Calponin-Like Chd64 Is Partly Disordered

Abstract

20-hydroxyecdysone (20E) and juvenile hormone (JH) signaling pathways interact to regulate insect development. Recently, two proteins, a calponin-like Chd64 and immunophilin FKBP39 have been found to play a pivotal role in the cross-talk between 20E and JH, although the molecular basis of interaction remains unknown. The aim of this work was to identify the structural features that would provide understanding of the role of Chd64 in multiple and dynamic complex that cross-links the signaling pathways. Here, we demonstrate the results of in silico and in vitro analyses of the structural organization of Chd64 from Drosophila melanogaster and its homologue from Tribolium castaneum. Computational analysis predicted the existence of disordered regions on the termini of both proteins, while the central region appeared to be globular, probably corresponding to the calponin homology (CH) domain. In vitro analyses of the hydrodynamic properties of the proteins from analytical size-exclusion chromatography and analytical ultracentrifugation revealed that DmChd64 and TcChd64 had an asymmetrical, elongated shape, which was further confirmed by small angle X-ray scattering (SAXS). The Kratky plot indicated disorderness in both Chd64 proteins, which could possibly be on the protein termini and which would give rise to specific hydrodynamic properties. Disordered tails are often involved in diverse interactions. Therefore, it is highly possible that there are intrinsically disordered regions (IDRs) on both termini of the Chd64 proteins that serve as platforms for multiple interaction with various partners and constitute the foundation for their regulatory function.

How did arthropod sesquiterpenoids and ecdysteroids arise? Comparison of hormonal pathway genes in non-insect arthropod genomes

“…Chd64 is another protein previously known to bind to the JHRE and modulates the responses to JH exposure ( Li et al 2007 ). Although Chd64 possesses a clear calponin homology (CH) domain (pfam id: pfam00307), its relationship to the wider family of other CH-containing genes is largely uncataloged ( Kozłowska et al 2014 ). In our analysis, we have also identified putative Chd64 orthologs in the noninsect arthropods ( supplementary fig.…”

Nucleoplasmin-like domain of FKBP39 from Drosophila melanogaster forms a tetramer with partly disordered tentacle-like C-terminal segments

“…The protein content and purity were evaluated after every stage of purification with SDS-PAGE57 and Coomassie Brilliant Blue R-250-stained gels58. The identification of the purified protein was performed by ESI-MS, as described previously4.…”

“…However, the molecular basis of the interactions between FKBP39, Chd64 and the other components of the multiprotein complex is not understood. Recently, Kozlowska et al 4. showed that the structure of Chd64 possesses a dual nature, consisting of a globular domain flanked by intrinsically disordered tails.…”

“…Thus, one IDP/IDR often binds a wide range of proteins67. For these reasons, the presence of disorderliness at the N- and C-termini of Chd64 may lay a foundation for its biological function because the labile, dynamic and highly adjustable nature of disordered regions could enable interaction with various partners4. The second key component of the putative multiprotein complex that crosslinks the 20E and JH signalling pathways is FKBP39.…”

Insight into the Unfolding Properties of Chd64, a Small, Single Domain Protein with a Globular Core and Disordered Tails

“…melanogaster and T . castaneum are partly disordered; however, the extent and position of this disorder have not been defined [ 8 ]. Intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs) have been the focus of recent structure-function relationship studies [ 9 ].…”