside of the pocket, making only 20 -OH accessible for the reaction (Figure 1B). In the end, Kato et al. went on to explain why DncV can’t be switched to cGAS-like synthase by an Ile257Arg mutation. They suggested that due to the different structural contexts of the DncV and cGAS acceptor pockets, the side chain of the mutated Arg257 is not fixed in the position close to the acceptor nucleotide as in the cGAS and thus failed to affect the orientation of the acceptor nucleotide. On the other hand, the corresponding Arg376Ile mutation of human cGAS did regain the 30 -50 cGAMP formation activity (Kranzusch et al., 2014). Several questions remain for DncV. The crystal structure of human cGAS revealed a positively charged cleft adjacent to the enzymatic scaffold. The positively charged cleft specifically engages dsDNA and induces structural rearrangement and dimerization of cGAS, which are important for the activation of cGAS (Kranzusch et al., 2013). Intriguingly, despite retaining the basic cleft, DncV does not require dsDNA binding to stimulate enzymatic ac-
tivity (Davies et al., 2012; Kranzusch et al., 2014). The possible role of the basic cleft in DncV remains unknown. Additionally, why DncV has evolved to make the 30 -50 , 30 -50 cGAMP and, in particular, whether it has downstream receptor(s) other than riboswitches (Nelson et al., 2015) remain a mystery. Nonetheless, the thorough structural analysis of DncV and deep understanding of its catalytic mechanism pave the way for the rational design of new antibacterial drugs targeting DncV, which could affect a broad spectrum of pathogenic bacteria.
ACKNOWLEDGMENTS M.G. is supported by NIH grants R01GM100136, R01GM106134, and R01CA178315.
Commichau, F.M., Dickmanns, A., Gundlach, J., Ficner, R., and Stulke, J. (2015). Mol. Microbiol. Published online April 13, 2015. http://dx.doi.org/ 10.1111/mmi.13026. Davies, B.W., Bogard, R.W., Young, T.S., and Mekalanos, J.J. (2012). Cell 149, 358–370. Kato, K., Ishii, R., Hirano, S., Ishitani, R., and Nureki, O. (2015). Structure 23, this issue, 843–850. Kranzusch, P.J., Lee, A.S., Berger, J.M., and Doudna, J.A. (2013). Cell Rep. 3, 1362–1368. Kranzusch, P.J., Lee, A.S., Wilson, S.C., Solovykh, M.S., Vance, R.E., Berger, J.M., and Doudna, J.A. (2014). Cell 158, 1011–1021. Mehne, F.M., Gunka, K., Eilers, H., Herzberg, C., Kaever, V., and Stu¨lke, J. (2013). J. Biol. Chem. 288, 2004–2017. Nelson, J.W., Sudarsan, N., Phillips, G.E., Stav, S., Lunse, C.E., McCown, P.J., and Breaker, R.R. (2015). Proc. Natl. Acad. Sci. USA. Published online April 6, 2015. http://dx.doi.org/10.1073/pnas. 1419264112.
REFERENCES Ablasser, A., Goldeck, M., Cavlar, T., Deimling, T., Witte, G., Ro¨hl, I., Hopfner, K.P., Ludwig, J., and Hornung, V. (2013). Nature 498, 380–384. Civril, F., Deimling, T., de Oliveira Mann, C.C., Ablasser, A., Moldt, M., Witte, G., Hornung, V., and Hopfner, K.P. (2013). Nature 498, 332–337.
802 Structure 23, May 5, 2015 ª2015 Elsevier Ltd All rights reserved
Romling, U., Galperin, M.Y., and Gomelsky, M. (2013). Microbiol. Mol. Biol. Rev. 77, 1–52. Sun, L., Wu, J., Du, F., Chen, X., and Chen, Z.J. (2013). Science 339, 786–791. Wu, J., Sun, L., Chen, X., Du, F., Shi, H., Chen, C., and Chen, Z.J. (2013). Science 339, 826–830.
