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Structures and function of the amino acid polymerase cyanophycin synthetase

Nature Chemical Biology volume 17, pages 1101–1110 (2021)Cite this article

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Abstract

Cyanophycin is a natural biopolymer produced by a wide range of bacteria, consisting of a chain of poly-l-Asp residues with l-Arg residues attached to the β-carboxylate sidechains by isopeptide bonds. Cyanophycin is synthesized from ATP, aspartic acid and arginine by a homooligomeric enzyme called cyanophycin synthetase (CphA1). CphA1 has domains that are homologous to glutathione synthetases and muramyl ligases, but no other structural information has been available. Here, we present cryo-electron microscopy and X-ray crystallography structures of cyanophycin synthetases from three different bacteria, including cocomplex structures of CphA1 with ATP and cyanophycin polymer analogs at 2.6 Å resolution. These structures reveal two distinct tetrameric architectures, show the configuration of active sites and polymer-binding regions, indicate dynamic conformational changes and afford insight into catalytic mechanism. Accompanying biochemical interrogation of substrate binding sites, catalytic centers and oligomerization interfaces combine with the structures to provide a holistic understanding of cyanophycin biosynthesis.

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Fig. 1: Cyanophycin and cyanophycin synthetase.
Fig. 2: Overall structure and activity of CphA1.
Fig. 3: Structure and mutagenesis of the G domain.
Fig. 4: Structure and mutagenesis of the M domain.
Fig. 5: Structure, conservation and mutagenesis of the N domain.
Fig. 6: Dimeric SuCphA1 mutants and model of cyanophycin synthesis.

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Data availability

The cryo-EM maps created in this study have been deposited to the EMDB: SuCphA1 bound with ATP (EMD-23311), SuCphA1 bound with ADPCP and (β-Asp-Arg)8-NH2 (EMD-23325), SuCphA1 bound with ATP and (β-Asp-Arg)8-Asn (EMD-23328), SuCphA1 with ATP and (β-Asp-Arg)16 (EMD-23326) and AbCphA1 with ATP (EMD-23327). The protein structures solved in this study have been deposited to the PDB: SuCphA1 with ATP (7LG5), SuCphA1 with ADPCP and (β-Asp-Arg)8-NH2 (7LGJ), SuCphA1 with ATP and (β-Asp-Arg)8-Asn (7LGQ), SuCphA1 with ATP, AbCphA1 with ATP (7LGM) and TmCphA1 (7LGN). Source data are provided with this paper.

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Acknowledgements

We thank members of the Schmeing laboratory for helpful advice and discussion, N. Rogerson for proofreading, staff at the McGill Facility of EM Research (M. Strauss, K. Basu and K. Sears) and APS (F. Murphy and S. Banarjee) for support during data collection. We thank the UCSD Cryo-Electron Microscopy Facility, which was supported in part by National Institutes of Health grants to T.S. Baker and a gift from the Agouron Institute to UCSD. This work was supported by a Canada Research Chair and NSERC Discovery grant no. 418420 to T.M.S., and by funding from the Swiss National Science Foundation and the ETH Zurich to D.H. We thank P. Emsley and R. Nicholls for help with restraints file generations. This work includes research conducted at the Northeastern Collaborative Access Team beamlines, which are funded by the National Institute of General Medical Sciences from the National Institutes of Health (grant no. P30 GM124165). The Eiger 16M detector on the 24-ID-E beamline is funded by a NIH-ORIP HEI grant (no. S10OD021527). This research used resources of the Advanced Photon Source, a US DOE Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under contract no. DE-AC02-06CH11357. Gene synthesis of TmCphA1 was conducted by the US DOE Joint Genome Institute, a DOE Office of Science User Facility, which is supported under contract no. DE-AC02-05CH11231, as part of JGI grant no. 503632 to T.M.S.

Author information

Authors and Affiliations

  1. Department of Biochemistry and Centre de Recherche en Biologie Structurale, McGill University, Montréal, Quebec, Canada

    Itai Sharon, Asfarul S. Haque & T. Martin Schmeing

  2. Laboratory of Organic Chemistry, ETH Zürich, Zürich, Switzerland

    Marcel Grogg, Dieter Seebach & Donald Hilvert

  3. Department of Cellular and Molecular Medicine, and Section of Molecular Biology, Division of Biological Sciences, University of California San Diego (UCSD), La Jolla, CA, USA

    Indrajit Lahiri & Andres E. Leschziner

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  1. Itai Sharon
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Contributions

T.M.S. and I.S. designed the study. I.S. performed all molecular biology and biochemical experiments. I.S., A.S.H., I.L. and T.M.S. performed cryo-EM data collection and initial structure determination. I.S. performed crystallography and structure determination, as well as modeling and refinement of all structures. M.G. performed chemical synthesis under the direction of D.S. and D.H. T.M.S. and I.S. wrote the paper and T.M.S., I.S., A.E.L., I.L. and D.H. edited it.

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Correspondence to T. Martin Schmeing.

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Peer review information Nature Chemical Biology thanks Elke Dittmann, Satish Nair and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 CphA1 distribution.

Phylogenetic tree of CphA1 sequences. A BLAST search found over 4000 CphA1-encoding gene sequences. Analysis of these sequences revealed that they are spread across most major bacterial phyla. Specific clades of particular interest were manually annotated and colored. The homologs used in this study are labeled in red. Gammaproteobacterial TmCphA1 and AbCphA1 share ~41% identity with cyanobacterial SuCphA1. There is evidence for both ancient horizontal gene transfer (alpha-, delta- and gammaproteobacterial CphA1s cluster together, but apart from betaproteobacteria1) and more recent transfer in the unlabeled, black clusters of CphA1s that are from several different bacterial groups.

Extended Data Fig. 2 CphA1 tetramerization.

a, SuCphA1 (a, c) and TmCphA1 (b, d) display different tetramer architectures, in which different monomers are responsible for tetramer-forming interactions. e, The EM map and structure of SuCphA1 showing the tetramer interface, which is centered on W672. f, Gel filtration chromatograms of all three CphA1 homologs used in this study, show they all form tetramers in solution.

Extended Data Fig. 3 Comparison of CphA1 active sites to homologous enzymes.

a, Overlay of SuCphA1 G domain and bifunctional glutathione synthetase from S. agalactiae (PDB code 3LN6) showing the similar ATP binding mode and conserved residues. b, Overlay of SuCphA1 G domain and glutathione synthetase from E. coli (PDB code 1GSA) showing the similar substrate orientation and overall structure. c, Overlay of SuCphA1 M domain and MurE ligase from M. tuberculosis residues (PDB code 2WTZ) showing the similar ATP binding mode and conserved, and (d) similar substrate orientation. e, The interactions made by cyanophycin with residues in the M domain of SuCphA1. f, The three versions of cyanophycin and cyanophycin analogs used for the determined structures of SuCphA1 presented in this study.

Extended Data Fig. 4 Flexibility, substrate binding and surface electrostatic potential of CphA1.

a, Local resolution estimates of the cryo-EM maps of tetrameric SuCphA1 (left) and dimeric AbCphA1 (right). b, Overlay of the two chains in the crystal structure of TmCphA1 (light blue) on the cryo-EM structure of SuCphA1 (colored), showing the different conformation adopted by Mlid of the crystal structure chain A and Glid in chain B. Mlid is not visible in chain B. c, Overlay of the unsharpened maps of SuCphA1 without cyanophycin substrate analogs (gray), and with (Asp-Arg)8-NH2 (red, right) and (Asp-Arg)8-Asn (blue, left). Clear extra density is visible in the maps calculated in the presence of substrate analogs, mostly near the active sites and the N domain. (Asp-Arg)8-Asn is also seen as product in the G domain active site, but no density is visible for the terminal Asn residue. d, Surface electrostatic potential maps of SuCphA1 and TmCphA1 dimers showing how the side that faces the active sites is lined with negatively and positively charged patched. The side facing the inner cavity, which is opposite the active sites, is mostly neutral. Active sites are marked with *, αa and αb are marked with rectangles.

Extended Data Fig. 5 CphA1 N domain structural homology, cyanophycin binding mode and mutants analysis.

a, Structure overlay of CphA1 with E. coli RNA polymerase alpha subunit (PDB code 4JK1), showing similarity in parts of their structures. b, Possible arrangement of cyanophycin that allows either its positive charges or negative charges to interact with αa or αb. c, Activity assays of TmCphA1 N domain mutants showing similar results to those observed for the equivalent SuCphA1 mutants (displayed in Fig. 5). n = 4 independent experiments. Data are presented as individual measurements and mean value, error bars represent SD values. d, Differential scanning fluorimetry melting curves and protein Tm values of CphA1 N domain mutants. The similarity of Tm values between wildtype and N domain mutants suggests that the observed differences in activity are a result of differences in interaction with cyanophycin rather than differences in protein stability. Additionally, the gel filtration profiles of the proteins during the purification process were all similar, again suggesting similar stability of wildtype and mutants. The values in the table represent the mean and SD of 3 independent measurements.

Source data

Extended Data Fig. 6 Dimeric CphA1 mutation schematics and activity in 100 mM sodium chloride.

a, The tetramerization interface, between W672 and residues 468–470, is disrupted in the obligate dimer W672A mutants. b, Cartoon representation of the CphA1 mutants. c, Both mutant combinations of dimer mutants (G+M+/G-M− and G+M−/G-M+) show the same decrease in activity level relative to wildtype. The ratio between the activity rate of the wildtype CphA1 and mutants is similar to that observed with no sodium chloride in the reaction buffer. n = 4 independent experiments. Data are presented as individual measurements and mean value, error bars represent SD values.

Source data

Extended Data Fig. 7 Fourier shell correlation for EM datasets.

FSC plots for all EM maps determined in this study.

Extended Data Fig. 8 Model of cyanophycin synthesis within wildtype and mutant CphA1.

Models of cyanophycin synthesis by WT CphA1 and the active site mutants used in the study.

Supplementary information

Supplementary Information

Supplementary Note, Fig. 1, Tables 1–6 and References.

Reporting Summary

Supplementary Video 1

The structure of SuCphA1. This video highlights the structure of the three domains, substrate binding interactions and tetramer interface.

Supplementary Video 2

Comparison of the structures of SuCphA1 and TmCphA1. This video focuses on the overall architectures of monomers, dimers and tetramers.

Supplementary Video 3

Four modes of 3D variability analysis of SuCphA1 bound with (β-Asp-Arg)8-Asn. This video shows the first mode from CryoSPARC2 3D variability analysis.

Supplementary Video 4

Model for the catalytic cycle of CphA1. This video shows the proposed model for biosynthesis of cyanophycin by SuCphA1.

Source data

Source Data Fig. 2

Statistical raw data Fig. 2a.

Source Data Fig. 2

Uncropped gel image Fig. 2b.

Source Data Fig. 3

Statistical raw data Fig. 3d.

Source Data Fig. 4

Statistical raw data Fig. 4d.

Source Data Fig. 5

Statistical raw data Fig. 5.

Source Data Fig. 6

Statistical raw data Fig. 6.

Source Data Extended Data Fig. 5

Statistical raw data Extended Data Fig. 5.

Source Data Extended Data Fig. 6

Statistical raw data Extended Data Fig. 6.

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Sharon, I., Haque, A.S., Grogg, M. et al. Structures and function of the amino acid polymerase cyanophycin synthetase. Nat Chem Biol 17, 1101–1110 (2021). https://doi.org/10.1038/s41589-021-00854-y

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