Biology subjects

Hoppe, N.

Publications and source records attributed to Hoppe, N..

7 recordsLinked to original sources

bioRxiv2024

Molecular basis of proton-sensing by G protein-coupled receptors

Three proton-sensing G protein-coupled receptors (GPCRs), GPR4, GPR65, and GPR68, respond to changes in extracellular pH to regulate diverse physiology and are implicated in a wide range of diseases. A central challenge in determining how protons activate these receptors is identifying the set of residues that bind protons. Here, we determine structures of each receptor to understand the spatial arrangement of putative proton sensing residues in the active state. With a newly developed deep mutational scanning approach, we determined the functional importance of every residue in proton activation for GPR68 by generating [~]9,500 mutants and measuring effects on signaling and surface expression. This unbiased screen revealed that, unlike other proton-sensitive cell surface channels and receptors, no single site is critical for proton recognition in GPR68. Instead, a network of titratable residues extend from the extracellular surface to the transmembrane region and converge on canonical class A GPCR activation motifs to activate proton-sensing GPCRs. More broadly, our approach integrating structure and unbiased functional interrogation defines a new framework for understanding the rich complexity of GPCR signaling. One-sentence summaryThe protonation networks governing activation of human pH-sensing GPCRs are uncovered by integrative cryo-EM and deep mutational scanning.

biochemistry↗

bioRxiv2023

GPR161 structure uncovers the redundant role of sterol-regulated ciliary cAMP signaling in the Hedgehog pathway

The orphan G protein-coupled receptor (GPCR) GPR161 is enriched in primary cilia, where it plays a central role in suppressing Hedgehog signaling1. GPR161 mutations lead to developmental defects and cancers2,3,4. The fundamental basis of how GPR161 is activated, including potential endogenous activators and pathway-relevant signal transducers, remains unclear. To elucidate GPR161 function, we determined a cryogenic-electron microscopy structure of active GPR161 bound to the heterotrimeric G protein complex Gs. This structure revealed an extracellular loop 2 that occupies the canonical GPCR orthosteric ligand pocket. Furthermore, we identify a sterol that binds to a conserved extrahelical site adjacent to transmembrane helices 6 and 7 and stabilizes a GPR161 conformation required for Gs coupling. Mutations that prevent sterol binding to GPR161 suppress cAMP pathway activation. Surprisingly, these mutants retain the ability to suppress GLI2 transcription factor accumulation in cilia, a key function of ciliary GPR161 in Hedgehog pathway suppression. By contrast, a protein kinase A-binding site in the GPR161 C-terminus is critical in suppressing GLI2 ciliary accumulation. Our work highlights how unique structural features of GPR161 interface with the Hedgehog pathway and sets a foundation to understand the broader role of GPR161 function in other signaling pathways.

biochemistry↗

bioRxiv2022

Autoantibody and hormone activation of the thyrotropin G protein-coupled receptor

Thyroid hormones are vital to growth and metabolism. Thyroid hormone synthesis is controlled by thyrotropin (TSH), which acts at the thyrotropin receptor (TSHR). Autoantibodies that activate the TSHR pathologically increase thyroid hormones in Graves disease. How autoantibodies mimic TSH function remains unclear. We determined cryogenic-electron microscopy structures of active and inactive TSHR. In inactive TSHR, the extracellular domain lies close to the membrane bilayer. TSH selects an upright conformation of the extracellular domain due to steric clashes between a conserved hormone glycan and the membrane bilayer. An activating autoantibody selects a similar upright conformation of the extracellular domain. Conformational changes in the extracellular domain are transduced to the seven transmembrane domain via a conserved hinge domain, a tethered peptide agonist, and a phospholipid that binds within the seven transmembrane domain. Rotation of the TSHR ECD relative to the membrane bilayer is sufficient for receptor activation, revealing a shared mechanism for other glycoprotein hormone receptors that may also extend to G protein-coupled receptors with large extracellular domains.

biochemistry↗

bioRxiv2021

Fusion protein strategies for cryo-EM study of G protein-coupled receptors

Single particle cryogenic-electron microscopy (cryo-EM) is used extensively to determine structures of activated G protein-coupled receptors (GPCRs) in complex with G proteins or arrestins. However, applying it to GPCRs without signaling proteins remains challenging because most receptors lack structural features in their soluble domains to facilitate image alignment. In GPCR crystallography, inserting a fusion protein between transmembrane helices 5 and 6 is a highly successful strategy for crystallization. Although the similar strategy has the potential to broadly facilitate cryo-EM structure determination of GPCRs alone without signaling protein, the critical determinants that make this approach successful are not yet clear. Here, we address this shortcoming by exploring different fusion protein designs, which led to structures of antagonist bound A2A adenosine receptor at 3.4[A] resolution and unliganded Smoothened at 3.7[A] resolution. The fusion strategies explored here are likely applicable to cryo-EM interrogation of other GPCRs and small integral membrane proteins.

biophysics↗

bioRxiv2020

Epistasis shapes the fitness landscape of an allosteric specificity switch

Epistasis is a major determinant in the emergence of novel protein function. In allosteric proteins, direct interactions between inducer-binding mutations propagate through the allosteric network, manifesting as epistasis at the level of biological function. Elucidating this relationship between local interactions and their global effects is essential to understanding evolution of allosteric proteins. We integrate computational design, structural and biophysical analysis to characterize the emergence of novel inducer specificity in an allosteric transcription factor. Adaptive landscapes of different inducers of the engineered mutant show that a few strong epistatic interactions constrain the number of viable sequence pathways, revealing ridges in the fitness landscape leading to new specificity. Crystallographic evidence shows a single mutation drives specificity by reshaping the binding pocket. Comparison of biophysical and functional landscapes emphasizes the nonlinear relationship between local inducer affinity and global function (allostery). Our results highlight the functional and evolutionary complexity of allosteric proteins.

biochemistry↗

bioRxiv2020

An ultra-high affinity synthetic nanobody blocks SARS-CoV-2 infection by locking Spike into an inactive conformation

Without an effective prophylactic solution, infections from SARS-CoV-2 continue to rise worldwide with devastating health and economic costs. SARS-CoV-2 gains entry into host cells via an interaction between its Spike protein and the host cell receptor angiotensin converting enzyme 2 (ACE2). Disruption of this interaction confers potent neutralization of viral entry, providing an avenue for vaccine design and for therapeutic antibodies. Here, we develop single-domain antibodies (nanobodies) that potently disrupt the interaction between the SARS-CoV-2 Spike and ACE2. By screening a yeast surface-displayed library of synthetic nanobody sequences, we identified a panel of nanobodies that bind to multiple epitopes on Spike and block ACE2 interaction via two distinct mechanisms. Cryogenic electron microscopy (cryo-EM) revealed that one exceptionally stable nanobody, Nb6, binds Spike in a fully inactive conformation with its receptor binding domains (RBDs) locked into their inaccessible down-state, incapable of binding ACE2. Affinity maturation and structure-guided design of multivalency yielded a trivalent nanobody, mNb6-tri, with femtomolar affinity for SARS-CoV-2 Spike and picomolar neutralization of SARS-CoV-2 infection. mNb6-tri retains stability and function after aerosolization, lyophilization, and heat treatment. These properties may enable aerosol-mediated delivery of this potent neutralizer directly to the airway epithelia, promising to yield a widely deployable, patient-friendly prophylactic and/or early infection therapeutic agent to stem the worst pandemic in a century.

biochemistry↗

bioRxiv2020

Robust Sequence Determinants of α-Synuclein Toxicity in Yeast Implicate Membrane Binding

Protein conformations are shaped by cellular environments, but how environmental changes alter the conformational landscapes of specific proteins in vivo remains largely uncharacterized, in part due to the challenge of probing protein structures in living cells. Here, we use deep mutational scanning to investigate how a toxic conformation of -synuclein, a dynamic protein linked to Parkinsons disease, responds to perturbations of cellular proteostasis. In the context of a course for graduate students in the UCSF Integrative Program in Quantitative Biology, we screened a comprehensive library of -synuclein missense mutants in yeast cells treated with a variety of small molecules that perturb cellular processes linked to -synuclein biology and pathobiology. We found that the conformation of -synuclein previously shown to drive yeast toxicity--an extended, membrane-bound helix--is largely unaffected by these chemical perturbations, underscoring the importance of this conformational state as a driver of cellular toxicity. On the other hand, the chemical perturbations have a significant effect on the ability of mutations to suppress -synuclein toxicity. Moreover, we find that sequence determinants of -synuclein toxicity are well described by a simple structural model of the membrane-bound helix. This model predicts that -synuclein penetrates the membrane to constant depth across its length but that membrane affinity decreases toward the C terminus, which is consistent with orthogonal biophysical measurements. Finally, we discuss how parallelized chemical genetics experiments can provide a robust framework for inquiry-based graduate coursework.

biochemistry↗