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As genetic code expansion advances beyond L-α-amino acids to backbone modifications and new polymerization chemistries, delineating what substrates the ribosome can accommodate remains a challenge. The Escherichia coli ribosome tolerates non-L-α-amino acids in vitro, but few structural insights that explain how are available, and the boundary conditions for efficient bond formation are so far unknown. Here we determine a high-resolution cryogenic electron microscopy structure of the E. coli ribosome containing α-amino acid monomers and use metadynamics simulations to define energy surface minima and understand incorporation efficiencies. Reactive monomers across diverse structural classes favour a conformational space where the aminoacyl-tRNA nucleophile is

Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) are a popular chemogenetic technology for manipulation of neuronal activity in uninstrumented awake animals with potential for human applications as well. The prototypical DREADD agonist clozapine N-oxide (CNO) lacks brain entry and converts to clozapine, making it difficult to apply in basic and translational applications. Here we report the development of two novel DREADD agonists, JHU37152 and JHU37160, and the first dedicated 18F positron emission tomography (PET) DREADD radiotracer, [18F]JHU37107. We show that JHU37152 and JHU37160 exhibit high in vivo DREADD potency. [18F]JHU37107 combined with PET allows for DREADD detection in locally-targeted neurons, and at their long-range projections, enabling noninvasive and longitudinal neuronal projection mapping.

Arylamide foldamers have been shown to have a number of biological and medicinal applications. For example, a class of pyrrole-imidazole polyamide foldamers is capable of binding specific DNA sequences and preventing development of various gene disorders, most importantly cancer. Molecular dynamics (MD) simulations can provide crucial details in understanding the atomic level events related to foldamer/DNA binding. An important first step in the accurate simulation of these foldamer/DNA systems is the reparametrization of force field parameters for torsion around the aryl-amide bonds. Here we highlight our Density Functional Theory (DFT) potential energy profiles and derived force field parameters for four aryl-amide bond types for the pyrrole and imidazole building blocks extensively used in foldamer design for the DNA-binding polyamides. These results contribute to developing of computational tools for an appropriate molecular modeling of pyrrole-imidazole polyamide/DNA binding, and provide an insight into the chemical factors that influence the flexibility of the pyrrole-imidazole polyamides, and their binding to DNA.

The dopamine D2 and D3 receptors (D2R and D3R) are important targets for antipsychotics and for the treatment of drug abuse. SB269652, a bitopic ligand that simultaneously binds both the orthosteric binding site (OBS) and a secondary binding pocket (SBP) in both D2R and D3R, was found to be a negative allosteric modulator. Previous studies identified Glu2.65 in the SBP to be a key determinant of both the affinity of SB269652 and the magnitude of its cooperativity with orthosteric ligands, as the E2.65A mutation decreased both of these parameters. However, the proposed hydrogen bond (H-bond) between Glu2.65 and the indole moiety of SB269652 is not a strong interaction, and a structure activity relationship study of SB269652 indicates that this H-bond may not be the only element that determines its allosteric properties. To understand the structural basis of the observed phenotype of E2.65A, we carried out molecular dynamics simulations with a cumulative length of ~77 μs of D2R and D3R wild-type and their E2.65A mutants bound to SB269652. In combination with Markov state model analysis and by characterizing the equilibria of ligand binding modes in different conditions, we found that in both D2R and D3R, whereas the tetrahydroisoquinoline moiety of SB269652 is stably bound in the OBS, the indole-2-carboxamide moiety is dynamic and only intermittently forms H-bonds with Glu2.65. Our results also indicate that the E2.65A mutation significantly affects the overall shape and size of the SBP, as well as the conformation of the N terminus. Thus, our findings suggest that the key role of Glu2.65 in mediating the allosteric properties of SB269652 extends beyond a direct interaction with SB269652, and provide structural insights for rational design of SB269652 derivatives that may retain its allosteric properties. [ABSTRACT FROM AUTHOR]

General anesthetics suppress CNS activity by modulating the function of membrane ion channels, in particular, by enhancing activity of GABAA receptors. In contrast, several volatile (isoflurane, desflurane) and i.v. (propofol) general anesthetics excite peripheral sensory nerves to cause pain and irritation upon administration. These noxious anesthetics activate transient receptor potential ankyrin repeat 1 (TRPA1), a major nociceptive ion channel, but the underlying mechanisms and site of action are unknown. Here we exploit the observation that pungent anesthetics activate mammalian but not Drosophila TRPA1. Analysis of chimeric Drosophila and mouse TRPA1 channels reveal a critical role for the fifth transmembrane domain (S5) in sensing anesthetics. Interestingly, we show that anesthetics share with the antagonist A-967079 a potential binding pocket lined by residues in the S5, S6, and the first pore helix; isoflurane competitively disrupts A-967079 antagonism, and introducing these mammalian TRPA1 residues into dTRPA1 recapitulates anesthetic agonism. Furthermore, molecular modeling predicts that isoflurane and propofol bind to this pocket by forming H-bond and halogen-bond interactions with Ser-876,Met-915, and Met-956. Mutagenizing Met-915 or Met-956 selectively abolishes activation by isoflurane and propofol without affecting actions of A-967079 or the agonist, menthol. Thus, our combined experimental and computational results reveal the potential binding mode of noxious general anesthetics at TRPA1. These data may provide a structural basis for designing drugs to counter the noxious and vasorelaxant properties of general anesthetics and may prove useful in understanding effects of anesthetics on related ion channels. [ABSTRACT FROM AUTHOR]

The programmed synthesis of sequence-defined biomaterials whose monomer backbones diverge from those of canonical α-amino acids represents the next frontier in protein and biomaterial evolution. Such next-generation molecules provide otherwise nonexistent opportunities to develop improved biologic therapies, bioremediation tools, and biodegradable plastic-like materials. One monomer family of particular interest for biomaterials includes β-hydroxy acids. Many natural products contain isolated β-hydroxy acid monomers, and polymers of β-hydroxy acids (β-esters) are found in polyhydroxyalkanoate (PHA) polyesters under development as bioplastics and drug encapsulation/delivery systems. Here we report that β 2 -hydroxy acids possessing both ( R ) and ( S ) absolute configuration are substrates for pyrrolysyl-tRNA synthetase (PylRS) enzymes in vitro and that ( S )-β 2 -hydroxy acids are substrates in cellulo . Using the orthogonal Ma PylRS/ Ma tRNA Pyl synthetase/tRNA pair, in conjunction with wild-type E. coli ribosomes and EF-Tu, we report the cellular synthesis of model proteins containing two ( S )-β 2 -hydroxy acid residues at internal positions. Metadynamics simulations provide a rationale for the observed preference for the ( S )-β 2 -hydroxy acid and provide mechanistic insights that inform future engineering efforts. As far as we know, this finding represents the first example of an orthogonal synthetase that acylates tRNA with a β 2 -hydroxy acid substrate and the first example of a protein hetero-oligomer containing multiple expanded-backbone monomers produced in cellulo ., Competing Interests: The authors declare the following competing financial interest(s): N.X.H. and A.S. are coinventors on an international patent application that incorporates methods outlined in this manuscript., (© 2024 The Authors. Published by American Chemical Society.)

Sodium ions (Na + ) allosterically modulate the binding of orthosteric agonists and antagonists to many class A G protein-coupled receptors, including the dopamine D 2 receptor (D 2 R). Experimental and computational evidences have revealed that this effect is mediated by the binding of Na + to a conserved site located beneath the orthosteric binding site (OBS). SB269652 acts as a negative allosteric modulator (NAM) of the D 2 R that adopts an extended bitopic pose, in which the tetrahydroisoquinoline moiety interacts with the OBS and the indole-2-carboxamide moiety occupies a secondary binding pocket (SBP). In this study, we find that the presence of a Na + within the conserved Na + -binding pocket is required for the action of SB269652. Using fragments of SB269652 and novel full-length analogues, we show that Na + is required for the high affinity binding of the tetrahydroisoquinoline moiety within the OBS, and that the interaction of the indole-2-carboxamide moiety with the SBP determines the degree of Na + -sensitivity. Thus, we extend our understanding of the mode of action of this novel class of NAM by showing it acts synergistically with Na + to modulate the binding of orthosteric ligands at the D 2 R, providing opportunities for fine-tuning of modulatory effects in future allosteric drug design efforts.

Metadynamics simulations were used to describe the conformational energy landscapes of several helically folded aromatic quinoline carboxamide oligomers bearing a single chiral group at either the C or N terminus. The calculations allowed the prediction of whether a helix handedness bias occurs under the influence of the chiral group and gave insight into the interactions (sterics, electrostatics, hydrogen bonds) responsible for a particular helix sense preference. In the case of camphanyl-based and morpholine-based chiral groups, experimental data confirming the validity of the calculations were already available. New chiral groups with a proline residue were also investigated and were predicted to induce handedness. This prediction was verified experimentally through the synthesis of proline-containing monomers, their incorporation into an oligoamide sequence by solid phase synthesis and the investigation of handedness induction by NMR spectroscopy and circular dichroism., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)