A slow-forming isopeptide bond in the structure of the major pilin SpaD from Corynebacterium diphtheriae has implications for pilus assembly

Kang, H.J.; Paterson, N.G.; Kim, C.U.; Middleditch, M.; Chang, C.; Ton-That, H.; Baker, E.N.

doi:10.1107/S1399004714001400

Figure 3
(a, b) Slow-forming isopeptide bond in SpaD. Mass spectrometry of freshly purified SpaD (a) revealed the presence of two protein species: one corresponding to SpaD with three isopeptide bonds (47 000.40 Da) and the other to SpaD with two isopeptide bonds (47 019.20 Da). When SpaD was incubated at 37°C for 24 h, the peak ratio changed and more of the three-isopeptide bond species were observed. The insets show charge envelopes before deconvolution. The observed charge envelope shifts to much higher m/z values when the third isopeptide bond forms as in (b), indicative of a tighter, more constrained protein structure with less surface area available for protonation. (c, d) LC-MS/MS of proteolytic products of SpaD containing peptides with and without the Lys58–Asn180 isopeptide-bond cross-link in domain D1. Fragmentation spectra of the parent ion at m/z 486.66³⁺ (c) indicate the presence of peptides cross-linked by the bond between SpaD Lys58 and Asn180, whereas the same sample also contained the ion at m/z 442.28²⁺ (d) showing a linear peptide containing Lys58. Daughter ions produced during MS/MS of these peptides are summarized in Tables 2

and 3

STRUCTURAL
BIOLOGY

ISSN: 2059-7983

Volume 70| Part 5| May 2014| Pages 1190-1201

https://doi.org/10.1107/S1399004714001400

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