Structures of a Nonribosomal Peptide Synthetase Module Bound to MbtH-like Proteins Support a Highly Dynamic Domain Architecture

Abstract

Nonribosomal peptide synthetases (NRPSs) produce a wide variety of peptide natural products. During synthesis, the multidomain NRPSs act as an assembly line, passing the growing product from one module to the next. Each module generally consists of an integrated peptidyl carrier protein, an amino acidloading adenylation domain, and a condensation domain that catalyzes peptide bond formation. Some adenylation domains interact with small partner proteins called MbtH-like proteins (MLPs) that enhance solubility or activity. A structure of an MLP bound to an adenylation domain has been previously reported using a truncated adenylation domain, precluding any insight that might be derived from understanding the influence of the MLP on the intact adenylation domain or on the dynamics of the entire NRPS module. Here, we present the structures of the full-length NRPS EntF bound to the MLPs from Escherichia coli and Pseudomonas aeruginosa. These new structures, along with biochemical and bioinformatics support, further elaborate the residues that define the MLP-adenylation domain interface. Additionally, the structures highlight the dynamic behavior of NRPS modules, including the module core formed by the adenylation and condensation domains as well as the orientation of the mobile thioesterase domain. Nonribosomal peptide synthetases (NRPSs)2 are fascinating modular enzymes that use an assembly line architecture to produce important peptide natural products (1-3). During synthesis, the amino acid building blocks are bound to peptidyl carrier protein (PCP) domains that migrate between catalytic active sites for the requisite steps in the biosynthetic pathway. Most NRPS modules contain an adenylation domain that activates the correct amino acid and loads the PCP domain. Internal modules contain a condensation domain that transfers the upstream amino acid or peptide to the newly loaded amino acid, extending the peptide length by one residue. Freed from the constraints of standard ribosomal synthesis, NRPS products display a wide range of chemical structures.The chemical diversity of NRPS products is further enhanced by the presence of additional internal domains or external proteins that modify the nascent peptide (4 -6). In addition to these tailoring enzymes, some NRPS biosynthetic clusters contain genes encoding small (ϳ70-residue) proteins. Named after the MbtH protein from the Mycobacterium tuberculosis mycobactin operon (7), these MbtH-like proteins (MLPs) were shown simultaneously by Thomas and co-workers (8) and Walsh and co-workers (9) to function as activators of acyladenylate formation. The MLP-NRPS interactions exhibit several interesting features. Some adenylation domains also require MLPs as chaperones and cannot be expressed without their MLP partner (10). Furthermore, genetic and biochemical studies have shown that MLPs can activate NRPS proteins in different biosynthetic clusters within a species and can be substituted heterologously in different species (11).Structural studies of MLPs and MLP-adenylation d...

Characterization of the Functional Variance in MbtH-like Protein Interactions with a Nonribosomal Peptide Synthetase

“…13,15,17 Residues involved in NRPS–MLP interaction in these structures were cross-referenced with the sequence alignments (Figure 3a). Among these residues, there is significant commonality between functional and nonfunctional MLPs.…”

“…More recent work has shown that the presence of YbdZ also has a minor influence on A domain affinity for the cosubstrate ATP. 13 …”

“…13 The critical Ala/Pro residue of the A domain coordinated by the Trp pocket of the MLP lies just downstream of the conserved A6 motif (GEx 10 GY) of A domains. 18 However, a majority of A domains, regardless of whether they were MLP-dependent or not, have a sequence that is compatible with MLP-NRPS interaction.…”

Structural insight into the necessary conformational changes of modular nonribosomal peptide synthetases

“…The most common are MbtH-like proteins (MLPs) that are small ~8 kDa proteins that activate adenylation domains [37]. The structures of MLPs with truncated adenylation domains [38] or with complete NRPS modules [39 •• ] identify a conserved binding motif but no obvious channel of communication between the interface and the catalytic residues. While most MLPs are independent proteins, some are covalently joined to adenylation domains in multidomain proteins.…”

“…The landmark structure of SrfA-C [17], a terminal module from the surfactin biosynthesis pathway, set the stage for the understanding of modular NRPSs. Several recent papers [39 •• ,44 •• ,45 •• ] provided new views that lead to a new model for the modular architecture of NRPS enzymes.…”