Biology subjects

Lerch, M. T.

Publications and source records attributed to Lerch, M. T..

5 recordsLinked to original sources

bioRxiv2025

A biased allosteric modulator functions as a molecular glue to induce β2AR dimerization

Family A G-protein coupled receptors (GPCRs) are typically described as monomers, yet growing evidence suggests they can form dimers with distinct signaling properties1-3. The mechanisms and therapeutic potential of such dimerization, however, remain poorly understood. Here, we show that AP-7-168, an optimized derivative of a {beta}-arrestin-biased negative allosteric modulator of the {beta}2-adrenergic receptor ({beta}2AR) that sustains bronchorelaxation in cell and tissue models4, functions as a molecular glue to promote {beta}2AR homodimerization. Cryo-EM structures reveal a unique binding mode in which two AP-7-168 molecules pack within a pocket formed by transmembrane helices 3, 4, and 5 of two protomers, stabilizing a dimeric conformation that selectively prevents {beta}-arrestin coupling. In cells, AP-7-168 robustly induces {beta}2AR dimerization and drives enlarged nanocluster formation. Combined with extensive functional studies, our findings unveil a novel allosteric mechanism by which a small molecule biases {beta}2AR signaling through dimerization, highlighting ligand-induced dimerization as a strategy for GPCR modulation.

biochemistry↗

bioRxiv2025

The role of intrinsically disordered domains in regulating G protein coupled receptor signaling

The 2A adrenergic receptor (2AAR) is a clinically important target for various diseases including hypertension, diabetes and chronic pain. Here, using single-molecule fluorescence resonance energy transfer imaging, we show how agonist-specific activation dynamics in both structured transmembrane domain (TMD) and intrinsically disorders regions (IDRs) of 2AAR lead to diverse signaling profiles. Through seven pairs of strategically designed fluorophore labels, we systematically investigate the real-time conformational changes of 2AAR. Our study reveals unique TM6 dynamics in 2AAR, featured by a high energy barrier for agonist-induced outward movements essential for activation. In contrast, we identify agonist-specific conformational dynamics of a partially disordered extracellular loop (ECL2), highlighting its role as a dynamic regulatory module that controls receptor function. Moreover, we characterize the conformational landscapes of the long third intracellular loop (ICL3), revealing its compact structural features and membrane-proximal localization in the basal state, where it acts as a negative allosteric regulator in transducer coupling. Furthermore, we identify multiful functional sub-states of ICL3 that are dynamically modulated by both kinase phosphorylation and drug efficacy. These findings offer previously underappreciated structural and dynamic insights into 2AAR function governed by both TMD and IDRs, and may open up new avenues for the development of better therapeutics.

biophysics↗

bioRxiv2025

ProGuide: a flexible framework for modeling global conformational rearrangements in proteins using DEER-derived distance restraints

Conformational heterogeneity is integral to protein function - ranging from enzyme catalysis to signal transduction - and visualizing distinct conformational states requires experimental techniques capable of providing such structural information. One particularly powerful method, double electron-electron resonance (DEER) spectroscopy, can provide a high-resolution, long-range ([~]15-80 [A]) probability distributions of distances between site-selected pairs of spin labels to resolve intra-protein distance parameters of unique protein conformations, as well as their respective likelihoods within a conformational ensemble. A current frontier in the field of DEER spectroscopy is utilizing this distance information in computational modeling to generate complete structural models of these multiple conformations. Although several methods have been developed for this purpose, modeling protein backbone structural rearrangements using multiple distance restraints remains challenging, due in part to the complexity provided by rotameric flexibility of the spin label side chain. Here, we overcome these challenges with ProGuide, a new framework for generating accurate structural models guided by DEER distance distribution information. Large conformational rearrangements are captured by performing iterative experimentally biased molecular dynamics simulations. In each iteration, spin-label rotameric heterogeneity is modeled using chiLife, and then C changes are calculated to capture distance-probability density present in the experimental DEER distributions and lacking from the modeled one. The resulting models of this process then go through a selection to generate the ensemble that best recapitulates the DEER data. We illustrate the power of this method using published DEER data from a study of biased agonism in the angiotensin II type 1 receptor (AT1R), a prototypical G protein coupled receptor (GPCR). The resulting AT1R models consist of both Gq- and {beta}-arrestin-biased conformations, including a completely novel {beta}-arrestin-biased conformation. These models reveal structural insights involving tertiary structural rearrangements as well as residue-level changes in crucial microswitch motifs. Taken together, the results demonstrate the power and flexibility of ProGuide to investigate conformational rearrangements of large, complex proteins using DEER-derived distance restraints.

biophysics↗

bioRxiv2025

Improved EPR sensitivity for aqueous biological samples using low-volume multi-channel cells and dielectric resonators

Reducing sample volumes for electron paramagnetic resonance (EPR) spectroscopy applications places increasing demands on hardware design to preserve or enhance EPR signal intensity. In this work, the design, fabrication, and testing of dielectric resonators and multi-channel aqueous sample cells for applications in X-band (nominally 9.5 GHz) EPR is presented. Our aim was to maximize the EPR signal intensity for sample sizes of 3-4 L and 200 nL. These advances are summarized as follows: single-crystal sapphire and rutile dielectric resonators with very low loss tangent and high resonator efficiency; minimum dielectric resonator coupling to radiation shield to reduce ohmic losses; 3D printed aqueous sample cells with thin multi-channel construction to minimize radio-frequency dissipation in the sample; and a Gordon coupler for maximum coupling range and minimum stored energy to eliminate frequency shifts during tuning. Sample cell geometries were designed by leveraging insights gained from analytic theory to inform finite-element modeling of electromagnetic fields. Experimental comparisons of multi-channel sample cells using a sapphire resonator exhibited a 2.2-fold increase in EPR signal intensity compared with a standard capillary at 3-4 L, while simulations predict an additional 23% improvement with further 3D printing advances. For samples at 200 nL, a rutile dielectric resonator with a multi-channel sample cell was simulated to improve EPR sensitivity by a 2.7-fold increase compared with a capillary at the same volume.

biophysics↗

bioRxiv2024

A pressure-jump EPR system to monitor millisecond conformational exchange rates of spin-labeled proteins

Site-directed spin labeling electron paramagnetic resonance (SDSL-EPR) using nitroxide spin labels is a well-established technology for mapping site-specific secondary and tertiary structure and for monitoring conformational changes in proteins of any degree of complexity, including membrane proteins, with high sensitivity. SDSL-EPR also provides information on protein dynamics in the time scale of ps-s using continuous wave lineshape analysis and spin lattice relaxation time methods. However, the functionally important time domain of s-ms, corresponding to large-scale protein motions, is inaccessible to those methods. To extend SDSL-EPR to the longer time domain, the perturbation method of pressure-jump relaxation is implemented. Here, we describe a complete high-pressure EPR system at Q-band for both static pressure and millisecond-timescale pressure-jump measurements on spin-labeled proteins. The instrument enables pressure jumps both up and down from any holding pressure, ranging from atmospheric pressure to the maximum pressure capacity of the system components ([~]3500 bar). To demonstrate the utility of the system, we characterize a local folding-unfolding equilibrium of T4 lysozyme. The results illustrate the ability of the system to measure thermodynamic and kinetic parameters of protein conformational exchange on the millisecond timescale.

biophysics↗