Manipulating Protein–Protein Interactions in Nonribosomal Peptide Synthetase Type II Peptidyl Carrier Proteins

Abstract

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 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 dynamic 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 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 PCPs significantly modulated their activities.

Dynamic visualization of type II peptidyl carrier protein recognition in pyoluteorin biosynthesis

“…Furthermore, previous molecular dynamic simulations revealed the relative flexibility of PltL loop 1, which supports loop 1 reorganization upon binding. 10 These data provide evidence that specific H-bonding and hydrophobic interactions allowed by the conformational flexibility of PltL loop 1 is responsible for its recognition towards PltF.…”

“…S7, ESI †), implicating their participation in, or response to, the binding event. 10 Of those residues, Ile19, Leu28, Leu35, Trp37, and Gly38 were seen at the protein-protein interface in the crystal structure (Fig. 2D).…”

“…9 PltL has been shown to exhibit specificity towards PltF and no interactivity towards homologous A domains. 10,11 This suggested the requirement of a specific protein-protein interaction motif for A domain activity. Studies have attempted to control the partner protein specificity of PltL; solution-phase nuclear magnetic resonance (NMR) titration experiments revealed a region of PltL, loop 1 (residues 19-41), that was postulated to form the protein-protein interface with PltF.…”

“…Studies have attempted to control the partner protein specificity of PltL; solution-phase nuclear magnetic resonance (NMR) titration experiments revealed a region of PltL, loop 1 (residues 19-41), that was postulated to form the protein-protein interface with PltF. 10 Mutagenesis of residues in this region disrupted activity, however, these studies could not resolve the PCP-A domain interface clearly enough to accurately manipulate PltL specificity.…”

The many faces and important roles of protein–protein interactions during non-ribosomal peptide synthesis

“…Indeed, NRPS activity is reduced when point mutations are introduced in the loop 1 motif. 40,79 Whilst the PA1221 PCP forms many interactions with its cognate A domain (Fig. 4A), the number of interactions reported in the case of LgrA in thiolation state is much lower.…”