Localized protein interaction surfaces on the EntB carrier protein revealed by combinatorial mutagenesis and selection

Jonathan R. Lai, Michael A. Fischbach, David R. Liu, Christopher T. Walsh

Research output: Contribution to journalArticle

38 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)11002-11003
Number of pages2
JournalJournal of the American Chemical Society
Volume128
Issue number34
DOIs
StatePublished - Aug 30 2006
Externally publishedYes

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Mutagenesis
Carrier Proteins
Membrane Proteins
Peptide Synthases
Polyketide Synthases
Proteins
Peptides
Iron plating
Enterobactin
Prosthetics
Elongation
Iron
Clone Cells
Mutation

ASJC Scopus subject areas

  • Chemistry(all)

Cite this

Localized protein interaction surfaces on the EntB carrier protein revealed by combinatorial mutagenesis and selection. / Lai, Jonathan R.; Fischbach, Michael A.; Liu, David R.; Walsh, Christopher T.

In: Journal of the American Chemical Society, Vol. 128, No. 34, 30.08.2006, p. 11002-11003.

Research output: Contribution to journalArticle

Lai, Jonathan R. ; Fischbach, Michael A. ; Liu, David R. ; Walsh, Christopher T. / Localized protein interaction surfaces on the EntB carrier protein revealed by combinatorial mutagenesis and selection. In: Journal of the American Chemical Society. 2006 ; Vol. 128, No. 34. pp. 11002-11003.
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