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

Abstract

Two major lipophilic hormones, 20-hydroxyecdysone (20E) and juvenile hormone (JH), govern insect development and growth. While the mode of action of 20E is well understood, some understanding of JH-dependent signalling has been attained only in the past few years, and the crosstalk of the two hormonal pathways remains unknown. Two proteins, the calponin-like Chd64 and immunophilin FKBP39 proteins, have recently been found to play pivotal roles in the formation of dynamic, multiprotein complex that cross-links these two signalling pathways. However, the molecular mechanism of the interaction remains unexplored. The aim of this work was to determine structural elements of Chd64 to provide an understanding of molecular basis of multiple interactions. We analysed Chd64 in two unrelated insect species, Drosophila melanogaster (DmChd64) and Tribolium castaneum (TcChd64). Using hydrogen-deuterium exchange mass spectrometry (HDX-MS), we showed that both Chd64 proteins have disordered tails that outflank the globular core. The folds of the globular cores of both Chd64 resemble the calponin homology (CH) domain previously resolved by crystallography. Monitoring the unfolding of DmChd64 and TcChd64 by far-ultraviolet (UV) circular dichroism (CD) spectroscopy, fluorescence spectroscopy and size-exclusion chromatography (SEC) revealed a highly complex process. Chd64 unfolds and forms of a molten globule (MG)—like intermediate state. Furthermore, our data indicate that in some conditions, Chd64 may exists in discrete structural forms, indicating that the protein is pliable and capable of easily acquiring different conformations. The plasticity of Chd64 and the existence of terminal intrinsically disordered regions (IDRs) may be crucial for multiple interactions with many partners.

Differential Expression of Drosophila Transgelins Throughout Development

“…While the cytoplasmic localization of Chd64 could be linked with its putative role in the modulation of actin cytoskeleton, its nuclear presence fits with previous studies reporting binding of Chd64 on Juvenile hormone receptor element 1 (JHRE1) and ecdysone response element (EcRE) ( Li et al, 2007 ), suggesting a putative role of Chd64 in transcription modulation. Interestingly, structural analysis indicated that Chd64 is a pliable protein-containing terminal intrinsically disordered regions (IDRs) that facilitate multiple molecular interactions ( Kozłowska et al, 2014 ; Tarczewska et al, 2015 ). Our data clearly demonstrate a complete absence of Chd64 expression in muscles during fly development.…”

Hemolymph Proteomics and Gut Microbiota of Horseshoe Crabs Tachypleus tridentatus and Carcinoscorpius rotundicauda

“…The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) are shown next to the branches. Tarczewska et al, 2015). To date, the hJHBP has only been identified and functionally tested in some lepidopterans (Orth et al, 2003;Suzuki et al, 2011), which was used to deliver the juvenile hormone to target tissue and protect it from degradation (Hidayat and Goodman, 1994;Debski et al, 2004).…”

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

“…Analytical SEC was performed as described previously60. Briefly, a Superdex 200 10/300 GL column was equilibrated with buffer C and calibrated using a set of standard proteins60.…”

“…Briefly, a Superdex 200 10/300 GL column was equilibrated with buffer C and calibrated using a set of standard proteins60. The column volume (V t ) was 24 ml, and the column void volume (V 0 ) was determined to be 7.86 ml using blue dextran.…”