Biology subjects

Erguven, M.

Publications and source records attributed to Erguven, M..

2 recordsLinked to original sources

bioRxiv2020

How far are we in the rapid prediction of drug resistance caused by kinase mutations?

Protein kinases regulate various cell signaling events in a diverse range of species through phosphorylation. The phosphorylation occurs upon transferring the terminal phosphate of an ATP molecule to a designated target residue. Due to the central role of protein kinases in proliferative pathways, point mutations occurring within or in the vicinity of ATP binding pocket can render the enzyme overactive, leading to cancer. Combatting such mutation-induced effects with the available drugs has been a challenge, since these mutations usually happen to be drug resistant. Therefore, the functional study of naturally and/or artificially occurring kinase mutations have been at the center of attention in diverse biology-related disciplines. Unfortunately, rapid experimental exploration of the impact of such mutations remains to be a challenge due to technical and economical limitations. Therefore, the availability of kinase-ligand binding affinity prediction tools is of great importance. Within this context, we have tested six state-of-the-art web-based affinity predictors (DSX-ONLINE, KDEEP, HADDOCK2.2, PDBePISA, Pose&Rank, and PRODIGY-LIG) in assessing the impact of kinase mutations with their ligand interactions. This assessment is performed on our structure-based protein kinase mutation benchmark, BINDKIN. BINDKIN contains 23 wild type-mutant pairs of kinase-small molecule complexes, together with their corresponding binding affinity data (in the form of IC50, Kd, and Ki). The web-server performances over BINDKIN show that the raw server predictions fail to produce good correlations with the experimental data. However, when we start looking in to the direction of change (whether a mutation improves/worsens the binding), we observe that over Ki data, DSX-ONLINE achieves a Pearsons R correlation coefficient of 0.97. When we used homology models instead of crystal structures, this correlation drops to 0.45. These results highlight that there is still room to improve the available web-based predictors to estimate the impact of protein kinase point mutations. We present our BINDKIN benchmark and all the related results online for the sake of aiding such improvement efforts. Our files can be reached at https://github.com/CSB-KaracaLab/BINDKIN

bioinformatics↗

bioRxiv2020

Investigating the determinants of mammalianMastl kinase activation

Mastl is a mitotic kinase that is essential for error-free chromosome segregation. It is an atypical member of the AGC kinase family, possessing a unique non-conserved middle region (NCMR). The mechanism of its activation prior to mitosis has been extensively studied in Xenopus egg extracts. These studies found several residues (corresponding to T193 and T206 in the activation loop, and S861 in the C-terminal tail, i.e., C-tail of mouse Mastl) whose phosphorylations are crucial for enzymatic activation. To date, the significance of these phosphosites was not confirmed in live mammalian cells. Here, we utilize a complementation cloning approach to determine the essentials of mammalian Mastl kinase activity. We employed a tamoxifen-inducible conditional knockout system to delete the endogenous Mastl in mouse embryonic fibroblasts (MEF) and screened various mutants for their ability to complement its loss. MEFs, ectopically expressing different phosphorylation site mutants, were induced to undergo recombination-mediated knockout in their endogenous Mastl loci. S861A and S861D mutants were able to complement endogenous Mastl loss with proliferation rates comparable to WT. In parallel, we examined the available protein kinase structures having a phosphorylated C-tail. Among the published states, two distinct positionings of the C-tail phosphoresidue were observed. Energetic analysis of these states revealed that only one conformation highly contributes to the C-tail docking. Our in-depth sequence and structure analysis showed that Mastl pS861 does not belong to the conformational state, where the phosphoresidue contributes to the C-tail docking. The C-tail of Mastl is relatively short and it lacks the hydrophobic (HF) motif. In other AGC kinases, the C-tail phosphosite aids the anchoring of this motif over the N-lobe, leading to the final step of kinase activation. Together with the lack of HF motif in Mastl, our results suggest that phosphorylation of the C-tail turn motif phosphosite (S861) is auxiliary and is dispensable for mammalian Mastl kinase function. Furthermore, we demonstrated that complementation cloning is a powerful approach for screening the determinants of an essential proteins functioning.

cell biology↗