Hydrogen sulfide production does not affect antibiotic resistance in Pseudomonas aeruginosa.

Hydrogen sulfide (H2S) has been proposed to protect bacteria from antibiotics, pointing to H2S-producing enzymes as possible targets for the development of antibiotic adjuvants. Here, MIC assays performed with Pseudomonas aeruginosa mutants producing altered H2S levels demonstrate that H2S does not affect antibiotic resistance in this bacterium. Moreover, correlation analyses in a large collection of P. aeruginosa cystic fibrosis isolates argue against the protective role of H2S from antibiotic activity during chronic lung infection.

Rapid kinetics of calcium dissociation from plant calmodulin and calmodulin-like proteins and effect of target peptides.

Calcium (Ca2+) signaling represents a universal information code in plants, playing crucial roles spanning developmental processes to stress responses. Ca2+ signals are decoded into defined plant adaptive responses by different Ca2+ sensing proteins, including calmodulin (CaM) and calmodulin-like (CML) proteins. Although major advances have been achieved in describing how these Ca2+ decoding proteins interact and regulate downstream target effectors, the molecular details of these processes remain largely unknown. Herein, the kinetics of Ca2+ dissociation from a conserved CaM and two CML isoforms from A. thaliana has been studied by fluorescence stopped-flow spectroscopy. Kinetic data were obtained for the isolated Ca2+-bound proteins as well as for the proteins complexed with different target peptides. Moreover, the lobe specific interactions between the Ca2+ sensing proteins and their targets were characterized by using a panel of protein mutants deficient in Ca2+ binding at the N-lobe or C-lobe. Results were analyzed and discussed in the context of the Ca2+-decoding and Ca2+-controlled target binding mechanisms in plants.

The interplay of self-assembly and target binding in centrin 1 from Toxoplasma gondii.

Centrins are conserved calcium (Ca2+)-binding proteins typically associated with centrosomes that have been implicated in several biological processes. In Toxoplasma gondii, a parasite that causes toxoplasmosis, three centrin isoforms have been recognized. We have recently characterized the metal binding and structural features of isoform 1 (TgCEN1), demonstrating that it possesses properties consistent with a role as a Ca2+ sensor and displays a Ca2+-dependent tendency to self-assemble. Herein, we expanded our studies, focusing on the self-association and target binding properties of TgCEN1 by combining biophysical techniques including dynamic light scattering, isothermal titration calorimetry, nuclear magnetic resonance, circular dichroism, and fluorescence spectroscopy. We found that the self-assembly process of TgCEN1 depends on different physicochemical factors, including Ca2+ concentration, temperature, and protein concentration, and is mediated by both electrostatic and hydrophobic interactions. The process is completely abolished upon removal of the first 21-residues of the protein and is significantly reduced in the presence of a binding target peptide derived from the human XPC protein (P17-XPC). Titration of P17-XPC to the intact protein and isolated domains showed that TgCEN1 possesses two binding sites with distinct affinities and Ca2+ sensitivity; a high-affinity site in the C-lobe which may be constitutively bound to the peptide and a low-affinity site in the N-lobe which is active only upon Ca2+ stimulus. Overall, our results suggest a specific mechanism of TgCEN1 for Ca2+-modulated target binding and support a N-to-C self-assembly mode, in which the first 21-residues of one molecule likely interact with the C-lobe of the other.

Folding Mechanism and Aggregation Propensity of the KH0 Domain of FMRP and Its R138Q Pathological Variant.

The K-homology (KH) domains are small, structurally conserved domains found in proteins of different origins characterized by a central conserved ßααß "core" and a GxxG motif in the loop between the two helices of the KH core. In the eukaryotic KHI type, additional αß elements decorate the "core" at the C-terminus. Proteins containing KH domains perform different functions and several diseases have been associated with mutations in these domains, including those in the fragile X mental retardation protein (FMRP). FMRP is an RNA-binding protein crucial for the control of RNA metabolism whose lack or mutations lead to fragile X syndrome (FXS). Among missense mutations, the R138Q substitution is in the KH0 degenerated domain lacking the classical GxxG motif. By combining equilibrium and kinetic experiments, we present a characterization of the folding mechanism of the KH0 domain from the FMRP wild-type and of the R138Q variant showing that in both cases the folding mechanism implies the accumulation of an on-pathway transient intermediate. Moreover, by exploiting a battery of biophysical techniques, we show that the KH0 domain has the propensity to form amyloid-like aggregates in mild conditions in vitro and that the R138Q mutation leads to a general destabilization of the protein and to an increased fibrillogenesis propensity.

Proteomic Investigation of the Role of Nucleostemin in Nucleophosmin-Mutated OCI-AML 3 Cell Line.

Nucleostemin (NS; a product of the GNL3 gene) is a nucleolar-nucleoplasm shuttling GTPase whose levels are high in stem cells and rapidly decrease upon differentiation. NS levels are also high in several solid and hematological neoplasms, including acute myeloid leukaemia (AML). While a role in telomere maintenance, response to stress stimuli and favoring DNA repair has been proposed in solid cancers, little or no information is available as to the role of nucleostemin in AML. Here, we investigate this issue via a proteomics approach. We use as a model system the OCI-AML 3 cell line harboring a heterozygous mutation at the NPM1 gene, which is the most frequent driver mutation in AML (approximately 30% of total AML cases). We show that NS is highly expressed in this cell line, and, contrary to what has previously been shown in other cancers, that its presence is dispensable for cell growth and viability. However, proteomics analysis of the OCI-AML 3 cell line before and after nucleostemin (NS) silencing showed several effects on different biological functions, as highlighted by ingenuity pathway analysis (IPA). In particular, we report an effect of down-regulating DNA repair through homologous recombination, and we confirmed a higher DNA damage rate in OCI-AML 3 cells when NS is depleted, which considerably increases upon stress induced by the topoisomerase II inhibitor etoposide. The data used are available via ProteomeXchange with the identifier PXD034012.

Nucleophosmin in Its Interaction with Ligands.

Nucleophosmin (NPM1) is a mainly nucleolar protein that shuttles between nucleoli, nucleoplasm and cytoplasm to fulfill its many functions. It is a chaperone of both nucleic acids and proteins and plays a role in cell cycle control, centrosome duplication, ribosome maturation and export, as well as the cellular response to a variety of stress stimuli. NPM1 is a hub protein in nucleoli where it contributes to nucleolar organization through heterotypic and homotypic interactions. Furthermore, several alterations, including overexpression, chromosomal translocations and mutations are present in solid and hematological cancers. Recently, novel germline mutations that cause dyskeratosis congenita have also been described. This review focuses on NPM1 interactions and inhibition. Indeed, the list of NPM1 binding partners is ever-growing and, in recent years, many studies contributed to clarifying the structural basis for NPM1 recognition of both nucleic acids and several proteins. Intriguingly, a number of natural and synthetic ligands that interfere with NPM1 interactions have also been reported. The possible role of NPM1 inhibitors in the treatment of multiple cancers and other pathologies is emerging as a new therapeutic strategy.

Nucleophosmin contains amyloidogenic regions that are able to form toxic aggregates under physiological conditions.

Nucleophosmin (NPM)-1 is a multifunctional protein involved in a variety of biologic processes and has been implicated in the pathogenesis of several human malignancies. To gain insight into the role of isolated fragments in NPM1 activities, we dissected the C-terminal domain (CTD) into its helical fragments. In this study, we observed the unexpected structural behavior of the peptide fragment corresponding to helix (H)2 (residues 264-277). This peptide has a strong tendency to form amyloidlike assemblies endowed with fibrillar morphology and ß-sheet structure, under physiologic conditions, as shown by circular dichroism, thioflavin T, and Congo red binding assays; dynamic light scattering; and atomic force microscopy. The aggregates are also toxic to neuroblastoma cells, as determined using 3-(4;5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction and Ca(2+) influx assays. We also found that the extension of the H2 sequence beyond its N terminus, comprising the connecting loop with H1, delayed aggregation and its associated cytotoxicity, suggesting that contiguous regions of H2 have a protective role in preventing aggregation. Our findings and those in the literature suggest that the helical structures present in the CTD are important in preventing harmful aggregation. These findings could elucidate the pathogenesis of acute myeloid leukemia (AML) caused by NPM1 mutants. Because the CTD is not properly folded in these mutants, we hypothesize that the aggregation propensity of this NPM1 region is involved in the pathogenesis of AML. Preliminary assays on NPM1-Cter-MutA, the most frequent AML-CTD mutation, revealed its significant propensity for aggregation. Thus, the aggregation phenomena should be seriously considered in studies aimed at unveiling the molecular mechanisms of this pathology.

Synergic role of nucleophosmin three-helix bundle and a flanking unstructured tail in the interaction with G-quadruplex DNA.

Nucleophosmin (NPM1) is a nucleocytoplasmic shuttling protein, mainly localized at nucleoli, that plays a number of functions in ribosome biogenesis and export, cell cycle control, and response to stress stimuli. NPM1 is the most frequently mutated gene in acute myeloid leukemia; mutations map to the C-terminal domain of the protein and cause its denaturation and aberrant cytoplasmic translocation. NPM1 C-terminal domain binds G-quadruplex regions at ribosomal DNA and at gene promoters, including the well characterized sequence from the nuclease-hypersensitive element III region of the c-MYC promoter. These activities are lost by the leukemic variant. Here we analyze the NPM1/G-quadruplex interaction, focusing on residues belonging to both the NPM1 terminal three-helix bundle and a lysine-rich unstructured tail, which has been shown to be necessary for high affinity recognition. We performed extended site-directed mutagenesis and measured binding rate constants through surface plasmon resonance analysis. These data, supported by molecular dynamics simulations, suggest that the unstructured tail plays a double role in the reaction mechanism. On the one hand, it facilitates the formation of an encounter complex through long range electrostatic interactions; on the other hand, it directly contacts the G-quadruplex scaffold through multiple and transient electrostatic interactions, significantly enlarging the contact surface.

Nucleophosmin mutations alter its nucleolar localization by impairing G-quadruplex binding at ribosomal DNA.

Nucleophosmin (NPM1) is an abundant nucleolar protein implicated in ribosome maturation and export, centrosome duplication and response to stress stimuli. NPM1 is the most frequently mutated gene in acute myeloid leukemia. Mutations at the C-terminal domain led to variant proteins that aberrantly and stably translocate to the cytoplasm. We have previously shown that NPM1 C-terminal domain binds with high affinity G-quadruplex DNA. Here, we investigate the structural determinants of NPM1 nucleolar localization. We show that NPM1 interacts with several G-quadruplex regions found in ribosomal DNA, both in vitro and in vivo. Furthermore, the most common leukemic NPM1 variant completely loses this activity. This is the consequence of G-quadruplex-binding domain destabilization, as mutations aimed at refolding the leukemic variant also result in rescuing the G-quadruplex-binding activity and nucleolar localization. Finally, we show that treatment of cells with a G-quadruplex selective ligand results in wild-type NPM1 dislocation from nucleoli into nucleoplasm. In conclusion, this work establishes a direct correlation between NPM1 G-quadruplex binding at rDNA and its nucleolar localization, which is impaired in the acute myeloid leukemia-associated protein variants.

Recognition and binding of apocytochrome c to P. aeruginosa CcmI, a component of cytochrome c maturation machinery.

The biogenesis of c-type cytochromes (Cytc) is a process that in Gram-negative bacteria demands the coordinated action of different periplasmic proteins (CcmA-I), whose specific roles are still being investigated. Activities of Ccm proteins span from the chaperoning of heme b in the periplasm to the specific reduction of oxidized apocytochrome (apoCyt) cysteine residues and to chaperoning and recognition of the unfolded apoCyt before covalent attachment of the heme to the cysteine thiols can occur. We present here the functional characterization of the periplasmic domain of CcmI from the pathogen Pseudomonas aeruginosa (Pa-CcmI*). Pa-CcmI* is composed of a TPR domain and a peculiar C-terminal domain. Pa-CcmI* fulfills both the ability to recognize and bind to P. aeruginosa apo-cytochrome c551 (Pa-apoCyt) and a chaperoning activity towards unfolded proteins, as it prevents citrate synthase aggregation in a concentration-dependent manner. Equilibrium and kinetic experiments with Pa-CcmI*, or its isolated domains, with peptides mimicking portions of Pa-apoCyt sequence allow us to quantify the molecular details of the interaction between Pa-apoCyt and Pa-CcmI*. Binding experiments show that the interaction occurs at the level of the TPR domain and that the recognition is mediated mainly by the C-terminal sequence of Pa-apoCyt. The affinity of Pa-CcmI* to full-length Pa-apoCyt or to its C-terminal sequence is in the range expected for a component of a multi-protein complex, whose task is to receive the apoCyt and to deliver it to other components of the apoCyt:heme b ligation protein machinery.

Conformational and dynamic properties of the KH1 domain of FMRP and its fragile X syndrome linked G266E variant.

The Fragile X messenger ribonucleoprotein (FMRP) is a multi-domain protein involved in interactions with various macromolecules, including proteins and coding/non-coding RNAs. The three KH domains (KH0, KH1 and KH2) within FMRP are recognized for their roles in mRNA binding. In the context of Fragile X syndrome (FXS), over-and-above CGG triplet repeats expansion, three specific point mutations have been identified, each affecting one of the three KH domains (R138QKH0, G266EKH1, and I304NKH2) resulting in the expression of non-functional FMRP. This study aims to elucidate the molecular mechanism underlying the loss of function associated with the G266EKH1 pathological variant. We investigate the conformational and dynamic properties of the isolated KH1 domain and the two KH1 site-directed mutants G266EKH1 and G266AKH1. Employing a combined in vitro and in silico approach, we reveal that the G266EKH1 variant lacks the characteristic features of a folded domain. This observation provides an explanation for functional impairment observed in FMRP carrying the G266E mutation within the KH1 domain, as it renders the domain unable to fold properly. Molecular Dynamics simulations suggest a pivotal role for residue 266 in regulating the structural stability of the KH domains, primarily through stabilizing the α-helices of the domain. Overall, these findings enhance our comprehension of the molecular basis for the dysfunction associated with the G266EKH1 variant in FMRP.

Catalytic specificity and crystal structure of cystathionine γ-lyase from Pseudomonas aeruginosa.

The escalating drug resistance among microorganisms underscores the urgent need for innovative therapeutic strategies and a comprehensive understanding of bacteria's defense mechanisms against oxidative stress and antibiotics. Among the recently discovered barriers, the endogenous production of hydrogen sulfide (H2S) via the reverse transsulfuration pathway, emerges as a noteworthy factor. In this study, we have explored the catalytic capabilities and crystal structure of cystathionine γ-lyase from Pseudomonas aeruginosa (PaCGL), a multidrug-opportunistic pathogen chiefly responsible for nosocomial infections. In addition to a canonical L-cystathionine hydrolysis, PaCGL efficiently catalyzes the production of H2S using L-cysteine and/or L-homocysteine as alternative substrates. Comparative analysis with the human enzyme and counterparts from other pathogens revealed distinct structural features within the primary enzyme cavities. Specifically, a distinctly folded entrance loop could potentially modulate the access of substrates and/or inhibitors to the catalytic site. Our findings offer significant insights into the structural evolution of CGL enzymes across different pathogens and provide novel opportunities for developing specific inhibitors targeting PaCGL.

Structure of nucleophosmin DNA-binding domain and analysis of its complex with a G-quadruplex sequence from the c-MYC promoter.

Nucleophosmin (NPM1) is a nucleocytoplasmic shuttling protein, mainly localized at nucleoli, that plays a key role in several cellular functions, including ribosome maturation and export, centrosome duplication, and response to stress stimuli. More than 50 mutations at the terminal exon of the NPM1 gene have been identified so far in acute myeloid leukemia; the mutated proteins are aberrantly and stably localized in the cytoplasm due to high destabilization of the NPM1 C-terminal domain and the appearance of a new nuclear export signal. We have shown previously that the 70-residue NPM1 C-terminal domain (NPM1-C70) is able to bind with high affinity a specific region at the c-MYC gene promoter characterized by parallel G-quadruplex structure. Here we present the solution structure of the NPM1-C70 domain and NMR analysis of its interaction with a c-MYC-derived G-quadruplex. These data were used to calculate an experimentally restrained molecular docking model for the complex. The NPM1-C70 terminal three-helix bundle binds the G-quadruplex DNA at the interface between helices H1 and H2 through electrostatic interactions with the G-quadruplex phosphate backbone. Furthermore, we show that the 17-residue lysine-rich sequence at the N terminus of the three-helix bundle is disordered and, although necessary, does not participate directly in the contact surface in the complex.

The folding pathway of a functionally competent C-terminal domain of nucleophosmin: protein stability and denatured state residual structure.

Nucleophosmin (NPM1) is a nucleolar protein implicated in ribosome biogenesis, centrosome duplication and cell cycle control; the NPM1 gene is the most frequent target for mutations in Acute Myeloid Leukemia. Mutations map to the C-terminal domain of the protein and cause its unfolding, loss of DNA binding properties and aberrant cellular localization. Here we investigate the folding pathway and denatured state properties of a NPM1 C-terminal domain construct encompassing the last 70 residues in the reference sequence. This construct is more stable than the previously characterized domain, which consisted of the last 53 residues. Data reveal that, similarly to what was discovered for the shorter construct, also the 70-residue construct of NPM1 displays a detectable residual structure in its denatured state. The higher stability of the latter domain allows us to conclude that the denatured state is robust to changes in solvent composition and that it consists of a discrete state in equilibrium with the expanded fully unfolded conformation. This observation, which might appear as a technicality, is in fact of general importance for the understanding of the folding of proteins. The implications of our results are discussed in the context of previous works on single domain helical proteins.

Role of myristoylation in modulating PCaP1 interaction with calmodulin.

Plasma membrane-associated Cation-binding Protein 1 (PCaP1) belongs to the plant-unique DREPP protein family with largely unknown biological functions but ascertained roles in plant development and calcium (Ca2+) signaling. PCaP1 is anchored to the plasma membrane via N-myristoylation and a polybasic cluster, and its N-terminal region can bind Ca2+/calmodulin (CaM). However, the molecular determinants of PCaP1-Ca2+-CaM interaction and the functional impact of myristoylation in the complex formation and Ca2+ sensitivity of CaM remained to be elucidated. Herein, we investigated the direct interaction between Arabidopsis PCaP1 (AtPCaP1) and CaM1 (AtCaM1) using both myristoylated and non-myristoylated peptides corresponding to the N-terminal region of AtPCaP1. ITC analysis showed that AtCaM1 forms a high affinity 1:1 complex with AtPCaP1 peptides and the interaction is strictly Ca2+-dependent. Spectroscopic and kinetic Ca2+ binding studies showed that the myristoylated peptide dramatically increased the Ca2+-binding affinity of AtCaM1 and slowed the Ca2+ dissociation rates from both the C- and N-lobes, thus suggesting that the myristoylation modulates the mechanism of AtPCaP1 recognition by AtCaM1. Furthermore, NMR and CD spectroscopy revealed that the structure of both the N- and C-lobes of Ca2+-AtCaM1 changes markedly in the presence of the myristoylated AtPCaP1 peptide, which assumes a helical structure in the final complex. Overall, our results indicate that AtPCaP1 biological function is strictly related to the presence of multiple ligands, i.e., the myristoyl moiety, Ca2+ ions and AtCaM1 and only a full characterization of their equilibria will allow for a complete molecular understanding of the putative role of PCaP1 as signal protein.

Molecular insights into RmcA-mediated c-di-GMP consumption: Linking redox potential to biofilm morphogenesis in Pseudomonas aeruginosa.

The ability of many bacteria to form biofilms contributes to their resilience and makes infections more difficult to treat. Biofilm growth leads to the formation of internal oxygen gradients, creating hypoxic subzones where cellular reducing power accumulates, and metabolic activities can be limited. The pathogen Pseudomonas aeruginosa counteracts the redox imbalance in the hypoxic biofilm subzones by producing redox-active electron shuttles (phenazines) and by secreting extracellular matrix, leading to an increased surface area-to-volume ratio, which favors gas exchange. Matrix production is regulated by the second messenger bis-(3',5')-cyclic-dimeric-guanosine monophosphate (c-di-GMP) in response to different environmental cues. RmcA (Redox modulator of c-di-GMP) from P. aeruginosa is a multidomain phosphodiesterase (PDE) that modulates c-di-GMP levels in response to phenazine availability. RmcA can also sense the fermentable carbon source arginine via a periplasmic domain, which is linked via a transmembrane domain to four cytoplasmic Per-Arnt-Sim (PAS) domains followed by a diguanylate cyclase (DGC) and a PDE domain. The biochemical characterization of the cytoplasmic portion of RmcA reported in this work shows that the PAS domain adjacent to the catalytic domain tunes RmcA PDE activity in a redox-dependent manner, by differentially controlling protein conformation in response to FAD or FADH2. This redox-dependent mechanism likely links the redox state of phenazines (via FAD/FADH2 ratio) to matrix production as indicated by a hyperwrinkling phenotype in a macrocolony biofilm assay. This study provides insights into the role of RmcA in transducing cellular redox information into a structural response of the biofilm at the population level. Conditions of resource (i.e. oxygen and nutrient) limitation arise during chronic infection, affecting the cellular redox state and promoting antibiotic tolerance. An understanding of the molecular linkages between condition sensing and biofilm structure is therefore of crucial importance from both biological and engineering standpoints.

Integration of evolutionary and desolvation energy analysis identifies functional sites in a plant immunity protein.

Plant immune responses often depend on leucine-rich repeat receptors that recognize microbe-associated molecular patterns or pathogen-specific virulence proteins, either directly or indirectly. When the recognition is direct, a molecular arms race takes place where plant receptors continually and rapidly evolve in response to virulence factor evolution. A useful model system to study ligand-receptor coevolution dynamics at the protein level is represented by the interaction between pathogen-derived polygalacturonases (PGs) and plant polygalacturonase-inhibiting proteins (PGIPs). We have applied codon substitution models to PGIP sequences of different eudicotyledonous families to identify putative positively selected sites and then compared these sites with the propensity of protein surface residues to interact with protein partners, based on desolvation energy calculations. The 2 approaches remarkably correlated in pinpointing several residues in the concave face of the leucine-rich repeat domain. These residues were mutated into alanine and their effect on the recognition of several PGs was tested, leading to the identification of unique hotspots for the PGIP-PG interaction. The combined approach used in this work can be of general utility in cases where structural information about a pattern-recognition receptor or resistance-gene product is available.

Characterization of two 1,3-ß-glucan-modifying enzymes from Penicillium sumatraense reveals new insights into 1,3-ß-glucan metabolism of fungal saprotrophs.

BACKGROUND: 1,3-ß-glucan is a polysaccharide widely distributed in the cell wall of several phylogenetically distant organisms, such as bacteria, fungi, plants and microalgae. The presence of highly active 1,3-ß-glucanases in fungi evokes the biological question on how these organisms can efficiently metabolize exogenous sources of 1,3-ß-glucan without incurring in autolysis. RESULTS: To elucidate the molecular mechanisms at the basis of 1,3-ß-glucan metabolism in fungal saprotrophs, the putative exo-1,3-ß-glucanase G9376 and a truncated form of the putative glucan endo-1,3-ß-glucosidase (ΔG7048) from Penicillium sumatraense AQ67100 were heterologously expressed in Pichia pastoris and characterized both in terms of activity and structure. G9376 efficiently converted laminarin and 1,3-ß-glucan oligomers into glucose by acting as an exo-glycosidase, whereas G7048 displayed a 1,3-ß-transglucanase/branching activity toward 1,3-ß-glucan oligomers with a degree of polymerization higher than 5, making these oligomers more recalcitrant to the hydrolysis acted by exo-1,3-ß-glucanase G9376. The X-ray crystallographic structure of the catalytic domain of G7048, solved at 1.9 Å of resolution, consists of a (ß/α)8 TIM-barrel fold characteristic of all the GH17 family members. The catalytic site is in a V-shaped cleft containing the two conserved catalytic glutamic residues. Molecular features compatible with the activity of G7048 as 1,3-ß-transglucanase are discussed. CONCLUSIONS: The antagonizing activity between ΔG7048 and G9376 indicates how opportunistic fungi belonging to Penicillium genus can feed on substrates similar for composition and structure to their own cell wall without incurring in a self-deleterious autohydrolysis.

Nucleophosmin C-terminal leukemia-associated domain interacts with G-rich quadruplex forming DNA.

Nucleophosmin (NPM1) is a nucleocytoplasmic shuttling phosphoprotein, mainly localized at nucleoli, that plays a key role in ribogenesis, centrosome duplication, and response to stress stimuli. Mutations at the C-terminal domain of NPM1 are the most frequent genetic lesion in acute myeloid leukemia and cause the aberrant and stable translocation of the protein in the cytoplasm. The NPM1 C-terminal domain was previously shown to bind nucleic acids. Here we further investigate the DNA binding properties of the NPM1 C-terminal domain both at the protein and nucleic acid levels; we investigate the domain boundaries and identify key residues for high affinity recognition. Furthermore, we demonstrate that the NPM1 C-terminal domain has a preference for G-quadruplex forming DNA regions and induces the formation of G-quadruplex structures in vitro. Finally we show that a specific sequence found at the SOD2 gene promoter, which was previously shown to be a target of NPM1 in vivo, is indeed folded as a G-quadruplex in vitro under physiological conditions. Our data extend considerably present knowledge on the DNA binding properties of NPM1 and suggest a general role in the transcription of genes characterized by the presence of G-quadruplex forming regions at their promoters.