Structure and chemistry of lysinoalanine crosslinking in the spirochaete flagella hook

Abstract

The flagellar hook protein FlgE from spirochaete bacteria self-catalyzes the formation of an unusual inter-subunit lysinoalanine (Lal) crosslink that is critical for cell motility. Unlike other known examples of Lal biosynthesis, conserved cysteine and lysine residues in FlgE spontaneously react to form Lal without the involvement of additional enzymes. Oligomerization of FlgE via its D0 and Dc domains drives assembly of the crosslinking site at the D1–D2 domain interface. Structures of the FlgED2 domain, dehydroalanine (DHA) intermediate and Lal crosslinked FlgE subunits reveal successive snapshots of the reaction. Cys178 flips from a buried configuration to release hydrogen sulfide (H2S/HS) and produce DHA. Interface residues provide hydrogen bonds to anchor the active site, facilitate β-elimination of Cys178 and polarize the peptide backbone to activate DHA for reaction with Lys165. Cysteine-reactive molecules accelerate DHA formation, whereas nucleophiles can intercept the DHA intermediate, thereby indicating a potential for Lal crosslink inhibitors to combat spirochaetal diseases.

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Fig. 1: Overview of lysinoalanine crosslinking reaction catalyzed by Td FlgE.
Fig. 2: Residue requirements for Lal crosslinking.
Fig. 3: Comparison of pre-crosslink, dehydroalanine and post-crosslink FlgE.
Fig. 4: Proposed model of Lal crosslink formation in Td FlgE.

Data availability

Coordinates and structure files for WT FlgED2, DHA FlgED2 and Lal crosslinked FlgED1D2:D2 have been deposited to the Protein Data Bank with the following accession codes: 6NDW (WT FlgED2), 6NDT (DHA FlgED2) and 6NDX (FlgED1D2:D2 Lal crosslinked dimer). Raw MS data for all mutants and X-ray diffraction images are available from the corresponding author upon reasonable request. Constructs encoding for full-length and truncated Td FlgE variants are available from the corresponding author upon reasonable request.

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Acknowledgements

This work was supported by NIH grant no. R35 122535 (B.R.C.), the CBI Training grant nos. T32 GM008500 (M.J.L. and B.R.C.), NIH R01-DE023431 (N.C., M.M. and C.L.), AI078958 (C.L.) and NIH SIG grant no. 1S10 OD017992-01 (S.Z.). CHESS is supported by the NSF and NIH/NIGMS (no. DMR-1332208). MacCHESS is supported by NIH/NIGMS (no. GM-103485). Remote data collection was performed at the NE-CAT beamlines (no. GM124165) using an Eiger detector (OD021527) at the Advanced Photon Source (DE-AC02-06CH11357). The authors would like to thank H. Le for assistance with LC–MS experiments, A. Bilwes-Crane for editing the manuscript, the Cornell Proteomic and MS Facility for providing the mass spectrometry data and E. Anderson and R. Bahwal for technical assistance with MS sample preparation, data acquisition and analysis.

Author information

B.R.C., M.J.L., N.W.C., C.L. and M.M. conceived the study. K.Z. generated an initial construct encoding for Td FlgE. M.J.L. performed the crystallography, X-ray diffraction experiments and associated structural analysis, site-directed mutagenesis, EDC crosslinking and Lal crosslinking SDS–PAGE assays. M.J. carried out preliminary work that identified NEM and DTNB as FlgE crosslink enhancers. M.J.L. prepared samples for MS. S.Z. performed MS data acquisition and associated samples analysis. M.J.L. and B.R.C. prepared figures. M.J.L., B.R.C. and N.W.C. wrote the manuscript. B.R.C., N.W.C., M.J.L., C.L., M.J., M.M. and S.Z. edited the manuscript. B.R.C., N.W.C., M.M. and C.L. supervised the study.

Correspondence to Brian R. Crane.

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