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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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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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.
The authors declare no competing interests.
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