Tandem SH3 Research Articles

Fold Evolution before LUCA: Common Ancestry of SH3 Domains and OB Domains.

SH3 and OB are the simplest, oldest, and most common protein domains within the translation system. SH3 and OB domains are β-barrels that are structurally similar but are topologically distinct. To transform an OB domain to a SH3 domain, β-strands must be permuted in a multistep and evolutionarily implausible mechanism. Here, we explored relationships between SH3 and OB domains of ribosomal proteins, initiation, and elongation factors using a combined sequence- and structure-based approach. We detect a common core of SH3 and OB domains, as a region of significant structure and sequence similarity. The common core contains four β-strands and a loop, but omits the fifth β-strand, which is variable and is absent from some OB and SH3 domain proteins. The structure of the common core immediately suggests a simple permutation mechanism for interconversion between SH3 and OB domains, which appear to share an ancestor. The OB domain was formed by duplication and adaptation of the SH3 domain core, or vice versa, in a simple and probable transformation. By employing the folding algorithm AlphaFold2, we demonstrated that an ancestral reconstruction of a permuted SH3 sequence folds into an OB structure, and an ancestral reconstruction of a permuted OB sequence folds into a SH3 structure. The tandem SH3 and OB domains in the universal ribosomal protein uL2 share a common ancestor, suggesting that the divergence of these two domains occurred before the last universal common ancestor.

Enhanced SH3:Linker Interaction Suppresses Activating Mutations of the c-Abl Protein-Tyrosine Kinase.

AbstractAbstract 1208Bcr-Abl, the chimeric protein-tyrosine kinase expressed as a result of the Philadelphia chromosome translocation, plays a pivotal role in the initiation and maintenance of chronic myelogenous leukemia (CML). Imatinib (Gleevec) is an ATP-competitive Bcr-Abl inhibitor that selectively kills Bcr-Abl+ CML cells. Despite its clinical success, imatinib is less effective in the advanced stages of CML due to the emergence of drug resistance caused by point mutations in the Abl kinase domain. Second generation Bcr-Abl inhibitors such as dasatinib and nilotinib are active against most imatinib-resistant forms of Bcr-Abl, with the exception of the T315I “gatekeeper” mutant. The Abl gatekeeper residue (Thr315) is located between the ATP-binding site and an adjacent hydrophobic pocket, and forms a key hydrogen bond with imatinib. Additionally, the T315I mutation produces a strong activating effect on the downregulated c-Abl “core,” consisting of the myristoylated N-terminal Ncap, tandem SH3 and SH2 regulatory domains, the SH2-kinase linker, which forms a polyproline type II helix for internal SH3 docking, and the tyrosine kinase domain. Using hydrogen-exchange mass spectrometry, we recently found that the T315I mutation not only induced conformational changes in the Abl kinase domain as expected, but also at a distance in the RT-loop of the SH3 domain. Such changes may allosterically contribute to kinase domain activation by disturbing the negative regulatory influence of SH3:linker interaction. Recently, a new class of allosteric Bcr-Abl inhibitors has been reported that targets the myristate-binding pocket of Abl, which localizes to C-lobe of the kinase domain and away from the active site. Together with our finding that the T315I mutation perturbs SH3:linker interaction, these inhibitors support the existence of an extensive network of allosteric interactions that work together to regulate Abl kinase activity. In this project, we investigated whether enhanced SH3:linker interaction can allosterically reverse the activating effects of the T315I imatinib resistance mutation as well as mutations of the N-terminal myristoylation site and myristic acid binding pocket. We created modified versions of Abl [High Affinity Linker proteins (HALs)] by mutating multiple residues within the SH2-kinase linker to proline, thereby enhancing the SH3 domain binding affinity. Using mammalian cell-based expression assays and immunoblotting with phosphospecific antibodies, we identified five of eleven Abl-HAL proteins that did not exhibit changes in basal kinase activity. The Abl-HAL protein with the greatest enhancement of SH3:linker interaction was then combined with the T315I mutation, a myristoylation-defective mutant, and a myristic acid binding pocket mutation. Remarkably, this HAL substitution completely reversed the activating effect of the myristic acid binding pocket mutation, while substantially suppressing the activity of Abl T315I and the myristoylation-defective mutant. These results indicate that stabilization of SH3:linker interaction allosterically represses Abl activation by a wide variety of mechanisms, and suggests a new approach to allosteric control of Bcr-Abl kinase activity. Disclosures:No relevant conflicts of interest to declare.