Article
Extended Data Fig. 11 | Effective molarity driven protein–protein
crosslinking with electrophile-containing side chains. a, A comparison of
the connectivity of a modified lysine side chain (via methylation) and
bromohomonorleucine (Bhn, 1u) used for crosslinking. b, Structure of histone
eH3.1-mimicking peptide binding to KDM4A. Identified cysteines that undergo
crosslinking (Cys306 and Cys234) are highlighted in orange and their distance
to H3-K9 is highlighted. c, Workf low of the crosslinking reaction products and
their identification by LC–MS/MS. Representative spectra are shown for each
crosslinking site (top, eH3.1-Bhn4; middle, eH3.1-Bhn9; bottom, eH3.1-Bhn27)
and captured cysteine residue. The bottom right spectrum shows an inter-
histone crosslink via ether formation as found in the in-solution digest.
d, Williamson C–O–C bond ether formation in an intermolecular fashion
between H3 proteins (Bhn4 in one linked to hydroxyl in another) is driven by
effective molarity, possibly suggesting a transient dimer model for KDM4A
function. e, The histone eH3.1-Bhn9 alkylator protein was incubated with
HeLa nuclear lysate to capture interaction partners via promixity-driven
crosslinking. After enrichment via the HA tag (on histone eH3.1), an α-FLAG
western blot reveals multiple higher molecular ‘weight’ (MW) bands
corresponding to the mass of the histone plus that of the captured interaction
partner. No higher MW bands were seen in conditions lacking Bhn.