Biology subjects

Muehle, J.

Publications and source records attributed to Muehle, J..

5 recordsLinked to original sources

bioRxiv2026

Discovery of a Cell-Permeable Gi-Signaling Inhibitor

Heterotrimeric G proteins regulate diverse physiological processes, yet selective small molecule modulators for the Gi subfamily remain scarce. Here, we employed native metabolomics to screen a fungal culture collection for G protein binders, which led to the discovery of N-hydroxyapiosporamide (NHAP), a fungal specialized metabolite that functionally inhibits Gi-signaling. NHAP selectively binds Gi1 and diminishes Gi1-mediated GTP turnover in biochemical assays. In primary ventricular cardiomyocytes, NHAP largely reversed acetylcholine-induced negative inotropy, demonstrating functional Gi-signaling blockade in a physiological context without acute cytotoxicity. This work establishes NHAP as a first-in-class Gi-selective inhibitor, providing a cell-permeable chemical scaffold for the optimization of next-generation agents to control Gi-signaling in cell-based systems and, ultimately, to target Gi1-driven pathologies.

biochemistry↗

bioRxiv2025

Mechanism of MEK1 phosphorylation by the N-terminal acidic motif mediated asymmetric BRAF dimer

The RAF/MEK/ERK signaling cascade regulates cell proliferation and differentiation and is frequently dysregulated in cancer. Approximately 90% of RAF-mutant cancers harbour mutations in B-type of Rapidly Accelerated Fibrosarcoma (BRAF). Its proto-oncogenicity has been attributed to a four-residue N-terminal acidic (NtA) motif. While a long-standing model proposes that the NtA promotes an activating asymmetric RAF dimerization, this idea has lacked structural support. Here, we present the first X-ray crystal structure of an NtA-mediated asymmetric BRAF dimer bound to its substrate Mitogen-activated Protein kinase Kinase (MEK1). Further cellular and biochemical analyses reveal that this asymmetric dimer is not an intermediate along the BRAF activation pathway, but rather a catalytically active state of the kinase. We validate this with the structure of BRAF bound to Ser222-phosphorylated MEK1, capturing the complex in a post-catalytic state. This combination of structural and cellular analysis establishes the relevance of NtA-driven asymmetry in BRAF activation and resolves a long-standing disconnect between RAF cancer genetics and structural biology.

biochemistry↗

bioRxiv2025

The structure of Egalitarian in complex with the K10 mRNA localization signal reveals a modular binding surface required for function

Asymmetric localization of mRNAs is a prevalent mechanism for spatial control of protein function and typically involves active transport by cytoskeletal motors. The mechanisms of recognition of localizing mRNAs by motor complexes are poorly understood. Egalitarian is an adaptor protein that binds localization signals in specific RNAs in Drosophila and recruits them to the dynein-dynactin complex for microtubule-based transport. We determined the crystal structure of Egalitarian in complex with the localization signal of the K10 mRNA. Three structural units of Egalitarian, a 3'-5'exonuclease domain, a linker and a C-terminal domain form shape-specific, base-directed and backbone interactions with the RNA. Based on the structure we identified conserved residues recognizing RNA in vitro. Genome-edited flies with mutations in these residues have deficits in Egalitarian function that are congruent with changes in in vitro RNA binding affinities. Our work demonstrates how a minimal RNA localization signal is recognized by an RNA localization factor.

biochemistry↗

bioRxiv2023

Cyclic peptide inhibitors stabilize Gq/11 heterotrimers

Heterotrimeric G proteins play a central role in cellular signaling, acting as switchable molecular regulators. Consequently, pharmacological agents to control G protein activity are of utmost importance to advance our understanding of this signal transduction system. The natural depsipeptides FR900359 (FR) and YM-254890 (YM) are two highly specific and widely used inhibitors of heterotrimeric Gq/11 proteins. These compounds have traditionally been understood to inhibit GDP dissociation by preventing the separation of the GTPase and -helical domains of the G subunit. In this work, we have determined the high-resolution crystal structures of FR and YM bound to heterotrimeric G11 and used them to explain the molecular basis underlying their efficient suppression of G protein signaling. Notably, our data show that FR and YM also function as stabilizers of the interface between the G and G{beta} subunits, acting as molecular adhesives that stabilize the entire heterotrimer. Our results reveal unrecognized mechanistic features that explain how FR and YM effectively blunt Gq/11 signaling in living cells.

biochemistry↗

bioRxiv2019

Cryo-EM structure of the rhodopsin-Gαi-βγ complex reveals binding of the rhodopsin C-terminal tail to the Gβ subunit

G protein-coupled receptors (GPCRs) are the largest class of integral membrane proteins and represent key targets for pharmacological research. GPCRs modulate cell physiology by engaging and activating a diversity of intracellular transducers, prominently heterotrimeric G proteins, but also G protein-receptor kinases (GRKs) and arrestins. The recent surge in the number of structures of GPCR-G protein complexes has expanded our understanding of G protein recognition and GPCR-mediated signal transduction. However, many aspects of these mechanisms, including the existence of transient interactions with transducers, have remained elusive. Here, we present the cryo-EM structure of the light-sensitive GPCR rhodopsin in complex with heterotrimeric Gi. In contrast to all reported structures, our density map reveals the receptor C-terminal tail bound to the G{beta} subunit of the G protein heterotrimer. This observation provides a structural foundation for the role of the C-terminal tail in GPCR signaling, and of G{beta} as scaffold for recruiting G subunits and GRKs. By comparing all available complex structures, we found a small set of common anchoring points that are G protein-subtype specific. Taken together, our structure and analysis provide new structural basis for the molecular events of the GPCR signaling pathway.

biochemistry↗