Redirecting RiPP biosynthetic enzymes to proteins and backbone-modified substrates

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are peptide-derived natural products that include the FDA-approved analgesic ziconotide1,2 as well as compounds with potent antibiotic, antiviral, and anticancer properties.3 RiPP enzymes known as cyclodehydratases and dehydrogenases represent an exceptionally well-studied enzyme class.3 These enzymes work together to catalyze intramolecular, interresidue condensation3,4 and aromatization reactions that install oxazoline/oxazole and thiazoline/thiazole heterocycles within ribosomally produced polypeptide chains. Here we show that the previously reported enzymes MicD-F and ArtGox accept backbone-modified monomers, including aramids and beta-amino acids, within leader-free polypeptides, even at positions immediately preceding or following the site of cyclization/dehydrogenation. The products are sequence-defined chemical polymers with multiple, diverse, non-alpha-amino acid subunits. We show further that MicD-F and ArtGox can install heterocyclic backbones within protein loops and linkers without disrupting the native tertiary fold. Calculations reveal the extent to which these heterocycles restrict conformational space; they also eliminate a peptide bond. Both features could improve the stability or add function to linker sequences now commonplace in emerging biotherapeutics. Moreover, as thiazoles and thiazoline heterocycles are replete in natural products,5,6,7 small molecule drugs,8,9 and peptide-mimetic therapeutics,10 their installation in protein-based biotherapeutics could improve or augment performance, activity, stability, and/or selectivity. This work represents a general strategy to expand the chemical diversity of the proteome beyond and in synergy with what can now be accomplished by expanding the genetic code.

Albumin–Methotrexate Prodrug Analogues That Undergo Intracellular Reactivation Following Entrance into Cancerous Glioma Cells

A family of monomodified bovine serum albumin (BSA) linked to methotrexate (MTX) through a variety of spacers was prepared. All analogues were found to be prodrugs having low MTX-inhibitory potencies toward dihydrofolate reductase in a cell-free system. The optimal conjugates regenerated their antiproliferative efficacies following entrance into cancerous glioma cell lines and were significantly superior to MTX in an insensitive glioma cell line. A BSA–MTX conjugate linked through a simple ethylene chain spacer, containing a single peptide bond located 8.7 Å distal to the protein back bone, and apart from the covalently linked MTX by about 12 Å, was most effective. The inclusion of an additional disulfide bond in the spacer neither enhanced nor reduced the killing potency of this analogue. Disrupting the native structure of the carrier protein in the conjugates significantly reduced their antiproliferative activity. In conclusion, we have engineered BSA–MTX prodrug analogues which undergo intracellular reactivation and facilitate antiproliferative activities following their entrance into glioma cells.

Origin of Increased Solvent Accessibility of Peptide Bonds in Mutual Synergetic Folding Proteins

Mutual Synergetic Folding (MSF) proteins belong to a recently discovered class of proteins. These proteins are disordered in their monomeric but ordered in their oligomeric forms. Their amino acid composition is more similar to globular proteins than to disordered ones. Our preceding work shed light on important structural aspects of the structural organization of these proteins, but the background of this behavior is still unknown. We suggest that solvent accessibility is an important factor, especially solvent accessibility of the peptide bonds can be accounted for this phenomenon. The side chains of the amino acids which form a peptide bond have a high local contribution to the shielding of the peptide bond from the solvent. During the oligomerization step, other non-local residues contribute to the shielding. We investigated these local and non-local effects of shielding based on Shannon information entropy calculations. We found that MSF and globular homodimeric proteins have different local contributions resulting from different amino acid pair frequencies. Their non-local distribution is also different because of distinctive inter-subunit contacts.

Peptidyl transferase center decompaction and structural constraints during early protein elongation on the ribosome

Peptide bond formation on the ribosome requires that aminoacyl-tRNAs and peptidyl-tRNAs are properly positioned on the A site and the P site of the peptidyl transferase center (PTC) so that nucleophilic attack can occur. Here we analyse some constraints associated with the induced-fit mechanism of the PTC, that promotes this positioning through a compaction around the aminoacyl ester orchestrated by U2506. The physical basis of PTC decompaction, that allows the elongated peptidyl-tRNA to free itself from that state and move to the P site of the PTC, is still unclear. From thermodynamics considerations and an analysis of published ribosome structures, the present work highlights the rational of this mechanism, in which the free-energy released by the new peptide bond is used to kick U2506 away from the reaction center. Furthermore, we show the evidence that decompaction is impaired when the nascent peptide is not yet anchored inside the exit tunnel, which may contribute to explain why the first rounds of elongation are inefficient, an issue that has attracted much interest for about two decades. Results in this field are examined in the light of the present analysis and a physico-chemical correlation in the genetic code, which suggest that elementary constraints associated with the size of the side-chain of the amino acids penalize early elongation events.

Orthoformimycin inhibits translation elongation by displacing the A-site tRNA and preventing peptide bond formation

The ribosome is a major target for antibiotics owing to its essential cellular role in protein synthesis. Structural analysis of ribosome-antibiotic complexes provides insight into the molecular basis for their inhibitory action and highlights possible avenues to improve their potential or overcome existing resistance mechanisms. Here we use X-ray crystallography and pre-steady state kinetics to detail the inhibitory mechanism of the antimicrobial on the large ribosomal subunit.

Cleavage of PGAM5 by the intramembrane protease PARL is governed by transmembrane helix dynamics and oligomeric state

The intramembrane protease PARL is a crucial mitochondrial safeguard by cleaving the mitophagy regulators PINK1 and PGAM5. PGAM5 substrate determinates have not been rigorously investigated and it is unclear how uncoupling the mitochondrial membrane potential regulates its processing inversely to PINK1. Here we show that in PGAM5 several hydrophilic residues distant from the cleavage site serve as key determinant for PARL-catalyzed cleavage. NMR analysis indicates that a short N-terminal amphipathic helix, followed by a kink and a C-terminal helix harboring the scissile peptide bond, is key for a productive interaction with PARL. In difference to PINK1, PGAM5 is stably inserted into the inner mitochondrial membrane until uncoupling the membrane potential triggers its disassembly into monomers that are vulnerable to PARL-catalyzed processing. We suggest a model in which PGAM5 is a slowly processed substrate with PARL-catalyzed cleavage that is influenced by multiple hierarchical substrate features including a membrane-potential-dependent oligomeric switch.

An Unbound Proline-Rich Signaling Peptide Frequently Samples Cis Conformations in Gaussian Accelerated Molecular Dynamics Simulations

Disordered proline-rich motifs are common across the proteomes of many species and are often involved in protein-protein interactions. Proline is a unique amino acid due to the covalent bond between the backbone nitrogen and the proline side chain. The resulting five-membered ring allows proline to sample the cis state about its peptide bond, which other residues cannot do as readily. Because proline-rich disordered sequences exist as ensembles that likely include structures with the proline peptide bond in cis, a robust methodology to accurately account for these conformations in the overall ensemble is crucial. Observing the cis conformations of proline in a disordered sequence is challenging both experimentally and computationally. Nitrogen-hydrogen NMR spectroscopy cannot directly observe proline residues, which lack an amide bond, and computational methods struggle to overcome the large kinetic barrier between the cis and trans states, since isomerization usually occurs on the order of seconds. In the current work, Gaussian accelerated molecular dynamics was used to overcome this free energy barrier and simulate proline isomerization in a tetrapeptide (KPTP) and in the 12-residue proline-rich SH3 binding peptide, ArkA. We found that Gaussian accelerated molecular dynamics, when combined with a lowered peptide bond dihedral angle potential energy barrier (15 kcal/mol), allowed sufficient sampling of the proline cis and trans states on a microsecond timescale. All ArkA prolines spend a significant fraction of time in cis, leading to a more compact ensemble with less polyproline II helix structure than an ArkA ensemble with all peptide bonds in trans. The ensemble containing cis prolines also matches more closely to in vitro circular dichroism data than the all-trans ensemble. The ability of the ArkA prolines to isomerize likely affects the peptide’s ability to bind its partner SH3 domain, and should be studied further. This is the first molecular dynamics simulation study of proline isomerization in a biologically relevant proline-rich sequence that we know of, and a similar protocol could be applied to study multi-proline isomerization in other proline-containing proteins to improve conformational diversity and agreement with in vitro data.