Structures of teixobactin-producing nonribosomal peptide synthetase condensation and adenylation domains ☆ Kemin Tan a, b , Min Zhou a, c , Robert P. Jedrzejczak a, b , Ruiying Wu a, b , Raul A. Higuera d , Dominika Borek e , Gyorgy Babnigg a, c , Andrzej Joachimiak a, b, f, * a Center for Structural Genomics of Infectious Diseases, University of Chicago, 5735 South Ellis Avenue, Chicago, IL 60637, USA b Structural Biology Center, X-ray Science Division, Advanced Photon Source, Argonne National Laboratory, 9700 S. Cass Ave. Lemont, IL 60439, USA c Biosciences, Argonne National Laboratory, Argonne, IL 60439, USA d BUILDing SCHOLARS, The University of Texas at El Paso, 500 W. University Ave, El Paso, TX 79968, USA e Department of Biophysics, UT Southwestern Medical Center, Dallas, TX 75390, USA f Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60367, USA ARTICLE INFO Keywords: Nonribosomal peptide synthetase Txo1 Txo2 Teixobactin Condensation domain Adenylation domain Conformational change α-helix regulation Mg 2þ -binding ABSTRACT The recently discovered antibiotic teixobactin is produced by uncultured soil bacteria. The antibiotic inhibits cell wall synthesis of Gram-positive bacteria by binding to precursors of cell wall building blocks, and therefore it is thought to be less vulnerable to development of resistance. Teixobactin is synthesized by two nonribosomal peptide synthetases (NRPSs), encoded by txo1 and txo2 genes. Like other NRPSs, the Txo1 and Txo2 synthetases are large, multifunctional, and comprised of several modules. Each module is responsible for catalysis of a distinct step of teixobactin synthesis and contains specific functional units, commonly including a condensation (C) domain, an adenylation (A) domain, and a peptidyl carrier protein (PCP) domain. Here we report the structures of the C-A bidomains of the two L-Ser condensing modules, from Txo1 and Txo2, respectively. In the structure of the C domain of the L-Ser subunit of Txo1, a large conformational change is observed, featuring an outward swing of its N-terminal α-helix. This repositioning, if functionally validated, provides the necessary conformational change for the condensation reaction in C domain, and likely represents a regulatory mechanism. In an A core subdomain, a well-coordinated Mg 2þ cation is observed, which is required in the adenylation reaction. The Mg 2þ -binding site is defined by a largely conserved amino acid sequence motif and is coordinated by the α-phosphate group of AMP (or ATP) when present, providing some structural evidence for the role of the metal cation in the catalysis of A domain. 1. Introduction Nonribosomal peptide synthetases (NRPSs) are large multifunctional enzymes, which consist of multiple modules that bind and catalyze in an assembly-line fashion the addition of amino acid monomers to create a variety of complex secondary metabolites (Marahiel et al., 1997; Fisch- bach & Walsh, 2006; Weissman, 2015; Miller & Gulick, 2016; Payne et al., 2016). The nonribosomal peptides produced in bacteria are of great in- terest to the pharmaceutical industry because of their antibacterial, anti- viral, and anticancer properties (Walsh, 2015; Gulick, 2017; Agrawal Abbreviations: NRPS, Nonribosomal peptide synthetase; C domain, Condensation domain; C Nterm subdomain, N-terminal subdomain of C domain; C Cterm sub- domain, C-terminal subdomain of C domain; A domain, Adenylation domain; A core subdomain, Large N-terminal subdomain of A domain; A sub subdomain, Small C- terminal subdomain of A domain; PCP domain, Peptidyl carrier domain; COM D domain, Donor communication-mediating domain; COM A domain, Acceptor communication-mediating domain; SAD, Single wavelength diffraction; RMSD, Root-mean-square deviation; SSM, Secondary-structure matching; MES, 2- morpho- linoethane sulfonic acid. ☆ The submitted manuscript has been created by UChicago Argonne, LLC, Operator of Argonne National Laboratory (“Argonne”). Argonne, a U.S. Department of Energy Office of Science laboratory, is operated under Contract No. DE-AC02-06CH11357. The U.S. Government retains for itself, and others acting on its behalf, a paid-up nonexclusive, irrevocable worldwide license in said article to reproduce, prepare derivative works, distribute copies to the public, and perform publicly and display publicly, by or on behalf of the Government. * Corresponding author. University of Chicago, Department of Biochemistry and Molecular Biology, Chicago IL 60367, United States. Tel.: 630 252 392. E-mail address: andrzejj@anl.gov (A. Joachimiak). Contents lists available at ScienceDirect Current Research in Structural Biology journal homepage: www.journals.elsevier.com/current-research-in-structural-biology https://doi.org/10.1016/j.crstbi.2020.01.002 Received 5 July 2019; Received in revised form 9 January 2020; Accepted 13 January 2020 2665-928X/© 2020 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by- nc-nd/4.0/). Current Research in Structural Biology 2 (2020) 14–24