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Remote on–off switching of protein activity by intrinsically disordered region

Nature Structural & Molecular Biology (2025)Cite this article

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Abstract

While the regulation of protein function theoretically encompasses alterations in both structural conformation and dynamic properties, the latter aspect, specifically conformational entropy, remains relatively unexplored. Here we show that an intrinsically disordered region (IDR), a prominent component of the proteome, can remotely switch protein activity on or off through a nonbinding, entropy-driven mechanism. Focusing on the disordered C-terminal tail of Sgt2, a chaperone in the guided entry of tail-anchored protein pathway, we demonstrate that it allosterically inhibits the N-terminal domain without direct contact, preventing unproductive chaperone–chaperone interactions. This inhibition is relieved upon client binding. These effects depend on specific IDR sequences but not the intervening regions. Beyond acting as a relay signal, the IDR also forms a dynamic complex with transmembrane domains of tail-anchored clients, serving as an entropic shelter. Moreover, the IDR-mediated activity of Sgt2 correlates with fast internal dynamics, establishing conformational entropy as a key regulatory principle. Our findings reveal IDRs as two-way entropic modulators, enabling distant, on-demand activity switching.

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Fig. 1: Autoinhibitory interaction of Sgt2 with the Get4/5 complex conferred by its disordered tail.
Fig. 2: Influence of the disordered tail length, composition, domain deletion and domain swapping on Sgt2 autoinhibition.
Fig. 3: Unlocking of the autoinhibitory activity upon dynamic capture of TA client loading by the disordered tail.
Fig. 4: Autoinhibitory mechanism of Sgt2 mediated by its disordered tail.
Fig. 5: Exploring the effects of IDR tails on the modulation of binding affinities in diverse protein systems.
Fig. 6: Dual functionality of the intrinsically disordered tail of Sgt2 in the GET pathway.

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Data availability

The atomic coordinates of Sgt2 were deposited to the Protein Data Bank under accession code 9LLV. NMR assignments for the C-terminal tail of Sgt2 are available from the BMRB under accession code 52887. Previously deposited BMRB entries 18341 (Sgt2N) and 27044 (Sgt2T) were used to support structural calculations. Additional data are available from the corresponding authors upon reasonable request. Source data are provided with this paper.

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Acknowledgements

We gratefully thank D. Li, X. Yao, J. Chou, Y. Xia and T. Xie for helpful discussions and the BioNMR Laboratory of the Division of Life Sciences and Medicine at the University of Science and Technology of China for NMR data collection. We thank the staff members of the NMR Spectroscopy System (https://cstr.cn/31125.02.SHMFF.SM3.NMR) at the Steady High Magnetic Field Facility, Chinese Academy of Sciences (https://cstr.cn/31125.02.SHMFF), for providing technical support and assistance in data collection and analysis. This work was supported by the National Natural Science Foundation of China (32471273, T2221005 and 31971144 to C.H.; 52025036 to Y.W.; 32170767 and 32470808 to C.W.) and the Global Select Project DJK-LX-2022011 of the Institute of Health and Medicine, Hefei Comprehensive National Science Center (to C.H.).

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  1. Ministry of Education Key Laboratory for Membrane-less Organelles and Cellular Dynamics, Center for Advanced Interdisciplinary Science and Biomedicine of IHM, Biomedical Sciences and Health Laboratory of Anhui Province, Hefei National Laboratory for Physical Sciences at the Microscale, School of Life Sciences, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, P. R. China

    Tuo Ji, Piao Ge, Shan Zhang, Chanjuan Wan, Hailong Liu, Xiaozhan Qu, Feng Zhu, Qingguo Gong, Weiya Xu, Chao Wang, Yucai Wang & Chengdong Huang

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Contributions

C.H. designed the research. T.J., P.G., S.Z., C. Wan, H.L. and X.Q. prepared the protein samples and conducted biochemical and biophysical experiments. T.J., P.G., C. Wan and C.H. performed the NMR experiments. C.H., T.J., P.G., H.L., F.Z., Q.G., W.X., C. Wang and Y.W. analyzed the data. C.H., T.J., C. Wang and Y.W. drafted the paper. All authors reviewed and approved the final manuscript.

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Correspondence to Chao Wang, Yucai Wang or Chengdong Huang.

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Extended data

Extended Data Fig. 1 Analysis of Sgt2 interaction with Get4/5 complex.

a, ITC analysis of Sgt2FL and Get4/5 in PBS buffer shows no interaction, consistent with the ITC data in Fig. 1c, which were obtained in MES buffer. b, ITC analysis of Sgt2FL with the UBL domain of Get5 also shows no interaction, suggesting that the autoinhibitory binding of Sgt2FL to Get4/5 is not due to steric clashes. c, SPR analysis of Sgt2FL and Get4/5 confirms the absence of interaction. Sgt2FL was immobilized on the chip, and interactions were assessed at varying concentrations of Get4/5. No significant binding response was observed. The experiment was performed in triplicate. d, SPR analysis of Sgt2NT and Get4/5 shows significant binding. Binding is depicted in response units (RU) over time (top). Values for kinetic rate constants (kon, koff) and dissociation constant (Kd) are listed in the table (bottom). The experiment was performed in triplicate, with error bars representing the standard deviation (s.d.). e-h, SEC-MALS analysis of Sgt2 variants with Get4/5. Measured molecular masses (with error bars) are color-coded, while theoretical masses (calculated from amino acid sequences) are shown in black and brackets. e, Sgt2N (purple) and Get4/5 (blue) form a stable complex (orange), indicating a heterotetrameric Get4/5 complex bound to two dimeric Sgt2N molecules. f, Sgt2NT (red) and Get4/5 (blue) form a stable complex (rose red) with the same stoichiometry. g,h, Full-length Sgt2FL (brown) does not interact with Get4/5, as no complex species were detected. The peak selection window used for MALS analysis is indicated as the dashed grey box. i, Methyl-TROSY NMR titrations of isotopically labeled Sgt2 (Ile, Val, Leu, Met and Ala) with unlabeled Get4/5-UBL domain shows no chemical shift perturbation or intensity change, further supporting the lack of interaction.

Source data

Extended Data Fig. 2 Disordered Conformation of Sgt2’s C-Terminal Tail.

a, 1H-15N HSQC NMR spectra of Sgt2C, revealing a lack of signal dispersion indicative of a disordered conformation, with assignments labeled. b, 1H-13C HMQC NMR spectra of Sgt2C, displaying limited signal dispersion consistent with a disordered conformation, with assignments labeled. c, Far-UV circular dichroism (CD) thermal denaturation of Sgt2C showing a prolonged transition, suggesting a non-cooperative unfolding process. d, Prediction of intrinsic disorder in Sgt2 using Metadisorder (top) and IUpred3 programs (bottom). In the Metadisorder plot, different colors indicate predictions from various methods: MetaDisorder3D (purple) uses structural alignments, MetaDisorder (blue) is a consensus-based predictor, MetaDisorderMD (green) integrates sequence- and structure-based predictions, and MetaDisorderMD2 (red) further optimizes these predictions. Both programs identify the C-terminal tail and the linker connecting Sgt2N and Sgt2T are disordered. e, Superposition of the 20 lowest-energy solution structures of Sgt2 with the Sgt2N domains aligned, revealing a highly dynamic “beads-on-string” conformation with minimal additional interdomain contacts beyond the N-domain dimerization. The color scheme for Sgt2’s domains is consistent with the schematic representation at the top. The inset highlights the well-aligned 20 structures of Sgt2N.

Source data

Extended Data Fig. 3 Dynamic architecture of dimeric Sgt2 with independent domains.

a, Superpositions of 1H-15N HSQC spectra and 1H-13C methyl-TROSY spectra of individual domains (or combinations) compared with those of full-length Sgt2 or domain combinations. The color scheme represents different domains as labeled. For clarity, the spectra on top are shown with outer contours only. Most residues with perturbations are located at domain interfaces (for example, labeled in the first three overlays in the top row). The similarity of signals from all three domains to their isolated counterparts suggests a highly dynamic architecture, with each domain behaving as an independent structural entity. The lower right plot in panel (a) compares the 1H-15N HSQC signal intensities of Sgt2C (I) with those in Sgt2FL (I0), plotted against residue numbers. Only well-separated peaks were analyzed. Most residues exhibit less than a 50% increase in intensity despite a 5.7-fold decrease in molecular weight, further suggesting the dynamic independence of Sgt2C. b, SAXS analysis of Sgt2NT, displaying the experimental SAXS curve along with the pair-probability, P(r), functions. The presence of multiple peaks in the P(r) function suggests the presence of multiple folded domains. c, SAXS analysis of Sgt2FL, showing the experimental SAXS curve and corresponding P(r) functions. This figure underscores the dynamic nature of dimeric Sgt2, wherein its three individual domains exhibit independent behavior. d, NMR-derived order parameter (S2) profiles of selected methyl residues in full-length Sgt2, highlighting fast (ps-ns) dynamics. e, Comparison of 15N R2 relaxation rates for isolated Sgt2C (green) and Sgt2C within full-length Sgt2 (orange), with error bars representing standard deviations from data fitting, and centre indicating fitted R₂ values. Only well-separated peaks were analyzed. The results indicate that Sgt2C remains highly dynamic and does not form strong interdomain interactions within full-length Sgt2.

Source data

Extended Data Fig. 4 Autoinhibitory activity of various Sgt2 constructs.

a-c, Amino acid sequences of domains used for Sgt2T replacement: GB1 (a), TRX (b), and SgtAT with sequence alignment to Sgt2T (c). d-g, Disordered sequences replacing Sgt2C tail: 65-residue Tau fragment (d), scrambled Sgt2C (e), 20-residue disordered L20 sequence (f), and C-terminal tail from homolog SgtA (SgtAC) (f), with sequence alignment (g).

Extended Data Fig. 5 Chaperoning of TA clients by Sgt2’s disordered tail.

a, ITC analysis of Sec22TMD-loaded Sgt2 binding to Get4/5 highlights the interaction potential induced by client loading. b, ITC analysis of Sgt2-Taufragment and Get4/5 shows no interaction, indicating that the non-relevant Tau fragment fails to promote Sgt2-Get4/5 binding. c. Methyl-TROSY NMR spectra comparison of Sgt2FL (gray) and Sgt2-Ysy6TMD (rose red) reveals signal broadening in Sgt2C upon client binding, indicating the involvement of Sgt2’s C-terminal tail in chaperoning the TA client Ysy6. d-e, Differential line broadening analysis identifies binding sites within Sgt2C for Ysy6TMD (d) and Sec22TMD (e), pinpointing residues involved in chaperoning. The overlap in these sites suggests a universal chaperoning mechanism for different TA clients. f, Sgt2 amino acid sequence highlighting domains and TA client binding site. g, Hydrophobicity plot of Sgt2C as a function of its primary sequence, with the TA client binding site annotation. The alignment of regions with high hydrophobicity score and the binding site indicates that Sgt2C likely uses its hydrophobic area to chaperone TA clients’ transmembrane domains.

Extended Data Fig. 6 Dynamic Conformation of Sgt2 Chaperoning Complex with TA Clients.

a, Far-UV CD spectra of Sgt2C (left) and Sgt2C-Ysy6TMD (right) in 5% (blue) and 10% TFE (green). TFE induces increased helical content in Sgt2C and Sgt2C-Ysy6TMD. b, 1H-15N HSQC spectra overlay of Sgt2C-Ysy6TMD under native conditions (Modena) and 10% TFE (azure). Presence of TFE enhances structural stability but fails to eliminate the high dynamic nature of the Sgt2C-Ysy6TMD complex, evidenced by missing signals. c, NMR characterization of various chaperoning complex fusion constructs (top cartoons) displayed in 1H-15N HSQC NMR spectra, revealing broadened signals with poor dispersion, characteristic of a molten-globule-like conformation. Structural instability persists despite changes in fusion orientation or Sgt2C supply. d, SAXS analysis of Ysy6TMD-loaded Sgt2 confirming the structural flexibility of the complex.

Source data

Extended Data Fig. 7 Sgt2C’s autoinhibition does not involve direct interaction with Sgt2N.

a, Overlay of 1H-15N-HSQC spectra of Sgt2C in the absence (gray) and presence (blue, outer contours only) of a 1:1 ratio of unlabeled Sgt2NT. Sgt2NT is calculated as a dimer, and Sgt2C as a monomer. The excellent spectral overlap indicates no direct interaction. b,c, Residue-resolved NMR signal attenuation (I/I0) of 15N-labeled Sgt2NT (b) and Sgt2C (c) upon titration with unlabeled Sgt2C and Sgt2NT, respectively. I0 and I represent signal intensities before and after titration. Minimal intensity changes in both cases confirm the absence of direct interaction between Sgt2NT and Sgt2C. d, ITC analysis of MTSL spin-labeled Sgt2 C39S/S294C with Get4/5, showing no interaction. Spin-labeled Sgt2 is calculated as a dimer, and Get4/5 as a heterotetramer. This result indicates that MTSL attachment does not interfere with the autoinhibitory activity of Sgt2C. e,f, Overlay of methyl-TROSY spectra of spin-labeled Sgt2 at residue 254 (e, red) and 294 (f, purple) in the paramagnetic state, compared with the corresponding spectra in the diamagnetic state (grey, bottom). The associated intensity plots are shown in Fig. 4d. The observed PRE effects are minimal, except for neighboring residue.

Source data

Extended Data Fig. 8 Slow-to-intermediate timescale exchange dynamics (µs-ms) profiles of Sgt2NT and Sgt2FL methyl groups.

a,b, Relaxation dispersion (CPMG) profiles of methyl groups within Sgt2NT (a) and Sgt2FL (b), measured at 600 MHz and 850 MHz. Representative residues from the Sgt2N and Sgt2T domains are shown. The error bars for R2eff rate values were calculated as twice the noise level measured in the spectra; data points reflect fitted R₂eff values. c,d, CEST profiles of methyl groups within Sgt2NT (c) and Sgt2FL (d), analyzed using ChemEX. Some residues (for example, I137 and I163) exhibit multiple dips, indicating conformational exchange; however, ChemEX failed to resolve the minor state, likely due to spectral noise. Both CPMG and CEST reveal slow conformational dynamics exclusively in the Sgt2T domain, with no detectable exchange in Sgt2N.

Source data

Extended Data Fig. 9 Differential dynamics of Sgt2FL and Sgt2NT.

a, NMR-derived order parameter profiles of methyl groups within Sgt2NT, elucidating its fast (ps-ns) dynamic properties. b, 1H-15N HSQC NMR spectra of Sgt2FL display poor quality with severely broadened signals, suggesting unfavorable dynamic property. c, Overlaid 1H-15N HSQC NMR spectra of Sgt2FL (gray) and Sgt2NT (green) further emphasize the stark contrast in spectral quality between the two proteins. This distinction suggests distinct dynamic properties for Sgt2FL and Sgt2NT, highlighting the potential impact of dynamics on their respective behaviors. d, ITC analysis of the chimeric construct of Sgt2N-Cshort with Get5-UBL, showing no detectable interaction. e, Comparison of the [1H]-15N-heteronuclear NOEs of Sgt2N and Sgt2N-Cshort, indicating a global decrease in NOE values upon attachment of the disordered Sgt2C fragment. Residues near the Sgt2N-Get4/5 binding interface, which show significant NOE reductions, are labeled. f, Saturated 1H-15N HSQC spectrum of Sgt2N-Cshort. The disordered Sgt2C fragment (pink) shows negative signals, while the structured Sgt2N domain (blue) remains positive, indicating that the disordered tail remains highly flexible and does not form stable contacts with Sgt2N. g, Peptide coverage in HDX-MS experiment. h, Heatmap representing the difference in HDX rates (ΔHDX), calculated as HDX rates of Sgt2FL minus those of Sgt2NT. Data is shown across three time points (0.5 min to 85 min) and color-scaled from blue (-20%) to pink (20%). Positive values indicate regions where Sgt2FL exhibits faster exchange rates than Sgt2NT, reflecting increased local flexibility.

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Extended Data Fig. 10 Dynamic modulation of Sgt2 by Ysy6TMD loading and associated IDR tail sequences.

a, Representative NMR-derived order parameters of methyl groups in Ysy6TMD-loaded Sgt2, illustrating fast (ps-ns) dynamics. Fitted profiles of selected residues are shown. b, Heatmap illustrating the difference in HDX rates (ΔHDX), calculated as HDX rates of Sgt2-Ysy6TMD minus those of Sgt2FL. Data is presented across three time points (30 s to 5000 s) and color-scaled from blue (-32%) to pink (32%). Negative values indicate regions where Sgt2-Ysy6TMD exhibits slower exchange rates than Sgt2NT, reflecting decreased local flexibility. c, Amino acid sequences of the proteins (domains) subjected to IDR-tailing and the sequences of IDR tails.

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Ji, T., Ge, P., Zhang, S. et al. Remote on–off switching of protein activity by intrinsically disordered region. Nat Struct Mol Biol (2025). https://doi.org/10.1038/s41594-025-01585-7

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