Samuel H. Gellman

Liquid–liquid phase separation mediated by proteins and/or nucleic acids is believed to underlie the formation of many distinct condensed phases, or membraneless organelles, within living cells. These condensates have been proposed to orchestrate a variety of important processes. Despite recent advances, the interactions that regulate the dynamics of molecules within a condensate remain poorly understood. We performed accumulated 564.7 μs all-atom molecular dynamics (MD) simulations (system size ∼200k atoms) of model condensates formed by a scaffold RNA oligomer and a scaffold peptide rich in arginine (Arg). These model condensates contained one of three possible guest peptides: the scaffold peptide itself or a variant in which six Arg residues were replaced by lysine (Lys) or asymmetric dimethyl arginine (ADMA). We found that the Arg-rich peptide can form the largest number of hydrogen bonds …

Parathyroid hormone 1 receptor (PTH1R) plays a key role in mediating calcium homeostasis and bone development, and aberrant PTH1R activity underlies several human diseases. Peptidic PTH1R antagonists and inverse agonists have therapeutic potential in treating these diseases, but their poor pharmacokinetics and pharmacodynamics undermine their in vivo efficacy. Herein, we report the use of a backbone-modification strategy to design a peptidic PTH1R inhibitor that displays prolonged activity as an antagonist of wild-type PTH1R and an inverse agonist of the constitutively active PTH1R–H223R mutant both in vitro and in vivo. This peptide may be of interest for the future development of therapeutic agents that ameliorate PTH1R malfunction.

Signal duration and subcellular location are emerging as important facets of G protein-coupled receptor (GPCR) function. The glucagon-like peptide-1 receptor (GLP-1R), a clinically relevant class B1 GPCR, stimulates production of the second messenger cAMP upon activation by the native hormone, GLP-1. cAMP production continues after the hormone-receptor complex has been internalized via endocytosis. Here, we report GLP-1 analogues that induce prolonged signaling relative to GLP-1. A single beta-amino acid substitution at position 18, with the residue derived from (S,S)-trans-2-aminocyclopentanecarboxylic acid (ACPC), enhances signaling duration with retention of receptor endocytosis. Pairing ACPC at position 18 with a second substitution, amino-aminoisobutyric acid (Aib) at position 16, abrogates endocytosis, but prolonged signaling is maintained. Prolonged signaling is sensitive to the structure of the beta residue at position 18. Cryo-electron microscopy (cryo-EM) structures of two GLP-1 analogues bound to the GLP-1R:Gs complex suggest substantial alterations to bound peptide structure and dynamics compared to the GLP-1:GLP-1R complex. These structural findings strengthen an emerging view that agonist dynamics in the receptor-bound state influence signaling profile. Our results advance understanding of the structural underpinnings of receptor activation and introduce new tools for exploring the impact of spatiotemporal signaling profiles following GLP-1R activation.

The gastric inhibitory polypeptide receptor (GIPR), a G protein-coupled receptor (GPCR) that regulates glucose metabolism and insulin secretion, is a target for the development of therapeutic agents to address type 2 diabetes and obesity. Signal transduction processes mediated by GPCR activation typically result in receptor phosphorylation, but very little is known about GIPR phosphorylation. Mass spectrometry (MS) is a powerful tool for detecting phosphorylation and other post-translational modifications of proteins and for identifying modification sites. However, applying MS methods to GPCRs is challenging because the native expression levels are low and the hydrophobicity of these proteins complicates isolation and enrichment. Here we use a widely available technique, trapped-ion-mobility spectrometry coupled to time-of-flight mass spectrometry (TIMS-TOF MS), to characterize the phosphorylation status of …

Peptide engineering efforts have delivered drugs for diverse human diseases. Side chain alteration is among the most common approaches to designing new peptides for specific applications. The peptide backbone can be modified as well, but this strategy has received relatively little attention. Here we show that new and favorable contacts between a His side chain on a target protein and an aromatic side chain on a synthetic peptide ligand can be engineered by rational and coordinated side chain modification and backbone extension. Side chain modification alone was unsuccessful. Binding measurements, high‐resolution structural studies and pharmacological outcomes all support the synergy between backbone and side chain modification in engineered ligands of the parathyroid hormone receptor‐1, which is targeted by osteoporosis drugs. These results should motivate other structure‐based designs …

VBUWHHLIZKOSMS-RIWXPGAOSA-N invicorp Chemical compound C ([C@@ H](C (= O) N [C@@ H](CC (C) C) C (= O) N [C@@ H](CC (N)= O) C (= O) N [C@@ H](CO) C (= O) N [C@@ H]([C@@ H](C) CC) C (= O) N [C@@ H](CC (C) C) C (= O) N [C@@ H](CC (N)= O) C (O)= O) NC (= O)[C@ H](CCCCN) NC (= O)[C@ H](CCCCN) NC (= O)[C@@ H](NC (= O)[C@ H](C) NC (= O)[C@ H](CCSC) NC (= O)[C@ H](CCC (N)= O) NC (= O)[C@ H](CCCCN) NC (= O)[C@ H](CCCNC (N)= N) NC (= O)[C@ H](CC (C) C) NC (= O)[C@ H](CCCNC (N)= N) NC (= O)[C@@ H](NC (= O)[C@ H](CC= 1C= CC (O)= CC= 1) NC (= O)[C@ H](CC (N)= O) NC (= O)[C@ H](CC (O)= O) NC (= O)[C@@ H](NC (= O)[C@ H](CC= 1C= CC= CC= 1) NC (= O)[C@@ H](NC (= O)[C@ H](C) NC (= O)[C@ H](CC (O)= O) NC (= O)[C@ H](CO) NC (= O)[C@@ H](N) CC= 1NC= NC= 1) C (C) C)[C@@ H](C) O)[C@@ H](C) O) C (C) C) C1= CC= C (O …

Liquid-liquid phase separation mediated by proteins and/or nucleic acids is believed to underlie the formation of many distinct condensed phases, or membraneless organelles, within living cells. These condensates have been proposed to orchestrate a variety of important processes. Despite recent advances, the interactions that regulate the dynamics of molecules within a condensate remain poorly understood. We performed an accumulated 564.7μs all-atom Molecular Dynamics (MD) simulations (system size ~200K atoms) of model condensates formed by a scaffold RNA oligomer and a scaffold peptide rich in arginine (Arg). These model condensates contained one of three possible guest peptides: the scaffold peptide itself or a variant in which six Arg residues were replaced by lysine (Lys) or asymmetric dimethyl arginine (ADMA). We found that the Arg-rich peptide can form the largest number of hydrogen bonds and bind the strongest to the scaffold RNA in the condensate, relative to the Lys- and ADMA-rich peptides. Our MD simulations also showed that the Arg-rich peptide diffused more slowly in the condensate relative to the other two guest peptides, which is consistent with a recent fluorescence microscopy study. There was no significant increase in the number of cation-π interactions between the Arg-rich peptide and the scaffold RNA compared to the Lys-rich and ADMA-rich peptides. Our results indicate that hydrogen bonds between the peptides and the RNA backbone, rather than cation-π interactions, play the major role in regulating peptide diffusion in the condensate.

We have applied an underexplored backbone modification strategy to generate new analogues of peptides that activate two clinically important class B1 G protein-coupled receptors (GPCRs). Most peptide modification strategies involve changing side chains or, less commonly, changing the configuration at side chain-bearing carbons (i.e., l residues replaced by d residues). In contrast, backbone modifications alter the number of backbone atoms and the identities of backbone atoms relative to a poly-α-amino acid backbone. Starting from the peptide agonists PTH(1–34) (the first 34 residues of the parathyroid hormone, used clinically as the drug teriparatide) and glucagon-like peptide-1 (7–36) (GLP-1(7–36)), we replaced native α-residue triads with a diad composed of a β-amino acid residue and a γ-amino acid residue. The β/γ diad retains the number of backbone atoms in the ααα triad. Because the β and γ …