Localized protein interaction surfaces on the EntB carrier protein revealed by combinatorial mutagenesis and selection.
Carrier proteins are 80- to 100-residue way stations that are central to polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) enzymatic assembly lines. Because the biosynthetic intermediates for catalytic operations are presented on carrier proteins as covalently attached thioesters (via a 4'-phosphopantetheine prosthetic group), the specific protein-protein interactions between carrier proteins and other NRPS/PKS domains are critical for high-fidelity conversion to the final product. Here we show by combinatorial mutagenesis and selection that the aryl carrier protein of EntB (EntB-ArCP) contains localized protein interaction surfaces. Our strategy involved random mutagenesis of N-terminal regions of EntB-ArCP, then selection for clones that produce enterobactin by plating onto iron-deficient media. We identified several residues that were highly conserved from our selection, two of which (G242 and D244) constitute an interaction surface on EntB-ArCP for the phosphopantetheinyl transferases (PPTases) EntD and Sfp. This PPTase interface is distinct from a previously characterized interface on EntB-ArCP for the downstream elongation module, EntF. These results suggest that different protein components recognize different faces of EntB-ArCP in the enterobactin synthetase and that the majority of EntB-ArCP surface residues are not involved in these interactions. Therefore, designing noncognate carrier protein interactions in PKS and NRPS systems should be possible with very few mutations on a particular carrier protein.
Lai
Jonathan R
JR
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Fischbach
Michael A
MA
Liu
David R
DR
Walsh
Christopher T
CT
D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D016296:Mutagenesis; D011485:Protein Binding; D012641:Selection, Genetic; D013499:Surface Properties
D029968:Escherichia coli Proteins