Solution Structure of a Nonribosomal Peptide Synthetase Carrier Protein Loaded with Its Substrate Reveals Transient, Well-Defined Contacts

Abstract

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.

Figure 1

Role of ArCP in yersiniabactin synthesis. a) ArCP is the first carrier protein domain of HMWP2, which also comprises two cyclization domains (Cy1 and Cy2), two peptidyl carrier proteins (PCP1 and PCP2), an adenylation domain for cysteine (A), and an epimerization domain embedded in the A-domain (E). b) The A-domain loads cysteine onto PCP1 and PCP2, and the stand alone A-domain YbtE loads salicylate onto ArCP. c) Cy1 catalyzes the condensation and cyclodehydration of Sal and Cys, forming 2-hydroxyphenylthiazoline on PCP1 and returning ArCP to its holo form. Not shown: Cy2, HMWP1 (a mixed PKS NRPS protein), and YbtU complete the synthesis of yersiniabactin (e). d) Nomenclature used to assign phosphopantetheine (J-U) and salicylate (V-AA) is shown in italics.

Figure 2

Solution structures of holo- (a,b) and loaded-ArCP (c,d). The lowest energy conformer of the NMR ensemble is shown for each form of the protein under two different views. e) Detail of loop1 shown for a superposition of holo- (pink) and loaded-ArCP (blue). Structures were aligned with each other using helices α1 through α4. f) Mean structures of Ser-PP from holo-ArCP (orange) and Ser-PP-Sal from loaded-ArCP (orange-red). The moieties were translated to overlay Ser 52 C^α of each form.

Figure 3

NMR dynamics of holo- and loaded-ArCP. (a–d) Residue-specific NMR relaxation parameters for holo (left, magenta) and loaded (right, cyan). The secondary structure of ArCP is illustrated below the plots. In (a–c) the relaxation parameters for the NN and NR positions of the PP are displayed at residues 99 and 100 respectively. A thick, colored line distinguishes them from the remaining residues. (a) R1 relaxation rates. (b) R2 relaxation rates. (c) Heteronuclear NOE parameterized by I_sat/I_ref where I_sat and I_ref are the amplitudes of signals in the proton-saturated and reference experiment, respectively. (d) Order parameter S². (e–h) Dynamics visualization of holo- (magenta) and loaded-ArCP (cyan), residues 16–90. A thicker ribbon corresponds to a reduced order parameter and increased flexibility. Colors represent the R_ex parameters fit during Lipari-Szabo analysis. Data are not available for residues in white due to overlap (e) holo-ArCP with PP in its bound state (f) loaded-ArCP with the PP in its bound state (g) holo-ArCP with PP in its unbound state (h) loaded-ArCP with PP in its unbound state

Figure 4

Comparison of holo- (a, in pink) and loaded-ArCP (c, in blue) in complex with an adenylation domain in A_T conformation (in white). The original structure of EntB-ArCP (brown) in complex with EntE is shown in b) (2ROG). In b), EntB-ArCP sidechains colored in red highlight interactions between A(N) and α2, those in blue show interactions between A(C) and ArCP, and those in green denote interactions between A(C) and loop1. The same color scheme was used for side-chains of EntE that are displayed in a–c). d) Detail showcasing changes in the conformation of loop1. The orientation in d is obtained by rotation of 30° around the Y vertical axis.