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

Tarczewska A, Kozłowska M, Dobryszycki P, Kaus-Drobek M, Dadlez M, Ożyhar A - PLoS ONE (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Faculty of Chemistry, Wrocław University of Technology, Wybrzeże Wyspiańskiego 27, 50-370, Wrocław, Poland.

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.

No MeSH data available.


Related in: MedlinePlus

Hydrogen-deuterium exchange of DmChd64 and TcChd64.(A), (C) The deuteration percentage of peptic fragments from DmChd64 (A) and TcChd64 (C) in an exchange time of 10 s. The positioning of peptides in the sequence is shown along the horizontal axis, represented by a horizontal bar with an equal length to that of the peptide. The position of the bar along the vertical axis marks the fraction exchanged after 10 sec. The y-axis error bars are standard deviations calculated from three independent experiments. The coloured rectangles correspond to groups of peptides with different exchange rates (I-VII). (B), (D) 3D models of DmChd64 and TcChd64, respectively. The putative structures were generated using the I-TASSER web-based tool [20] using sequences of recombinant DmChd64 and TcChd64 as described previously [8] and the results were visualised using PyMOL [30]. The coloured regions correspond to groups of peptides with different exchange rates (I-VII): region I (DmChd64 1–39, TcChd64 1–27; blue), region II (DmChd64: 40–50, TcChd64: 28–45; turquoise), region III (DmChd64: 46–65, TcChd64: 44–53; grey), region IV (DmChd64: 66–74, TcChd64: 54–63; green), region V (DmChd64: 76–101, TcChd64: 63–89; yellow), region VI (DmChd64: 98–135, TcChd64 90–122; orange), region VII (DmChd64: 136–211, TcChd64 123–199; red). The arrows point to the W residues, marked in black.
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pone.0137074.g001: Hydrogen-deuterium exchange of DmChd64 and TcChd64.(A), (C) The deuteration percentage of peptic fragments from DmChd64 (A) and TcChd64 (C) in an exchange time of 10 s. The positioning of peptides in the sequence is shown along the horizontal axis, represented by a horizontal bar with an equal length to that of the peptide. The position of the bar along the vertical axis marks the fraction exchanged after 10 sec. The y-axis error bars are standard deviations calculated from three independent experiments. The coloured rectangles correspond to groups of peptides with different exchange rates (I-VII). (B), (D) 3D models of DmChd64 and TcChd64, respectively. The putative structures were generated using the I-TASSER web-based tool [20] using sequences of recombinant DmChd64 and TcChd64 as described previously [8] and the results were visualised using PyMOL [30]. The coloured regions correspond to groups of peptides with different exchange rates (I-VII): region I (DmChd64 1–39, TcChd64 1–27; blue), region II (DmChd64: 40–50, TcChd64: 28–45; turquoise), region III (DmChd64: 46–65, TcChd64: 44–53; grey), region IV (DmChd64: 66–74, TcChd64: 54–63; green), region V (DmChd64: 76–101, TcChd64: 63–89; yellow), region VI (DmChd64: 98–135, TcChd64 90–122; orange), region VII (DmChd64: 136–211, TcChd64 123–199; red). The arrows point to the W residues, marked in black.

Mentions: Disordered regions of native DmChd64 and TcChd64 were mapped via HDX-MS. HDX exploits variations in the chemical exchange rate of backbone amide hydrogens in proteins. Hydrogens that are deeply buried in the protein core or that form hydrogen bonds only exchange if the protein undergoes a conformational change. Regions of ID exchange rapidly, and the exchange is completed after only a few seconds of exposure to D2O buffers [19]. To better visualise the HDX results (Fig 1A and 1C), we generated 3D models of the putative structures of DmChd64 and TcChd64 using I-TASSER [20]. The different regions of amide hydrogen exchange were superimposed on the I-TASSER model via colour coding (Fig 1B and 1D). DmChd64 and TcChd64 are homologues with 74% sequence identity. Recombinant DmChd64 and TcChd64 contain additional amino acids from affinity tags, which facilitate their purification [8]. As shown in Fig 1A and 1C, fragments in both Chd64 proteins rapidly exchange after 10 seconds of incubation in D2O buffer. Peptides covering the first 39 residues in the N-terminal region in Drosophila and 27 residues in Tribolium Chd64 exchange almost all of the amide hydrogens. This denotes high fragment flexibility due to the presence of ID (Fig 1A–1D, region I, marked in blue). Additionally, the presence of a larger region with less frequent exchange indicates well-ordered structure. This region comprises residues 40–139 in Drosophila and 28–122 in Tribolium and most likely corresponds to the CH domain, which is a well-defined protein module. Proteins containing the CH domain are abundant in all metazoans and are involved in actin binding and signal transduction [7]. An X-ray of the CH domain of human spectrin reveals its well-ordered helical structure [6]. Interestingly, the pepsin digestion patterns in the central regions of DmChd64 and TcChd64 are very similar. These globular fragments contain five distinct regions with significantly varied exchange rates. Consequently, a helix and loops in the CH domain from both Chd64 proteins are predicted to exist based on the crystallographic structure of the human spectrin CH domain. Pepsin cleavage generated a series of short peptides (Fig 1A and 1C, region II) at the beginning of the globular module between residues 40–50 and 28–45 in Drosophila and Tribolium, respectively, where the exchange rate was nearly zero, corresponding to a stable helical fragment (Fig 1B–1D, marked in turquois). The next series included peptides in which the exchange rate was increased, comprises residues 46–65 in Drosophila and 44–53 in Tribolium (Fig 1A and 1C, region III). These peptides mainly correspond to a loop (Fig 1B and 1D, marked in grey). However, another stable, strongly protected helical region was identified in residues 66–74 and 54–63 of DmChd64 and TcChd64, respectively (Fig 1A–1D, region IV, marked in green). In residues 76–101 of Drosophila and 63–89 of Tribolium, deuteration occurred at a rate of 60–80% (Fig 1A and 1D, region V), indicating the presence of a moderately flexible region, corresponding to a long loop in the I-TASSER model (Fig 1B and 1D, marked in yellow). The last distinct region within the globular core, located between residues 98–135 in DmChd64 and 90–122 in TcChd64, was also more protected (Fig 1A and 1C, region VI) and corresponded to a helix probably located along the edge of the CH domain (Fig 1B and 1D, marked in orange). Peptides covering the C-termini underwent rapid exchange, and deuteration was completed after 10 s of exposure to D2O buffer (Fig 1A–1D, region VII, marked in red). These regions, beginning at residue 130 in DmChd64 and 125 in TcChd64, are disordered. Overall ID accounts for 47% of the DmChd64 and 49% of the TcChd64 sequence.


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

Tarczewska A, Kozłowska M, Dobryszycki P, Kaus-Drobek M, Dadlez M, Ożyhar A - PLoS ONE (2015)

Hydrogen-deuterium exchange of DmChd64 and TcChd64.(A), (C) The deuteration percentage of peptic fragments from DmChd64 (A) and TcChd64 (C) in an exchange time of 10 s. The positioning of peptides in the sequence is shown along the horizontal axis, represented by a horizontal bar with an equal length to that of the peptide. The position of the bar along the vertical axis marks the fraction exchanged after 10 sec. The y-axis error bars are standard deviations calculated from three independent experiments. The coloured rectangles correspond to groups of peptides with different exchange rates (I-VII). (B), (D) 3D models of DmChd64 and TcChd64, respectively. The putative structures were generated using the I-TASSER web-based tool [20] using sequences of recombinant DmChd64 and TcChd64 as described previously [8] and the results were visualised using PyMOL [30]. The coloured regions correspond to groups of peptides with different exchange rates (I-VII): region I (DmChd64 1–39, TcChd64 1–27; blue), region II (DmChd64: 40–50, TcChd64: 28–45; turquoise), region III (DmChd64: 46–65, TcChd64: 44–53; grey), region IV (DmChd64: 66–74, TcChd64: 54–63; green), region V (DmChd64: 76–101, TcChd64: 63–89; yellow), region VI (DmChd64: 98–135, TcChd64 90–122; orange), region VII (DmChd64: 136–211, TcChd64 123–199; red). The arrows point to the W residues, marked in black.
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pone.0137074.g001: Hydrogen-deuterium exchange of DmChd64 and TcChd64.(A), (C) The deuteration percentage of peptic fragments from DmChd64 (A) and TcChd64 (C) in an exchange time of 10 s. The positioning of peptides in the sequence is shown along the horizontal axis, represented by a horizontal bar with an equal length to that of the peptide. The position of the bar along the vertical axis marks the fraction exchanged after 10 sec. The y-axis error bars are standard deviations calculated from three independent experiments. The coloured rectangles correspond to groups of peptides with different exchange rates (I-VII). (B), (D) 3D models of DmChd64 and TcChd64, respectively. The putative structures were generated using the I-TASSER web-based tool [20] using sequences of recombinant DmChd64 and TcChd64 as described previously [8] and the results were visualised using PyMOL [30]. The coloured regions correspond to groups of peptides with different exchange rates (I-VII): region I (DmChd64 1–39, TcChd64 1–27; blue), region II (DmChd64: 40–50, TcChd64: 28–45; turquoise), region III (DmChd64: 46–65, TcChd64: 44–53; grey), region IV (DmChd64: 66–74, TcChd64: 54–63; green), region V (DmChd64: 76–101, TcChd64: 63–89; yellow), region VI (DmChd64: 98–135, TcChd64 90–122; orange), region VII (DmChd64: 136–211, TcChd64 123–199; red). The arrows point to the W residues, marked in black.
Mentions: Disordered regions of native DmChd64 and TcChd64 were mapped via HDX-MS. HDX exploits variations in the chemical exchange rate of backbone amide hydrogens in proteins. Hydrogens that are deeply buried in the protein core or that form hydrogen bonds only exchange if the protein undergoes a conformational change. Regions of ID exchange rapidly, and the exchange is completed after only a few seconds of exposure to D2O buffers [19]. To better visualise the HDX results (Fig 1A and 1C), we generated 3D models of the putative structures of DmChd64 and TcChd64 using I-TASSER [20]. The different regions of amide hydrogen exchange were superimposed on the I-TASSER model via colour coding (Fig 1B and 1D). DmChd64 and TcChd64 are homologues with 74% sequence identity. Recombinant DmChd64 and TcChd64 contain additional amino acids from affinity tags, which facilitate their purification [8]. As shown in Fig 1A and 1C, fragments in both Chd64 proteins rapidly exchange after 10 seconds of incubation in D2O buffer. Peptides covering the first 39 residues in the N-terminal region in Drosophila and 27 residues in Tribolium Chd64 exchange almost all of the amide hydrogens. This denotes high fragment flexibility due to the presence of ID (Fig 1A–1D, region I, marked in blue). Additionally, the presence of a larger region with less frequent exchange indicates well-ordered structure. This region comprises residues 40–139 in Drosophila and 28–122 in Tribolium and most likely corresponds to the CH domain, which is a well-defined protein module. Proteins containing the CH domain are abundant in all metazoans and are involved in actin binding and signal transduction [7]. An X-ray of the CH domain of human spectrin reveals its well-ordered helical structure [6]. Interestingly, the pepsin digestion patterns in the central regions of DmChd64 and TcChd64 are very similar. These globular fragments contain five distinct regions with significantly varied exchange rates. Consequently, a helix and loops in the CH domain from both Chd64 proteins are predicted to exist based on the crystallographic structure of the human spectrin CH domain. Pepsin cleavage generated a series of short peptides (Fig 1A and 1C, region II) at the beginning of the globular module between residues 40–50 and 28–45 in Drosophila and Tribolium, respectively, where the exchange rate was nearly zero, corresponding to a stable helical fragment (Fig 1B–1D, marked in turquois). The next series included peptides in which the exchange rate was increased, comprises residues 46–65 in Drosophila and 44–53 in Tribolium (Fig 1A and 1C, region III). These peptides mainly correspond to a loop (Fig 1B and 1D, marked in grey). However, another stable, strongly protected helical region was identified in residues 66–74 and 54–63 of DmChd64 and TcChd64, respectively (Fig 1A–1D, region IV, marked in green). In residues 76–101 of Drosophila and 63–89 of Tribolium, deuteration occurred at a rate of 60–80% (Fig 1A and 1D, region V), indicating the presence of a moderately flexible region, corresponding to a long loop in the I-TASSER model (Fig 1B and 1D, marked in yellow). The last distinct region within the globular core, located between residues 98–135 in DmChd64 and 90–122 in TcChd64, was also more protected (Fig 1A and 1C, region VI) and corresponded to a helix probably located along the edge of the CH domain (Fig 1B and 1D, marked in orange). Peptides covering the C-termini underwent rapid exchange, and deuteration was completed after 10 s of exposure to D2O buffer (Fig 1A–1D, region VII, marked in red). These regions, beginning at residue 130 in DmChd64 and 125 in TcChd64, are disordered. Overall ID accounts for 47% of the DmChd64 and 49% of the TcChd64 sequence.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Faculty of Chemistry, Wrocław University of Technology, Wybrzeże Wyspiańskiego 27, 50-370, Wrocław, Poland.

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.

No MeSH data available.


Related in: MedlinePlus