Solution Structure of a Nonribosomal Peptide Synthetase Carrier Protein Loaded with Its Substrate Reveals Transient, Well-Defined Contacts.
Nonribosomal peptide synthetases (NRPSs) are microbial enzymes that produce a wealth of important natural products by condensing substrates in an assembly line manner. The proper sequence of substrates is obtained by tethering them to phosphopantetheinyl arms of holo carrier proteins (CPs) via a thioester bond. CPs in holo and substrate-loaded forms visit NRPS catalytic domains in a series of transient interactions. A lack of structural information on substrate-loaded carrier proteins has hindered our understanding of NRPS synthesis. Here, we present the first structure of an NRPS aryl carrier protein loaded with its substrate via a native thioester bond, together with the structure of its holo form. We also present the first quantification of NRPS CP backbone dynamics. Our results indicate that prosthetic moieties in both holo and loaded forms are in contact with the protein core, but they also sample states in which they are disordered and extend in solution. We observe that substrate loading induces a large conformational change in the phosphopantetheinyl arm, thereby modulating surfaces accessible for binding to other domains. Our results are discussed in the context of NRPS domain interactions.
Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine , Hunterian 701, 725 North Wolfe Street, Baltimore, Maryland 21205, United States ;
Andrew C
AC
Goodrich
Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine , Hunterian 701, 725 North Wolfe Street, Baltimore, Maryland 21205, United States ;
Bradley J
BJ
Harden
Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine , Hunterian 701, 725 North Wolfe Street, Baltimore, Maryland 21205, United States ;
Dominique P
DP
Frueh
D000213:Acyl Carrier Protein / Q000378:metabolism; D020134:Catalytic Domain; D020035:Holoenzymes / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D010453:Peptide Synthases / Q000737:chemistry* / Q000378:metabolism*; D020156:Salicylic Acid / Q000378:metabolism; D012996:Solutions
D000213:Acyl Carrier Protein; D020035:Holoenzymes; D012996:Solutions; D010453:Peptide Synthases; C104385:non-ribosomal peptide synthase; D020156:Salicylic Acid