Human Glucosylceramide Synthase at Work as Provided by "In Silico" Molecular Docking, Molecular Dynamics, and Metadynamics.

Glucosylceramide synthase (GCS) is an enzyme that catalyzes the first reaction of ceramide glycosylation in sphingolipid metabolism. It represents a primary target in the pharmacological treatment of some lysosomal storage diseases (LSDs), such as Gaucher and Niemann-Pick syndromes. In this study, starting from the model reported in the AlphaFold Protein Structure Database, the location and conformations of GCS substrates and cofactors have been provided by a step-by-step in silico procedure, by which the functional manganese ion and the substrates have been inserted in the GCS structure through combined molecular docking and full-atomistic molecular dynamics approaches, including metadynamics. A detailed analysis by structural dynamics of the complete model system, i.e., the enzyme anchored to the plasma membrane, containing the manganese ion and the two substrates, has been carried out to identify its complex conformational landscape by means of well-tempered metadynamics. A final structure was selected, in which both substrates were present in the active site of the enzyme at minimum distance, thus giving support to a SNi-type reaction mechanism for catalysis. Asp236, Glu235, and Asp144 are found to interact with the metal cofactor, which is able to trap the phosphates of UDP-glucose, while Gly210, Trp276, and Val208 cooperate to provide its correct orientation. Phe205, Cys207, Tyr237, and Leu284 form a pocket for the polar head of the ceramide, which is transiently placed in position to determine the catalytic event, when His193 interacts with the head of the ceramide, thus anchoring the substrate to the active site.

Novel Mixed NOP/Opioid Receptor Peptide Agonists.

The nociceptin/orphanin FQ (N/OFQ)/N/OFQ receptor (NOP) system controls different biological functions including pain and cough reflex. Mixed NOP/opioid receptor agonists elicit similar effects to strong opioids but with reduced side effects. In this work, 31 peptides with the general sequence [Tyr/Dmt1,Xaa5]N/OFQ(1-13)-NH2 were synthesized and pharmacologically characterized for their action at human recombinant NOP/opioid receptors. The best results in terms of NOP versus mu opioid receptor potency were obtained by substituting both Tyr1 and Thr5 at the N-terminal portion of N/OFQ(1-13)-NH2 with the noncanonical amino acid Dmt. [Dmt1,5]N/OFQ(1-13)-NH2 has been identified as the most potent dual NOP/mu receptor peptide agonist so far described. Experimental data have been complemented by in silico studies to shed light on the molecular mechanisms by which the peptide binds the active form of the mu receptor. Finally, the compound exerted antitussive effects in an in vivo model of cough.

Structural determinants driving the binding process between PDZ domain of wild type human PALS1 protein and SLiM sequences of SARS-CoV E proteins.

Short Linear Motifs (SLiMs) are functional protein microdomains that typically mediate interactions between a short linear region in one protein and a globular domain in another. Surface Plasmon Resonance assays have been performed to determine the binding affinity between PDZ domain of wild type human PALS1 protein and tetradecapeptides representing the SLiMs sequences of SARS-CoV-1 and SARS-CoV-2 E proteins (E-SLiMs). SARS-CoV-2 E-SLiM binds to the human target protein with a higher affinity compared to SARS-CoV-1, showing a difference significantly greater than previously reported using the F318W mutant of PALS1 protein and shorter target peptides. Moreover, molecular dynamics simulations have provided clear evidence of the structural determinants driving this binding process. Specifically, the Arginine 69 residue in the SARS-CoV-2 E-SLiM is the key residue able to both enhance the specific polar interaction with negatively charged pockets of the PALS1 PDZ domain and reduce significantly the mobility of the viral peptide. These experimental and computational data are reinforced by the comparison of the interaction between the PALS1 PDZ domain with the natural ligand CRB1, as well as the corresponding E-SLiMs of other coronavirus members such as MERS and OCF43. Our results provide a model at the molecular level of the strategies used to mimic the endogenous SLiM peptide in the binding of the tight junctions of the host cell, explaining one of the possible reasons of the severity of the infection and pulmonary inflammation by SARS-CoV-2.

Improved binding of SARS-CoV-2 Envelope protein to tight junction-associated PALS1 could play a key role in COVID-19 pathogenesis.

The Envelope (E) protein of SARS-CoV-2 is the most enigmatic protein among the four structural ones. Most of its current knowledge is based on the direct comparison to the SARS E protein, initially mistakenly undervalued and subsequently proved to be a key factor in the ER-Golgi localization and in tight junction disruption. We compared the genomic sequences of E protein of SARS-CoV-2, SARS-CoV and the closely related genomes of bats and pangolins obtained from the GISAID and GenBank databases. When compared to the known SARS E protein, we observed a significant difference in amino acid sequence in the C-terminal end of SARS-CoV-2 E protein. Subsequently, in silico modelling analyses of E proteins conformation and docking provide evidences of a strengthened binding of SARS-CoV-2 E protein with the tight junction-associated PALS1 protein. Based on our computational evidences and on data related to SARS-CoV, we believe that SARS-CoV-2 E protein interferes more stably with PALS1 leading to an enhanced epithelial barrier disruption, amplifying the inflammatory processes, and promoting tissue remodelling. These findings raise a warning on the underestimated role of the E protein in the pathogenic mechanism and open the route to detailed experimental investigations.

Ligand pathways in neuroglobin revealed by low-temperature photodissociation and docking experiments.

A combined biophysical approach was applied to map gas-docking sites within murine neuroglobin (Ngb), revealing snapshots of events that might govern activity and dynamics in this unique hexacoordinate globin, which is most likely to be involved in gas-sensing in the central nervous system and for which a precise mechanism of action remains to be elucidated. The application of UV-visible microspectroscopy in crystallo, solution X-ray absorption near-edge spectroscopy and X-ray diffraction experiments at 15-40 K provided the structural characterization of an Ngb photolytic intermediate by cryo-trapping and allowed direct observation of the relocation of carbon monoxide within the distal heme pocket after photodissociation. Moreover, X-ray diffraction at 100 K under a high pressure of dioxygen, a physiological ligand of Ngb, unravelled the existence of a storage site for O2 in Ngb which coincides with Xe-III, a previously described docking site for xenon or krypton. Notably, no other secondary sites were observed under our experimental conditions.

Microswitches for the Activation of the Nociceptin Receptor Induced by Cebranopadol: Hints from Microsecond Molecular Dynamics.

Cebranopadol (CBP) is a novel analgesic acting as agonist at the nociceptin (NOP) and µ-opioid (MOP) receptors, exhibiting high potency and efficacy as an antinociceptive and antihypersensitive drug. The binding conformation and the dynamical interactions of CBP with the NOP receptor have been investigated by molecular docking, molecular dynamics (MD) in the microsecond time scale, and hybrid quantum mechanics/molecular mechanics (QM/MM). CBP binds to the NOP receptor as a bidentate ligand of the aspartate D1303,32 by means of both its fluoroindole and dimethyl nitrogens. Starting from the known crystal structure of the inactive state of the receptor, in complex with the antagonist compound-24 (NOP-C24) the comparative analysis of 1 µs MD trajectories of the NOP-C24 complex itself and the NOP_free and NOP-CBP complexes provides new insights on the already known microswitches related to receptor activation, in the frame of the extended ternary complex model. The agonist acts by destabilizing the inactive conformation of the NOP receptor, by inducing a conformational change of M1343,36, which allows W2766,48 to flip around its χ2 dihedral, going in close proximity to the receptor hydrophobic core (T1383,40, P2275,50, F2726,44), which is known to be fundamental for the activation of the opioid receptors. A complete rational picture is also provided for the role of N1333,35 and W2766,48 undergoing critical conformational changes related to an anticooperativity effect, i.e. the well-known role of sodium as negative modulator of agonist binding. Finally, the movement of residue Y3197,53 belonging to the NPxxY motif is also induced by the binding of the agonist in the inactive state, opening a gate for a water channel just as upon receptor activation.

BBS9 gene in nonsyndromic craniosynostosis: Role of the primary cilium in the aberrant ossification of the suture osteogenic niche.

Nonsyndromic craniosynostosis (NCS) is the premature ossification of skull sutures, without associated clinical features. Mutations in several genes account for a small number of NCS patients; thus, the molecular etiopathogenesis of NCS remains largely unclear. Our study aimed at characterizing the molecular signaling implicated in the aberrant ossification of sutures in NCS patients. Comparative gene expression profiling of NCS patient sutures identified a fused suture-specific signature, including 17 genes involved in primary cilium signaling and assembly. Cells from fused sutures displayed a reduced potential to form primary cilia compared to cells from control patent sutures of the same patient. We identified specific upregulated splice variants of the Bardet Biedl syndrome-associated gene 9 (BBS9), which encodes a structural component of the ciliary BBSome complex. BBS9 expression increased during in vitro osteogenic differentiation of suture-derived mesenchymal cells of NCS patients. Also, Bbs9 expression increased during in vivo ossification of rat sutures. BBS9 functional knockdown affected the expression of primary cilia on patient suture cells and their osteogenic potential. Computational modeling of the upregulated protein isoforms (observed in patients) predicted that their binding affinity within the BBSome may be affected, providing a possible explanation for the aberrant suture ossification in NCS.

"In silico" study of the binding of two novel antagonists to the nociceptin receptor.

Antagonists of the nociceptin receptor (NOP) are raising interest for their possible clinical use as antidepressant drugs. Recently, the structure of NOP in complex with some piperidine-based antagonists has been revealed by X-ray crystallography. In this study, a multi-flexible docking (MF-docking) procedure, i.e. docking to multiple receptor conformations extracted by preliminary molecular dynamics trajectories, together with hybrid quantum mechanics/molecular mechanics (QM/MM) simulations have been carried out to provide the binding mode of two novel NOP antagonists, one of them selective (BTRX-246040, formerly named LY-2940094) and one non selective (AT-076), i.e. able to inactivate NOP as well as the classical µ- k- and δ-opioid receptors (MOP KOP and DOP). According to our results, the pivotal role of residue D1303,32 (upper indexes are Ballesteros-Weinstein notations) is analogous to that enlighten by the already known X-ray structures of opioid receptors: binding of the molecules are predicted to require a slight readjustment of the hydrophobic pocket (residues Y1313,33, M1343,36, I2195,43, Q2806,52 and V2836,55) in the orthosteric site of NOP, accommodating either the pyridine-pyrazole (BTRX-246040) or the isoquinoline (AT-076) moiety of the ligand, in turn allowing the protonated piperidine nitrogen to maximize interaction (salt-bridge) with residue D1303,32 of the NOP, and the aromatic head to be sandwiched in optimal π-stacking between Y1313,33 and M1343,36. The QM/MM optimization after the MF-docking procedure has provided the more likely conformations for the binding to the NOP receptor of BTRX-246040 and AT-076, based on different pharmacophores and exhibiting different selectivity profiles. While the high selectivity for NOP of BTRX-246040 can be explained by interactions with NOP specific residues, the lack of selectivity of AT-076 could be associated to its ability to penetrate into the deep hydrophobic pocket of NOP, while retaining a conformation very similar to the one assumed by the antagonist JDTic into the K-opioid receptor. The proposed binding geometries fit better the binding pocket environment providing clues for experimental studies aimed to design selective or multifunctional opioid drugs.

A Dynamic Picture of the Early Events in Nociceptin Binding to the NOP Receptor by Metadynamics.

Nociceptin (NCC, also known as FQ (N/OFQ)) is the 17-amino acid neuropeptide, endogenous ligand for the G-protein-coupled receptor (NOP, also known as ORL-1). In this study, starting from the recently reported x-ray structure at pH 7 of NOP in complex with an antagonist, new insights, to our knowledge, on the binding geometry of NCC to NOP have been provided in silico. After a rigid docking of NCC in an α-helix conformation, molecular dynamics (MD) and metadynamics (METAD), a method for the analysis of free-energy surfaces (FES), were performed on the protein-peptide complex. Free-energy profiles were obtained as a function of the α-helix content of different segments of the 17-mer ligand, and a structural ensemble of conformations of NCC, corresponding to the minimum of the FES, was extracted, thus representing the NCC bound to the inactive form of NOP. The structural features were compared with many known experimental data. The pose of the "message" domain (residues 1-4) of NCC differs from that of the known NOP antagonists, as being slightly slipped deeper inside the protein core. A residual α-helix content in the central part of the peptide (residues 4-9) is maintained, whereas the C-terminal segment (residues 13-17) is unstructured and highly flexible. An important stabilization due to interactions with residues D130 and D110 of the receptor has been found, in agreement with the large decrease in agonist potency reported for the D130A and D110A mutants. The importance of the extracellular domain 2 (ECL2) in the selectivity toward the endogenous ligand has been confirmed. A pivotal role for the conserved residue N133 is suggested and further supported by a study of the N133A in silico mutant. Accordingly, N133 can work as a molecular microswitch driving the change between the inactive and active NOP conformations, in the framework of an extended H-bond and water network rearrangement in the deep binding site.

Vanadium K-edge XANES in vanadium-bearing model compounds: a full multiple scattering study.

A systematic study is presented on a set of vanadium-bearing model compounds, representative of the most common V coordination geometries and oxidation states, analysed by means of vanadium K-edge X-ray absorption near-edge spectroscopy calculations in the full multiple scattering (FMS) framework. Analysis and calibration of the free parameters of the theory under the muffin-tin approximation (muffin-tin overlap and interstitial potential) have been carried out by fitting the experimental spectra using the MXAN program. The analysis shows a correlation of the fit parameters with the V coordination geometry and oxidation state. By making use of this correlation it is possible to approach the study of unknown V-bearing compounds with useful preliminary information.

The non-octarepeat copper binding site of the prion protein is a key regulator of prion conversion.

The conversion of the prion protein (PrP(C)) into prions plays a key role in transmissible spongiform encephalopathies. Despite the importance for pathogenesis, the mechanism of prion formation has escaped detailed characterization due to the insoluble nature of prions. PrP(C) interacts with copper through octarepeat and non-octarepeat binding sites. Copper coordination to the non-octarepeat region has garnered interest due to the possibility that this interaction may impact prion conversion. We used X-ray absorption spectroscopy to study copper coordination at pH 5.5 and 7.0 in human PrP(C) constructs, either wild-type (WT) or carrying pathological mutations. We show that mutations and pH cause modifications of copper coordination in the non-octarepeat region. In the WT at pH 5.5, copper is anchored to His96 and His111, while at pH 7 it is coordinated by His111. Pathological point mutations alter the copper coordination at acidic conditions where the metal is anchored to His111. By using in vitro approaches, cell-based and computational techniques, we propose a model whereby PrP(C) coordinating copper with one His in the non-octarepeat region converts to prions at acidic condition. Thus, the non-octarepeat region may act as the long-sought-after prion switch, critical for disease onset and propagation.

Intermediate states in the binding process of folic acid to folate receptor α: insights by molecular dynamics and metadynamics.

Folate receptor α (FRα) is a cell surface, glycophosphatidylinositol-anchored protein which has focussed attention as a therapeutic target and as a marker for the diagnosis of cancer. It has a high affinity for the dietary supplemented folic acid (FOL), carrying out endocytic transport across the cell membrane and delivering the folate at the acidic pH of the endosome. Starting from the recently reported X-ray structure at pH 7, 100 ns classical molecular dynamics simulations have been carried out on the FRα-FOL complex; moreover, the ligand dissociation process has been studied by metadynamics, a recently reported method for the analysis of free-energy surfaces (FES), providing clues on the intermediate states and their energy terms. Multiple dissociation runs were considered to enhance the configurational sampling; a final clustering of conformations within the averaged FES provides the representative structures of several intermediate states, within an overall barrier for ligand escape of about 75 kJ/mol. Escaping of FOL to solvent occurs while only minor changes affect the FRα conformation of the binding pocket. During dissociation, the FOL molecule translates and rotates around a turning point located in proximity of the receptor surface. FOL at this transition state assumes an "L" shaped conformation, with the pteridin ring oriented to optimize stacking within W102 and W140 residues, and the negatively charged glutamate tail, outside the receptor, interacting with the positively charged R103 and R106 residues, that contrary to the bound state, are solvent exposed. We show that metadynamics method can provide useful insights at the atomistic level on the effects of point-mutations affecting functionality, thus being a very promising tool for any study related to folate-targeted drug delivery or cancer therapies involving folate uptake.

Quantitative analysis of deconvolved X-ray absorption near-edge structure spectra: a tool to push the limits of the X-ray absorption spectroscopy technique.

A deconvolution procedure has been applied to K-edge X-ray absorption near-edge structure (XANES) spectra of lanthanoid-containing solid systems, namely, hexakis(dmpu)praseodymium(III) and -gadolinium(III) iodide. The K-edges of lanthanoids cover the energy range 38 (La)-65 (Lu) keV, and the large widths of the core-hole states lead to broadening of spectral features, reducing the content of structural information that can be extracted from the raw X-ray absorption spectra. Here, we demonstrate that deconvolution procedures allow one to remove most of the instrumental and core-hole lifetime broadening in the K-edge XANES spectra of lanthanoid compounds, highlighting structural features that are lost in the raw data. We show that quantitative analysis of the deconvolved K-edge XANES spectra can be profitably used to gain a complete local structural characterization of lanthanoid-containing systems not only for the nearest neighbor atoms but also for higher-distance coordination shells.

Distal-proximal crosstalk in the heme binding pocket of the NO sensor DNR.

In the opportunistic pathogen Pseudomonas aeruginosa the denitrification process is triggered by nitric oxide (NO) and plays a crucial role for the survival in chronic infection sites as a microaerobic-anaerobic biofilm. This respiratory pathway is transcriptionally induced by DNR, an heme-based gas sensor which positively responds to NO. Molecular details of the NO sensing mechanism employed by DNR are now emerging: we recently reported an in vitro study which dissected, for the first time, the heme-iron environment and identified one of the heme axial ligand (i.e. His187), found to be crucial to respond to NO. Nevertheless, the identification of the second heme axial ligand has been unsuccessful, given that a peculiar phenomenon of ligand switching around the heme-iron presumably occurs in DNR. The unusual heme binding properties of DNR could be due to the remarkable flexibility in solution of DNR itself, which, in turns, is crucial for the sensing activity; protein flexibility and dynamics indeed represent a common strategy employed by heme-based redox sensors, which present features deeply different from those of "canonical" hemeproteins. The capability of DNR to deeply rearrange around the heme-iron as been here demonstrated by means of spectroscopic characterization of the H167A/H187A DNR double mutant, which shows unusual kinetics of binding of NO and CO. Moreover, we show that the alteration (such as histidines mutations) of the distal side of the heme pocket is perceived by the proximal one, possibly via the DNR protein chain.

Synthesis and characterization of different immunogenic viral nanoconstructs from rotavirus VP6 inner capsid protein.

In order to deliver low-cost viral capsomeres from a large amount of soluble viral VP6 protein from human rotavirus, we developed and optimized a biotechnological platform in Escherichia coli. Specifically, three different expression protocols were compared, differing in their genetic constructs, ie, a simple native histidine-tagged VP6 sequence, VP6 fused to thioredoxin, and VP6 obtained with the newly described small ubiquitin-like modifier (SUMO) fusion system. Our results demonstrate that the histidine-tagged protein does not escape the accumulation in the inclusion bodies, and that SUMO is largely superior to the thioredoxin-fusion tag in enhancing the expression and solubility of VP6 protein. Moreover, the VP6 protein produced according to the SUMO fusion tag displays well-known assembly properties, as observed in both transmission electron microscopy and atomic force microscopy images, giving rise to either VP6 trimers, 60 nm spherical virus-like particles, or nanotubes a few microns long. This different quaternary organization of VP6 shows a higher level of immunogenicity for the elongated structures with respect to the spheres or the protein trimers. Therefore, the expression and purification strategy presented here - providing a large amount of the viral capsid protein in the native form with relatively simple, rapid, and economical procedures - opens a new route toward large-scale production of a more efficient antigenic compound to be used as a vaccination tool or as an adjuvant, and also represents a top-quality biomaterial to be further modified for biotechnological purposes.

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.

Structural and functional insights on folate receptor α (FRα) by homology modeling, ligand docking and molecular dynamics.

Folate receptor α (FRα) is a cell surface, glycophosphatidylinositol (GPI)-anchored protein with a high affinity for its ligand partner, which is highly expressed in malignant cells and has been selected as a therapeutic target and marker for the diagnosis of cancer. No direct structural information is available from either X-ray diffraction or NMR on the post-translational structure of this disulfide-rich protein. Three-dimensional models of the FRα structure have been derived with the recent homology modeling packages, using the crystal structure of the riboflavin-binding protein (RfBP) as a template. Molecular dynamics trajectories have been exploited allowing successfully the formation of a full disulfide bridge network, which was expected based on the similarities between FRα and RfBP. After the selection of the best model, a folic acid molecule was docked "in silico" onto the putative binding site and its binding mode was compared with that of vintafolide, a much larger molecule designed as a chemotherapy agent targeting specifically FRα. In both cases, a 40ns molecular dynamics trajectory was calculated, providing suggestions regarding the key structural determinants driving the affinity and specificity of FRα for folic acid with respect to other folate homologues. Moreover, some other crucial experimental results related to the structure of the receptor are discussed, such as the expected location/accessibility of known immune epitopes, the set of N-linked glycosylation sites and the effect of point mutations on the impairment of folate binding. Our results may provide useful insights for studies related to folate-targeted drug delivery or cancer therapies involving folate uptake.

Dioxygen oxidation Cu(II) → Cu(III) in the copper complex of cyclo(Lys-dHis-ßAla-His): a case study by EXAFS and XANES approach.

A former spectroscopic study of Cu(II) coordination by the 13-membered ring cyclic tetrapeptide c(Lys-dHis-ßAla-His) (DK13), revealed the presence, at alkaline pH, of a stable peptide/Cu(III) complex formed in solution by atmospheric dioxygen oxidation. To understand the nature of this coordination compound and to investigate the role of the His residues in the Cu(III) species formation, Cu K-edge XANES, and EXAFS spectra have been collected for DK13 and two other 13-membered cyclo-peptides: the diastereoisomer c(Lys-His-ßAla-His) (LK13), and c(Gly-ßAla-Gly-Lys) (GK13), devoid of His residues. Comparison of pre-edge peak features with those of Cu model compounds, allowed us to get information on copper oxidation state in two of the three peptides, DK13 and GK13: DK13 contains only Cu(III) ions in the experimental conditions, while GK13 binds only with Cu(II). For LK13/Cu complex, EXAFS spectrum suggested and UV-vis analysis confirmed the presence of a mixture of Cu(II) and Cu(III) coordinated species. Theoretical XANES spectra have been calculated by means of the MXAN code. The good agreement between theoretical and experimental XANES data collected for DK13, suggests that the refined structure, at least in the first coordination shell around Cu, is a good approximation of the DK13/Cu(III) coordination species present at strongly alkaline pH. All the data are consistent with a slightly distorted pyramidal CuN(4) unit, coming from the peptide bonds. Surprisingly, the His side-chains seemed not involved in the final, stable, Cu(III) scaffold.

Effects of the pathological Q212P mutation on human prion protein non-octarepeat copper-binding site.

Prion diseases are a class of fatal neurodegenerative disorders characterized by brain spongiosis, synaptic degeneration, microglia and astrocytes activation, neuronal loss and altered redox control. These maladies can be sporadic, iatrogenic and genetic. The etiological agent is the prion, a misfolded form of the cellular prion protein, PrP(C). PrP(C) interacts with metal ions, in particular copper and zinc, through the octarepeat and non-octarepeat binding sites. The physiological implication of this interaction is still unclear, as is the role of metals in the conversion. Since prion diseases present metal dyshomeostasis and increased oxidative stress, we described the copper-binding site located in the human C-terminal domain of PrP-HuPrP(90-231), both in the wild-type protein and in the protein carrying the pathological mutation Q212P. We used the synchrotron-based X-ray absorption fine structure technique to study the Cu(II) and Cu(I) coordination geometries in the mutant, and we compared them with those obtained using the wild-type protein. By analyzing the extended X-ray absorption fine structure and the X-ray absorption near-edge structure, we highlighted changes in copper coordination induced by the point mutation Q212P in both oxidation states. While in the wild-type protein the copper-binding site has the same structure for both Cu(II) and Cu(I), in the mutant the coordination site changes drastically from the oxidized to the reduced form of the copper ion. Copper-binding sites in the mutant resemble those obtained using peptides, confirming the loss of short- and long-range interactions. These changes probably cause alterations in copper homeostasis and, consequently, in redox control.