Biology subjects

Macdonald, C. B.

Publications and source records attributed to Macdonald, C. B..

8 recordsLinked to original sources

bioRxiv2026

Mapping the sequence determinants of constitutive activity in GPCRs

G protein-coupled receptors (GPCRs) signal not only in response to ligands but also in their absence. This basal, or constitutive, activity has distinct physiological consequences and is the only functional measurement available for orphan receptors. It is also hard to compare receptors because each is reported relative to its own baseline. Here, we measure basal activity on a single scale across the human GPCR family. Pairing a barcoded library of all human GPCRs with a transcriptional cAMP reporter, we scored 901 receptor constructs in one pool experiment. Basal activity forms a continuous distribution, and many of the highest-scoring recipes have prior reports of ligand-independent activity, with orphan being strongly represented. We then exhaustively mutated one of them, GPR161, and measured its effects on protein abundance, surface expression, and cAMP signaling. Of the 528 positions, only 10 specifically alter signaling independent of expression. Constraints on basal signaling are dominated by reepotro biogenesis, which requires multiphenotype scanning to resolve. Together, these datasets place basal activity on a common scale across the GPCR family and provide a template for dissecting it at residue resolution.

biochemistry↗

bioRxiv2024

Cell geometry, microtubule anchoring and anisotropic dynamic instability: challenges and solutions to transverse cortical array organization

The self-organization of cortical microtubule arrays within plant cells is an emergent phenomenon with important consequences for the synthesis of the cell wall, cell shape, and subsequently the structure of plants. Mathematical modelling and experiments have elucidated the underlying processes involved. There has been recent interest in the influence of geometric cues on array orientation, be it direct (cell shape) or indirect (tension in the membrane). However, the mechanical influence of membrane curvature on these elastic filaments has largely been ignored. A previous model was proposed to describe how the anchoring process may control the deflection of individual microtubules seeking to minimize bending on a cylindrical cell. We incorporate this process into a model of interacting microtubules and find the cell curvature influence to be significant: the array favours orientations parallel to the direction of elongation rather than the expected transverse direction. Even without elasticity, the geometry of large cells hinders robust microtubule organization. These results suggest the necessity of additional processes to overcome these factors. We propose an orientation-dependent catastrophe rate, hypothetically caused by cellulose microfibrils impeding microtubule polymerization. We find a combination of anchoring and impedance to be sufficient to generate transverse arrays despite the geometric influences. Significance StatementThe organization of microtubule (MT) polymers into parallel arrays along the two-dimensional cortex of plant cells is crucial for directional cell growth and plant development. Despite decades of experimentation and more recent computational modelling, understanding the mechanisms that orient cortical MTs remains incomplete. With computational modelling, we have re-examined an assumption common to many models: that MTs grow along straight (geodesic) paths rather than minimizing bending. We model MT bending, and find a significant disruption of transverse MT ordering, especially in larger cells. We find that angle-dependent MT behaviour can counteract the effect of bending in certain contexts.

plant biology↗

bioRxiv2024

Deep mutational scanning of EccD3 reveals the molecular basis of its essentiality in the mycobacterium ESX secretion system

Tuberculosis remains the deadliest infectious disease in the world and requires novel therapeutic targets. The ESX-3 secretion system, which is essential for iron and zinc homeostasis and thus M. tuberculosis survival, is a promising target. In this study, we perform a deep mutational scan on the ESX-3 core protein EccD3 in the model organism M. smegmatis. We systematically investigated the functional roles of 145 residues across the soluble ubiquitin-like domain, the conformationally distinct flexible linker, and selected transmembrane helices of EccD3. Our data combined with structural comparisons to ESX-5 complexes support a model where EccD3 stabilizes the complex, with the hinge motif within the linker being particularly sensitive to disruption. Our study is the first deep mutational scan in mycobacteria, which could help guide drug development toward novel treatment of tuberculosis. This study underscores the importance of context-specific mutational analyses for discovering essential protein interactions within mycobacterial systems.

microbiology↗

bioRxiv2024

Mapping kinase domain resistance mechanisms for the MET receptor tyrosine kinase via deep mutational scanning

Mutations in the kinase and juxtamembrane domains of the MET Receptor Tyrosine Kinase are responsible for oncogenesis in various cancers and can drive resistance to MET-directed treatments. Determining the most effective inhibitor for each mutational profile is a major challenge for MET-driven cancer treatment in precision medicine. Here, we used a deep mutational scan (DMS) of [~]5,764 MET kinase domain variants to profile the growth of each mutation against a panel of 11 inhibitors that are reported to target the MET kinase domain. We validate previously identified resistance mutations, pinpoint common resistance sites across type I, type II, and type I [1/2] inhibitors, unveil unique resistance and sensitizing mutations for each inhibitor, and verify non-cross-resistant sensitivities for type I and type II inhibitor pairs. We augment a protein language model with biophysical and chemical features to improve the predictive performance for inhibitor-treated datasets. Together, our study demonstrates a pooled experimental pipeline for identifying resistance mutations, provides a reference dictionary for mutations that are sensitized to specific therapies, and offers insights for future drug development.

cancer biology↗

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

Conserved regulatory motifs in the juxtamembrane domain and kinase N-lobe revealed through deep mutational scanning of the MET receptor tyrosine kinase domain.

MET is a receptor tyrosine kinase (RTK) responsible for initiating signaling pathways involved in development and wound repair. MET activation relies on ligand binding to the extracellular receptor, which prompts dimerization, intracellular phosphorylation, and recruitment of associated signaling proteins. Mutations, which are predominantly observed clinically in the intracellular juxtamembrane and kinase domains, can disrupt typical MET regulatory mechanisms. Understanding how juxtamembrane variants, such as exon 14 skipping (MET{Delta}Ex14), and rare kinase domain mutations can increase signaling, often leading to cancer, remains a challenge. Here, we perform a parallel deep mutational scan (DMS) of the MET intracellular kinase domain in two fusion protein backgrounds: wild type and MET{Delta}Ex14. Our comparative approach has revealed a critical hydrophobic interaction between a juxtamembrane segment and the kinase C-helix, pointing to potential differences in regulatory mechanisms between MET and other RTKs. Additionally, we have uncovered a {beta}5 motif that acts as a structural pivot for the kinase domain in MET and other TAM family of kinases. We also describe a number of previously unknown activating mutations, aiding the effort to annotate driver, passenger, and drug resistance mutations in the MET kinase domain.

molecular biology↗

bioRxiv2022

A stochastic model of cortical microtubule anchoring and mechanics provides regulatory control of microtubule shape

The organization of cortical microtubule arrays play an important role in the development of plant cells. Until recently, the direct mechanical influence of cell geometry on the constrained microtubule (MT) trajectories have been largely ignored in computational models. Modelling MTs as thin elastic rods constrained on a surface, a previous study examined the deflection of MTs using a fixed number of segments and uniform segment lengths between MT anchors. It is known that the resulting MT curves converge to geodesics as the anchor spacing approaches zero. In the case of long MTs on a cylinder, buckling was found for transverse trajectories. There is a clear interplay between two factors in the problem of deflection: curvature of the membrane and the lengths of MT segments. We examine the latter in detail, in the backdrop of a circular cylinder. In reality, the number of segments are not predetermined and their lengths are not uniform. We present a minimal, realistic model treating the anchor spacing as a stochastic process and examine the net effect on deflection. We find that, by tuning the ratio of growth speed to anchoring rate, it is possible to mitigate MT deflection and even prevent buckling for lengths significantly larger than the previously derived critical buckling length. We suggest that this mediation of deflection by anchoring might provide cells with a means of preventing arrays from deflecting away from the transverse orientation.

biophysics↗

bioRxiv2022

Deep Insertion, Deletion, and Missense Mutation Libraries for Exploring Protein Variation in Evolution, Disease, and Biology

Insertions and deletions (indels) are a major source of genetic variation in evolution and the cause of nearly 30% of Mendelian disease. Despite their importance, indels are left out of nearly every systematic mutational scan to date due to technical challenges associated with making indel-containing libraries, limiting our understanding of indels in disease, biology, and evolution. Here we present a library generation method, DIMPLE, that generates deletions, insertions, and missense at similar frequencies within any gene. To benchmark DIMPLE, we generated libraries within four genes (Kir2.1, VatD, TRPV1, and OPRM1) of varying length and evolutionary origin. DIMPLE produces libraries that are near complete, low cost, and low bias. We measured how missense mutations and indels of varying length impact the potassium channel Kir2.1 surface expression. Across all Kir2.1s secondary structure, deletions are more disruptive than insertions, beta sheets are extremely sensitive to large deletions, and flexible loops allow insertions far more frequently than deletions. DIMPLEs low bias, ease of use, and low cost will enable high throughput probing of the importance of indels in disease and evolution.

genomics↗