NFAT5, Which Protects Against Hypertonicity, Is Activated by That Stress Via Structuring of Its Intrinsically Disordered Domain

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NFAT5, which protects against hypertonicity, is activated by that stress via structuring of its intrinsically disordered domain

Raj Kumara, Jenna F. DuMondb, Shagufta H. Khanc, E. Brad Thompsond,YiHee, Maurice B. Burgb,1, and Joan D. Ferrarisb

aDepartment of Biomedical Sciences, College of Medicine, University of Houston, Houston, TX 77204; bSystems Biology Center, Division of Intramural Research, National Heart, Lung and Blood Institute, Bethesda, MD 20892; cDepartment of Medical Education, Geisinger Commonwealth School of Medicine, Scranton, PA 18509; dDepartment of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX 77555; and eBiochemistry and Biophysics Center, Division of Intramural Research, National Heart, Lung and Blood Institute, Bethesda, MD 20892

Contributed by Maurice B. Burg, June 20, 2020 (sent for review July 19, 2019; reviewed by Prakash Kulkarni, S. Stoney Simons, Jr., and Vladimir N. Uversky) Nuclear Factor of Activated T cells 5 (NFAT5) is a transcription factor extracellular tonicity (13–15). NFAT5 is a multidomain transcrip- (TF) that mediates protection from adverse effects of hypertonicity tion factor (TF) in which the ID N-terminal domain (NTD) in- by increasing transcription of genes, including those that lead to cludes a tonicity-dependent auxiliary export region responsible for cellular accumulation of protective organic osmolytes. NFAT5 has nuclear and cytoplasmic localization and AD1 (amino acids 1–76), three intrinsically ordered (ID) activation domains (ADs). Using the one of NFAT5’s three activation domains (16–22). NFAT5 ID NFAT5 N-terminal domain (NTD), which contains AD1, as a model, C-terminal domain (CTD) contains two tonicity-dependent trans- we demonstrate by biophysical methods that the NTD senses osmo- activation domains, AD2 and AD3. Although AD1 alone is the lytes and hypertonicity, resulting in stabilization of its ID regions. In weakest of the three, its inclusion in test constructs doubles the the presence of sufficient NaCl or osmolytes, trehalose and sorbitol, hypertonicity response of AD2 or AD2AD3 (18, 19). the NFAT5 NTD undergoes a disorder-to-order shift, adopting higher ID regions are disproportionately higher in cell-signaling average secondary and tertiary structure. Thus, NFAT5 is activated by proteins, giving them an advantage over proteins with ordered the stress that it protects against. In its salt and/or osmolyte-induced conformations, since proteins with ID regions can more effi- PHYSIOLOGY more ordered conformation, the NTD interacts with several proteins, ciently and selectively interact with appropriate target binding including HMGI-C, which is known to protect against apoptosis. – These findings raise the possibility that the increased intracellular partner proteins and enhance allosteric responses (23 33). The ID ionic strength and elevated osmolytes caused by hypertonicity acti- regions of many TFs are known to undergo disorder-to-order vate and stabilize NFAT5. conformational transition upon interacting with organic osmolytes and encounters with specific target binding molecules (26, NFAT5 | hypertonicity | osmolytes | intrinsically disordered region | 27). Due to their lack of stable structure, the detailed mecha- protein–protein interactions nism behind the functions of NFAT5’s ID regions are unknown. We chose to investigate first the simpler of NFATs two major ammalian cells have adaptive responses that enhance sur- Mvival during various forms of stress (1). Among these, un- Significance compensated extracellular hyperosmotic stress results in osmotic outflow of water with a concomitant reduction in cell volume and NFAT5 mediates protection from adverse effects of hyperto- an increase in intracellular ionic strength. This leads to cell cycle nicity. Cells, e.g. kidney, in hypertonic conditions take up salts delay, DNA breakage, oxidative stress, and apoptotic death (2–4). and protective organic osmolytes. By mechanisms not fully Cells adapt to hypertonic stress by accumulating organic osmolytes, understood, NFAT5 is activated and induces genes including which are known to compensate for the cell volume reduction in- those that code enzymes responsible for synthesizing protec- duced by the hyperosmotic environment by allowing for the osmotic tive osmolytes. We show that in solution the NFAT5 NTD, influx of water into cells. Under isosmotic conditions, a reduction or which contains one of NFAT5′s three transcription activation loss of intracellular osmolytes mayalsoleadtoafunctionalre- domains, undergoes hypertonicity- and/or osmolyte-induced duction in cell volume and an increase in ionic strength sufficient to disorder-to-order conformational rearrangement. In its more compromise normal cellular metabolic and biochemical function folded conformation, interaction of NFAT5 NTD with specific (5–8). Another known function of such osmolytes is to protect proteins is enhanced, including that with high mobility group proteins from denaturation by preserving their native structure in protein (HMGI-C), which has been shown to protect against the face of potentially denaturing conditions. The possibility that apoptosis. These findings suggest that in vivo, increased in- this function may apply in hyperosmotic stress has, to our knowl- tracellular ionic strength, coupled with osmolytes, may directly edge, not been investigated, nor has the possibility that high salt activate NFAT5. They encourage further pursuit of this possibility. alone can stabilize any intrinsically ordered (ID) region of Nuclear Author contributions: R.K., J.F.D., E.B.T., Y.H., M.B.B., and J.D.F. designed research; R.K., Factor of Activated T cells 5 (NFAT5). J.F.D., S.H.K., and Y.H. performed research; R.K., J.F.D., S.H.K., E.B.T., Y.H., and J.D.F. The osmosensitive TF NFAT5/TonEBP/OREBP plays a cru- contributed new reagents/analytic tools; R.K., J.F.D., S.H.K., E.B.T., Y.H., M.B.B., and cial role protecting cells against deleterious effects of hyper- J.D.F. analyzed data; and R.K., J.F.D., E.B.T., Y.H., M.B.B., and J.D.F. wrote the paper. osmotic stress upon urinary concentration, the adaptive immune Reviewers: P.K., City of Hope National Medical Center; S.S.S., National Institutes of Health; response, and other physiological systems, particularly in tissues and V.N.U., University of South Florida. that experience large fluctuations in tonicity, such as the renal The authors declare no competing interest. medulla (9–12). NFAT5 modulates cellular response to osmotic Published under the PNAS license. changes by enhancing the expression of target genes, including 1To whom correspondence may be addressed. Email: [email protected]. those responsible for synthesis and/or transport of multiple organic This article contains supporting information online at https://www.pnas.org/lookup/suppl/ osmolytes, such that the intracellular concentration of compatible doi:10.1073/pnas.1911680117/-/DCSupplemental. protective osmolytes is increased and compensates for increased

www.pnas.org/cgi/doi/10.1073/pnas.1911680117 PNAS Latest Articles | 1of6 Downloaded by guest on September 29, 2021 ID regions, hypothesizing that in the presence of appropriate Results concentrations of compatible osmolyte(s) or simple inorganic NFAT5 NTD Shows ID Characteristics. Secondary structural analysis salts, the ID NTD of NFAT5 would undergo disorder-to-order of the NFAT5 NTD (amino acids 2–220) predicted a large conformational rearrangements that would enhance protein–protein amount of sequence in random coil (ID) configuration (SI Ap- interactions. pendix, Supplementary Methods), with more than 75% of the In this study, we show our hypothesis to be correct: When NTD sequence random coil and only a small proportion as helix incubated with the natural organic osmolytes trehalose or sor- or sheet (SI Appendix, Fig. S1). We used several independent bitol, NFAT5 NTD changes from ID to more ordered conforma- predictors to analyze the ID nature of NFAT5 NTD. Analysis by tion, consistent with a two-state transition. In this folded conformation, IUPred showed ID regions in NFAT5 (SI Appendix, Fig. S2). We the interaction of NFAT5 NTD with specific proteins, e.g., high also applied ANCHOR, which predicts potential binding regions mobility group protein (HMGI-C), is significantly enhanced. within ID proteins. Such regions function by undergoing a We also found that ID NTD can adopt higher secondary/ter- disorder-to-order transition upon binding to a protein partner. tiary structure in the presence of NaCl. Sorbitol and NaCl are ANCHOR identifies segments in a generally disordered region known to work together to maintain cellular tonicity (6), and that cannot form enough favorable intrachain interactions to fold our data suggest that their structural effects are greater than the spontaneously and have the energetic capability to gain structure additive. These data show that NFAT5’sIDNTDcanbecome by interacting with a globular partner protein (SI Appendix, Fig. more structured in the presence of osmolytes and inorganic salt, S2). Analysis by three different versions of PONDR for ID pre- and that this enhances important protein–protein interactions. diction further strengthened the case for the ID nature of NFAT5 SI Appendix Such interactions are the natural function of TFs. These results NTD, evident from PONDR scores over 0.5 ( ,Fig. provide proof of principle of a physical basis for salt and S3). Further, computational analysis of ID predisposition of the osmolyte actions on NFAT5 structure and function. NFAT5 NTD was extended to include the CH-CDF plot method, which is based on the combined use of the Charge/Hydropathy Materials and Methods and CDF (Cumulative Distribution Function) binary disorder Bacterial Expression, Protein Purification, and Biophysical Analyses. Plasmid of predictors (SI Appendix, Figs. S4 and S5). In addition to providing human NFAT5 NTD (amino acids 2–220) in pET41b was transformed into plots showing ID predisposition of NTD, we utilized IUPRED HMS174 Escherichia coli competent cells. Recombinant protein was expressed and PONDR analyses to show the per-residue disorder predis- and purified to near homogeneity as described (SI Appendix, Supplemental position of the full-length NFAT5 protein (SI Appendix, Figs. S6 Methods). The far-ultraviolet (UV) and near-UV circular dichroism (CD) spectra and S7). These plots provide the overall disorder status of NFAT5 of the purified recombinant NFAT5 NTD protein were recorded at 22 °C on a protein. Together, these data support the belief that NFAT5 NTD Jasco 815 spectropolarimeter by using a 0.1-cm and 1.0-cm quartz cell, re- possesses characteristics of an ID protein. Solution biophysical spectively, as described (34–37). The spectra were recorded at a fixed protein data confirm this notion. concentration. All spectra recorded were corrected for the contribution of solute concentrations. Each spectrum is a result of five spectra accumulated, Trehalose Induces Secondary/Tertiary Structure in NFAT5 NTD. We averaged, and smoothed. Fluorescence emission spectra of protein in solution expressed and purified NFAT5 NTD protein to near homoge- were monitored at excitation wavelength of 278 or 295 nm as described (36, SI Appendix – 37). All measurements were made using 1-cm rectangular cuvettes at 22 °C, neity ( , Figs. S8 S12). The effects of increasing concentrations of trehalose on purified NFAT5 NTD were an- and all data were corrected for the contribution of the respective solute SI concentrations. alyzed using far-UV CD spectroscopy (Fig. 1A). As predicted ( Appendix, Figs. S1–S7), the spectrum of NFAT5 NTD in buffer Protein–Protein Interactions. Pulldowns were performed from HEK293 cell shows little secondary structural content, as evident from a lysates comprising a 1:1 mix of cytoplasm and membrane extracts or a 1:1 mix minimum around 200 nm in the CD spectrum (black circle; of soluble and chromatin bound nuclear extracts as prepared (SI Appendix, Fig. 1A). Increasing concentrations of trehalose (0.20–1.40 M) Supplemental Methods). After a detergent removal and buffer exchange as resulted in a concentration-dependent increase in minima described (20), NFAT5 NTD and trehalose (1.4 M) were added to the proteins around 220 nm, a pattern indicative of increased helical content (including a control without trehalose) and incubated overnight at 4 °C with (Fig. 1A). The trehalose-induced conformational transition in rotation. The samples were passed through a 0.22-μm filter and mixed with the NFAT5 NTD appears to be cooperative, from the sigmoidal preequilibrated Co2+ resin (GE Health Care Life Sciences) to purify NFAT5 curve of ellipticity at 220 nm (Φ220), suggestive of folding to a NTD and any associated proteins. The resin was first washed with 10 column natural configuration (Fig. 1B). Limited proteolytic digestions of volumes (CVs) of 25 mM NaPi, pH 7.4, 150 mM NaCl, 1 mM ethyl- NFAT5 NTD in the presence and absence of trehalose show that enediaminetetraacetic acid (EDTA), 20 mM imidazole buffer. The His-tag at 0.0 M trehalose, the protein is nearly completely digested NFAT5 NTD and associated proteins were eluted with 5 CVs of 25 mM ; SI Appendix NaPi, pH 7.4, 150 mM NaCl, 1 mM EDTA, 250 mM imidazole buffer. All (compare lanes 2 and 3 , Fig. S13), whereas it is fractions were first analyzed using 4–12% Bis-Tris gels (Life Tech) to deter- increasingly protected by 0.2–1.0 M trehalose (lanes 3–7). The mine the pulled down NFAT5 NTD and the associated proteins in the pres- effect appears to reach saturation beyond 1.0 M trehalose (lanes ence and absence of trehalose. Samples were buffer exchanged as described 7–9; SI Appendix, Fig. S13). This suggests that NFAT5 NTD has (20). Protease digestion was achieved by incubation at 37 °C overnight. The regions that have folded into a tertiary structure that secludes the digested supernatant was removed to a clean protein LoBind tube and ad- residues attacked by the protease to positions not easily reached. justed to pH 3, using 1% trifluoracetic acid (TFA), and concentrated via To acquire further evidence for tertiary structure occurring in SpeedVac before being resuspended in 50 μL of 1% TFA. The tryptic peptides NFAT5 NTD, we recorded the near-UV CD spectra of this were then desalted, dried, and resuspended in 0.1% formic acid for liquid protein in the presence and absence of 1 M NaCl, sorbitol, or – chromatography mass spectrometry (LC-MS/MS) analysis as described (20). trehalose (SI Appendix, Fig. S14). The spectra show changes typical of those arising from adding differing solutes. Such LC-MS/MS Analysis and Protein Identification. Tryptic peptides were analyzed changes may arise from movement of residues from polar to on an Orbitrap Elite mass spectrometer as described (20). Briefly, samples were purified using an Agilent Zorbax 300SB-C18 trap column in a buffer more hydrophobic environments in the protein. These data containing 0.1% formic acid in water at a flow rate of 6 μL/min for 10 min suggest that trehalose induces, on average, a compact tertiary (20). Trapped peptides were separated using reversed-phase C18 column structural arrangement in NFAT5 NTD. followed by MS analysis as described (20). The Mascot search algorithm and relative protein abundance were used to identify proteins from the raw MS Trehalose Causes the NFAT5 NTD Protein to Fold into a Conformation data, and peptides were searched against a target-decoy protein (Swiss-Prot that Interacts with Specific Proteins. To monitor the environment human) as described (20). around the Trp and Tyr residues of the NFAT5 NTD, we

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1911680117 Kumar et al. Downloaded by guest on September 29, 2021 A osmo-stressed cell nuclear extracts, the MS data showed that 0 trehalose facilitates interaction of NFAT5 NTD with high mobility group protein (HMGI-C) and other proteins (Fig. 3). Of 0.0M -5 note, high mobility group protein HMGI-C/HMGA-2 was pre- 0.2M sent in bioreplicates of nuclear proteins plus NFAT5 NTD when 0.4M trehalose was present (Fig. 3C). HMGI-C has previously been -10 0.6M shown to be present in N-terminal NFAT5 peptide affinity 0.8M pulldown from nuclear extracts (20, 22). -15 1.0M 1.2M NaCl and Sorbitol Produce Independent, Greater than Additive Effects

Ellipticity [mdeg] Ellipticity 1.4M to Fold NFAT5 NTD Protein. -20 Since many cells adapt to an increase in intracellular ionic strength by accumulating sorbitol, we deter- 200 220 240 260 Wavelength [nm] mined whether exposure to NaCl or sorbitol can cause folding of B 6.5 ID NFAT5 NTD. In concentrations of NaCl up to 2.0 M, NFAT5 NTD shows an increased level of secondary structural

) 6.0 elements, as evident from a dip in the CD spectrum around T 5.5 220 nm (Fig. 4A). Similar results were observed in the presence of sorbitol (Fig. 4B). Thus, at higher concentrations of either 5.0 NaCl or sorbitol, ID NFAT5 NTD adopts, on average, a more 4.5 ordered conformation. Further, in 1 M NaCl or sorbitol alone, the degree of NFAT5 NTD structure is similar (Fig. 4C).

Ellipticity ( Ellipticity 4.0 T However, when both solutes are present, the level of secondary 3.5 structural elements appears to be higher (Fig. 4C). A comparison of the theoretical sum of the change due to the two treatments 0.0 0.4 0.8 1.2 singly to experimental CD spectra collected from a mixture of Trehalose [M] both solutes present together indicates that the effects of NaCl and sorbitol on the folding of NFAT5 NTD protein appear to be Fig. 1. Presence of trehalose induces secondary structure in ID NFAT5 NTD. greater than additive (Fig. 4D). Interactive effects of mixed (A) Far-UV CD spectra of NFAT5 NTD protein in the absence and presence of osmolytes have been previously explored (40). PHYSIOLOGY increasing concentrations of trehalose [0.0–1.4 M]. Each spectrum represents an average of three spectra recorded, corrected for the contribution of the Discussion buffer, and smoothed. (B) Trehalose-induced conformational transition of Hypertonicity increases NFAT5 expression and activates it, NFAT5 NTD as assessed by increase in ellipticity at 220-nm wavelength. resulting in NFAT5-dependent expression of target genes important for cell survival, such as aldose reductase, BGT1, SMIT, HSP70, and TAUT (41–51). In addition to its osmoadaptive employed inherent fluorescence emission, with excitation at responses in the kidney, NFAT5 also regulates the expression of 278 nm to monitor both amino acids and at 295 nm to monitor SI Appendix the urea transporter, aquaporins and serum- and glucocorticoid- Trp only ( , Figs. S15 and S16). In both sets of spectra, inducible kinase (Sgk1). NFAT5 is also expressed in many the quantum yield of the fluorescence significantly increases with respect to increasing concentrations of trehalose (SI Appendix, Figs. S15 and S16). Trp residues buried in nonpolar regions of the protein can be followed at 329 nm (38, 39). Our results based A C 1.0 0.9 on emission at 329 nm (Fig. 2 C and D) showed that in the F/F0 F/F max 0329nm presence of trehalose, Trp may be buried, strongly suggestive of 0.9 0.8 acquired tertiary structure. There was a similar trend for the shift 0.8 0.7 in fluorescence emission maximum after excitation at 278 or 0.7

ac @ 278 nm 295 nm in the presence of trehalose (Fig. 2 A and B). These @ 278 nm 0.6 0 0 fluorescence emission studies, together with the CD data, thus 0.6 F/F indicate that the trehalose causes, on average in the conformer F/F 0.5 0.5 ensemble, secondary and tertiary structures to form coopera- 0.0 0.4 0.8 1.2 0.0 0.4 0.8 1.2 tively in NFAT5 NTD. We estimated the degree of exposure of Trehalose [M] Trehalose [M] Trp residues to solvent in native NFAT5 NTD by measuring the B D efficiency with which their fluorescence was quenched by acryl- 0.70 1.0 F/F F/F 0329nm amide, a dynamic quencher of Trp fluorescence. The fluores- 0max 0.9 0.65 cence emission spectra of NFAT5 NTD recorded in the absence 0.60 and presence of increasing concentrations of acrylamide showed 0.8 0.55 that the fluorescence intensity for the Trp maximum was sig- 0.7 b d @ 295 nm @ 295 nm 0.50 0 nificantly reduced (≥40%) as sufficient acrylamide was added (SI 0 0.6 Appendix 0.45

, Fig. S17), suggesting that without osmolyte, the Trp F/F

F/F 0.5 residues in NFAT5 NTD are readily accessible. 0.40 0.0 0.4 0.8 1.2 The NFAT5 NTD is known to interact with several cellular 0.0 0.4 0.8 1.2 proteins important for NFAT5-mediated transcriptional activity. Trehalose [M] Trehalose [M] Since disorder-order transition in ID regions often facilitates their interactions with specific proteins, we tested for such in- Fig. 2. (A and B) Reversible trehalose-induced conformational transition of NFAT5 NTD monitored at maximum wavelength (F/F0 max) at an excitation teractions of NFAT5 NTD in nuclear and cytoplasmic extracts of wavelength of 278 and 295 nm, respectively. (C and D) Reversible trehalose- HEK293 cells by use of peptide affinity chromatography fol- induced conformational transition of NFAT5 NTD monitored at 329-nm – lowed by sodium dodecyl sulfate polyacrylamide gel electro- emission wavelength (F/F0 329 nm) after exciting at a wavelength of 278 and phoresis (Fig. 3 A and B) and mass spectrometry (Fig. 3C). In 295 nm, respectively.

Kumar et al. PNAS Latest Articles | 3of6 Downloaded by guest on September 29, 2021 A B results demonstrate that the ID NTD of NFAT5 undergoes such a transition in the presence of natural osmolytes, trehalose and sorbitol, or even simply high NaCl. The presence of AD1 doubles the hyperosmotic response of AD2 or AD2AD3-containing constructs. This effect is significant because physiologic activation of NFAT5 regulates the expression of genes that can synthesize cellular osmolytes in response to tonicity. These findings therefore suggest a mechanism for controlling the synthesis of organic osmolytes by the allosteric response of NFAT5 to the cellular osmotic state. Since NFAT5-mediated regulation of NaCl (ionic strength) C and sorbitol (osmolyte) are involved in counterbalancing cellular Protein N C N+T C+T hypertonicity in the renal medulla and possibly in other cells/ tissues, we tested whether these two solutes could work in con- NFAT5 junction to maintain the disorder-order transition in NTD con- Prothymosin alpha formation. Our biophysical data showing the folding of the ID NTD in the presence of each solute alone strongly suggests that Granulins these specific solutes may be involved in providing structural stability to ID NFAT5 protein, specifically its NTD. Further, the PEST proteolyc signal- combined effects of these solutes suggest that under hypertonic containing nuclear protein and even physiological conditions, the activity of NFAT5 may be regulated by the fine-tuning response to concentrations of NaCl High mobility group protein and sorbitol. HMGI-C Due to their structural flexibility, the ID regions of many TFs play important roles in their biological functions. In the large Fig. 3. HEK293 cells under osmotic stress (500 mOsm NaCl), followed by ensembles of transient structures that compose ID regions are nuclear (N) and cytoplasmic (C) protein extraction and addition of N-term – (amino acids 2–220) NFAT5, incubated both with and without trehalose (T) some that facilitate multiple, specific protein protein interac- and His-tag purification, using a Co2+ column. (A) Nuclear extraction. (B) tions with coregulatory proteins. These cofactors are critical for Cytoplasmic extraction. Lane 1: MW marker. Lane 2: Flow through. Lane 3: initiation and maintenance of transcriptional activity (27, 63–66). 20 mM imidazole wash. Lane 4: 250 mM imidazole elution. Lane 5: Flow To investigate whether increased natural structure in its NTD through + 1.4 M trehalose. Lane 6: 20 mM imidazole wash + 1.4 M trehalose. affects relevant cellular protein–protein interactions, we looked Lane 7: 250 mM imidazole elution + 1.4 M trehalose. Lane 8: bovine serum for a correlation between change in NFAT5 NTD structure and albumin std, 0.1 mg/mL. (C) In-solution digestion followed by LC-MS/MS determination of proteins present in each extraction (Nuclear ± 1.4 M tre- association with other proteins. MS identified several NFAT5 halose, Cytoplasmic ± 1.4 M trehalose). NTD protein binding partners. Among these, HMGI-C was present only when NFAT5 NTD was incubated with nuclear

nonrenal tissues or cells in which osmoadaptation is less critical under physiological conditions (52–57). Such protection could A B become important in extreme conditions, e.g., severe dehydra- 0 0 tion. Hypertonic activation of NFAT5 also increases the ex- -10 pression of target genes that synthesize or transport multiple -10 0.0M 0.0M organic osmolytes (12). Certain organic osmolytes are known to 0.1M 0.1M -20 0.5M -20 0.5M act as chemical chaperones to fold/stabilize proteins including ID 1.0M 1.0M regions of many transcription factors (58–61). NFAT5 contains -30 NaCl 2.0M -30 Sorbitol 2.0M Ellipticity [m.deg] Ellipticity [mdeg] multiple regions predicted to be ID, including its NTD and CTD, 200 220 240 260 200 220 240 260 both important for NFAT5 function. The NTD contains an Wavelength [nm] Wavelength [nm] auxiliary export region, involved in nuclear and cytoplasmic locali- C D

zation, a monopartite nuclear localization signal, and transcription 0 0 activation function AD1 (20, 62). The CTD contains transactivation domains AD2 and AD3 (18, 19, 22). Since several predictors of -10 -10 protein structure agreed that all these domains are ID, we tested -20 0.0M -20 the hypothesis that protective organic osmolytes and/or NaCl could 1.0M N N+S (1.0M) Theo 1.0M S promote their folding. To the best of our knowledge, the existence -30 1.0M N+1.0M S -30 N+S (1.0M) Exp Ellipticity [m.deg] Ellipticity

of these ID regions and their capacity to take on secondary and [m.degree] Ellipticity tertiary structure has not been experimentally validated. 200 210 220 230 240 250 260 200 210 220 230 240 250 260 We selected the NFAT5 NTD for the present study, as proof Wavelength [nm] Wavelength [nm] of principle. Our biophysical analyses provide physical proof that Fig. 4. Incubation of NFAT5 NTD with NaCl (N) or sorbitol (S) induces sec- the NFAT5 NTD is ID. CD, fluorescence emission, and limited ondary structure in ID NFAT5 NTD. Far-UV CD spectra of NFAT5 NTD protein proteolytic digestion data clearly demonstrate that in aqueous in the absence and presence of increasing concentrations of NaCl (A)and solution, the NTD possesses little or no secondary/tertiary struc- sorbitol (B), [0.0–2.0 M], respectively. Each spectrum represents an average ture. We further show that protective osmolytes often found in of three spectra recorded, corrected for the contribution of the buffer, and cells, as well as NaCl, can cause the protein to fold. smoothed. (C) Far-UV CD spectra of NFAT5 NTD protein in buffer (black), 1.0 M NaCl (red), 1.0 M sorbitol, and 1.0 M NaCl + 1.0 M sorbitol. (D) Far UV It has been reported that the ID regions of several TF proteins CD spectra (black) showing the average of individually recorded NFAT5 NTD undergo disorder-order conformational transition in the pres- in the presence of 1.0 M NaCl and 1.0 M sorbitol (theoretical sum [theo]), and ence of organic osmolytes, such that an ordered conformation average when recorded in the presence of 1.0 M NaCl + 1.0 M sorbitol to- with expected function is acquired in the protein (23, 24). Our gether (experimental [exp]).

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1911680117 Kumar et al. Downloaded by guest on September 29, 2021 extracts in the presence of osmolyte. We thus have demonstrated the NTD is unstructured in the full-length NFAT5 protein and that the increase in NFAT5 structure caused by osmolyte en- how its ID/folded domains interact in vivo. hances physiologically relevant protein–protein interactions. Un- In sum, we have demonstrated that, in solution: the NFAT5 compensated osmotic stress damages DNA, interrupts cellular NTD is ID; protective natural osmolytes or NaCl increase its proliferation, and kills cells (3, 12). This is prevented by accu- secondary and tertiary structure and salt plus osmolyte effects mulation of compatible organic osmolytes, whose function is are additive (or greater than additive); the dose–response curves shared by trehalose. Our finding that trehalose induces association of NFAT5 to these agents suggests a two-state transition (with- of NFAT5 with HMGI-C contributes to explaining the association out excluding the possibility of intermediate transient states), a since HMGI-C suppresses apoptosis (67, 68) and is involved with – hallmark of a switch from disorder to ordered structure; the proliferation of cancer cells (68 70). more structured ensemble of NFAT5 isomers enhanced binding It has been demonstrated thermodynamically that in multido- of HMGI-C, a physiologically relevant protein. The degree of main proteins, the presence of ID domains enhances the allosteric dynamic behavior in proteins ranges widely, from slightly varying response of binding (e.g., a protein or DNA). The quantitative nature of interdomain coupling in holoNFAT5 will determine the interactions between well-ordered subunits to complete ID. This extent to which this occurs (29, 63). The allosteric model, which gives the opportunity for varying regional and quantitative explains why ID enhances allosteric responses, shows that inter- changes in structure to have important discriminatory effects on – domain coupling can enhance or repress folding of other domains, protein function (71, 72, 75, 76). A few other stress response thus altering their function. This has been verified experimentally transcription factors are believed to respond directly to the stress for a two-domain version of the GR/NR3C1 (37). Even a brief itself or its immediate products, e.g., the responders to heat consideration of the implications of the ensemble allosteric model shock, unfolded proteins in the ER, and oxidative stress (77–80). leads to the conclusion that in NFAT5, with its three separate, Our results suggest a physical mechanism by which NFAT5 re- interactive, ID activation domains the coupling responses will be sponds to hypertonic stress by direct structural reactions to salt complex (71, 72). However, based on observations with many and osmolytes. other proteins (73, 74), it is likely that the NFAT NTD and CTD exist in an ID conformation in the full-length protein. Extensive Data Availability. All study data are included in the article and future studies will be required to determine the degree to which SI Appendix.

1. E. K. Hoffmann, I. H. Lambert, S. F. Pedersen, Physiology of cell volume regulation in 20. J. F. DuMond et al., Peptide affinity analysis of proteins that bind to an unstructured PHYSIOLOGY vertebrates. Physiol. Rev. 89, 193–277 (2009). NH2-terminal region of the osmoprotective transcription factor NFAT5. Physiol. Ge- 2. F. Lang et al., Functional significance of cell volume regulatory mechanisms. Physiol. nomics 48, 290–305 (2016). Rev. 78, 247–306 (1998). 21. J. F. DuMond, Y. He, M. B. Burg, J. D. Ferraris, Expression, fermentation and purifi- 3. N. I. Dmitrieva, Q. Cai, M. B. Burg, Cells adapted to high NaCl have many DNA breaks cation of a predicted intrinsically disordered region of the transcription factor, and impaired DNA repair both in cell culture and in vivo. Proc. Natl. Acad. Sci. U.S.A. NFAT5. Protein Expr. Purif. 115, 141–145 (2015). 101, 2317–2322 (2004). 22. J. F. Dumond et al., Peptide affinity analysis of proteins that bind to an unstructured 4. N. I. Dmitrieva, L. F. Michea, G. M. Rocha, M. B. Burg, Cell cycle delay and apoptosis in region containing the transactivating domain of the osmoprotective transcription – response to osmotic stress. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 130, factor NFAT5. Physiol. Genomics 48, 835 849 (2016). 23. H. J. Dyson, P. E. Wright, Intrinsically unstructured proteins and their functions. Nat. 411–420 (2001). Rev. Mol. Cell Biol. 6, 197–208 (2005). 5. P. H. Yancey, M. E. Clark, S. C. Hand, R. D. Bowlus, G. N. Somero, Living with water 24. P. E. Wright, H. J. Dyson, Intrinsically disordered proteins in cellular signalling and stress: Evolution of osmolyte systems. Science 217, 1214–1222 (1982). regulation. Nat. Rev. Mol. Cell Biol. 16,18–29 (2015). 6. M. Gallazzini, J. D. Ferraris, M. Kunin, R. G. Morris, M. B. Burg, Neuropathy target 25. R. Kumar, I. J. McEwan, Allosteric modulators of steroid hormone receptors: Struc- esterase catalyzes osmoprotective renal synthesis of glycerophosphocholine in re- tural dynamics and gene regulation. Endocr. Rev. 33, 271–299 (2012). sponse to high NaCl. Proc. Natl. Acad. Sci. U.S.A. 103, 15260–15265 (2006). 26. S. H. Khan, R. Kumar, An overview of the importance of conformational flexibility in 7. A. Garcia-Perez, M. B. Burg, Renal medullary organic osmolytes. Physiol. Rev. 71, gene regulation by the transcription factors. J. Biophys. 2009, 210485 (2009). – 1081 1115 (1991). 27. A. S. Garza, N. Ahmad, R. Kumar, Role of intrinsically disordered protein regions/ 8. M. B. Burg, E. D. Kwon, D. Kültz, Regulation of gene expression by hypertonicity. domains in transcriptional regulation. Life Sci. 84 , 189–193 (2009). – Annu. Rev. Physiol. 59, 437 455 (1997). 28. A. M. Garza, S. H. Khan, R. Kumar, Site-specific phosphorylation induces functionally 9. C. Lopez-Rodríguez, J. Aramburu, A. S. Rakeman, A. Rao, NFAT5, a constitutively active conformation in the intrinsically disordered N-terminal activation function nuclear NFAT protein that does not cooperate with Fos and Jun. Proc. Natl. Acad. Sci. (AF1) domain of the glucocorticoid receptor. Mol. Cell. Biol. 30, 220–230 (2010). U.S.A. 96, 7214–7219 (1999). 29. V. J. Hilser, E. B. Thompson, Intrinsic disorder as a mechanism to optimize allosteric 10. H. Miyakawa, S. K. Woo, S. C. Dahl, J. S. Handler, H. M. Kwon, Tonicity-responsive coupling in proteins. Proc. Natl. Acad. Sci. U.S.A. 104, 8311–8315 (2007). enhancer binding protein, a rel-like protein that stimulates transcription in response 30. S. H. Khan et al., Binding of the N-terminal region of coactivator TIF2 to the intrin- to hypertonicity. Proc. Natl. Acad. Sci. U.S.A. 96, 2538–2542 (1999). sically disordered AF1 domain of the glucocorticoid receptor is accompanied by 11. B. C. Ko, C. W. Turck, K. W. Lee, Y. Yang, S. S. Chung, Purification, identification, and conformational reorganizations. J. Biol. Chem. 287, 44546–44560 (2012). characterization of an osmotic response element binding protein. Biochem. Biophys. 31. R. Kumar et al., Regulation of the structurally dynamic N-terminal domain of pro- Res. Commun. 270,52–61 (2000). gesterone receptor by protein-induced folding. J. Biol. Chem. 288, 30285–30299 12. M. B. Burg, J. D. Ferraris, N. I. Dmitrieva, Cellular response to hyperosmotic stresses. (2013). Physiol. Rev. 87, 1441–1474 (2007). 32. S. H. Khan, J. A. Arnott, R. Kumar, Naturally occurring osmolyte, trehalose induces 13. M. B. Burg, J. D. Ferraris, Intracellular organic osmolytes: Function and regulation. functional conformation in an intrinsically disordered activation domain of gluco- J. Biol. Chem. 283, 7309–7313 (2008). corticoid receptor. PLoS One 6, e19689 (2011). 14. J. D. Ferraris et al., ORE, a eukaryotic minimal essential osmotic response element. The 33. R. Kumar, G. Litwack, Structural and functional relationships of the steroid hormone receptors’ N-terminal transactivation domain. Steroids 74, 877–883 (2009). aldose reductase gene in hyperosmotic stress. J. Biol. Chem. 271, 18318–18321 (1996). 34. I. V. Baskakov et al., Trimethylamine N-oxide-induced cooperative folding of an in- 15. B. C. Ko, B. Ruepp, K. M. Bohren, K. H. Gabbay, S. S. Chung, Identification and trinsically unfolded transcription-activating fragment of human glucocorticoid re- characterization of multiple osmotic response sequences in the human aldose receptor. J. Biol. Chem. 274, 10693–10696 (1999). ductase gene. J. Biol. Chem. 272, 16431–16437 (1997). 35. A. S. Garza, S. H. Khan, C. M. Moure, D. P. Edwards, R. Kumar, Binding-folding in- 16. C. Y. Cheung, B. C. Ko, NFAT5 in cellular adaptation to hypertonic stress–Regulations duced regulation of AF1 transactivation domain of the glucocorticoid receptor by a and functional significance. J. Mol. Signal. 8, 5 (2013). cofactor that binds to its DNA binding domain. PLoS One 6, e25875 (2011). 17. Y. Izumi et al., Mutations that reduce its specific DNA binding inhibit high NaCl- 36. R. Kumar et al., Interdomain signaling in a two-domain fragment of the human induced nuclear localization of the osmoprotective transcription factor NFAT5. Am. glucocorticoid receptor. J. Biol. Chem. 274, 24737–24741 (1999). – J. Physiol. Cell Physiol. 303, C1061 C1069 (2012). 37. R. Kumar, J. C. Lee, D. W. Bolen, E. B. Thompson, The conformation of the gluco- 18. S. D. Lee, E. Colla, M. R. Sheen, K. Y. Na, H. M. Kwon, Multiple domains of TonEBP corticoid receptor af1/tau1 domain induced by osmolyte binds co-regulatory proteins. cooperate to stimulate transcription in response to hypertonicity. J. Biol. Chem. 278, J. Biol. Chem. 276, 18146–18152 (2001). 47571–47577 (2003). 38. T. L. Bushueva, A. G. Tonevitsky, The effect of pH on the conformation and stability of 19. J. D. Ferraris et al., Activity of the TonEBP/OREBP transactivation domain varies di- the structure of plant toxin-ricin. FEBS Lett. 215, 155–159 (1987). rectly with extracellular NaCl concentration. Proc. Natl. Acad. Sci. U.S.A. 99, 739–744 39. K. Watanabe, E. Honjo, T. Tsukamoto, G. Funatsu, Fluorescence studies on the in- (2002). teraction of adenine with ricin A-chain. FEBS Lett. 304, 249–251 (1992).

Kumar et al. PNAS Latest Articles | 5of6 Downloaded by guest on September 29, 2021 40. L. M. Holthauzen, D. W. Bolen, Mixed osmolytes: The degree to which one osmolyte 60. F. D. Rumjanek, Osmolyte induced tumorigenesis and metastasis: Interactions with affects the protein stabilizing ability of another. Protein Sci. 16, 293–298 (2007). intrinsically disordered proteins. Front. Oncol. 8, 353 (2018). 41. C. López-Rodríguez et al., Loss of NFAT5 results in renal atrophy and lack of tonicity- 61. A. L. Mansouri et al., Folding propensity of intrinsically disordered proteins by osmotic responsive gene expression. Proc. Natl. Acad. Sci. U.S.A. 101, 2392–2397 (2004). stress. Mol. Biosyst. 12, 3695–3701 (2016). 42. H. Miyakawa et al., Cis- and trans-acting factors regulating transcription of the BGT1 62. E. H. Tong et al., Regulation of nucleocytoplasmic trafficking of transcription factor – gene in response to hypertonicity. Am. J. Physiol. 274, F753 F761 (1998). OREBP/TonEBP/NFAT5. J. Biol. Chem. 281, 23870–23879 (2006). 43. K. Y. Na, S. K. Woo, S. D. Lee, H. M. Kwon, Silencing of TonEBP/NFAT5 transcriptional 63. J. Li, V. J. Hilser, Assessing allostery in intrinsically disordered proteins with ensemble – activator by RNA interference. J. Am. Soc. Nephrol. 14, 283 288 (2003). allostery model. Methods Enzymol. 611, 531–557 (2018). 44. T. Ito et al., Expression of taurine transporter is regulated through the TonE 64. J. Liu et al., Intrinsic disorder in transcription factors. Biochemistry 45, 6873–6888 (tonicity-responsive element)/TonEBP (TonE-binding protein) pathway and contrib- (2006). – utes to cytoprotection in HepG2 cells. Biochem. J. 382, 177 182 (2004). 65. V. Narayan et al., A multiprotein binding interface in an intrinsically disordered re- 45. S. K. Woo, S. D. Lee, K. Y. Na, W. K. Park, H. M. Kwon, TonEBP/NFAT5 stimulates gion of the tumor suppressor protein interferon regulatory factor-1. J. Biol. Chem. transcription of HSP70 in response to hypertonicity. Mol. Cell. Biol. 22, 5753–5760 286, 14291–14303 (2011). (2002). 66. K. Sugase, H. J. Dyson, P. E. Wright, Mechanism of coupled folding and binding of an 46. J. Lakshmipathi et al., Identification of NFAT5 as a transcriptional regulator of the intrinsically disordered protein. Nature 447, 1021–1025 (2007). EDN1 gene in collecting duct. Am. J. Physiol. Renal Physiol. 316, F481–F487 (2019). 67. R. Fujikane, K. Komori, M. Sekiguchi, M. Hidaka, Function of high-mobility group A 47. W. Y. Go, X. Liu, M. A. Roti, F. Liu, S. N. Ho, NFAT5/TonEBP mutant mice define osmotic stress as a critical feature of the lymphoid microenvironment. Proc. Natl. Acad. proteins in the DNA damage signaling for the induction of apoptosis. Sci. Rep. 6, Sci. U.S.A. 101, 10673–10678 (2004). 31714 (2016). 48. R. Reeves, Nuclear functions of the HMG proteins. Biochim. Biophys. Acta 1799,3–14 68. J. Sun, Y. Qiao, T. Song, H. Wang, MiR-495 suppresses cell proliferation by directly – (2010). targeting HMGA2 in lung cancer. Mol. Med. Rep. 19, 1463 1470 (2019). 49. A. Garcia-Perez et al., Molecular cloning of cDNA coding for kidney aldose reductase. 69. I. El Ayachi et al., The WNT10B network is associated with survival and metastases in Regulation of specific mRNA accumulation by NaCl-mediated osmotic stress. J. Biol. chemoresistant triple-negative breast cancer. Cancer Res. 79, 982–993 (2019). Chem. 264, 16815–16821 (1989). 70. Q. Zou et al., RKIP suppresses the proliferation and metastasis of breast cancer cell 50. D. Sheikh-Hamad, A. García-Pérez, J. D. Ferraris, E. M. Peters, M. B. Burg, Induction of lines through up-regulation of miR-185 targeting HMGA2. Arch. Biochem. Biophys. gene expression by heat shock versus osmotic stress. Am. J. Physiol. 267, F28–F34 610,25–32 (2016). (1994). 71. H. N. Motlagh, J. O. Wrabl, J. Li, V. J. Hilser, The ensemble nature of allostery. Nature 51. J. D. Ferraris, M. B. Burg, C. K. Williams, E. M. Peters, A. García-Pérez, Betaine trans- 508, 331–339 (2014). porter cDNA cloning and effect of osmolytes on its mRNA induction. Am. J. Physiol. 72. H. N. Motlagh, J. Li, E. B. Thompson, V. J. Hilser, Interplay between allostery and in- 270, C650–C654 (1996). trinsic disorder in an ensemble. Biochem. Soc. Trans. 40, 975–980 (2012). 52. Y. Nakayama, T. Peng, J. M. Sands, S. M. Bagnasco, The TonE/TonEBP pathway me- 73. B. Xue, C. J. Brown, A. K. Dunker, V. N. Uversky, Intrinsically disordered regions of p53 diates tonicity-responsive regulation of UT-A urea transporter expression. J. Biol. family are highly diversified in evolution. Biochim. Biophys. Acta 1834, 725–738 – Chem. 275, 38275 38280 (2000). (2013). 53. M. A. Lanaspa et al., The expression of aquaporin-1 in the medulla of the kidney is 74. I. M. Kuznetsova, O. I. Povarova, V. N. Uversky, K. K. Turoverov, Native globular actin dependent on the transcription factor associated with hypertonicity, TonEBP. J. Biol. has a thermodynamically unstable quasi‐stationary structure with elements of in- Chem. 285, 31694–31703 (2010). trinsic disorder. FEBS J. 283, 438–445 (2016). 54. A. K. M. Lam et al., Osmotic response element-binding protein (OREBP) is an essential 75. V. N. Uversky, p53 proteoforms and intrinsic disorder: An illustration of the protein regulator of the urine concentrating mechanism. J. Biol. Chem. 279, 48048–48054 structure-function continuum concept. Int. J. Mol. Sci. 17, 1874 (2016). (2004). 76. P. Kulkarni et al., Phosphorylation-induced conformational dynamics in an intrinsi- 55. U. Hasler et al., Tonicity-responsive enhancer binding protein is an essential regulator cally disordered protein and potential role in phenotypic heterogeneity. Proc. Natl. of aquaporin-2 expression in renal collecting duct principal cells. J. Am. Soc. Nephrol. – 17, 1521–1531 (2006). Acad. Sci. U.S.A. 114, E2644 E2653 (2017). 56. S. Chen et al., Tonicity-dependent induction of Sgk1 expression has a potential role in 77. S. Chen, X. Wang, M. F. Nisar, M. Lin, J. L. Zhong, Heme oxygenases: Cellular multi- dehydration-induced natriuresis in rodents. J. Clin. Invest. 119, 1647–1658 (2009). functional and protective molecules against UV-induced oxidative stress. Oxid. Med. 57. J. Trama, Q. Lu, R. G. Hawley, S. N. Ho, The NFAT-related protein NFATL1 (TonEBP/ Cell. Longev. 2019, 5416728 (2019). NFAT5) is induced upon T cell activation in a calcineurin-dependent manner. 78. M. Valera-Alberni, C. Canto, Mitochondrial stress management: A dynamic journey. J. Immunol. 165, 4884–4894 (2000). Cell Stress 2, 253–274 (2018). 58. S. H. Khan, N. Ahmad, F. Ahmad, R. Kumar, Naturally occurring organic osmolytes: 79. K. Dokladny, O. B. Myers, P. L. Moseley, Heat shock response and autophagy– From cell physiology to disease prevention. IUBMB Life 62, 891–895 (2010). Cooperation and control. Autophagy 11, 200–213 (2015). 59. R. Kumar, Role of naturally occurring osmolytes in protein folding and stability. Arch. 80. H. O. Rashid, R. K. Yadav, H. R. Kim, H. J. Chae, ER stress: Autophagy induction, in- Biochem. Biophys. 491,1–6 (2009). hibition and selection. Autophagy 11, 1956–1977 (2015).

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