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. 2016:1401:3-29.
doi: 10.1007/978-1-4939-3375-4_1.

Structural Biology of Nonribosomal Peptide Synthetases

Bradley R Miller  1   2 , Andrew M Gulick  3   4
Affiliations

Structural Biology of Nonribosomal Peptide Synthetases

Bradley R Miller et al. Methods Mol Biol. 2016.

Abstract

The nonribosomal peptide synthetases are modular enzymes that catalyze synthesis of important peptide products from a variety of standard and non-proteinogenic amino acid substrates. Within a single module are multiple catalytic domains that are responsible for incorporation of a single residue. After the amino acid is activated and covalently attached to an integrated carrier protein domain, the substrates and intermediates are delivered to neighboring catalytic domains for peptide bond formation or, in some modules, chemical modification. In the final module, the peptide is delivered to a terminal thioesterase domain that catalyzes release of the peptide product. This multi-domain modular architecture raises questions about the structural features that enable this assembly line synthesis in an efficient manner. The structures of the core component domains have been determined and demonstrate insights into the catalytic activity. More recently, multi-domain structures have been determined and are providing clues to the features of these enzyme systems that govern the functional interaction between multiple domains. This chapter describes the structures of NRPS proteins and the strategies that are being used to assist structural studies of these dynamic proteins, including careful consideration of domain boundaries for generation of truncated proteins and the use of mechanism-based inhibitors that trap interactions between the catalytic and carrier protein domains.

Keywords: Enzymology; Metabolic pathways; Modular enzymes; Nonribosomal peptide synthetase; Peptides; Siderophores; Structural biology.

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Figures

Figure 1
Figure 1
Chemical structure of the phosphopantetheine cofactor attached to a conserved serine residue of the peptidyl carrier protein.
Figure 2
Figure 2
Structures of core NRPS domains. A. The structure of the Type II PCP domain BlmI (PDB 4I4D). The four helices are shown along with Ser44, the site of phosphopantetheinylation. B. The active site of PheA (PDB 1AMU), the adenylation domain from the gramicidin synthetase NRPS. The ligand molecules AMP and phenylalanine are shown in ball-and-stick represenatation. Protein side chains are labeled including the residues that form the phenylalanine binding pocket and residues that interact with the nucleotide. C. The condensation domain of the CDA synthetase (PDB 4JN3) is shown in ribbon representation. The two subdomains are shown, along with active site residues His156, His157, and Asp161, which is partially obscured by His157. D. The active site of the thioesterase domain from EntF is shown (PDB 3TEJ). The pantetheine, covalently bound to Ser1006, is directed from the PCP domain to the active site, which is composed of the catalytic triad Asp1165, His1271, and Ser1138. The different NRPS domains are shown in specific colors, which will be maintained through the chapter. PCP domains are shown in blue. Adenylation domains are shown in pink for the N-terminal sub-domain and maroon for the C-terminal sub-domain. Condensation domain is shown in light green and the thioesterase domain is shown in yellow.
Figure 3
Figure 3
Reaction catalyzed by the NRPS adenylation domain.
Figure 4
Figure 4
Domain alternation of NRPS adenylation domains. The structures of two free-standing adenylation domains are shown from two bacterial siderophore synthesis, A. DhbE from the bacillibactin NRPS of B. subtilis and B. EntE from the enterobactin NRPS of E. coli. The DhbE structure (PDB 1MDB) is in the adenlyate-forming conformation, with the A10 motif of the C-terminal subdomain directed towards the active site. The EntE structure (PDB 4IZ6) adopts the thioester-forming conformation with the A8 motif near the active site. The carrier protein and the pantetheine cofactor of structure 4IZ6 are not shown for clarity.
Figure 5
Figure 5
Reaction catalyzed by the NRPS condensation domain.
Figure 6
Figure 6
Reaction catalyzed by the NRPS thioesterase domain.
Figure 7
Figure 7
Crystal Structures of multi-domain NRPS proteins. A. The structure of the complete termination module of SrfA-C (PDB 2VSQ) shows the PCP interactions with the downstream side of the condensation domain. B. The adenylation-PCP structure of PA1221 (PDB 4DG9) illustrates a functional interface between the PCP and the thioester-forming conformation of the adenylation domain. C. The structure of the SlgN1 protein (PDB 4GN5) shows the interaction between the MbtH-like domain (forest green) interacting with the N-terminal subdomain of the adenylation domain. The conserved tryptophan residues are highlighted in the MLP. D. The PCP-thioesterase domain of EntF (PDB 3TEJ) is shown illustrating the binding of the pantetheine into the active site of the thioesterase domain.
Figure 8
Figure 8
Model for the delivery of the PCP to the adenylation domain active site. The SrfA-C protein is shown in A. A model conformation that adopts the thioester-forming conformation where the PCP is bound to the adenylation domain and B. the crystallographic structure where the PCP interacts with the condensation domain. The N-terminal subdomain of the adenylation domain, as well as the condensation and thioesterase domains, are all shown in surface representation, while the PCP and the C-terminal subdomain of the adenlyation domain are shown as ribbons. The structure in panel A is derived by modeling the SrfA-C (PDB 2VSQ) adenylation and PCP domains onto the thioester-forming conformation observed with EntE-B (PDB 4IZ6) or PA1221 (PDB 4DG9). The serine residue that is the site of phosphopantetheinylation is highlighted with the yellow sphere.
Figure 9
Figure 9
Mechanism-based inhibitors used to crystallize NRPS multi-domain structures. A. The α-chloroacetyl-CoA derivative was designed to react with the catalytic serine of thioesterase domain. The catalytic reaction, the inhibitor rationale, and the observed structure are shown. B. The vinylsulfonamide inhibitor is shown, along with the two-step reaction catalyzed by the adenylation domain. The covalent inhibitor complex has been observed in two crystal structures.
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