Abstract

BinAB is a naturally occurring paracrystalline larvicide distributed worldwide to combat the devastating diseases borne by mosquitoes. These crystals are composed of homologous molecules, BinA and BinB, which play distinct roles in the multi-step intoxication process, transforming from harmless, robust crystals, to soluble protoxin heterodimers, to internalized mature toxin, and finally to toxic oligomeric pores. The small size of the crystals—50 unit cells per edge, on average—has impeded structural characterization by conventional means. Here we report the structure of Lysinibacillus sphaericus BinAB solved de novo by serial-femtosecond crystallography at an X-ray free-electron laser. The structure reveals tyrosine- and carboxylate-mediated contacts acting as pH switches to release soluble protoxin in the alkaline larval midgut. An enormous heterodimeric interface appears to be responsible for anchoring BinA to receptor-bound BinB for co-internalization. Remarkably, this interface is largely composed of propeptides, suggesting that proteolytic maturation would trigger dissociation of the heterodimer and progression to pore formation.

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Data deposits

Atomic coordinates and structure factors have been deposited in the Protein Data Bank under accession codes 5FOY (pH 7 structure), 5FOZ (pH 10 structure) and 5G37 (pH 5 structure).

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Acknowledgements

We acknowledge the help of the following people during data collection: S. Lee, J. Koralek, R. Shoeman, S. Botha, B. Doak and O. Zeldin. We thank A. Volveda for advice regarding sequence-wise Fourier difference map integration; J. Brooks-Bartlett and E. Garman for help with dose calculations; and M. Weik for discussions and continuing support. We thank the HCIA program of HHMI, the W.M. Keck Foundation (grant 2843398), the NIH (grant AG-029430), National Science Foundation (grant MCB 0958111) and DOE (DE-FC02-02ER63421) (to D.S.E.), the France Alzheimer Foundation (FA-AAP-2013-65-101349) and the Agence Nationale de la Recherche (ANR-12-BS07-0008-03) (to J.-P.C.), NIH grants GM095887 and GM102520 for data-processing methods (to N.K.S.), and NIH grant AI45817 (to B.A.F.). Support by the CNRS (PEPS-SASLELX-2013, PEPS-SASLELX-2014) funded travel to LCLS. Use of the LCLS at SLAC National Accelerator Laboratory, is supported by the US Department of Energy, Office of Science, and Office of Basic Energy Sciences under contract no. DE-AC02-76SF00515. The CXI instrument was funded by the Linac Coherent Light Source Ultrafast Science Instruments project, itself funded by the DOE Office of Basic Energy Sciences. Parts of the sample injector used at LCLS for this research were funded by the National Institutes of Health, P41GM103393, formerly P41RR001209.

Author information

Author notes

    • Jacques-Philippe Colletier
    • , Michael R. Sawaya
    •  & Mari Gingery

    These authors contributed equally to this work.

Affiliations

  1. Institut de Biologie Structurale (IBS), Univ. Grenoble Alpes, CEA, CNRS, 38044 Grenoble, France

    • Jacques-Philippe Colletier
    •  & Nicolas Coquelle
  2. UCLA-DOE Institute for Genomics and Proteomics, Department of Biological Chemistry, University of California, Los Angeles, California 90095-1570, USA

    • Michael R. Sawaya
    • , Mari Gingery
    • , Jose A. Rodriguez
    • , Duilio Cascio
    •  & David S. Eisenberg
  3. Howard Hughes Medical Institute, University of California, Los Angeles, California 90095-1570, USA

    • Michael R. Sawaya
    •  & David S. Eisenberg
  4. Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA

    • Aaron S. Brewster
    • , Tara Michels-Clark
    •  & Nicholas K. Sauter
  5. Department of Entomology and Graduate Program in Cell, Molecular and Developmental Biology, University of California, Riverside, California 92521, USA

    • Robert H. Hice
    • , Hyun-Woo Park
    • , Dennis K. Bideshi
    •  & Brian A. Federici
  6. Linac Coherent Light Source, SLAC National Accelerator Laboratory, Menlo Park, California 94025, USA

    • Sébastien Boutet
    • , Garth J. Williams
    • , Marc Messerschmidt
    • , Daniel P. DePonte
    • , Raymond G. Sierra
    • , Hartawan Laksmono
    • , Jason E. Koglin
    •  & Mark S. Hunter
  7. Department of Biological Sciences, California Baptist University, Riverside, California 92504, USA

    • Hyun-Woo Park
    •  & Dennis K. Bideshi
  8. Molecular and Cellular Physiology, and Howard Hughes Medical Institute, Stanford University, Stanford, California 94305, USA

    • Monarin Uervirojnangkoorn
    •  & Axel T. Brunger

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Contributions

J.-P.C., M.R.S., M.G., J.A.R., D.C., B.A.F. and D.S.E designed and coordinated the project. M.G. carried out the in vivo production of BinAB nano-crystals. H.-W.P., D.K.B. and B.A.F. engineered bacterial strains to produce enlarged BinAB crystals. R.H.H. and H.-W.P. performed toxicity assays. R.H.H. designed the mutagenesis protocol. D.K.B. performed crystal solubilization assays. M.R.S. performed heavy atom derivatizations. J.-P.C., M.R.S., J.A.R., D.C., A.S.B., T.M.-C., S.B., G.J.W., M.M., D.P.D., R.G.S., H.L., J.E.K., M.S.H., N.C., M.U. and N.K.S. acquired and processed data. J.-P.C. and M.R.S carried out the MIRAS and MR phasing, and built and refined the atomic models. R.G.S. and H.L. developed the MESH-on-a-stick injector. J.-P.C., M.R.S., and D.S.E prepared the manuscript with input from M.G., J.A.R., D.C., A.S.B., T.M.-C., R.G.S., M.S.H., A.T.B., B.A.F., and N.K.S.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Jacques-Philippe Colletier or David S. Eisenberg.

Reviewer Information Nature thanks C. Berry, H. Chapman and P. Wang for their contribution to the peer review of this work.

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