Biosynthesis of a clickable pyoverdine via in vivo enzyme engineering of an adenylation domain

Abstract The engineering of non ribosomal peptide synthetases (NRPS) for new substrate specificity is a potent strategy to incorporate non-canonical amino acids into peptide sequences, thereby creating peptide diversity and broadening applications. The non-ribosomal peptide pyoverdine is the primary siderophore produced by Pseudomonas aeruginosa and holds biomedical promise in diagnosis, bio-imaging and antibiotic vectorization. We engineered the adenylation domain of PvdD, the terminal NRPS in pyoverdine biosynthesis, to accept a functionalized amino acid. Guided by molecular modeling, we rationally designed mutants of P. aeruginosa with mutations at two positions in the active site. A single amino acid change results in the successful incorporation of an azido-l-homoalanine leading to the synthesis of a new pyoverdine analog, functionalized with an azide function. We further demonstrated that copper free click chemistry is efficient on the functionalized pyoverdine and that the conjugated siderophore retains the iron chelation properties and its capacity to be recognized and transported by P. aeruginosa. The production of clickable pyoverdine holds substantial biotechnological significance, paving the way for numerous downstream applications

The molecular mechanisms underlying the ERalpha-36-mediated signaling in breast cancer

Alterations in estrogen-mediated cellular signaling have largely been implicated in the pathogenesis of breast cancer. Here, we investigated the signaling regulation of a splice variant of the estrogen receptor, namely estrogen receptor (ERalpha-36), associated with a poor prognosis in breast cancers. Coupling in vitro and in vivo approaches we determined the precise sequential molecular events of a new estrogen signaling network in an ERalpha-negative cell line and in an original patient-derived xenograft. After estrogen treatment, ERalpha-36 rapidly associates with Src at the level of the plasma membrane, initiating downstream cascades, including MEK1/ERK activation and paxillin phosphorylation on S126, which in turn triggers a higher expression of cyclin D1. Of note, the direct binding of ERalpha-36 to ERK2 prevents its dephosphorylation by MKP3 and enhances the downstream signaling. These findings improve our understanding of the regulation of non-genomic estrogen signaling and open new avenues for personalized therapeutic approaches targeting Src or MEK in ERalpha-36-positive patients.PMC542271

J Med Chem

The multimeric DNA sliding clamps confer high processivity to replicative DNA polymerases and are also binding platforms for various enzymes involved in DNA metabolism. These enzymes interact with the clamp through a small peptide that binds into a hydrophobic pocket which is a potential target for the development of new antibacterial compounds. Starting from a generic heptapeptide, we used a structure-based strategy to improve the design of new peptide ligands. Chemical modifications at specific residues result in a dramatic increase of the interaction as measured by SPR and ITC. The affinity of our best hits was improved by 2 orders of magnitude as compared to the natural ligand, reaching 10(-8) M range. The molecular basis of the interactions was analyzed by solving the co-crystal structures of the most relevant peptides bound to the clamp and reveals how chemical modifications establish new contacts and contributes to an increased affinity of the ligand

The Asymmetric Binding of PGC-1α to the ERRα and ERRγ Nuclear Receptor Homodimers Involves a Similar Recognition Mechanism

Background: PGC-1a is a crucial regulator of cellular metabolism and energy homeostasis that functionally acts togetherwith the estrogen-related receptors (ERRa and ERRc) in the regulation of mitochondrial and metabolic gene networks.Dimerization of the ERRs is a pre-requisite for interactions with PGC-1a and other coactivators, eventually leading totransactivation. It was suggested recently (Devarakonda et al) that PGC-1a binds in a strikingly different manner to ERRcligand-binding domains (LBDs) compared to its mode of binding to ERRa and other nuclear receptors (NRs), where itinteracts directly with the two ERRc homodimer subunits.Methods/Principal Findings: Here, we show that PGC-1a receptor interacting domain (RID) binds in an almost identicalmanner to ERRa and ERRc homodimers. Microscale thermophoresis demonstrated that the interactions between PGC-1aRID and ERR LBDs involve a single receptor subunit through high-affinity, ERR-specific L3 and low-affinity L2 interactions.NMR studies further defined the limits of PGC-1a RID that interacts with ERRs. Consistent with these findings, the solutionstructures of PGC-1a/ERRa LBDs and PGC-1a/ERRc LBDs complexes share an identical architecture with an asymmetricbinding of PGC-1a to homodimeric ERR.Conclusions/Significance: These studies provide the molecular determinants for the specificity of interactions betweenPGC-1a and the ERRs, whereby negative cooperativity prevails in the binding of the coactivators to these receptors. Ourwork indicates that allosteric regulation may be a general mechanism controlling the binding of the coactivators tohomodimers