Structural and mutational analysis of the nonribosomal peptide synthetase heterocyclization domain provides insight into catalysis.

Nonribosomal peptide synthetases (NRPSs) are a family of multidomain, multimodule enzymes that synthesize structurally and functionally diverse peptides, many of which are of great therapeutic or commercial value. The central chemical step of peptide synthesis is amide bond formation, which is typically catalyzed by the condensation (C) domain. In many NRPS modules, the C domain is replaced by the heterocyclization (Cy) domain, a homologous domain that performs two consecutive reactions by using hitherto unknown catalytic mechanisms. It first catalyzes amide bond formation, and then the intramolecular cyclodehydration between a Cys, Ser, or Thr side chain and the backbone carbonyl carbon to form a thiazoline, oxazoline, or methyloxazoline ring. The rings are important for the form and function of the peptide product. We present the crystal structure of an NRPS Cy domain, Cy2 of bacillamide synthetase, at a resolution of 2.3 Å. Despite sharing the same fold, the active sites of C and Cy domains have important differences. The structure allowed us to probe the roles of active-site residues by using mutational analyses in a peptide synthesis assay with intact bacillamide synthetase. The drastically different effects of these mutants, interpreted by using our structural and bioinformatic results, provide insight into the catalytic mechanisms of the Cy domain and implicate a previously unexamined Asp-Thr dyad in catalysis of the cyclodehydration reaction.

Structural and functional aspects of the nonribosomal peptide synthetase condensation domain superfamily: discovery, dissection and diversity.

Nonribosomal peptide synthetases (NRPSs) are incredible macromolecular machines that produce a wide range of biologically- and therapeutically-relevant molecules. During synthesis, peptide elongation is performed by the condensation (C) domain, as it catalyzes amide bond formation between the nascent peptide and the amino acid it adds to the chain. Since their discovery more than two decades ago, C domains have been subject to extensive biochemical, bioinformatic, mutagenic, and structural analyses. They are composed of two lobes, each with homology to chloramphenicol acetyltransferase, have two binding sites for their two peptidyl carrier protein-bound ligands, and have an active site with conserved motif HHxxxDG located between the two lobes. This review discusses some of the important insights into the structure, catalytic mechanism, specificity, and gatekeeping functions of C domains revealed since their discovery. In addition, C domains are the archetypal members of the C domain superfamily, which includes several other members that also function as NRPS domains. The other family members can replace the C domain in NRP synthesis, can work in concert with a C domain, or can fulfill diverse and novel functions. These domains include the epimerization (E) domain, the heterocyclization (Cy) domain, the ester-bond forming C domain, the fungal NRPS terminal C domain (CT), the ß-lactam ring forming C domain, and the X domain. We also discuss structural and function insight into C, E, Cy, CT and X domains, to present a holistic overview of historical and current knowledge of the C domain superfamily. This article is part of a Special Issue entitled: Biophysics in Canada, edited by Lewis Kay, John Baenziger, Albert Berghuis and Peter Tieleman.

Crystallization and preliminary crystallographic analysis of the first condensation domain of viomycin synthetase.

Nonribosomal peptide synthetases (NRPSs) are large multimodular enzymes that synthesize important secondary metabolites such as antibiotics. NRPSs follow a modular synthetic logic whereby each successive amino-acid monomer is added to the peptide chain by successive multi-domain modules. The condensation domain catalyzes the central chemical event in the synthetic cycle, peptide-bond formation, and is present in every elongation module of the NRPS. Viomycin is an antituberculosis nonribosomal peptide that is synthesized by a series of four NRPS proteins and then modified by tailoring proteins. In order to study the mechanisms of peptide-bond formation in viomycin and in NRPSs in general, a structural study of the first condensation domain of the viomycin synthetase protein VioA (VioA-C1) was initiated. The gene for VioA-C1 was cloned from genomic DNA of Streptomyces vinaceus, expressed as an octahistidine-tagged construct and purified by column chromatography. VioA-C1 was crystallized using the sitting-drop vapor-diffusion method. X-ray diffraction data were collected on a rotating-anode source to 2.9 Å resolution. The data could be indexed in the orthorhombic space group P212121, with unit-cell parameters a = 46.165, b = 68.335, c = 146.423 Å. There is likely to be one monomer in the asymmetric unit, giving a solvent content of 49.2% and a Matthews coefficient (VM) of 2.42 Å(3) Da(-1). Structural determination is in progress.

Structures and function of a tailoring oxidase in complex with a nonribosomal peptide synthetase module.

Nonribosomal peptide synthetases (NRPSs) are large modular enzymes that synthesize secondary metabolites and natural product therapeutics. Most NRPS biosynthetic pathways include an NRPS and additional proteins that introduce chemical modifications before, during or after assembly-line synthesis. The bacillamide biosynthetic pathway is a common, three-protein system, with a decarboxylase that prepares an NRPS substrate, an NRPS, and an oxidase. Here, the pathway is reconstituted in vitro. The oxidase is shown to perform dehydrogenation of the thiazoline in the peptide intermediate while it is covalently attached to the NRPS, as the penultimate step in bacillamide D synthesis. Structural analysis of the oxidase reveals a dimeric, two-lobed architecture with a remnant RiPP recognition element and a dramatic wrapping loop. The oxidase forms a stable complex with the NRPS and dimerizes it. We visualized co-complexes of the oxidase bound to the elongation module of the NRPS using X-ray crystallography and cryo-EM. The three active sites (for adenylation, condensation/cyclization, and oxidation) form an elegant arc to facilitate substrate delivery. The structures enabled a proof-of-principle bioengineering experiment in which the BmdC oxidase domain is embedded into the NRPS.

Author Correction: Evasion of immunosurveillance by genomic alterations of PPARγ/RXRα in bladder cancer.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Evasion of immunosurveillance by genomic alterations of PPARγ/RXRα in bladder cancer.

Muscle-invasive bladder cancer (MIBC) is an aggressive disease with limited therapeutic options. Although immunotherapies are approved for MIBC, the majority of patients fail to respond, suggesting existence of complementary immune evasion mechanisms. Here, we report that the PPARγ/RXRα pathway constitutes a tumor-intrinsic mechanism underlying immune evasion in MIBC. Recurrent mutations in RXRα at serine 427 (S427F/Y), through conformational activation of the PPARγ/RXRα heterodimer, and focal amplification/overexpression of PPARγ converge to modulate PPARγ/RXRα-dependent transcription programs. Immune cell-infiltration is controlled by activated PPARγ/RXRα that inhibits expression/secretion of inflammatory cytokines. Clinical data sets and an in vivo tumor model indicate that PPARγHigh/RXRαS427F/Y impairs CD8+ T-cell infiltration and confers partial resistance to immunotherapies. Knockdown of PPARγ or RXRα and pharmacological inhibition of PPARγ significantly increase cytokine expression suggesting therapeutic approaches to reviving immunosurveillance and sensitivity to immunotherapies. Our study reveals a class of tumor cell-intrinsic "immuno-oncogenes" that modulate the immune microenvironment of cancer.Muscle-invasive bladder cancer (MIBC) is a potentially lethal disease. Here the authors characterize diverse genetic alterations in MIBC that convergently lead to constitutive activation of PPARgamma/RXRalpha and result in immunosurveillance escape by inhibiting CD8+ T-cell recruitment.

Chemical Probes Allow Structural Insight into the Condensation Reaction of Nonribosomal Peptide Synthetases.

Nonribosomal peptide synthetases (NRPSs) synthesize a vast variety of small molecules, including antibiotics, antitumors, and immunosuppressants. The NRPS condensation (C) domain catalyzes amide bond formation, the central chemical step in nonribosomal peptide synthesis. The catalytic mechanism and substrate determinants of the reaction are under debate. We developed chemical probes to structurally study the NRPS condensation reaction. These substrate analogs become covalently tethered to a cysteine introduced near the active site, to mimic covalent substrate delivery by carrier domains. They are competent substrates in the condensation reaction and behave similarly to native substrates. Co-crystal structures show C domain-substrate interactions, and suggest that the catalytic histidine's principle role is to position the α-amino group for nucleophilic attack. Structural insight provided by these co-complexes also allowed us to alter the substrate specificity profile of the reaction with a single point mutation.

Crystal structures of the first condensation domain of CDA synthetase suggest conformational changes during the synthetic cycle of nonribosomal peptide synthetases.

Nonribosomal peptide synthetases (NRPSs) are large modular macromolecular machines that produce small peptide molecules with wide-ranging biological activities, such as antibiotics and green chemicals. The condensation (C) domain is responsible for amide bond formation, the central chemical step in nonribosomal peptide synthesis. Here we present two crystal structures of the first condensation domain of the calcium-dependent antibiotic (CDA) synthetase (CDA-C1) from Streptomyces coelicolor, determined at resolutions 1.8Å and 2.4Å. The conformations adopted by CDA-C1 are quite similar in these two structures yet distinct from those seen in other NRPS C domain structures. HPLC-based reaction assays show that this CDA-C1 construct is catalytically active, and small-angle X-ray scattering experiments suggest that the conformation observed in these crystal structures could faithfully represent the conformation in solution. We have performed targeted molecular dynamics simulations, normal mode analyses and energy-minimized linear interpolation to investigate the conformational changes required to transition between the observed structures. We discuss the implications of these conformational changes in the synthetic cycle and of the observation that the "latch" that covers the active site is consistently formed in all studied C domains.