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In humans, N[H.sub.3] transport across cell membranes is facilitated by the Rh (rhesus) family of proteins. Human Rh C glycoprotein (RhCG) forms a trimeric complex that plays an essential role in ammonia excretion and renal pH regulation. The X-ray crystallographic structure of human RhCG, determined at 2.1 A resolution, reveals the mechanism of ammonia transport. Each monomer contains 12 transmembrane helices, one more than in the bacterial homologs. Reconstituted into proteoliposomes, RhCG conducts N[H.sub.3] to raise internal pH. Models of the erythrocyte Rh complex based on our RhCG structure suggest that the erythrocytic Rh complex is composed of stochastically assembled heterotrimers of RhAG, RhD, and RhCE. ammonia channel | comparative modeling | membrane protein | rhesus factor | X-ray structure www.pnas.org/cgi/doi/10.1073/pnas.1003587107

Aquaporin (AQP) 4 is the predominant water channel in the mammalian brain, abundantly expressed in the blood-brain and brain-cerebrospinal fluid interfaces of glial cells. Its function in cerebral water balance has implications in neuropathological disorders, including brain edema, stroke, and head injuries. The 1.8-[Angstrom] crystal structure reveals the molecular basis for the water selectivity of the channel. Unlike the case in the structures of water-selective AQPs AqpZ and AQP1, the asparagines of the 2 Asn-Pro-Ala motifs do not hydrogen bond to the same water molecule; instead, they bond to 2 different water molecules in the center of the channel. Molecular dynamics simulations were performed to ask how this observation bears on the proposed mechanisms for how AQPs remain totally insulating to any proton conductance while maintaining a single file of hydrogen bonded water molecules throughout the channel. brain edema | inhibitor discovery | NPA motif

Tirzepatide (LY3298176) is a fatty-acid-modified, dual incretin receptor agonist that exhibits pharmacology similar to native GIP at the glucose-dependent insulinotropic polypeptide receptor (GIPR) but shows bias toward cyclic adenosine monophosphate signaling at the glucagon-like peptide-1 receptor (GLP-1R). In addition to GIPR signaling, the pathway bias at the GLP-1R may contribute to the efficacy of tirzepatide at improving glucose control and body weight regulation in type 2 diabetes mellitus. To investigate the structural basis for the differential signaling of tirzepatide, mechanistic pharmacology studies were allied with cryogenic electron microscopy. Here, we report high-resolution structures of tirzepatide in complex with the GIPR and GLP-1R. Similar to the native ligands, tirzepatide adopts an α-helical conformation with the N terminus reaching deep within the transmembrane core of both receptors. Analyses of the N-terminal tyrosine (Tyr1Tzp) of tirzepatide revealed a weak interaction with the GLP-1R. Molecular dynamics simulations indicated a greater propensity of intermittent hydrogen bonding between the lipid moiety of tirzepatide and the GIPR versus the GLP-1R, consistent with a more compact tirzepatide-GIPR complex. Informed by these analyses, tirzepatide was deconstructed, revealing a peptide structure-activity relationship that is influenced by acylation-dependent signal transduction. For the GIPR, Tyr1Tzp and other residues making strong interactions within the receptor core allow tirzepatide to tolerate fatty acid modification, yielding an affinity equaling that of GIP. Conversely, high-affinity binding with the extracellular domain of the GLP-1R, coupled with decreased stability from the Tyr1Tzp and the lipid moiety, foster biased signaling and reduced receptor desensitization. Together, these studies inform the structural determinants underlying the function of tirzepatide. [ABSTRACT FROM AUTHOR]

In the current study, cellular and molecular approaches have been used to analyze the biophysical nature of T cell receptor (TCR)--peptide MHC (pMHC) interactions for two autoreactive TCRs. These two TCRs recognize the N-terminal epitope of myelin basic protein (MBP1-11) bound to the MHC class II protein, I-[A.sup.u], and are associated with murine experimental autoimmune encephalomyelitis. Mice transgenic for the TCRs have been generated and characterized in other laboratories. These analyses indicate that the mice either develop encephalomyelitis spontaneously (172.10 TCR) or only if immunized with autoantigen in adjuvant (1934.4 TCR). Here, we show that the 172.10 TCR binds MBP1-11:I-[A.sup.u] with a 4-5-fold higher affinity than the 1934.4 TCR. Consistent with the higher affinity, 172.10 T hybridoma cells are significantly more responsive to autoantigen than 1934.4 cells. The interaction of the 172.10 TCR with cognate ligand is more entropically unfavorable than that of the 1934.4 TCR, indicating that the 172.10 TCR undergoes greater conformational rearrangements upon ligand binding. The studies therefore suggest a correlation between the strength and plasticity of a TCR-pMHC interaction and the frequency of spontaneous disease in the corresponding TCR transgenic mice. The comparative analysis of these two TCRs has implications for understanding autoreactive T cell recognition and activation.

SignificanceTirzepatide is a dual agonist of the glucose-dependent insulinotropic polypeptide receptor (GIPR) and the glucagon-like peptide-1 receptor (GLP-1R), which are incretin receptors that regulate carbohydrate metabolism. This investigational agent has proven superior to selective GLP-1R agonists in clinical trials in subjects with type 2 diabetes mellitus. Intriguingly, although tirzepatide closely resembles native GIP in how it activates the GIPR, it differs markedly from GLP-1 in its activation of the GLP-1R, resulting in less agonist-induced receptor desensitization. We report how cryogenic electron microscopy and molecular dynamics simulations inform the structural basis for the unique pharmacology of tirzepatide. These studies reveal the extent to which fatty acid modification, combined with amino acid sequence, determines the mode of action of a multireceptor agonist.