Sanghavi, H. M. | Search bioRxiv

bioRxiv2020

Oligomerisation of THAP9 transposase: role of DNA and amino-terminal domains

Active DNA transposases like the Drosophila P element transposase (DmTNP) undergo oligomerisation as a prerequisite for transposition. Human THAP9 (hTHAP9) is a catalytically active but functionally uncharacterised homologue of DmTNP. Here we report (using co-IP, pull down, co-localization, PLA) that both the full length as well as truncated hTHAP9 and DmTNP (corresponding to amino-terminal DNA binding and Leucine-rich coiled coil domains) undergo homo-oligomerisation, predominantly in the nuclei of HEK293T cells. Interestingly, the oligomerisation is shown to be partially mediated by DNA. However, mutating the leucines (either individually or together) or deleting the predicted coiled coil region did not significantly affect oligomerisation. Thus, we highlight the importance of DNA as well as the amino-terminal regions of both hTHAP9 and DmTNP, for their ability to form higher order oligomeric states. We also report that Hcf-1, THAP1, THAP10 and THAP11 are possible protein interaction partners of hTHAP9. These studies lead to several questions about the different putative oligomeric states of hTHAP9 and how they may be related to its yet unknown physiological role as well as interaction partners.

biochemistry↗

bioRxiv2019

G-domain prediction across the diversity of G protein families

Guanine nucleotide binding proteins are characterized by a structurally and mechanistically conserved GTP-binding domain, indispensable for binding GTP. The G domain comprises of five adjacent consensus motifs called G boxes, which are separated by amino acid spacers of different lengths. Several G proteins, discovered over time, are characterized by diverse function and sequence. This sequence diversity is also observed in the G box motifs (specifically the G5 box) as well as the inter-G box spacer length. The Spacers and Mismatch Algorithm (SMA) introduced in this study, can predict G-domains in a given G protein sequence, based on user-specified constraints for approximate G-box patterns and inter-box gaps in each G protein family. The SMA parameters can be customized as more G proteins are discovered and characterized structurally. Family-specific G box motifs including the less characterized G5 motif as well as G domain boundaries were predicted with higher precision. Overall, our analysis suggests the possible classification of G protein families based on family-specific G box sequences and lengths of inter-G box spacers. Significance StatementIt is difficult to define the boundaries of a G domain as well as predict G boxes and important GTP-binding residues of a G protein, if structural information is not available. Sequence alignment and phylogenetic methods are often unsuccessful, given the sequence diversity across G protein families. SMA is a unique method which uses approximate pattern matching as well as inter-motif separation constraints to predict the locations of G-boxes. It is able to predict all G boxes including the less characterized G5 motif which marks the carboxy-terminal boundary of a G domain. Thus, SMA can be used to predict G domain boundaries within a large multi-domain protein as long as the user-specified constraints are satisfied. ClassificationBiological Sciences/Biophysics and Computational Biology

bioinformatics↗