Biology subjects

Macdonald, C. B.

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

3 recordsLinked to original sources

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↗