Structure of PA1221, a nonribosomal peptide synthetase containing adenylation and peptidyl carrier protein domains

Abstract

Many bacteria use large modular enzymes for the synthesis of polyketide and peptide natural products. These multidomain enzymes contain integrated carrier domains that deliver bound substrates to multiple catalytic domains, requiring coordination of these chemical steps. Nonribosomal peptide synthetases (NRPSs) load amino acids onto carrier domains through the activity of an upstream adenylation domain. Our lab recently determined the structure of an engineered two-domain NRPS containing fused adenylation and carrier domains. This structure adopted a domain-swapped dimer that illustrated the interface between these two domains. To continue our investigation, we now examine PA1221, a natural two-domain protein from Pseudomonas aeruginosa. We have determined the amino acid specificity of this new enzyme and used domain specific mutations to demonstrate that loading the downstream carrier domain within a single protein molecule occurs more quickly than loading of a nonfused carrier domain intermolecularly. Finally, we have determined crystal structures of both apo- and holo-PA1221 proteins, the latter using a valine-adenosine vinylsulfonamide inhibitor that traps the adenylation domain-carrier domain interaction. The protein adopts an interface similar to that seen with the prior adenylation domain-carrier protein construct. A comparison of these structures with previous structures of multidomain NRPSs suggests that a large conformational change within the NRPS adenylation domains guides the carrier domain into the active site for thioester formation.

Figure 1

Multidomain catalysis of peptide synthesis by the NRPSs. (A) Reactions catalyzed by the most common NRPS catalytic domains. The adenylation domain catalyzes a two step reaction to first adenylate the amino acid and then covalently load the downstream carrier domain, represented by the blue oval. The pantetheine cofactor is represented by the thiol SH. The domain alternation hypothesis suggests a 140° rotation of a small C-terminal subdomain within the adenylation domain is used to adopt the catalytic conformations for the two partial reactions. The condensation domain catalyzes peptide bond formation, transferring the loaded amino acid from an upstream carrier domain (green) to form the dipeptide on the downstream carrier (blue). The terminal thioesterase domain catalyzes thioester hydrolysis, releasing the peptide. (B) Schematic representation of SrfA-C highlighting the structural problem of the domain rearrangements required to enable the phosphopantetheine cofactor to reach the neighboring active sites. The schematic represents the domain orientation observed in the SrfA-C structure (PDBID:2VSQ). The condensation domain is grey, the adenylation domain is red, the PCP domain is light blue, and the thioesterase is dark blue.

Figure 2

Substrate specificity of PA1221. Specificity was tested utilizing the ³²P-PPi exchange assay. Amino acids tested are reported as single letter code and background activity, lacking a carboxylate substrate, is labeled “0”. Additional acids were tested and labeled a, b, and c, representing acetate, 4-chlorobenzate, and 2-amino-benzoate, respectively. Three other assays were performed in a series of alternate experiments (Alt.). Two mutant enzymes were tested with valine as the amino acid substrate, the K499L A10 adenylation domain mutant (A) and the Ser533Ala phosphopantetheinyl attachment site mutant (S). Finally, the impact of 1.2-fold molar excess of the MbtH-like protein PA2412 is shown in the final bar (2).

Figure 3

Structures of apo- and holo-PA1221. (A) Ribbon diagram of apo-PA1221 with N-terminal domain colored grey, black, and wheat, the C-terminal subdomain highlighted with orange helices. The A8 loop, an antiparallel sheet that immediately follows the hinge at Asp417, is shown in yellow and the N- and C-termini are indicated with blue and brown spheres. (B) Ribbon diagram of holo-PA1221, colored as in panel A with the PCP colored red and the four helices labeled 1–4. Lys499, the catalytic lysine from the A10 motif, is shown in stick representation. The same loop, Ala496-Leu500, is disordered in the apo-structure and is shown with a dotted line in panel A. (C) Stereo image of apo-PA1221 active site with AMP and (2R,3R)-(−)-butanediol and F_o-F_c difference density contoured at 3 σ. (D) Stereo image of holo-PA1221 phosphopantetheine tunnel and active site with phosphopantetheine and Val-AVS with F_o-F_c difference density contoured at 3 σ. Both electron density maps were created with coefficients calculated prior to inclusion of ligands in the model.

Figure 4

Stereorepresentations of holo-PA1221. (A) Ribbon diagram of the interaction between the Adenylation and PCP domains. The PCP is shown in red, while the adenylation domain is colored as in Figure 3B. Several residues from the adenylation domain are labeled in black to orient the viewer. The PCP residue Ala561 is labeled red; this residue is located on helix 2. The residues that form the hydrophobic interface with this helix include Thr264 and Leu268. Leu 261 and Leu265 are shown but not labeled for clarity. Val455 is shown interacting with Leu554 and Leu555, which are not labeled. Residues that contribute to the hydrogen bonding network include Arg450, Arg452, Asn453, and Gln457 are shown. These residues interact primarily with main chain atoms of the PCP. (B) Phe213, the conserved aromatic residue of the A4 motif, is stabilized by interactions with aromatic residues Tyr425 and Trp480 of the C-terminal domain. Rotation of Phe213 opens the phosphopantetheine tunnel allowing proper binding of the pantetheine for thioester formation.

Figure 5

Functional analysis of intra- vs. inter-molecular loading with the PA1221 protein. The top panel shows the initial loading of ³H-Valine onto PA1221 at 37°C. The individual bars represent averages of two assays with wild-type enzyme in the absence (WT-) and presence (WT) of ATP, the K499L mutant (A10-), the S553A mutant (PCP-) and a co-incubation reaction containing equal amounts of the K499L and the S553A mutant enzymes (Comp). Results are expressed as nmol valine incorporated per nmol of functional PCP as the experiment with the compensatory mutants used equal amounts of total protein, or half as much of each functional domain. In the bottom panel, the reaction was monitored on ice, slowing the reaction to observe differences between the wt and the combination of the K499L and S553A mutant enzymes. Charging with ³H-valine was monitored over two hours. Loading of the WT enzyme is represented by the filled squares. The inter-molecular reaction, forced through the reaction containing equal amounts of the two compensatory mutants, K499L and S553A, is represented by the open circle. All data points, reflecting duplicate reactions at each time point, are shown. The filled grey circle is an anomalous data point from the compensatory mutant experiment that was omitted from the linear regression plot to better represent the rate of incorporation. Results are expressed as nmol of valine incorporated into the 3 nmol of holo-PCP domain used in both experiments.

Figure 6

Comparison of holo-PA1221 and EntE-B. (A) The interaction of the EntE-B protein (PDBID:3RG2) with N-terminal domain in grey, C-terminal subdomain is green, and PCP in forest green. The N- and C-termini, as well as the hinge residue, are shown with blue, brown, and red spheres respectively. The orientation is similar to the PA1221 orientation in Figure 3 (B) C-terminal subdomains and PCP domains of holo-PA1221 and EntE-B structurally aligned by least squares fitting of N-terminal domains, which are not shown for clarity. The N-terminal domain helices that interact with helix 2 of the PCP are shown in grey for both PA1221 and EntE-B and labeled N’ helix.

Figure 7

Comparison of holo-PA1221 with SrfA-C adenylation and PCP domains. (A) The PA1221 and SrfA-C proteins were aligned on the basis of the N-terminal subdomain for comparison of the overall organization of C-terminal subdomains and PCPs. Where the (left) PA1221 carrier domain packs against the adenylation domains, the SrfA-C protein (right) extends the PCP to interact with the neighboring condensation domain. (B) The SrfA-C (blue) and PA1221 (orange and red) C-terminal subdomain and PCPs were aligned on the basis of the central sheet in the C-terminal subdomain. A stereo representation illustrates the differences in rotations of the PCP domain relative to the C-terminal subdomain. The pantetheine attachment site at the start of helix 2 of the PCP is highlighted in yellow. (C) Schematic model for the role of domain alternation in the movement of modular PCPs. Each panel illustrates the SrfA-C adenylation and PCP domains. The N- and C-terminal subdomains are grey and orange; the PCP is red. The adenylate-forming model is the orientation of the experimentally determined adenylation and PCP domains from 2VSQ. The middle model was created by superposing the C-terminal subdomain and PCP of 2VSQ as a rigid body onto PA1221, using the C-terminal subdomains for alignment. In this model, the PCP and N-terminal subdomains overlap, illustrating that a second component of rotation is needed to adopt the assumed thioester-forming model (right panel), which was created by superimposing both the SrfA-C C-terminal subdomain and the PCP onto the similar domain orientation in PA1221.