2 research outputs found
Sulfonyl 3‑Alkynyl Pantetheinamides as Mechanism-Based Cross-Linkers of Acyl Carrier Protein Dehydratase
Acyl carrier proteins (ACPs) play
a central role in acetate biosynthetic
pathways, serving as tethers for substrates and growing intermediates.
Activity and structural studies have highlighted the complexities
of this role, and the protein–protein interactions of ACPs
have recently come under scrutiny as a regulator of catalysis. As
existing methods to interrogate these interactions have fallen short,
we have sought to develop new tools to aid their study. Here we describe
the design, synthesis, and application of pantetheinamides that can
cross-link ACPs with catalytic β-hydroxy-ACP dehydratase (DH)
domains by means of a 3-alkynyl sulfone warhead. We demonstrate this
process by application to the Escherichia coli fatty acid synthase and apply it to probe protein–protein
interactions with noncognate carrier proteins. Finally, we use solution-phase
protein NMR spectroscopy to demonstrate that sulfonyl 3-alkynyl pantetheinamide
is fully sequestered by the ACP, indicating that the <i>crypto</i>-ACP closely mimics the natural DH substrate. This cross-linking
technology offers immediate potential to lock these biosynthetic enzymes
in their native binding states by providing access to mechanistically
cross-linked enzyme complexes, presenting a solution to ongoing structural
challenges
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Manipulating Protein–Protein Interactions in Nonribosomal Peptide Synthetase Type II Peptidyl Carrier Proteins
In an effort to elucidate
and engineer interactions in type II
nonribosomal peptide synthetases, we analyzed biomolecular recognition
between the essential peptidyl carrier proteins and adenylation domains
using nuclear magnetic resonance (NMR) spectroscopy, molecular dynamics,
and mutational studies. Three peptidyl carrier proteins, PigG, PltL,
and RedO, in addition to their cognate adenylation domains, PigI,
PltF, and RedM, were investigated for their cross-species activity.
Of the three peptidyl carrier proteins, only PigG showed substantial
cross-pathway activity. Characterization of the novel NMR solution
structure of holo-PigG and molecular dynamics simulations of holo-PltL
and holo-PigG revealed differences in structures and dynamics of these
carrier proteins. NMR titration experiments revealed perturbations
of the chemical shifts of the loop 1 residues of these peptidyl carrier
proteins upon their interaction with the adenylation domain. These
experiments revealed a key region for the protein–protein interaction.
Mutational studies supported the role of loop 1 in molecular recognition,
as mutations to this region of the peptidyl carrier proteins significantly
modulated their activities