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

Bradley R Miller,Eric J Drake,Ce Shi,Courtney C Aldrich,Andrew M Gulick

doi:10.1074/jbc.m116.746297

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 acid-loading 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.

Highlights

Nonribosomal peptide synthetases (NRPSs)2 are fascinating modular enzymes that use an assembly line architecture to produce important peptide natural products [1,2,3]
The crystal structure of an MbtH-like proteins (MLPs) from Pseudomonas aeruginosa called PA2412 [13] and the solution structures of MLPs from M. tuberculosis [14] and three other organisms (Mycobacterium marinum (Protein Data Bank code 2MYY), Mycobacterium avium (Protein Data Bank code 2N6G), and Burkholderia pseudomallei (Protein Data Bank code 2LPD)) that have not been published all show a flat architecture with three ␤-strands and an ␣-helix that lies across the ␤-sheet
We present the structures of EntF bound to two MLPs, its true partner and a homolog from P. aeruginosa

Summary

Results

Structures of EntF Bound to MLPs—We determined the crystal structures of EntF in complex with two MLPs in a new crystal form. Crystals of EntF bound to the E. coli YbdZ diffracted to 3.0 Å. Crystals were obtained by incubating EntF with the serine adenosine vinylsulfonamide (SerAVS) mechanism-based inhibitor used in the original EntF structure [21]. This resulted in covalent trapping of the PCP in an interaction with the adenylation domain in the thioesterforming conformation. In contrast to the previous EntF structure, the thioesterase domain was ordered in the new crystal form (Fig. 2). Crystallization data r.m.s.d., root mean square deviation; Avg, average; Aden, adenylation domain; Cond, condensation domain; TE, thioesterase domain. Correlation coefficients CC1/2 and CC* are from iMOSFLM/Scala

Diffraction data

EntF EntF ϩ YbdZ

Discussion

Experimental Procedures