Evolutionary Pro-To-Thr Mutation in the Intrinsically Disordered Domain of ANP32 Family Members Modulates Their Target Binding Modes

RESEARCH ARTICLE www.advancedscience.com Evolutionary Pro-To-Thr Mutation in the Intrinsically Disordered Domain of ANP32 Family Members Modulates Their Target Binding Modes Blanca Baños-Jaime, Ana B. Uceda-Mayo, Francisco Rivero-Rodríguez, Miguel Á. Casado-Combreras, Alejandro Velázquez-Cruz, Adrián Velázquez-Campoy, Laura Corrales-Guerrero,* Miguel A. De la Rosa, and Irene Díaz-Moreno* Gene duplication has allowed protein evolution toward novel functions and mechanisms. The differences between paralogous genes frequently rely on the sequence of disordered regions. For instance, in mammals, the chaperones ANP32A and ANP32B share a common evolutionary line and have some exchangeable functions based on their similar N-terminal domains. Nevertheless, their C-terminal low-complexity-acidic-regions (LCARs) display substantial sequence differences, unveiling some degree of variability between them, in agreement with their different tissue-specific expression patterns. These structural and computational results indicate that a substitution in the vicinity of the nuclear localization signal (NLS), of Pro in ANP32A for Thr in ANP32B, determines the overall compactness of the C-terminal LCAR. The different structural properties of the disordered region affect the binding mode of ANP32 members to their targets. This type of divergent binding mode is exemplified with the extra-mitochondrial cytochrome c(Cc), a well-known ANP32B partner and which now determine also binds to ANP32A; and with the RNA binding protein HuR, whose export to the cytoplasm is mediated by ANP32 proteins under stress. Therefore, differential expression patterns of ANP32A or ANP32B may affect the regulation of Ccand HuR and can help to explain the distinct roles of these proteins in diseases. B. Baños-Jaime, A. B. Uceda-Mayo, F. Rivero-Rodríguez, M. Á. Casado-Combreras, A. Velázquez-Cruz, L. Corrales-Guerrero[+], M. A. De la Rosa, I. Díaz-Moreno Institute for Chemical Research (IIQ) Scientific Research Center “Isla de la Cartuja” (cicCartuja) University of Seville-CSIC Avda. Americo Vespucio 49, Seville 41092, Spain E-mail: [email protected];[email protected] The ORCID identification number(s) for the author(s) of this article can be found under https://doi.org/10.1002/advs.202415566 [+]Present address: Institute of Plant Biochemistry and Photosynthesis (IBVF), Scientific Research Center “Isla de la Cartuja” (cicCartuja), University of Seville-CSIC, Avda. Americo Vespucio 49, Seville 41092, Spain © 2025 The Author(s). Advanced Science published by Wiley-VCH GmbH. 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. DOI: 10.1002/advs.202415566 1. Introduction The acidic leucine-rich nuclear phosphoprotein 32 (ANP32) family comprises evolutionary conserved histone chaperones that are present in different organisms of the animal kingdom, with homologous proteins in plants and protists.[1]All ANP32 proteins share a common domain arrangement, with a structured N-terminal leucinerich region (LRR) and a C-terminal intrinsically disordered domain (IDD) (Figure 1A). This IDD is called low-complexity-acidicregion (LCAR), as it contains highly repetitive acidic stretches with no remarkable pattern.[2] The human ANP32 family consists of eight members (A to H), of which the proteins A and B are highly conserved (with 71% sequence identity and 81% similarity between the two human proteins).[2,3] ANP32B is derived from a gene duplication of ANP32A that evolved independently during the early origin of vertebrates.[1,2] This kind of diversification after gene A. Velázquez-Campoy Institute for Biocomputation and Physic of Complex Systems (BIFI), Joint Unit GBsC-CSIC-BIFI University of Zaragoza C. Mariano Esquillor, Zaragoza 50018, Spain A. Velázquez-Campoy Departament of Biochemistry and Molecular and Cellular Biology University of Zaragoza C. Miguel Servet 177, Zaragoza 50013, Spain A. Velázquez-Campoy Institute for Health Research of Aragón (IIS Aragon) Avda. San Juan Bosco 13, Zaragoza 50009, Spain A. Velázquez-Campoy Centre for Biomedical Research Network of Hepatic and Digestive Diseases (CIBERehd) Av. Monforte de Lemos 3–5, Madrid 28029, Spain Adv. Sci. 2025,12, 2415566 2415566 (1 of 14) © 2025 The Author(s). Advanced Science published by Wiley-VCH GmbH www.advancedsciencenews.com www.advancedscience.com Figure 1. ANP32 protein family structure and alignment. A) Schematic representation of ANP32 protein family domain architecture. The LRR of the ANP32 protein family is shown as a ribbon representation using the X-ray diffraction model of ANP32A LRR domain (PDB ID: 4XOS), whereas the LCAR is represented as an orange curvy line. The inset indicates the localization of the NLS. Residue numbers corresponding to ANP32A or ANP32B are colored in violet or blue respectively. B) Sequence alignment of the NLS nearby sequence of ANP32 family members from human and related organisms using MUSCLE.[36]Both the tree and the organisms are colored in violet, blue or grey according to the indicated protein. Uniprot code for each protein are indicated in the figure. Residues belonging to the NLS are highlighted in pink. The highly conserved residues Gln235 and Pro241 of ANP32A are shaded in violet, whereas Glu237 and Thr244 of ANP32B are in blue. For mammals, Gln235 and Pro241 of ANP32A and Glu237 and Thr244 of ANP32B are in bold letters. Residue numbers corresponding to ANP32A are colored in violet, and to ANP32B, in blue. duplication often relies on structurally disordered regions of the proteins.[4–6]In some cases, retention of gene duplicates— paralogs—is associated with tissue expression divergence.[7–9]Indeed, ANP32A and ANP32B exhibit a differential expression pattern: while both members are synthesized in brain, placenta, and pancreas, only ANP32A is present in kidney and skeletal muscle, and only ANP32B in heart, lungs, and thymus.[10–12] Besides their role in the transcription regulation derived from their histone chaperone activity,[13–16]ANP32A and ANP32B share multiple functions, for instance in the RNA metabolism of viruses, such as influenza or human immunodeficiency virus (HIV).[17–21]They are also responsible for the nucleo-cytoplasmic transport of the oncoprotein human antigen R (HuR) upon heat shock. Under regular conditions, ANP32A and ANP32B interact with the RNA-binding protein (RBP) HuR in the nucleus, and HuR shuttles to the cytoplasm through its shuttling sequence.[3]Stress induced by heat shock increases the interaction of HuR with ANP32A and ANP32B and enables HuR to be exported via the nuclear export factor CRM1, which is also responsible for ANP32 protein transport. Moreover, the same domain of HuR that regulates its interactions with the LCAR of ANP32 family members hosts the HuR nucleocytoplasmic sequence (HNS) for HuR transport.[22]Further, ANP32A, along with the histone chaperone SET/TAF-I𝛽,[23–29]is a well-known inhibitor of protein phosphatase 2A (PP2A), whose RNA is, in turn, encoded by RBPs as KH-Splicing Regulator Protein (KSRP).[30]We recently reported that ANP32B is also a PP2A inhibitor, regulated by the extra-mitochondrial hemeprotein Cc.[31]Ccmigrates from the mitochondria to the cell nucleus under DNA damage conditions.[32–34]In the nucleus, Cc targets the disordered C-terminal LCAR of ANP32B, inducing long-range allosteric movements into the structured N-terminal LRR of the histone chaperone and thereby hampering PP2A inhibition.[31] Adv. Sci. 2025,12, 2415566 2415566 (2 of 14) © 2025 The Author(s). Advanced Science published by Wiley-VCH GmbH 21983844, 2025, 12, Downloaded from https://advanced.onlinelibrary.wiley.com/doi/10.1002/advs.202415566 by Readcube (Labtiva Inc.), Wiley Online Library on [24/04/2025]. 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 www.advancedsciencenews.com www.advancedscience.com Figure 2. Structural models of ANP32A230-249 and ANP32B232-251 peptides. A) Ribbon representations for the structural models of ANP32A230-249 (left) and ANP32B232-251 peptides (right). Upper panels show the overlapping of the top ten models calculated by CYANA for each construct. Lower panels show the #0.1 solution provided by CYANA. Pro241 is highlighted in pink in the ANP32A230-249 peptide model, and Thr244, in blue in the ANP32B232-251 peptide model. B) Residue-specific Neighbor Corrected Structural Propensity Calculator (ncSPC) scores obtained for ANP32A230-249 and ANP32B232-251 peptides. “+1″indicates the maximum propensity to form a full 𝛼-helix, and “0” indicates disorder. C) 𝛼-helix propensity scores obtained for ANP32A230-249 and ANP32B232-251 peptides. The values were obtained using the CSI3.0 web server. D) Mean of the solvent-accessible surface area (SASA) of the ten selected structures of ANP32A230-249 and ANP32B232-251 peptides. E) Atomic fluctuations (Root mean square fluctuation, RMSF) values of backbone atoms between the ten selected structures of each peptide as a function of residue number. ANP32A230-249 data are represented in violet, and ANP32B232-251 data, in blue. Residues of each peptide are indicated in graphs B, C, and E. Residues belonging to the NLS are highlighted in pink. Gln235 and Pro241 of ANP32A are shaded in violet, whereas Glu237 and Thr244 of ANP32B are in blue. Adv. Sci. 2025,12, 2415566 2415566 (3 of 14) © 2025 The Author(s). Advanced Science published by Wiley-VCH GmbH 21983844, 2025, 12, Downloaded from https://advanced.onlinelibrary.wiley.com/doi/10.1002/advs.202415566 by Readcube (Labtiva Inc.), Wiley Online Library on [24/04/2025]. 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 www.advancedsciencenews.com www.advancedscience.com Figure 3. ANP32A interaction with Cc.A)Upper panel: Western blot against c-myc tag, at the C-terminus of ANP32A, after performing a pulldown assay with extracts from cells transfected with either the empty vector (control) or the pCDNA3.1-ANP32A-c-myc. Middle panel: Western blot against Cc demonstrating that the hemeprotein was captured within the carboxymethyl cellulose matrix. Lower panel: Western blot against c-myc tag of HEK293T lysates as a loading and transfection control. B) Schematic representation of the ANP32A and ANP32A1-167 constructs, used in D and E. C) ITC titrations of ANP32A with Ccat low (left panel) and moderate (center panel) ionic strength; and of ANP32A1-167 and Ccat low ionic strength (right panel). Thermograms and binding isotherms are shown in the upper and lower panels, respectively. D) Detailed view of the superimposed 2D 1H-15N HSQC spectra of 15Nlabeled Ccduring titrations with increasing concentrations of ANP32A (upper panel)orANP32A 1-167 (lower panel) at low ionic strength. The color guide of ANP32A constructs concentration is shown in the panel. E) Curves representing the best global fit of several amide signals in the direct (1H) dimension with a global KDvalue for ANP32A binding with Cc. F) Representation of averaged chemical-shift perturbations (Δ𝛿AVG) and broadening of Ccamide signals upon Cc-ANP32A complex formation at a 1:1 ratio (Cc:ANP32A). Δ𝛿AVG is represented by a blue color gradient, ranging from white Adv. Sci. 2025,12, 2415566 2415566 (4 of 14) © 2025 The Author(s). Advanced Science published by Wiley-VCH GmbH 21983844, 2025, 12, Downloaded from https://advanced.onlinelibrary.wiley.com/doi/10.1002/advs.202415566 by Readcube (Labtiva Inc.), Wiley Online Library on [24/04/2025]. 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 www.advancedsciencenews.com www.advancedscience.com Despite the high degree of conservation and the similar functions between ANP32A and ANP32B, the differential tissue expression and the distinct evolution of these proteins suggest that they have (at least in part) distinct cellular functions. This work aims to explain how small differences in an IDD can affect the whole protein dynamics and function. In particular, we demonstrate that the presence of a Pro residue in the LCAR of ANP32A induces a more open and binding-favorable conformation as compared to its ANP32B counterpart. This Pro-to-Thr substitution affects the binding of ANP32 family members with other proteins, such as Ccand HuR, as evidenced by the point mutation of P241T in ANP32A peptide. Moreover, the ANP32A complexes with Ccand HuR behave as structurally heterogeneous ensembles (so-called fuzzy complexes), allowing a high degree of plasticity. Overall, our results suggest distinct regulatory patterns for ANP32A and ANP32B based on their C-terminal LCAR conformational arrangements. 2. Results 2.1. Pro-To-Thr Substitution Alters the Overall Structure and Dynamics of ANP32 LCAR All the ANP32 family members except C and D contain a NLS in the C-terminus of the LCAR, the most variable region between ANP32A and ANP32B in sequence, besides the fact that ANP32A LCAR has a higher proportion of aromatic residues. The functional relevance of the C-terminal LCAR region has been recently highlighted, since the deletion of the last 20 residues in ANP32B was enough to alter the thermodynamics of the binding to Cc leading to differences in its functionality.[31]The NLS stands out because it contains numerous basic residues, and in particular Lys and Arg,[1,35]in contrast to the highly acidic nature of the remaining LCAR. Thus, the mobility of the C-terminal region may be affected by electrostatic attractions between the NLS and the negative residues along the LCAR, with a plausible impact on protein-protein interactions involving the LCAR. Therefore, these structural differences between LCARs could represent a reason for the functional variations between ANP32 family members. To understand the evolution of the ANP32 family LCAR, we aligned the sequences of ANP32 family members A, B, and E in diverse organisms, with a special focus on the NLS nearby sequence (Figure 1B). Although some of the residues surrounding the NLS vary according to the organisms, we found two highly conserved residues in mammals: Gln235 and Pro241 in human ANP32A, and the equivalent residues of Glu237 and Thr244 in human ANP32B, respectively. The highly conserved changes in ANP32A and ANP32B in the evolutionary branch where mammals arose suggest a specialization of each ANP32 protein. Moreover, given the conformational restraint of Pro residues, we wondered if the Pro-to-Thr substitution might modify the dynamic properties of the C-terminal region of the LCAR, thus affecting the function and/or regulation of ANP32B. To address these questions, we designed synthetic peptides containing the last 20 amino acids of the ANP32A or ANP32B human protein (ANP32A230-249 and ANP32B232-251). Structural features of these peptides were studied in detail by Nuclear Magnetic Resonance (NMR) 2D 1H-1H Total Correlation Spectroscopy (TOCSY) and nuclear Overhauser effect spectroscopy (NOESY) (Figure S1, Supporting Information). Using NOEs corresponding to the C-end NLS-containing stretch of ANP32A or ANP32B, we obtained model ensembles for the peptides comprising the ten structures with the lowest energy values. Structure calculations with CYANA included the interatomic/interresidue distances inferred from NOESY cross-correlation peak intensities as restraints. Nevertheless, such structural models partially represent the total conformational landscape of each peptide, including those with a preferential pattern of contacts among residues. The results obtained after structural refinement–consisting of several rounds of distance violation analysis and filtering–showed that both peptides substantially differed in their conformational arrangements (Figure 2A). Specifically, the ANP32A230-249 peptide exhibited a broader, more diverse ensemble of conformations, with a higher backbone root-meansquare deviation (RMSD) between the ten selected models with respect to that of the ANP32B232-251 peptide (Table S1, Supporting Information). In addition, the ANP32A230-249 peptide showed open, extended conformations, in which Pro241 keeps the Nand C-ends separated from each other (Figure 2A). In contrast, the ANP32B232-251 peptide predominantly adopted closed, compact conformations, likely driven by the formation of Thr-enhanced transient contacts between the acidic Nand Cends with the basic NLS stretch. Moreover, the ANP32B232-251 peptide exhibited a larger tendency for 𝛼-helix formation than ANP32A230-249 peptide, supporting the idea of a more compact and best-defined conformation for ANP32B LCAR (Figure 2B,C). We further analyzed the solvent-accessible surface area (SASA) of the top 10 lowest-energy conformers of each peptide, as well as their atomic fluctuations (root-mean-square-fluctuation, RMSF) (Figure 2D,E). ANP32A230-249 exhibited a significantly higher exposure to solvent than ANP32B232-251. Moreover, the ANP32B232-251 peptide showed a decreased inter-model RMSF as compared to ANP32A230-249, in agreement with the lower backbone RMSD values and higher compaction degree inferred from CYANA-calculated NMR models. Altogether, our NMR structural models revealed that the ANP32A230-249 stretch mainly adopts open conformations with a large global mobility, as inferred from backbone RMSD and RMSF values in addition to solvent accessibility, whereas the ANP32B paralog mainly adopts closed conformations. A different conformation propensity of the LCAR could affect the manner in which ANP32 proteins interact with their different binding partners. Therefore, we first compared the binding capacity of ANP32A and ANP32B to histones. To test that, we performed a coimmunoprecipitation experiment after transfection of HeLa cells with expression vectors for c-myc-tagged ANP32A (Δ𝛿AVG =0.0 ppm) to dark blue (Δ𝛿AVG >0.075 ppm). Residues exhibiting Δ𝛿AVG >0.075 ppm are marked with blue labels. Residues showing a linebroadening larger than the average plus two SDs (15.96 Hz) are in green. Residues labeled with an asterisk show both significant Δ𝛿AVG and broadening. The heme group is in red. G) Biolayer Interferometry analysis of the interaction between ANP32A or ANP32B with Cc. The wavelength shifts at equilibrium were plotted against Ccconcentration. Adv. Sci. 2025,12, 2415566 2415566 (5 of 14) © 2025 The Author(s). Advanced Science published by Wiley-VCH GmbH 21983844, 2025, 12, Downloaded from https://advanced.onlinelibrary.wiley.com/doi/10.1002/advs.202415566 by Readcube (Labtiva Inc.), Wiley Online Library on [24/04/2025]. 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 www.advancedsciencenews.com www.advancedscience.com Table 1. Thermodynamic parameters of the cytochrome c:ANP32A complex at low or moderate ionic strength. Ionic strength [mM] KD[μM] ΔG[kcal mol−1]ΔH[kcal mol−1]–TΔS[kcal mol−1]n 25 3.6 −7.4 0.4 −7.8 0.65 34.0 −6.1 8.6 −14.1 0.65 100 32.0 −6.1 1.5 −7.6 0.60 Thermodynamic parameters for the interaction of ANP32A with Ccat low and moderate ionic strength. Enthalpy (ΔH), entropic contribution (–TΔS), equilibrium dissociation constant (KD), Gibbs free energy (ΔG), and reaction stoichiometry (n) are shown. Relative errors: KD, 20%; ΔG, 0.1 kcal/mol; ΔHand –TΔS,0.4kcal/mol;andn, 0.03. or ANP32B. The presence of histone H3 in the immunoprecipitated sample with anti-c-myc, detected by western blot, confirmed that H3 binds both ANP32 proteins in a similar manner (Figure S2A, Supporting Information). Thus, LCAR differences do not affect histone binding of ANP32 proteins, in agreement with that being mediated by the LRR domains.[14] 2.2. The LCAR of ANP32A Interacts with Cytochrome cUnder DNA Damage Conditions To determine if the Pro/Thr and/or the Gln/Glu substitution could enable binding discrimination through the LCAR region, we tested as a binding partner the hemeprotein Cc, which specifically recognizes the ANP32B C-terminal NLScontaining stretch.[31]We first tested whether Cccan also recognize the ANP32A LCAR by a pulldown assay, using extracts from HEK293T cells transfected with either the empty pCDNA3.1 vector or that encoding ANP32A-c-myc, and incubated with recombinant Cc. The resulting Cc:ANP32A complex was co-purified, revealing that Ccinteracts with ANP32A (Figure 3A). After treating Heltog cells (HeLa cells constitutively expressing the Ccgene fused to green fluorescent protein [GFP])[37]with camptothecin (CPT) for 1 or 4 h, to induce DNA double-strand breaks,[38]we observed that Cc-GFP translocated into the cell nucleus, where also localizes ANP32A (Figure S2B, Supporting Information). Colocalization of ANP32A and Ccin the cell nucleus under DNA damage conditions supports the possible interaction between these proteins in the cellular context, as it was previously demonstrated for ANP32B.[31,39] Due to the high sequence similarity between ANP32A and ANP32B, it seems plausible that Ccrecognizes both proteins at the same region. Since the interaction of the LCAR of ANP32B with Ccis well established,[31]we performed Isothermal Titration Calorimetry (ITC) assays with Ccand either the fulllength ANP32A or a construct lacking its LCAR (ANP32A1-167) (Figure 3B). ITC analyses showed that ANP32A binds to Ccat both low (25 mM) and moderate (100 mM) ionic strength, although one of the two binding sites was lost in the latter case (Figure 3C). Interestingly, ANP32A1-167 was unable to interact with Cceven at low ionic strength. This result suggests that Ccexclusively recognizes the LCAR of ANP32A. Titration of ANP32A with Ccat low ionic strength indicated that two molecules of Cc bind the ANP32A LCAR at independent binding sites, with no observed cooperativity (Table 1). Notably, Ccbinding to ANP32ALCAR is entropically driven, similar to many other complexes involving IDDs.[40–42]At moderate ionic strength, data are consistent with a single site for Ccwith low affinity (ca. 32 μM) and have enthalpic and entropic values similar to those obtained at low ionic strength. Additionally, we recorded 2D 1H-15N heteronuclear single quantum correlation (HSQC) spectra of 15NCc,eitherfreeorin the presence of increasing amounts of 14N full-length ANP32A or ANP32A1-167, under low ionic strength conditions. Ccamide signals showed no significant chemical-shift perturbations in titrations with ANP32A1-167, further corroborating the ITC findings (Figure 3D). A thorough analysis of the Cc:ANP32A complex at a 1:1 ratio shows that particular Ccresonances experience specific chemical-shift perturbations and substantial broadening (Figure S3, Supporting Information), consistent with an intermediate chemical exchange rate within the NMR timescale and in agreement with the transitory nature of the complex and its dissociation constant (KD) within the micromolar range (Table 1and Figure 3E). To delimit the interaction surface between Ccand ANP32A, we represented the averaged chemical-shift perturbations (Δ𝛿AVG ) and linewidth broadening of Ccamide resonances on the Cc surface (Figure 3F). Most residues with large Δ𝛿AVG and significant broadening map close to the heme crevice. In addition, an additional cluster of perturbed residues with either significant Δ𝛿AVG or broadening are located at the opposite face of the Cc heme group. This spread pattern of interacting residues supports the formation of structurally heterogeneous fuzzy ensembles, in which the chaperone´s LCAR plays a crucial role.[43–45]Thus, the ANP32A LCAR is likely represented by an ensemble of exchanging conformations, which in turn facilitate its interaction with Cc. 2.3. Cytochrome cDifferentiates Between the LCARs of ANP32A and ANP32B Although both ANP32A and ANP32B interact with Ccvia their Cterminal region, there are remarkable differences in the thermodynamic parameters and binding surfaces between full-length ANP32A and ANP32B in complex with Cc(Figure 3C,F).[31]At low ionic strength, Ccbinds to ANP32A with higher affinity (KD ca. 3.6 μM) than the one previously reported for ANP32B (KDca. 9.5 μM). Strikingly, the ANP32A and ANP32B interactions with Ccpresent very different thermodynamic profiles (Cc-ANP32A, ΔH: 0.4 kcal mol−1and -TΔS: -7.8 kcal mol−1;Cc-ANP32B, ΔH: -10.7 kcal mol−1and -TΔS: 3.9 kcal mol−1; Table 1).[31]Moreover, ANP32A contacts Ccthrough an extended surface. Therefore, Cclikely presents different modes of recognizing the LCAR of both chaperones. To confirm the differences in binding, we performed Biolayer Interferometry assays by attaching biotinylated ANP32 proteins to a streptavidin-coated sensor and incubating with increasing concentrations of Cc(Figure 3G). Again, Ccinteracted with ANP32A with higher affinity than with ANP32B (Cc-ANP32A, KDca. 2.3 μM; Cc-ANP32B, KDca. 4.2 μM). Also, the total number of Ccmolecules that interact with the sensor Adv. Sci. 2025,12, 2415566 2415566 (6 of 14) © 2025 The Author(s). Advanced Science published by Wiley-VCH GmbH 21983844, 2025, 12, Downloaded from https://advanced.onlinelibrary.wiley.com/doi/10.1002/advs.202415566 by Readcube (Labtiva Inc.), Wiley Online Library on [24/04/2025]. 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 www.advancedsciencenews.com www.advancedscience.com Table 2. Thermodynamic parameters of the cytochrome c:ANP32A/B peptide complexes. Protein complex KD[μM] ΔG[kcal mol−1]ΔH[kcal mol−1]−TΔS [kcal mol−1] n Cc:ANP32A230-249 69 −5.7 −2.6 −3.1 0.76 Cc:ANP32B232-251 710 −4.3 −8.0 3.7 1.20 Cc:ANP32A230-249 P241T 190 −5.1 −6.9 1.8 0.77 Cc:ANP32A230-249 Q235E/P241T 190 −5.1 −8.2 3.1 0.77 Thermodynamic parameters for the interactions of ANP32A/B peptides with Cc. Enthalpy (ΔH), entropic contribution (–TΔS), equilibrium dissociation constant (KD), Gibbs free energy (ΔG), and reaction stoichiometry (n) are shown. Relative errors: KD, 20%; ΔG, 0.1 kcal/mol; ΔHand –TΔS,0.4kcal/mol;andn, 0.03. (Bmax) was greater for Cc-ANP32A complex, in consonance with the broader interaction surface. To corroborate our hypothesis that the divergent conformations and dynamics of the NLS-containing peptides are responsible for the different interactions with protein partners, we scaled-down the system by performing ITC assays between the NLS-containing peptides (ANP32A230-249 and ANP32B232-251) and Cc. This simplification of the binding model facilitated a picture of the thermodynamic profile of Ccbinding to the NLS-containing regions. ITC-derived data were fitted to a single binding-site model (Figure S4, Supporting Information). Both peptides exhibited much higher KDvalues than the full-length ANP32A/B protein when interacting with Cc, indicating the relevance of the acidic content of the entire LCAR. The interaction between Ccand ANP32A230-249 was mainly entropically driven (–TΔS: -3.1 kcal mol−1; Table 2), as for the full-length protein. Surprisingly, Ccpresented a 10-fold higher affinity for ANP32A230-249 than for ANP32B232-251 (Table 2); our structural models suggest that this could be due to the ANP32A peptide adopting more appropriate conformations for Ccbinding (Figure 2A). We then explored the specific contributions of the major amino acid substitutions between the ANP32A and ANP32B peptides (i.e., Gln235 and Pro241) to the binding event. We designed two additional synthetic 20-residue peptides, using the ANP32A230-249 wild-type stretch as a template, to suppress differences between the ANP32A and ANP32B LCARs. One of these peptides contains a P241T single mutation, and the other, a Q235E/P241T double mutation. Of note, the single substitution of Pro-to-Thr in ANP32A230-249 was necessary and sufficient to: i) weaken the binding between Ccand the peptide, and ii) cause a loss of conformational entropy and render the binding into an entropically-driven one (Table 2and Figure S4, Supporting Information). The double Q235E/P241T ANP32A230-249 mutant reproduced the thermodynamic profile of ANP32B232-251 complexed with Cc(Table 2and Figure S4, Supporting Information). Although complex affinity was diminished for both the single and the double mutants, neither showed KDvalues as high as those determined for the Cc:ANP32B232-251 complex. A plausible explanation is the presence of an additional Gly-Gly pair in the ANP32B232-251 peptide, which provides a slightly different molecular context and is likely responsible for changes in the global mobility and internal dynamics of the molecule.[46] To explore the impact of different conformational arrangements of ANP32A230-249 or ANP32B232-251 in binding to Cc,weperformed ab initio flexible Brownian dynamics (BD) simulations. The 10 best conformers of each peptide—inferred from CYANA calculations from NOESY experiments (Figure 2A)—were selected and set as targets of Cc.Ccapproaching trajectories to the alternating conformations of both paralog peptides were monitored. Our results showed that Ccpreferably explores the C-end of the ANP32A230-249 or ANP32B232-251 peptide (Figure 4A), where negatively-charged residues accumulate. However, the area explored by Ccon the ANP32A230-249 peptide was wider than that on the ANP32B232-251. This finding agrees with the open local conformation of the ANP32A230-249 stretch and its larger SASA, which may facilitate Ccbinding. NMR titrations of 15N-labeled Ccwith both ANP32A230-249 and ANP32B232-251 peptides showed that the chemical-shift perturbations spread over the whole surface of Cc–including the opposite face to the heme group–was induced mainly by the ANP32A230-249 peptide (Figure 4B). To visualize the Ccsurface sampled by both ANP32 C-terminal peptides, we performed NMR-driven docking simulations. Ccwas set as the target of the most represented conformation of ANP32A230-249 or ANP32B232-251 peptides among the complexes provided by the aforementioned flexible BD calculations, and experimental restrictions based on NMR titrations (Figure 4B) were imposed. Simulations rendered mass centers for ANP32A230-249 showing a widely distributed sampling of Cc, from its heme-surrounding region to its opposite side (Figure 4C), resembling the NMR spectra obtained with full length ANP32A on Cc(Figure 3F). Such distribution of complexes was narrowed down to a more limited area for the ANP32B232-251 peptide (Figure 4C), in agreement with the map of NMR chemical-shift perturbations (Figure 4B). This difference is likely due to a more compact conformation of the ANP32B232-251 peptide limiting its ability to sample the hemeprotein. Notably, ab initio BD simulations yielded very similar complex distributions for both interactions (Figure S5, Supporting Information), confirming that the obtained complexes are the most energetically favorable. To further corroborate differences in the dynamic behavior of 15N-Cc/ANP32A230-249 and 15NCc/ANP32B232-251 complexes, NMR relaxation measurements were recorded (Figure 5A,B). Binding to ANP32A and ANP32B peptides did not significantly alter the rotational correlational time (𝜏c)ofCcwith respect to free hemeprotein (free Cc6.0 ns, ANP32A230-249-bound Cc5.9 ns, ANP32A232-251-bound Cc5.8 ns) likely due to the small molecular weight of both peptides. Comparing the differences in R1,R2, and 15N{1H} NOE parameters revealed that the interface of Nterminal and C-terminal 𝛼-helixes in foldon I of ANP32A-bound Ccexhibited higher mobility in the ps-to-ns timescale, as the 15N{1H} NOE values for the V3, K5, K8, D93, L94, I95 residues Adv. Sci. 2025,12, 2415566 2415566 (7 of 14) © 2025 The Author(s). Advanced Science published by Wiley-VCH GmbH 21983844, 2025, 12, Downloaded from https://advanced.onlinelibrary.wiley.com/doi/10.1002/advs.202415566 by Readcube (Labtiva Inc.), Wiley Online Library on [24/04/2025]. 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 www.advancedsciencenews.com www.advancedscience.com Figure 4. Cytochrome cdifferentiates between the LCAR of ANP32A and ANP32B. A) Simulated flexible ab-initio Brownian dynamics solutions for Cc sampling of the ANP32A230-249 (upper panels) and ANP32B232-251 (lower panels) peptides. Representations of the peptide structures show ribbons colored in black for the best-fitted solution provided by CYANA for each peptide. Mass centers of Cc(corresponding to the 50000 registered complexes for each simulation) are shown as red spheres. B) Representation of Δ𝛿AVG of Ccamide signals upon Cc-ANP32A230-249 (upper panel)orCc-ANP32B232-251 (lower panel) complex formation at 1:6 ratio (Cc:peptide). Δ𝛿AVG is represented as a blue color gradient, ranging from white (Δ𝛿AVG =0.0 ppm) to dark blue, whereby Δ𝛿AVG >0.035 ppm or 0.050 ppm for Cc:ANP32A230-249 and Cc:ANP32B232-251, respectively. The heme group is represented in red. C) NMR-driven docking solutions for ANP32A230-249 and ANP32B232-251 complexes with Cc. Mass centers distributions of ANP32A230-249 (upper panels)or ANP32B232-251 (lower panels), representing the 10000 lowest energy conformations of both complexes with Cc. Mass centers are shown as violet spheres for ANP32A230-249 and blue spheres for ANP32B232-251. Ribbon and surface areas of Cc(PDB entry 2N9I) are shown in red; the heme group, in light green. are smaller for ANP32A-bound Ccprotein. Similar behavior is observed for the most of 60´s helix of Cc(foldon II) in complex with ANP32A. Altogether, this finding supports the highly dynamic behavior of Ccupon binding to the ANP32A230-249 peptide, in line with a fuzzy recognition manner. In addition, Ccresidues in complex with ANP32A230-249 experienced larger R2rates than in the presence of ANP32B232-251, due to conformational exchange in the μs-to-ms timescale that constraints local motions. The scattered pattern of perturbed residues on the Ccsurface perfectly matches with NMR chemical-shift perturbation maps and NMR-restraint docking calculations (Figure 4B,C). 2.4. Pro-To-Thr Substitution also Affects ANP32 Binding to HuR The LCARs of ANP32A and ANP32B are implicated in the binding of these proteins with multiple targets, such as the polymerase and nucleoprotein of the influenza A virus or the RNA-binding protein HuR.[3,20,21]As the Pro241 substitution alters their affinity for Cc, we explored the possibility that the interaction with other ligands could also be affected. To test our hypothesis, we studied HuR interactions with the ANP32A230-249 and ANP32B232-251 peptides. We designed an HuR construct containing the RRM domains 2 and 3, separated by the Adv. Sci. 2025,12, 2415566 2415566 (8 of 14) © 2025 The Author(s). Advanced Science published by Wiley-VCH GmbH 21983844, 2025, 12, Downloaded from https://advanced.onlinelibrary.wiley.com/doi/10.1002/advs.202415566 by Readcube (Labtiva Inc.), Wiley Online Library on [24/04/2025]. 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 www.advancedsciencenews.com www.advancedscience.com Figure 5. Differences in the dynamics of Ccin the presence of ANP32 peptides. A) Differences in longitudinal relaxation rate R1(top), transversal relaxation rate R2(middle), and heteronuclear 15N{1H} NOE (bottom) between the experimental values at 500 MHz for the complexes formed between the reduced form of Ccand ANP32A230-249 or ANP32B232-251 peptides, plotted as a function of the residue number. Asterisks (*) indicate prolines and non-assigned residues. A scheme of the secondary structure elements of Ccis included at the top. B) Ribbon structure of Cc(PDB: 2N9I) colored according to the difference in its dynamic properties when forming complexes with ANP32A230-249 (red) or ANP32B232-251 (blue) peptides. Prolines, non-assigned residues and the heme group are in grey. Models has been calculated with CHIMERA 1.14. hinge region of HuR (HuR106-326), which interacts with ANP32 proteins.[3]We then performed ITC assays with HuR106-326 and the ANP32A230-249 or ANP32B232-251 peptides (Figure 6A). Similar to Cc’s affinity, HuR’s affinity was ca. 5-fold higher for ANP32A230-249 than for ANP32B232-251 at low ionic strength, suggesting that the open conformation of the ANP32A peptide enhances interactions with ANP32 target proteins through the Cterminal LCAR. ITC results indicate that two molecules of each ANP32 peptide bind to HuR106-326, with the interaction driven by enthalpy (Table S2, Supporting Information). This finding perfectly matches with rigid-body/docking abinitio BD simulations, with HuR106-326 as the target of each ANP32 peptide. Both ANP32A230-249 and ANP32B232-251 peptides mainly explored the hinge region of HuR (Figure 6B), in agreement with previous reports.[3,22]While the HuR/ANP32B complex showed two preferred, most delimited binding sites for the ANP32B232-251 peptide (in agreement with the stoichiometry HuR:ANP32B of 1:2 inferred by ITC), the ANP32A230-249 peptide widely sampled most of the positively-charged HuR106-326.Thesedifferences in modes of interaction resembled those described for the Cc/ANP32 complexes mentioned above, with the ANP32B232-251 peptide exhibiting a most limited flanking of HuR, probably accounting for a reduced interacting surface. Both interactions were characterized by a negative enthalpy due to long-range electrostatic interactions during HuR-involving complexes formation under low ionic strength. The highly positively-charged HuR hinge—with a pIof ca. 11.5 and several His residues susceptible of protonation and deprotonation at physiological pH—makes electrostatics key in HuR/ANP32 adducts (Figure 6B). Flexible BDs of HuR106-326 and the ANP32A230-249 or ANP32B232-251 peptide revealed that HuR explores the C-end of both peptides, although the contacts with the ANP32A230-249 peptide map to a broader region (Figure 6C). Thus, the open conformation found in the ANP32A C-terminal region might represent a binding favorable arrangement, influencing the protein contacts and subsequent process regulation. 3. Discussion In this work, we demonstrate the importance of punctual modifications in IDDs for the dynamics and function of the proteins, using the Pro/Thr evolution in ANP32A/ANP32B as an example. Molecular models of the C-terminal regions of ANP32A and ANP32B substantially differ in their structure and dynamics Adv. Sci. 2025,12, 2415566 2415566 (9 of 14) © 2025 The Author(s). Advanced Science published by Wiley-VCH GmbH 21983844, 2025, 12, Downloaded from https://advanced.onlinelibrary.wiley.com/doi/10.1002/advs.202415566 by Readcube (Labtiva Inc.), Wiley Online Library on [24/04/2025]. 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