Phosphorylation of cytochrome c at tyrosine 48 finely regulates its binding to the histone chaperone SET/TAF-Iβ in the nucleus

RESEARCH ARTICLE Phosphorylation of cytochrome cat tyrosine 48 finely regulates its binding to the histone chaperone SET/TAF-Iβ in the nucleus Joaquin Tamargo-Azpilicueta 1 | Miguel ´ A. Casado-Combreras 1 | Rafael L. Giner-Arroyo 1 | Adri an Vel azquez-Campoy 2,3,4,5 | Inmaculada M arquez 6 | José L. Olloqui-Sariego 6 | Miguel A. De la Rosa 1 | Irene Diaz-Moreno 1 1 Institute for Chemical Research (IIQ), Scientific Research Center “Isla de la Cartuja”(cicCartuja), University of Seville –CSIC, Seville, Spain 2 Institute for Biocomputation and Physic of Complex Systems (BIFI), Joint Unit GBsC-CSIC-BIFI, University of Zaragoza, Zaragoza, Spain 3 Department of Biochemistry and Molecular and Cellular Biology, University of Zaragoza, Zaragoza, Spain 4 Institute for Health Research Arag on (IIS Aragon), Zaragoza, Spain 5 Centre for Biomedical Research Network of Hepatic and Digestive Diseases (CIBERehd), Madrid, Spain 6 Department of Physical-Chemistry, University of Seville, Seville, Spain Correspondence Irene Diaz-Moreno, Institute for Chemical Research (IIQ), Scientific Research Center “Isla de la Cartuja”(cicCartuja), University of Seville –CSIC, C. Américo Vespucio, 49 (cicCartuja), 41092 Seville, Spain. Email: [email protected] Funding information MICIU/AEI/10.13039/501100011033 and by ERDF/EU, Grant/Award Numbers: PID2021-126663NB-I00, 10.13039/501100011033; Junta de Andalucía, Grant/Award Number: BIO- 198; Fundaci on Ram on Areces Review Editor: Nir Ben-Tal Abstract Post-translational modifications (PTMs) of proteins are ubiquitous processes present in all life kingdoms, involved in the regulation of protein stability, subcellular location and activity. In this context, cytochrome c(Cc) is an excellent case study to analyze the structural and functional changes induced by PTMS as Ccis a small, moonlighting protein playing different roles in different cell compartments at different cell-cycle stages. Ccis actually a key component of the mitochondrial electron transport chain (ETC) under homeostatic conditions but is translocated to the cytoplasm and even the nucleus under apoptotic conditions and/or DNA damage. Phosphorylation does specifically alter the Cc redox activity in the mitochondria and the Ccnon-redox interaction with apoptosis-related targets in the cytoplasm. However, little is known on how phosphorylation alters the interaction of Ccwith histone chaperones in the nucleus. Here, we report the effect of CcTyr48 phosphorylation by examining the protein interaction with SET/TAF-Iβin the nuclear compartment using a combination of molecular dynamics simulations, biophysical and structural approaches such as isothermal titration calorimetry (ITC) and nuclear magnetic resonance (NMR) and in cell proximity ligation assays. From these experiments, we infer that Tyr48 phosphorylation allows a fine-tuning of the Cc-mediated inhibition of SET/TAF-Iβhistone chaperone activity in vitro. Received: 18 June 2024 Revised: 9 October 2024 Accepted: 22 October 2024 DOI: 10.1002/pro.5213 This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2024 The Author(s). Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society. Protein Science. 2024;33:e5213. wileyonlinelibrary.com/journal/pro 1of18 https://doi.org/10.1002/pro.5213 Our findings likewise reveal that phosphorylation impacts the nuclear, stressresponsive functions of Cc, and provide an experimental framework to explore novel aspects of Ccpost-translational regulation in the nucleus. KEYWORDS cytochrome c, DNA damage response, gene code expansion, histone chaperones, nuclear magnetic resonance, phosphorylation, post-translational modifications, protein–protein interaction, SET/TAF-Iβ 1|INTRODUCTION Tissue homeostasis and normal cell physiology rely on a plethora of interconnected, dynamic mechanisms that are activated in response to endogenous (e.g., oxidative stress, DNA replication stress) and exogenous (e.g., ionizing radiation, oxygen–glucose deprivation) sources of stress (Galluzzi et al. 2018). Most signaling factors participating in these pathways undergo post-translational modifications (PTMs) that are fundamental to modulate their structure, activity, and localization (Dunphy et al. 2021). Mitochondria, essential organelles for the production of energy in eukaryotic cells, act as hubs of many of the stress response pathways by (i) producing biosynthetic intermediates and free radicals that act as signaling molecules in the cytosol and nuclei; (ii) sensing and initiating the response to protein misfolding or mistargeting; and (iii) regulating the release of pleiotropic proteins that target the cytoplasm or the nucleus (Bohovych and Khalimonchuk 2016). In this context, cytochrome c(Cc) stands out as a multifunctional factor that (i) acts as a soluble electron carrier in the mitochondrial electron transport chain (ETC) in homeostasis (Alvarez-Paggi et al. 2017; Díaz-Quintana et al. 2020; Moore 1990), (ii) is required for apoptosis progression in the cytoplasm (Alvarez-Paggi et al. 2017; Liu et al. 1996), and (iii) participates as a genotoxic stress response element upon translocation to the cell nucleus under DNA damage (Arif et al. 2016; Nolin et al. 2016; Nur-E-Kamal et al. 2004; Xiang et al. 2021). When shuttled to the nucleus, Ccbinds to a number of targets involved in DNA damage response (DDR) that share some structural features although encompassing diverse functions (Gonz alez-Arzola et al. 2022; Martínez- F abregas et al. 2014a,2014b). Among them, the oncoprotein SET/template-activating factor Iβ(SET/TAF-Iβ) participates in the control of chromatin dynamics as a histone chaperone (Karetsou et al. 2009; Kato et al. 2011; Kawase et al. 1996; Muto et al. 2007; Park et al. 2023; Seo et al. 2001). Furthermore, SET/TAF-Iβregulates the activity of protein phosphatase 2A (PP2A), a master regulator of DDR (Di Mambro and Esposito 2022; Li et al. 1996; Ramos et al. 2019) and retains DDR factors at DNA repair foci (Kalousi et al. 2015; Kim et al. 2014). In this context, nuclear translocated Cchas been reported to hamper SET/TAF-Iβbinding to histones, thus limiting nucleosome (dis)assembly activity (Gonz alez-Arzola et al. 2015) and its modulatory action on PP2A (Casado- Combreras et al. 2022). Besides its localization-dependent function, Cccan be post-translationally modified in vivo, both under homeostatic or pathogenic states (García-Heredia et al. 2012; Guerra-Castellano et al. 2020; Kalpage et al. 2020). Among the most well-described PTMs of Cc, Tyr48 (i.e., the second of the two Tyr residues in the GYSY motif) phosphorylation was first identified in bovine liver tissue (Yu et al. 2008), where reactive oxygen species (ROS) and toxic byproducts are constantly generated as a consequence of this organ's metabolism and detoxifying activity (Conde de la Rosa et al. 2022). Interestingly, a Cc phosphomimetic mutant generated by substituting Tyr48 by a non-canonical amino acid (namely, p-carboxy- methyl-L-phenylalanine, pCMF, a phenylalanine residue with a carboxymethyl group bonded to its ζcarbon) through the genetic code expansion technology, was found to have a reduced electron transfer rate and to preclude Cc-mediated caspase activation (Gomila et al. 2022; Guerra-Castellano et al. 2015,2018; Moreno-Beltr an et al. 2017). In line with this, other authors have described that Tyr48 phosphorylation has a cytoprotective role, as it reduces the electron flow in the ETC (thus preventing electrons from leaking and generating ROS), while impeding the progression of apoptosis (Pecina et al. 2010). In this work, isothermal titration calorimetry (ITC) and nuclear magnetic resonance (NMR) experiments with the phosphomimetic Y48pCMF mutant allow to conclude that Ccphosphorylation on Tyr48 leads to (i) a lower binding affinity and a change in the binding mode of Cctoward SET/TAF-Iβ; (ii) and, ultimately, to a decreased ability of Ccto inhibit the histone chaperone activity. Further details on phosphorylation-induced changes in Ccstructure and subsequent SET/TAF-Iβrec- ognition are provided. Immunofluorescence microscopy 2of18 TAMARGO-AZPILICUETA ET AL. 1469896x, 2024, 12, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/pro.5213 by Readcube (Labtiva Inc.), Wiley Online Library on [21/11/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License and proximity ligation assays (PLA) confirm that Cc phosphomimetic mutants are located at the cell nuclei upon DNA damage response. Based on these findings, we conclude that CcTyr48 phosphorylation might modulate cell tolerance to apoptosis not only by impairing the activation of Ccpro-apoptotic targets in the cytoplasm, but also by modulating its binding to the histone chaperone SET/TAF-Iβin the nucleus. 2|RESULTS 2.1 |Tyr48 phosphorylation modifies the heme environment by increasing intramolecular cytochrome cdynamics Phosphorylation of Tyr48 changes the affinity of Cc toward components of the ETC and abolishes apoptosome activation in the cytoplasm. We thus sought to determine if such a PTM also modifies Ccfunctioning in the nucleus. Due to strong yield limitations to obtain phosphorylated proteins from tissues and the lack of knowledge about the activity and specificity of kinases catalyzing this modification, we resorted to test three Tyr48 site-directed substitutions of Cc: the two classical phosphomimetic substitutions Tyr-to-Asp (Y48D) and Tyr-to-Glu (Y48E) and the substitution by p-carboxy- methyl-L-phenylalanine (pCMF) non-canonical amino acid (Y48pCMF), which more accurately resembles the volume and hydropathy of the phosphorylated Tyr (Gomila et al. 2022; Guerra-Castellano et al. 2015; Moreno-Beltr an et al. 2017; Pérez-Mejías et al. 2020; Xie et al. 2007). To determine the structural resemblance of phosphomimetic Y48pCMF, Y48D, and Y48E Ccspecies with respect to the Tyr48-phosphorylated protein, we resorted to molecular dynamics (MDs) simulations. Substitutions by phosphorylated Tyr (pTyr), pCMF, Asp, or Glu were introduced to the wild-type (WT) CcNMR-based model in its reduced state (Imai et al. 2016). Strikingly, whereas the unphosphorylated CcTyr48 sidechain remained facing inwards and contacting with the heme propionate groups, phosphorylation of this residue triggered its rotation toward the solvent (Figure 1a). This reorientation of Tyr48 was also observed in all the phosphomimetic mutants, suggesting that the addition of a negative charge induced by phosphorylation or after its substitution by pCMF, Asp or Glu causes its side-chain exclusion from the heme crevice (Figure 1a). Radius of gyration (R g ) and backbone rootmean-square deviation (RMSD) values were stable during all the simulations, whereas slight increases were observed for the Y48E mutant (Figures 1b and S1a,b, Supporting Information). The secondary structure of all the species analyzed remained mostly stable throughout the entire simulation (Figure S2), although the foldon V (residues 40–57) exhibited increased flexibility in the phosphorylated and phosphomimetic species compared to the unmodified WT variant (Figures 1c,d and S1c). Among the phosphomimetics, average RMSF values of the foldon V of the Y48pCMF phosphomimetic were comparable to those of the Tyr48-phosphorylated variant, followed by Y48D Cc. However, dynamics of the Y48E mutants in this region were markedly enhanced with respect to the phosphorylated variant. For all that, the close proximity (<2.5 Å) of the heme moiety was more accessible for water molecules in the phosphorylated and phosphomimetic variants than in WT Cc(Figure 1e). A deeper scrutiny of the distances between the mass centers of the residue at position 48 and the heme iron indicated that this distance is significantly higher in the phosphorylated and phosphomimetic variants than in the unmodified Cc(Figure S3a,b). Further, the distance RMSD (dRMSD), which provides insights on the changes in the distance of the residue with the rest of the protein, was calculated using the structure at t=0 s as the reference. Interestingly, while the distance and dRMSD values remained stable in the WT, unmodified Cc, the phosphorylation or phosphomimetic substitution induced a rearrangement in the conformation during the initial steps of the simulation (Figure S3a) that lead to larger distances (around 5 Å further than the WT ones) between the residues at position 48 and the heme iron (Figure S3c). Similarly to the reduced form, running a 1-μs simulation of the ferric form of Ccwas enough to reach convergence in RMSD and R g values (Figure S4a). Likewise, we found that the RMSF values of the foldon V (Figure S4b), the water molecules radial distribution near the heme group (Figure S4c) and the distance of the Tyr48 sidechain to the heme iron (Figure S4d) were enhanced in all the phosphomimetics and in the phosphorylated form compared to the WT, and that the Y48E was the most variable. In fact, secondary structure (Figure S4e) and overall flexibility of the molecule (Figure S4f), was comparable to the reduced form as well. Then, Y48pCMF, Y48D, and Y48E phosphomimetic mutants were recombinantly expressed to biophysically characterize them and validate the results extracted from MD simulations. Circular dichroism (CD) spectra in the far-UV region were recorded for all the phosphomimetic mutants in the oxidized state (Figure 2a), showing that the substitution of Tyr48 by pCMF, Asp or Glu barely affected the overall secondary structure of Cc(Table S1), in agreement with MD simulations (Figures S2 and S4e). At physiological pH values, Ccheme iron is hexacoordinated with N δ1 of His18 and S δ of Met80 as axial TAMARGO-AZPILICUETA ET AL.3of18 1469896x, 2024, 12, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/pro.5213 by Readcube (Labtiva Inc.), Wiley Online Library on [21/11/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License ligands. At higher pH values, the S δ -Met80-coordination of ferric heme iron band is replaced to Lys72, Lys73, or Lys79, in a well-described process known as alkaline transition (Alvarez-Paggi et al. 2017; Assfalg et al. 2003; Boffi et al. 2001). Tyr48 substitutions by pCMF or Glu are known to disrupt the H-bond network around the porphyrin ring, thereby facilitating alkaline transition. Indeed, the pK a value for alkaline transition has been calculated as 9.3 ± 0.1 for WT Cc(Guerra-Castellano et al. 2015), while it decreases to 6.7 ± 0.1 for Y48pCMF Cc (Guerra-Castellano et al. 2015) and 7.0 ± 0.5 for Y48E Cc (García-Heredia et al. 2011). To check if Y48D Ccfollows this behavior, we measured its pK a value. A pH titration was performed while recording the charge-transfer band at 699 nm, which is lost upon alkaline transition (Figure S5) (Schejter and George 1964). In line with the shown decrease in alkaline transition pK a by Y48pCMF and Y48E phosphomimetic mutants, Y48D CcpK a shifts to 7.3 ± 0.1. This suggests that intramolecular changes experienced by phosphomimetic Ccmutants are also reproduced by the Y48D one, in good agreement with the local perturbations predicted by the MD simulations. To gather information about the heme environment of the phosphomimetic mutants in the oxidized state (Blauer et al. 1993; Santucci and Ascoli 1997), the visible (B-band, 350–450 nm) CD spectra of WT, Y48pCMF, Y48D, and Y48E Ccwere recorded at pH 7.4, which is near to (i) the expected physiological pH of the nucleus and cytoplasm (Casey et al. 2010) and (ii) to the alkaline transition pK a values of the phosphomimetic mutants (Figure S5). In fact, while WT Ccshows a Cotton effect at 410 nm—that is, a positive-in-sign and negative-in-sign bands typical of the native state in low spin cytochromes (Blauer et al. 1993)—the B-band splitting is lost in the phosphomimetic mutants (Figure 2a). This splitting is caused by the inner electric field of the protein around the heme porphyrin, and its loss can be attributed to changes in FIGURE 1 Molecular dynamics simulations of WT, Tyr48-phosphorylated and phosphomimetic cytochrome cspecies. (a) Overlapping of 10 trajectory snapshots, taken each 100 ns, from representative 1-μsMD simulations. Tyr48 side chains in unphosphorylated and phosphorylated Ccare zoomed-in to illustrate their orientation change induced by phosphorylation. (b) Average backbone atomic rootmean-square deviations (RMSD, bottom panel) and radius of gyration (R gyr , top panel) for the three MD simulation runs. (c) Average atomic root-mean-square fluctuations (RMSF) per Ccresidue. Region corresponding to foldon V (residues 40–57), which contains Tyr48, is shadowed in gray. (d) Ribbon representation of Ccshowing its five foldons. Residues involved in each foldon are below the foldon name. (e) Radial distribution function, grðÞ, of water molecules around the Cc porphyrin ring. A spherical region of 10Å radius around the heme group was analyzed. Average values are represented as thick lines, and standard deviation (±standard error for n=3 replicas) for each replica is represented by a shaded envelope. 4of18 TAMARGO-AZPILICUETA ET AL. 1469896x, 2024, 12, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/pro.5213 by Readcube (Labtiva Inc.), Wiley Online Library on [21/11/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License the coordination of the heme iron. Taken together, these results indicate that phosphorylation induces a modification of the heme environment that leads to a drop of the alkaline transition pK a to a neutral pH, what highlights a potential biological relevance of the phosphorylation-induced alkaline transition. Then, to determine the redox state reversibility of the phosphomimetic mutants, UV/Vis absorption spectra were recorded for WT, Y48pCMF, Y48D, and Y48E Cc, in both their reduced and oxidized state, at pH 7.4 (Figure 2b). All the spectra exhibited the typical hallmarks of c-type cytochromes. In the reduced species, an intense maximum at ca. 415 nm (i.e., Soret band) as well as two lower maximums at 550 and 520 nm (i.e., αand β bands, respectively) were observed. In the oxidized state the αand βbands merged into one single maximum at ca. 530 nm. In all the phosphomimetic species studied, a slight shift on the Soret band maximum was observed in the oxidized state from 409 to 406 nm (Table S2), as previously described in the literature (García-Heredia et al. 2011). In this context, previous reports pointed out that the Y48pCMF mutant exhibited milder differences in redox functionality than the Y48E mutant when compared to WT Cc, which is in line with the reduced flexibility of foldon V in the former phosphomimetic mutant (Figure 1c) (Guerra-Castellano et al. 2015; Moreno-Beltr an et al. 2017). Thus, we attempted to define the magnitude of redox functionality differences between WT and Y48D Cc. To this end, the thermodynamics and kinetics of the interfacial electron transfer of WT or Y48D Ccadsorbed onto a gold electrode modified with an 8-mercaptooctanoic acid (MOA) self-assembled monolayer (SAM) were determined by performing variable temperature cyclic voltammetry (Figure S6 and Table S3). This monolayer allowed us to simulate the biological protein binding scenario. The standard redox potentials at 25C associated with the Fe(III)/Fe(II) redox conversion of the immobilized WT and Y48D Ccheme group were estimated from the midpoint redox potential values (E 1/2 ) of the raw voltammograms (Figure S6a), resulting in 160 ± 7 mV and 98 ± 5 mV, respectively. Notably, the E 1/2 of immobilized human and horse Ccat 25C and pH 7 (Table S3) were substantially lower than the value of 260 mV vs. NHE determined for both proteins in solution (Oviedo-Rouco et al. 2022; Rodríguez-Rold an et al. 2006) due to the relative stabilization of the protein ferric form following its adsorption on a negatively charged thiol monolayer, as observed before for other mammalian, yeast, and bacterial Cc(de Groot et al. 2007; Kranich et al. 2009; Molinas et al. 2011; Todorovic et al. 2006). Notably, the Y48D mutant showed a lower redox potential, which is consistent with a higher stabilization of the ferric form upon immobilization. Similar redox potentials were obtained for the previously synthesized Y48pCMF and Y48H Ccphosphomimetic species immobilized under the same binding conditions (Table S3), suggesting that the negative potential shift of Y48D Ccarise from its higher sensitivity to the electrostatic binding event (Olloqui-Sariego et al. 2022). The variation of E 1/2 with temperature (Figure S6b) provides the entropic and enthalpic contributions (ΔS0 rc and ΔH0 rc) to the E 1/2 values of the immobilized proteins, by the expression (Battistuzzi et al. 2001; Olloqui-Sariego et al. 2018,2020), ΔS0 rc ¼nF ∂E1=2 ∂T  P,xi ,ð1Þ ΔH0 rc ¼nF ∂E1=2=T  ∂1=TðÞ  P,xi ,ð2Þ where n=1, and Fhas its usual meaning. Comparison of the estimated reduction entropies and enthalpies (Table S3) values for both proteins revealed similar ΔH0 rc, and higher ΔS0 rc absolute values for the Y48D mutant. The lower redox potential of Y48D is thus entropic in origin, suggesting that the tyrosine mutation increases the FIGURE 2 Biophysical characterization of phosphomimetic cytochrome cmutants. (a) Far-UV (left panel) and visible (right panel) CD spectra of oxidized WT and phosphomimetic Ccmutants Y48pCMF, Y48D and Y48E. (b) UV/visible absorption spectra of oxidized (solid lines) and reduced (dashed lines) WT and phosphomimetic Ccvariants. AU, arbitrary units. TAMARGO-AZPILICUETA ET AL.5of18 1469896x, 2024, 12, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/pro.5213 by Readcube (Labtiva Inc.), Wiley Online Library on [21/11/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License structure and solvation differences between its oxidized and reduced states. The effect of the Y48H mutation on the kinetics of the interfacial electron transfer has previously been investigated (Olloqui-Sariego et al. 2022). For this purpose, the standard electron transfer rate constants (k s )of the proteins were determined from the scan rate dependence of the voltametric peak potentials using the Butler-Volmer formalism (Laviron 1979), at variable temperature (Figure S6c). Interestingly, the Y48D mutant displayed slightly lower k s values at lower temperatures, but became higher at increasing temperatures, thereby suggesting different activation parameters to the interfacial electron transfer rate between WT and Y48D Cc. These activation parameters, the pre-exponential factors (A) and activation enthalpies (ΔH# ET) associated with k s can be obtained from the corresponding Arrhenius plot (Figure S6b) according to ln ks¼ln AΔH# ET RT ,ð3Þ where the intercept and slope of these plots provide the A and ΔH# ET values, respectively, which are collected in Table S3. Interestingly, the tyrosine mutation provokes a significant increase in both the pre-exponential factor and the activation enthalpy. This switch of activation parameters was similar to those reported for other Tyr48 mutants (Olloqui-Sariego et al. 2022), suggesting that such a change is largely independent of tyrosine mutation. The orientation of the adsorbed Cconto SAM carboxylated organic surfaces is ruled by interactions with Lys residues 13, 72, 73, 79, 86, 87, and 88 (Xu and Bowden 2006), which are predicted to be unaffected by the mutation (Figures S4f and S6d). Therefore, the substantial rise in the pre-exponential factor can arise from a structural protein rearrangement that brings the heme ring closer to the electrode. Further, the two-fold higher activation enthalpy of Y48D compared to that of WT Cc points toward a greater contribution of the solvent in the activation process. These results are in agreement with the higher mobility of foldon V in the Y48D mutant inferred from MDs, enhancing the accessibility of water molecules to the heme moiety and enabling a large approach between the heme crevice and the electrode. Altogether, these findings suggest that the redox state of phosphomimetic Ccretains its reversibility, although the porphyrin environment and its solvent accessibility were perturbed. Furthermore, our observations indicate that the Y48D Ccphosphomimetic mutant is closer to replicate the structural and biophysical characteristics of the Tyr48-phosphorylated and Y48pCMF phosphomimetic species than the Y48E phosphomimetic variant. 2.2 |Tyr48 phosphorylation weakens and modifies the binding mode of cytochrome cto SET/TAF-Iβ In order to check whether Tyr48 phosphorylation changes the Ccactivity in the nucleus, as is the case at the mitochondrial (Gomila et al. 2022; Guerra-Castellano et al. 2018; Lee et al. 2006; Moreno-Beltr an et al. 2017;Yu et al. 2008) and cytoplasmic (García-Heredia et al. 2011; Moreno-Beltr an et al. 2017; Pecina et al. 2010) spaces, we analyzed the phosphorylation effect on the binding of Cc to SET/TAF-Iβ, a nuclear histone chaperone involved in DDR at multiple levels (Kalousi et al. 2015; Karetsou et al. 2009; Kato et al. 2011; Li et al. 1996). The interface used by Ccto recognize SET/TAF-Iβencompasses residues around the heme crevice, including many residues from foldon V (i.e., Ser47, Tyr48, Ala50, Ala51, Lys53, Gln54, Lys55, Gly56) and the 20–35 loop (i.e., Val20, Lys22, Gly23, Gly24, Lys25, Gln31, His33, Leu35) (Casado-Combreras et al. 2022,2024; Gonz alez-Arzola et al. 2015), which were predicted to have an enhanced flexibility within the Tyr48-phosphorylated and phosphomimetic species Y48pCMF, Y48D, and Y48E (Figure 1). ITC experiments showed that the interaction between reduced WT Ccand SET/TAF-Iβis entropically driven (binding enthalpy, ΔH=1.3 kcal mol 1 ; entropic term, – TΔS=8.8 kcal mol 1 ) with a dissociation constant (K D ) of 3.4 μM, a Cc:SET/TAF-Iβstoichiometry of 2:1, and a binding mode with modest positive cooperativity at 25C; in fact, the affinity coupling between the two sites is quite small (ρ=1.1, Δg=0.1 kcal mol 1 ), but the enthalpic coupling is considerable, reflected in a biphasic profile in the binding isotherm (Figure S7 and Table 1), as previously reported (Gonz alez-Arzola et al. 2015). Both binding events are entropically driven, but the second one shows a much larger entropic contribution and a more unfavorable enthalpy. Remarkably, all Ccphosphomimetic mutants exhibited a complete loss of the cooperative binding to SET/TAF-Iβ, as inferred from the monophasic profile of binding isotherms (Figure S7), although the Cc:SET/TAF-Iβbinding stoichiometry was conserved, remaining close to 2:1 (Figure S7 and Table 1). Additionally, there was a notable decrease in the binding affinity by 7-fold with Y48pCMF and Y48D Cc, and by 30-fold with Y48E Cc(Table 1). Moreover, the binding of Y48pCMF, Y48D, and Y48E Ccto SET/TAF-Iβbecame enthalpically driven (ΔH<4.6 kcal mol 1 ;TΔS= 13.6 kcal mol 1 ). This is consistent with a strong disruption of the electrostatics between the highly acidic surface of SET/TAF-Iβand the positively charged heme surroundings of WT Cc—as reflected by the entropically driven binding mode (Cooper 2005)—induced by the additional negative charge in the phosphomimetic 6of18 TAMARGO-AZPILICUETA ET AL. 1469896x, 2024, 12, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/pro.5213 by Readcube (Labtiva Inc.), Wiley Online Library on [21/11/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License species. It is noteworthy that the decrease in the affinity for SET/TAF-Iβexhibited by Ccmutants (Table 1) is proportional to the increase in mobility experienced at foldon V, likely penalizing the affinity through a larger conformational entropic loss (Figure 1). Taken together, these results suggest a contribution of phosphorylationinduced intramolecular dynamics that could explain the observed binding changes. In line with this finding, the Y48E mutant showed the most different thermodynamic profile and highest structural distortions, as previously mentioned (Figure 1), as well as a significant increase in K D (Table 1). In summary, these findings point out that Tyr48 phosphorylation—when it is mimicked by pCMF, Asp, or Glu—causes a drastic effect on the binding of Cc toward SET/TAF-Iβ. 2.3 |Tyr48-phosphorylated cytochrome c is less capable of inhibiting SET/TAF-Iβ histone chaperone activity To test whether the changes in structure and binding affinity of Cctoward SET/TAF-Iβby Tyr48 phosphorylation affected its nuclear function, we performed a competition assay recording 1D 1 H NMR spectra of reduced Cc in the presence of SET/TAF-Iβand increasing concentrations of a mixture of histones (Figure 3). First, the differences in binding of WT and Y48pCMF Ccto SET/TAF-Iβwere confirmed by monitoring the broadening of the Met80-εCH 3 NMR signal of the heme Fe(II) axial ligand at different SET/TAF-Iβ:Ccratios (Figures 3a and S8a). The addition of SET/TAF-Iβto a Cc sample at a 1:0.14 molar ratio (Cc:SET/TAF-Iβ) yielded a complete broadening of the Met80-εCH 3 signal beyond the detection limit with WT Cc,butnotwiththeY48pCMF species. The prominent signal in the Y48pCMF Ccspectra at the indicated ratio denotes fast exchanging populations between the unbound and bound states of Y48pCMF Cc. On the other hand, WT Ccsignal remained at the baseline level at this ratio, suggesting that WT Ccis mostly bound. Increasing the concentration of SET/TAF-Iβup to a 1:0.27 ratio (Cc:SET/TAF-Iβ) yielded a total broadening of the Met80-εCH 3 signal with both WT and Y48pCMF species. These results are consistent with the calorimetry data, which indicated a lower binding affinity of the phosphomimetic Cctoward SET/TAF-Iβthan the unmodified, WT Cc (Table 1). As a control of binding specificity, the linewidth of the CcMet80-εCH 3 signal was monitored and found to be unaffected upon addition of BSA (Figure S8b). TABLE 1 Thermodynamic parameters for SET/TAF-Iβcomplexes with reduced WT and mutant cytochrome cspecies. Protein complex K D1 (μM) ΔG (kcal mol 1 ) ΔH 1 (kcal mol 1 ) TΔS 1 (kcal mol 1 ) K D2 (μM) ΔH 2 (kcal mol 1 ) TΔS 2 (kcal mol 1 )n WT Cc:SET/TAF-Iβ3.4 7.5 1.3 8.8 3.1 6.9 14.4 1.2 Y48pCMF Cc:SET/TAF-Iβ23 6.3 4.6 1.7 - - - 2.2* Y48D Cc:SET/TAF-Iβ22 6.3 7.5 1.2 - - - 2.2* Y48E Cc:SET/TAF-Iβ110 5.4 19.0 13.6 - - - 2.3* Note: Calculated equilibrium dissociation constant (K D ), Gibbs free energy (ΔG), binding enthalpy (ΔH), entropic term (TΔS). nstands for (i) the protein active fraction in the WT Cc:SET/TAF-Iβcomplex fitted to a two-binding-sites binding model, or (ii) the binding stoichiometry of complexes involving Cc phosphomimetic mutants with independent binding sites, indicated using an asterisk (*). FIGURE 3 NMR titration of WT and phosphomimetic cytochrome cspecies with SET/TAF-Iβand histones. (a) 1D 1 H NMR spectra monitoring the Met80-εCH 3 signal of reduced WT (left) or Y48pCMF (right) Cc(13 μM) in free state and in the presence of increasing concentrations of SET/TAF-Iβ(0 μM, Cc: SET/TAF-Iβratio 1:0; 1.75 μM, Cc:SET/TAF-Iβratio 1:0.14; 3.5 μM, ratio 1:0.27). (b) 1D 1 H NMR spectra monitoring the WT (left) and Y48pCMF (right) CcMet80-εCH 3 signal in the presence of saturating concentration of SET/TAF-Iβand upon addition of increasing amounts of calf thymus histones. TAMARGO-AZPILICUETA ET AL.7of18 1469896x, 2024, 12, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/pro.5213 by Readcube (Labtiva Inc.), Wiley Online Library on [21/11/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License To corroborate that the phosphomimetic species of Cc retained the ability to inhibit SET/TAF-Iβhistone chaperone activity, we measured the recovery of the Met80-εCH 3 signal upon addition of increasing histones amounts (Figure 3b). The increase in the signal intensity served as an indicator of Ccrelease from its complex with SET/TAF-Iβ, hence revealing a competition between the hemeprotein and histones. Both WT and Y48pCMF Cc are completely displaced from the complex with SET/- TAF-Iβwhen adding 40 μg of a calf thymus histones mixture. Notably, the Met80-εCH 3 signal intensity recovery is gradual upon adding increasing amounts of histones, indicating that Y48pCMF Ccstill retain the ability to compete with histones for SET/TAF-Iβbinding sites. According to the fitting of the normalized maximum intensity of the Met80-εCH 3 NMR signal, the histone/Cc (His/Cc) mass ratio required to inhibit the histone chaperone activity of SET/TAF-Iβto a half (i.e., the half maximal inhibition concentration, EC50), was slightly reduced from 0.77 (±0.026) for WT to 0.69 (±0.046) for Y48pCMF Cc(Figure S8c). Altogether, these results indicate that phosphorylation does not fully impair the Cc-mediated inhibition of SET/TAF-Iβhistone chaperone activity but modulates the hemeprotein binding to SET/TAF-Iβand the ability to compete against histones (Figure 3b). 2.4 |Tyr48-phosphomimetic cytochrome cis able to shuttle to the cell nucleus under basal DNA damage and interact with SET/TAF-Iβ Tyr48 phosphomimetic Ccvariants undergo structural and dynamical modifications that affect their recognition of SET/TAF-Iβ, as reflected in their mode of competition with histones for binding sites on the chaperone. We then wanted to test whether phosphomimetic Ccspecies are also able to translocate to the nucleus and recognize SET/TAF-Iβin cell. First, to test whether phosphomimetic Ccspecies can redistribute to the nucleus, we monitored the Ccsubcellular localization by immunofluorescence in mouse embryonic fibroblasts (MEFs) deprived of both copies of the Ccgenes (Cc / ) and transfected with constructs encoding WT or phosphomimetic Y48D human Cc. Notably, we found that both WT and Cc / MEF cells showed basal formation of γ-H2AX foci, an indicator of the activation of the DNA damage response (DDR; Mah et al. 2010; Paull et al. 2000). Basal DNA damage under homeostatic conditions with subsequent formation of DNA foci has been observed for other cell lines (Guard et al. 2019). In consequence, endogenous Ccwas mainly localized in mitochondria of WT MEFs but was also detected in the nuclei that contained γ-H2AX foci (Figure 4a). As a control, no fluorescence was observed when using the polyclonal anti-Ccantibody in Cc / MEF cells when transfected with an empty vector. FIGURE 4 Interaction of WT and Y48D cytochrome cwith SET/TAF-Iβin the nucleus of MEF cells on basal DNA damage levels. (a) Immunofluorescence analyses of γ-H2AX and Ccin WT (endogenous Cc) and Cc / (transfected or not with WT or Y48D Ccspecies) MEF cells. The subcellular distribution of Ccand γ-H2AX was visualized using anti-Ccand anti-γ-H2AX antibodies—namely, FITC and Alexa Fluor 568, respectively. NT stands for non-transfected cells but treated equally to the transfected and WT control cells, including incubation with both primary and secondary antibodies. Scale bars are 20 μm. (b) In situ PLA detection of Cc-SET/TAF-Iβcomplexes and immunofluorescence labeling of Ccin Cc / MEF transfected with an empty vector, WT Ccor Y48D Cc. Representative projections of each condition with PLA spots in red and nuclei in blue are shown. Scale bars are 20 μm. (c) Quantitation of PLA spots per nucleus (n=27 for empty vector, n=28 for WT Cc, and n=27 for Y48D Cc). Black lines and whiskers represent the mean and the SD, respectively. Statistical significance was calculated using the Mann– Whitney test (***p< 0.001; “ns”stands for nonsignificant). 8of18 TAMARGO-AZPILICUETA ET AL. 1469896x, 2024, 12, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/pro.5213 by Readcube (Labtiva Inc.), Wiley Online Library on [21/11/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License The Y48D phosphomimetic variant was chosen for in cell experiments instead of the Y48pCMF mutant, as the introduction of pCMF species in eukaryotes using the gene code expansion remains challenging in hemeproteins because of their particular folding route that involves entering in the mitochondria (Dumont et al. 1988,1991;Grasso et al. 2022;Heetal.2022;NiuandGuo2023). From our observations herein reported, Y48D Ccis structurally and biophysically closer to pY48 and Y48pCMF Ccthan the Y48E mutant (Figures 1and S1–S4) and further reproduces the Y48pCMF interplay with SET/TAF-Iβ(Table 1). Cc / MEFs transfected with WT or Y48D Ccexpression vectors also exhibited γ-H2AX foci formation, as did WT MEFs. They also showed a mainly mitochondrial localization of Cc, as well as a nuclear pool of unmodified or phosphomimetic Cc. In this framework, we wanted to determine if the Y48D Ccphosphomimetic was also able to physically interact with endogenous SET/TAF-Iβin cell.Tothis end, PLA was performed in these cells (Figure 4b,c). Following transfection, PLA spots were detected in the nucleus, evidencing interaction between SET/TAF-Iβ and both variants of the heme protein. No significant differences were found between the number of nuclear PLA spots of WT and Y48D Cc, which could be ascribed to changes in transfection or protein expression levels, among others. The interaction between SET/TAF-Iβand Ccwas also widely detected in the cytosol. This interaction may take place following the cytosolic localization of SET/TAF-Iβupon being posttranslationally modified (Nagata et al. 1998;Veraetal. 2007;Yuetal.2013), if there is an available Cccytosolic pool originating from the mitochondrial release. Altogether, these results show that a pool of phosphomimetic Ccreaches the nucleus of cells under active DDR, and that such a post-translationally modified Cc population are still able to interact with SET/TAF-Iβ. 3|DISCUSSION As a pleiotropic regulator of cell life and death, Ccis comprehensively subjected to PTMs that affect its redox activity in the mitochondria and pro-apoptotic function in the cytosol (García-Heredia et al. 2011; Gomila et al. 2022; Guerra-Castellano et al. 2020; Kalpage et al. 2020; Moreno-Beltr an et al. 2015,2017; Olloqui-Sariego et al. 2022; Pérez-Mejías et al. 2020). In this work, we broaden the functional scope of PTMs over Ccto the cell nucleus, where the hemeprotein is translocated under genotoxic stress (Alvarez-Paggi et al. 2017; Arif et al. 2016; Casado- Combreras et al. 2022; Gonz alez-Arzola et al. 2015,2022; Martínez-F abregas et al. 2014a,2014b; Moreno-Beltr an et al. 2015; Nolin et al. 2016; Nur-E-Kamal et al. 2004; Rivero-Rodríguez et al. 2021; Xiang et al. 2021; Zhou et al. 2024), demonstrating that Tyr48 phosphorylation modifies its binding toward its nuclear partner SET/TAF- Iβin vitro. Tyr48, along with Gly41, Gly45, Ala51, Asn42, and Lys55, are the most evolutionary conserved residues of the Ccfoldon V (residues 40–57; Figure S9), which is the Ccsubstructure with the lowest unfolding free energy. Interestingly, four out of the six described pathogenic, naturally occurring Ccmissense mutations (i.e., G41S, Y48H, A51V, A51T) fall within this foldon (De Rocco et al. 2014; Marzollo et al. 2024; Morison et al. 2008; Ong et al. 2017). Our MD simulations suggest that the sidechains of phosphorylated Tyr and phosphomimetic pCMF at position 48 are excluded from the heme environment and turn outwards, enlarging the heme crevice and thus altering the alkaline transition and redox properties of the protein. This is consistent with marked differences in the orientation of Tyr48 sidechain and the local structure of foldon V between NMR-resolved structures of WT (PDB ID: 2N9I; Imai et al. 2016) and Y48pCMF (2N3Y; Moreno-Beltr an et al. 2017). Likewise, the Y48H thrombocytopenia-related Ccmutant was found to have increased dynamics in the 40–57 and 71– 85 loops (Deacon et al. 2017), along with a drop in the alkaline transition pK a (Deacon et al. 2018; Deng et al. 2019). These findings indicate that Tyr48 controls the rigidity of the foldon V and, consequently, the alkaline transition. However, unlike the Y48H missense mutation, PTMs are reversible, allowing cells to switch between two active configurations of Ccbased on the cellular status. Tyr48 phosphorylation plays a cytoprotective role by decelerating the electron flux in mitochondrial ETC (Gomila et al. 2022; Pecina et al. 2010; Yu et al. 2008), decreasing ROS production (Moreno-Beltr an et al. 2017; Pecina et al. 2010), and impeding caspase activation (Moreno-Beltr an et al. 2017; Pecina et al. 2010). Here, we show that Tyr48 phosphorylation further modulates the binding to one of its nuclear partners (namely, the histone chaperone SET/TAF-Iβ). ITC experiments revealed a ca. 10-fold reduction in the binding affinity of Y48pCMF and Y48D Ccphosphomimetic mutants toward SET/TAF-Iβ(Figure 3and Table 1). These results suggest that the increase in foldon-V mobility and the addition of a negative phosphate charge—mimicked by pCMF, Asp or Glu mutations—hampers the binding to SET/TAF-Iβ, by altering orientation of residues important for the chaperone recognition and distorting the charge complementarity. This leads to a shift of the thermodynamic binding profile from an entropically driven binding to an enthalpically driven binding and to the loss of binding cooperativity (Table 1). Despite reduced binding, 1D 1 HNMRexperimentsof WT and Y48pCMF Ccshow that the phosphomimetic Cc TAMARGO-AZPILICUETA ET AL.9of18 1469896x, 2024, 12, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/pro.5213 by Readcube (Labtiva Inc.), Wiley Online Library on [21/11/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License of SET/TAF-Iβby cytochrome c. 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