Biology subjects

Gorday, K.

Publications and source records attributed to Gorday, K..

3 recordsLinked to original sources

bioRxiv2024

Autophosphorylation of the oncogenic protein TEL-ABL confers resistance to the allosteric ABL inhibitor asciminib

Chromosomal translocations that fuse the ABL1 gene to BCR and TEL cause human leukemias. Oligomerization and the loss of an inhibitory myristoylation modification lead to unregulated kinase activity of the BCR-ABL and TEL-ABL fusion proteins. ATP-competitive ABL inhibitors, such as imatinib and ponatinib, are effective against both fusion proteins. We discovered that asciminib, an allosteric inhibitor of BCR-ABL that binds to the myristoyl binding site in the ABL kinase domain, is >2000-fold less potent against TEL-ABL than BCR-ABL in cell-growth assays. This is surprising because the ABL components of the two fusion proteins, including the asciminib binding sites, have identical sequence. We deleted a short helical segment in the ABL kinase domain that closes over asciminib when it is bound. This deletion results in asciminib resistance in BCR-ABL, but has no effect on TEL-ABL, suggesting that the native autoinhibitory mechanism that asciminib engages in BCR-ABL is disrupted in TEL-ABL. We show, using mammalian cell expression and single-molecule microscopy, that BCR-ABL is mainly dimeric while TEL-ABL forms higher-order oligomers. Oligomerization can promote trans-autophosphorylation of ABL, and we find that a regulatory phosphorylation site in the SH3 domain of ABL (Tyr 89) is highly phosphorylated in TEL-ABL. This phosphorylation is expected to disassemble the autoinhibited conformation of ABL, thereby preventing asciminib binding. We show that TEL-ABL is intrinsically susceptible to inhibition by asciminib, but that increased phosphorylation results in resistance. Our results demonstrate that different ABL fusion proteins can have dramatically different responses to allosteric inhibitors due to differential phosphorylation. One Sentence SummaryWhen TEL-ABL is phosphorylated, it is insensitive to asciminib. However, when TEL-ABL is dephosphorylated by a phosphatase, asciminib sensitivity is restored.

biochemistry↗

bioRxiv2022

A Saturation-Mutagenesis Analysis of the Interplay Between Stability and Activation in Ras

Cancer mutations in Ras occur predominantly at three hotspots: Gly 12, Gly 13, and Gln 61. Previously, we reported that deep mutagenesis of H-Ras using a bacterial assay identified many other activating mutations (Bandaru et al. eLife, 2017). We now show that the results of saturation mutagenesis of H-Ras in mammalian Ba/F3 cells correlate well with results of bacterial experiments in which H-Ras or K-Ras are co-expressed with a GTPase-activating protein (GAP). The prominent cancer hotspots are not dominant in the Ba/F3 data. We used the bacterial system to mutagenize Ras constructs of different stabilities and discovered a feature that distinguishes the cancer hotspots. While mutations at the cancer hotspots activate Ras regardless of construct stability, mutations at lower-frequency sites (e.g., at Val 14 or Asp 119) can be activating or deleterious, depending on the stability of the Ras construct. We characterized the dynamics of three non-hotspot activating Ras mutants by using NMR to monitor hydrogen-deuterium exchange (HDX). These mutations result in global increases in HDX rates, consistent with the destabilization of Ras. An explanation for these observations is that mutations that destabilize Ras increase nucleotide dissociation rates, enabling activation by spontaneous nucleotide exchange. A further stability decrease can lead to insufficient levels of folded Ras - and subsequent loss of function. In contrast, the cancer hotspot mutations are mechanism-based activators of Ras that interfere directly with the action of GAPs. Our results demonstrate the importance of GAP surveillance and protein stability in determining the sensitivity of Ras to mutational activation.

biophysics↗

bioRxiv2021

Allosteric communication in DNA polymerase clamp loaders relies on a critical hydrogen-bonded junction

Clamp loaders are AAA+ ATPases that load sliding clamps onto DNA. We mapped the mutational sensitivity of the T4 bacteriophage sliding clamp and clamp loader by deep mutagenesis, and found that residues not involved in catalysis or binding display remarkable tolerance to mutation. An exception is a glutamine residue in the AAA+ module (Gln 118) that is not located at a catalytic or interfacial site. Gln 118 forms a hydrogen-bonded junction in a helical unit that we term the central coupler, because it connects the catalytic centers to DNA and the sliding clamp. A suppressor mutation indicates that hydrogen bonding in the junction is important, and molecular dynamics simulations reveal that it maintains rigidity in the central coupler. The glutamine-mediated junction is preserved in diverse AAA+ ATPases, suggesting that a connected network of hydrogen bonds that links ATP molecules is an essential aspect of allosteric communication in these proteins.

biophysics↗