A short,
flexible, and unstructured peptide tag that has versatile
and facile use in protein labeling applications is highly desirable.
Here, we report an 11-residue peptide tag with an internal cysteine
(a W-tag, derived from a Comm PY peptide motif that is known to bind
with Nedd4 WW3* domain) that can be installed at different regions
of the target protein without compromising its covalent reactivity
with the reactive label (a 35-residue synthetic Nedd4 WW3* domain
derivative). This versatility is explained by the unique structural
features of the reaction. NMR analysis reveals that both the W-tag
peptide and reactive Nedd4 WW3* protein are unstructured before they
encounter each other. The binding interaction of the two induces noticeable
structural changes and promotes global folding. Consequently, the
reactive cysteine residue at W-tag and the electrophilic chloroacetyl
group at Nedd4 WW3* domain are positioned to be in close proximity,
inducing an intermolecular covalent cross-linking. The covalent linkage
in turn stabilizes the folding of the protein complex. This unique
multistep mechanism renders this labeling reaction amenable to different
sites of the proteins of interest: installation of the tag at N- and
C-termini, in the flexible linker region, in the loop region, and
the extracellular terminus of target proteins exhibited comparable
reactivity. This work therefore represents the first proximity-induced
cysteine reaction based on the unique binding features of WW domains
that demonstrates unprecedented versatility
