Targeting NUPR1-dependent stress granules formation to induce synthetic lethality in KrasG12D-driven tumors.

We find that NUPR1, a stress-associated intrinsically disordered protein, induced droplet formation via liquid-liquid phase separation (LLPS). NUPR1-driven LLPS was crucial for the creation of NUPR1-dependent stress granules (SGs) in pancreatic cancer cells since genetic or pharmacological inhibition by ZZW-115 of NUPR1 activity impeded SGs formation. The KrasG12D mutation induced oncogenic stress, NUPR1 overexpression, and promoted SGs development. Notably, enforced NUPR1 expression induced SGs formation independently of mutated KrasG12D. Mechanistically, KrasG12D expression strengthened sensitivity to NUPR1 inactivation, inducing cell death, activating caspase 3 and releasing LDH. Remarkably, ZZW-115-mediated SG-formation inhibition hampered the development of pancreatic intraepithelial neoplasia (PanINs) in Pdx1-cre;LSL-KrasG12D (KC) mice. ZZW-115-treatment of KC mice triggered caspase 3 activation, DNA fragmentation, and formation of the apoptotic bodies, leading to cell death, specifically in KrasG12D-expressing cells. We further demonstrated that, in developed PanINs, short-term ZZW-115 treatment prevented NUPR1-associated SGs presence. Lastly, a four-week ZZW-115 treatment significantly reduced the number and size of PanINs in KC mice. This study proposes that targeting NUPR1-dependent SGs formation could be a therapeutic approach to induce cell death in KrasG12D-dependent tumors.

NUPR1 protects against hyperPARylation-dependent cell death.

Proteomic, cellular and biochemical analysis of the stress protein NUPR1 reveals that it binds to PARP1 into the nucleus and inhibits PARP1 activity in vitro. Mutations on residues Ala33 or Thr68 of NUPR1 or treatment with its inhibitor ZZW-115 inhibits this effect. PARylation induced by 5-fluorouracil (5-FU) treatment is strongly enhanced by ZZW-115 and associated with a decrease of NAD+/NADH ratio and rescued by the PARP inhibitor olaparib. Cell death induced by ZZW-115 treatment of pancreas cancer-derived cells is rescued by olaparib and improved with PARG inhibitor PDD00017273. The mitochondrial catastrophe induced by ZZW-115 treatment or by genetic inactivation of NUPR1 is associated to a hyperPARylation of the mitochondria, disorganization of the mitochondrial network, mitochondrial membrane potential decrease, and with increase of superoxide production, intracellular level of reactive oxygen species (ROS) and cytosolic levels of Ca2+. These features are rescued by olaparib or NAD+ precursor nicotinamide mononucleotide in a dose-dependent manner and partially by antioxidants treatments. In conclusion, inactivation of NUPR1 induces a hyperPARylation, which in turn, induces a mitochondrial catastrophe and consequently a cell death through a non-canonical Parthanatos, since apoptosis inducing-factor (AIF) is not translocated out of the mitochondria.

Structural insights into choline-O-sulfatase reveal the molecular determinants for ligand binding.

Choline-O-sulfatase (COSe; EC 3.1.6.6) is a member of the alkaline phosphatase (AP) superfamily, and its natural function is to hydrolyze choline-O-sulfate into choline and sulfate. Despite its natural function, the major interest in this enzyme resides in the landmark catalytic/substrate promiscuity of sulfatases, which has led to attention in the biotechnological field due to their potential in protein engineering. In this work, an in-depth structural analysis of wild-type Sinorhizobium (Ensifer) meliloti COSe (SmeCOSe) and its C54S active-site mutant is reported. The binding mode of this AP superfamily member to both products of the reaction (sulfate and choline) and to a substrate-like compound are shown for the first time. The structures further confirm the importance of the C-terminal extension of the enzyme in becoming part of the active site and participating in enzyme activity through dynamic intra-subunit and inter-subunit hydrogen bonds (Asn146A-Asp500B-Asn498B). These residues act as the `gatekeeper' responsible for the open/closed conformations of the enzyme, in addition to assisting in ligand binding through the rearrangement of Leu499 (with a movement of approximately 5 Å). Trp129 and His145 clamp the quaternary ammonium moiety of choline and also connect the catalytic cleft to the C-terminus of an adjacent protomer. The structural information reported here contrasts with the proposed role of conformational dynamics in promoting the enzymatic catalytic proficiency of an enzyme.

NUPR1 inhibitor ZZW-115 induces ferroptosis in a mitochondria-dependent manner.

Ferroptosis is an iron-dependent cell death characterized by the accumulation of hydroperoxided phospholipids. Here, we report that the NUPR1 inhibitor ZZW-115 induces ROS accumulation followed by a ferroptotic cell death, which could be prevented by ferrostatin-1 (Fer-1) and ROS-scavenging agents. The ferroptotic activity can be improved by inhibiting antioxidant factors in pancreatic ductal adenocarcinoma (PDAC)- and hepatocellular carcinoma (HCC)-derived cells. In addition, ZZW-115-treatment increases the accumulation of hydroperoxided lipids in these cells. We also found that a loss of activity and strong deregulation of key enzymes involved in the GSH- and GPX-dependent antioxidant systems upon ZZW-115 treatment. These results have been validated in xenografts induced with PDAC- and HCC-derived cells in nude mice during the treatment with ZZW-115. More importantly, we demonstrate that ZZW-115-induced mitochondrial morphological changes, compatible with the ferroptotic process, as well as mitochondrial network disorganization and strong mitochondrial metabolic dysfunction, which are rescued by both Fer-1 and N-acetylcysteine (NAC). Of note, the expression of TFAM, a key regulator of mitochondrial biogenesis, is downregulated by ZZW-115. Forced expression of TFAM is able to rescue morphological and functional mitochondrial alterations, ROS production, and cell death induced by ZZW-115 or genetic inhibition of NUPR1. Altogether, these results demonstrate that the mitochondrial cell death mediated by NUPR1 inhibitor ZZW-115 is fully rescued by Fer-1 but also via TFAM complementation. In conclusion, TFAM could be considered as an antagonist of the ferroptotic cell death.

Targeting the Stress-Induced Protein NUPR1 to Treat Pancreatic Adenocarcinoma.

Cancer cells activate stress-response mechanisms to adapt themselves to a variety of stressful conditions. Among these protective mechanisms, those controlled by the stress-induced nuclear protein 1 (NUPR1 ) belong to the most conserved ones. NUPR1 is an 82-residue-long, monomeric, basic and intrinsically disordered protein (IDP), which was found to be invariably overexpressed in some, if not all, cancer tissues. Remarkably, we and others have previously showed that genetic inactivation of the Nupr1 gene antagonizes the growth of pancreatic cancer as well as several other tumors. With the use of a multidisciplinary strategy by combining biophysical, biochemical, bioinformatic, and biological approaches, a trifluoperazine-derived compound, named ZZW-115, has been identified as an inhibitor of the NUPR1 functions. The anticancer activity of the ZZW-115 was first validated on a large panel of cancer cells. Furthermore, ZZW-115 produced a dose-dependent tumor regression of the tumor size in xenografted mice. Mechanistically, we have demonstrated that NUPR1 binds to several importins. Because ZZW-115 binds NUPR1 through the region around the amino acid Thr68, which is located into the nuclear location signal (NLS) region of the protein, we demonstrated that treatment with ZZW-115 inhibits completely the translocation of NUPR1 from the cytoplasm to the nucleus by competing with importins.

Inactivation of NUPR1 promotes cell death by coupling ER-stress responses with necrosis.

It was already described that genetic inhibition of NUPR1 induces tumor growth arrest. In this paper we studied the metabolism changes after NUPR1 downregulation in pancreatic cancer cells, which results in a significant decrease of OXPHOS activity with a concomitant lower ATP production which precedes the necrotic cell death. We demonstrated that NUPR1 downregulation induces a mitochondrial failure with a loss of the mitochondrial membrane potential, a strong increase in ROS production and a concomitant relocalization of mitochondria to the vicinity of the endoplasmic reticulum (ER). In addition, the transcriptomic analysis of NUPR1-deficient cells shows a decrease in the expression of some ER stress response-associated genes. Indeed, in ER stressors-treated cells with thapsigargin, brefeldin A or tunicamycin, a greater increase in necrosis and decrease of ATP content was observed in NUPR1-defficent cells. Finally, in vivo experiments, using acute pancreatitis which induces ER stress as well as NUPR1 activation, we observed that NUPR1 expression protects acinar cells from necrosis in mice. Importantly, we also report that the cell death observed after knocking-down NUPR1 expression is completely reversed by incubation with Necrostatin-1, but not by inhibiting caspase activity with Z-VAD-FMK. Altogether, these data enable us to describe a model in which inactivation of NUPR1 in pancreatic cancer cells results in an ER stress that induces a mitochondrial malfunction, a deficient ATP production and, as consequence, the cell death mediated by a programmed necrosis.

Cysteine Mutational Studies Provide Insight into a Thiol-Based Redox Switch Mechanism of Metal and DNA Binding in FurA from Anabaena sp. PCC 7120.

AIMS: The ferric uptake regulator (Fur) is the main transcriptional regulator of genes involved in iron homeostasis in most prokaryotes. FurA from Anabaena sp. PCC 7120 contains five cysteine residues, four of them arranged in two redox-active CXXC motifs. The protein needs not only metal but also reducing conditions to remain fully active in vitro. Through a mutational study of the cysteine residues present in FurA, we have investigated their involvement in metal and DNA binding. RESULTS: Residue C101 that belongs to a conserved CXXC motif plays an essential role in both metal and DNA binding activities in vitro. Substitution of C101 by serine impairs DNA and metal binding abilities of FurA. Isothermal titration calorimetry measurements show that the redox state of C101 is responsible for the protein ability to coordinate the metal corepressor. Moreover, the redox state of C101 varies with the presence or absence of C104 or C133, suggesting that the environments of these cysteines are mutually interdependent. INNOVATION: We propose that C101 is part of a thiol/disulfide redox switch that determines FurA ability to bind the metal corepressor. CONCLUSION: This mechanism supports a novel feature of a Fur protein that emerges as a regulator, which connects the response to changes in the intracellular redox state and iron management in cyanobacteria. Antioxid. Redox Signal. 00, 000-000.

The carboxy-terminal domain of Erb1 is a seven-bladed ß-propeller that binds RNA.

Erb1 (Eukaryotic Ribosome Biogenesis 1) protein is essential for the maturation of the ribosomal 60S subunit. Functional studies in yeast and mammalian cells showed that altogether with Nop7 and Ytm1 it forms a stable subcomplex called PeBoW that is crucial for a correct rRNA processing. The exact function of the protein within the process remains unknown. The N-terminal region of the protein includes a well conserved region shown to be involved in PeBoW complex formation whereas the carboxy-terminal half was predicted to contain seven WD40 repeats. This first structural report on Erb1 from yeast describes the architecture of a seven-bladed ß-propeller domain that revealed a characteristic extra motif formed by two α-helices and a ß-strand that insert within the second WD repeat. We performed analysis of molecular surface and crystal packing, together with multiple sequence alignment and comparison of the structure with other ß-propellers, in order to identify areas that are more likely to mediate protein-protein interactions. The abundance of many positively charged residues on the surface of the domain led us to investigate whether the propeller of Erb1 might be involved in RNA binding. Three independent assays confirmed that the protein interacted in vitro with polyuridilic acid (polyU), thus suggesting a possible role of the domain in rRNA rearrangement during ribosome biogenesis.

Functional Characterization of Nupr1L, A Novel p53-Regulated Isoform of the High-Mobility Group (HMG)-Related Protumoral Protein Nupr1.

We have previously demonstrated a crucial role of nuclear protein 1 (NUPR1) in tumor development and progression. In this work, we report the functional characterization of a novel Nupr1-like isoform (NUPR1L) and its functional interaction with the protumoral factor NUPR1. Through the use of primary sequence analysis, threading, and homology-based molecular modeling, as well as expression and immunolocalization, studies reveal that NUPR1L displays properties, which are similar to member of the HMG-like family of chromatin regulators, including its ability to translocate to the cell nucleus and bind to DNA. Analysis of the NUPR1L promoter showed the presence of two p53-response elements at positions -37 and -7, respectively. Experiments using reporter assays combined with site-directed mutagenesis and using cells with controllable p53 expression demonstrate that both of these sequences are responsible for the regulation of NUPR1L expression by p53. Congruently, NUPR1L gene expression is activated in response to DNA damage induced by oxaliplatin treatment or cell cycle arrest induced by serum starvation, two well-validated methods to achieve p53 activation. Interestingly, expression of NUPR1L downregulates the expression of NUPR1, its closely related protumoral isoform, by a mechanism that involves the inhibition of its promoter activity. At the cellular level, overexpression of NUPR1L induces G1 cell cycle arrest and a decrease in their cell viability, an effect that is mediated, at least in part, by downregulating NUPR1 expression. Combined, these experiments constitute the first functional characterization of NUPR1L as a new p53-induced gene, which negatively regulates the protumoral factor NUPR1.

Biophysical analysis of the MHR motif in folding and domain swapping of the HIV capsid protein C-terminal domain.

Infection by human immunodeficiency virus (HIV) depends on the function, in virion morphogenesis and other stages of the viral cycle, of a highly conserved structural element, the major homology region (MHR), within the carboxyterminal domain (CTD) of the capsid protein. In a modified CTD dimer, MHR is swapped between monomers. While no evidence for MHR swapping has been provided by structural models of retroviral capsids, it is unknown whether it may occur transiently along the virus assembly pathway. Whatever the case, the MHR-swapped dimer does provide a novel target for the development of anti-HIV drugs based on the concept of trapping a nonnative capsid protein conformation. We have carried out a thermodynamic and kinetic characterization of the domain-swapped CTD dimer in solution. The analysis includes a dissection of the role of conserved MHR residues and other amino acids at the dimerization interface in CTD folding, stability, and dimerization by domain swapping. The results revealed some energetic hotspots at the domain-swapped interface. In addition, many MHR residues that are not in the protein hydrophobic core were nevertheless found to be critical for folding and stability of the CTD monomer, which may dramatically slow down the swapping reaction. Conservation of MHR residues in retroviruses did not correlate with their contribution to domain swapping, but it did correlate with their importance for stable CTD folding. Because folding is required for capsid protein function, this remarkable MHR-mediated conformational stabilization of CTD may help to explain the functional roles of MHR not only during immature capsid assembly but in other processes associated with retrovirus infection. This energetic dissection of the dimerization interface in MHR-swapped CTD may also facilitate the design of anti-HIV compounds that inhibit capsid assembly by conformational trapping of swapped CTD dimers.

Electrostatic effects in the folding of the SH3 domain of the c-Src tyrosine kinase: pH-dependence in 3D-domain swapping and amyloid formation.

The SH3 domain of the c-Src tyrosine kinase (c-Src-SH3) aggregates to form intertwined dimers and amyloid fibrils at mild acid pHs. In this work, we show that a single mutation of residue Gln128 of this SH3 domain has a significant effect on: (i) its thermal stability; and (ii) its propensity to form amyloid fibrils. The Gln128Glu mutant forms amyloid fibrils at neutral pH but not at mild acid pH, while Gln128Lys and Gln128Arg mutants do not form these aggregates under any of the conditions assayed. We have also solved the crystallographic structures of the wild-type (WT) and Gln128Glu, Gln128Lys and Gln128Arg mutants from crystals obtained at different pHs. At pH 5.0, crystals belong to the hexagonal space group P6522 and the asymmetric unit is formed by one chain of the protomer of the c-Src-SH3 domain in an open conformation. At pH 7.0, crystals belong to the orthorhombic space group P212121, with two molecules at the asymmetric unit showing the characteristic fold of the SH3 domain. Analysis of these crystallographic structures shows that the residue at position 128 is connected to Glu106 at the diverging ß-turn through a cluster of water molecules. Changes in this hydrogen-bond network lead to the displacement of the c-Src-SH3 distal loop, resulting also in conformational changes of Leu100 that might be related to the binding of proline rich motifs. Our findings show that electrostatic interactions and solvation of residues close to the folding nucleation site of the c-Src-SH3 domain might play an important role during the folding reaction and the amyloid fibril formation.

Biomechanical considerations of foot-ground contact in T'ai Chi Chuan.

Although numerous studies have linked t'ai chi chuan (TCC) practice with benefits for balance, reduction in the number of falls, and in the fear of falling, most of them did not address the causes of these benefits in depth. Some studies, however, sought to determine the causes from the biomechanical point of view. This article aims to thoroughly describe and critically review recent papers on foot-ground contact in TCC practice, one of the parameters involved in balance biomechanics in TCC performance. No previous review on this subject has been found. Nine electronic databases were searched for publications between 1996 and 2013. Studies were excluded if they were not published in English or were abstracts, posters, or summaries from conferences. From a total of 195 articles identified, 4 randomized controlled trials and 3 non-randomized controlled trials were eligible for the analysis. The number of studies that assessed foot-ground contact in TCC and effects on normal gait, postural control improvement, and fall prevention is still quite small. These studies were based on intervention protocols and used populations that were too heterogeneous to allow reliable comparisons. According to the studies analyzed, TCC practice clearly improved parameters associated with foot-ground contact. Nevertheless, the manner in which these benefits are transferred to daily displacement habits still remains unclear.

Evidence supporting the existence of a NUPR1-like family of helix-loop-helix chromatin proteins related to, yet distinct from, AT hook-containing HMG proteins.

NUPR1, a small chromatin protein, plays a critical role in cancer development, progression, and resistance to therapy. Here, using a combination of structural bioinformatics and molecular modeling methods, we report several novel findings that enhance our understanding of the biochemical function of this protein. We find that NUPR1 has been conserved throughout evolution, and over time it has undergone duplications and transpositions to form other transcriptional regulators. Using threading, homology-based molecular modeling, molecular mechanics calculations, and molecular dynamics simulations, we generated structural models for four of these proteins: NUPR1a, NUPR1b, NUPR2, and the NUPR-like domain of GTF2-I. Comparative analyses of these models combined with extensive linear motif identification reveal that these four proteins, though similar in their propensities for folding, differ in size, surface changes, and sites amenable for posttranslational modification. Lastly, taking NUPR1a as the paradigm for this family, we built models of a NUPR-DNA complex. Additional structural comparisons revealed that NUPR1 defines a new family of small-groove-binding proteins that share structural features with, yet are distinct from, helix-loop-helix AT-hook-containing HMG proteins. These models and inferences should lead to a better understanding of the function of this group of chromatin proteins, which play a critical role in the development of human malignant diseases.

Rationally designed interfacial peptides are efficient in vitro inhibitors of HIV-1 capsid assembly with antiviral activity.

Virus capsid assembly constitutes an attractive target for the development of antiviral therapies; a few experimental inhibitors of this process for HIV-1 and other viruses have been identified by screening compounds or by selection from chemical libraries. As a different, novel approach we have undertaken the rational design of peptides that could act as competitive assembly inhibitors by mimicking capsid structural elements involved in intersubunit interfaces. Several discrete interfaces involved in formation of the mature HIV-1 capsid through polymerization of the capsid protein CA were targeted. We had previously designed a peptide, CAC1, that represents CA helix 9 (a major part of the dimerization interface) and binds the CA C-terminal domain in solution. Here we have mapped the binding site of CAC1, and shown that it substantially overlaps with the CA dimerization interface. We have also rationally modified CAC1 to increase its solubility and CA-binding affinity, and designed four additional peptides that represent CA helical segments involved in other CA interfaces. We found that peptides CAC1, its derivative CAC1M, and H8 (representing CA helix 8) were able to efficiently inhibit the in vitro assembly of the mature HIV-1 capsid. Cocktails of several peptides, including CAC1 or CAC1M plus H8 or CAI (a previously discovered inhibitor of CA polymerization), or CAC1M+H8+CAI, also abolished capsid assembly, even when every peptide was used at lower, sub-inhibitory doses. To provide a preliminary proof that these designed capsid assembly inhibitors could eventually serve as lead compounds for development of anti-HIV-1 agents, they were transported into cultured cells using a cell-penetrating peptide, and tested for antiviral activity. Peptide cocktails that drastically inhibited capsid assembly in vitro were also able to efficiently inhibit HIV-1 infection ex vivo. This study validates a novel, entirely rational approach for the design of capsid assembly interfacial inhibitors that show antiviral activity.

Conformational stability of hepatitis C virus NS3 protease.

The hepatitis C virus NS3 protease is responsible for the processing of the nonstructural region of viral precursor polyprotein in infected hepatic cells. NS3 has been considered a target for drug discovery for a long time. NS3 is a zinc-dependent serine protease. However, the zinc ion is not involved in the catalytic mechanism, because it is bound far away from the active site. Thus, zinc is essential for the structural integrity of the protein and it is considered to have a structural role. The first thermodynamic study on the conformational equilibrium and stability of NS3 and the effect of zinc on such equilibrium is presented here. In agreement with a previous calorimetric study on the binding of zinc to NS3, the global unfolding heat capacity is dominated by the zinc dissociation step, suggesting that the binding of zinc induces a significant structural rearrangement of the protein. In addition, contrary to other homologous zinc-dependent proteases, the zinc-free NS3 protease is not completely unstructured. It is apparent that the conformational landscape of hepatitis C virus NS3 protease is fairly complex due to its intrinsic plasticity, and to the interactions with its different effectors (zinc and the accessory viral protein NS4A) and their modulation of the population of the different conformational states.

Insights into the functionality of the putative residues involved in enterocin AS-48 maturation.

AS-48 is a 70-residue, α-helical, cationic bacteriocin produced by Enterococcus faecalis and is very singular in its circular structure and its broad antibacterial spectrum. The AS-48 preprotein consists of an N-terminal signal peptide (SP) (35 residues) followed by a proprotein moiety that undergoes posttranslational modifications to yield the mature and active circular protein. For the study of the specificity of the region of AS-48 that is responsible for maturation, three single mutants have been generated by site-directed mutagenesis in the as-48A structural gene. The substitutions were made just in the residues that are thought to constitute a recognition site for the SP cleavage enzyme (His-1, Met1) and in those involved in circularization (Met1, Trp70). Each derivative was expressed in the enterococcal JH2-2 strain containing the necessary native biosynthetic machinery for enterocin production. The importance of these derivatives in AS-48 processing has been evaluated on the basis of the production and structural characterization of the corresponding derivatives. Notably, only two of them (Trp70Ala and Met1Ala derivatives) could be purified in different forms and amounts and are characterized for their bactericidal activity and secondary structure. We could not detect any production of AS-48 in JH2-2(pAM401-81(His-1Ile)) by using the conventional chromatographic techniques, despite the high efficiency of the culture conditions applied to produce this enterocin. Our results underline the different important roles of the mutated residues in (i) the elimination of the SP, (ii) the production levels and antibacterial activity of the mature proteins, and (iii) protein circularization. Moreover, our findings suggest that His-1 is critically involved in cleavage site recognition, its substitution being responsible for the blockage of processing, thereby hampering the production of the specific protein in the cellular culture supernatant.

p8/nupr1 regulates DNA-repair activity after double-strand gamma irradiation-induced DNA damage.

The stress protein p8 is a small, highly basic, unfolded, and multifunctional protein. We have previously shown that most of its functions are exerted through interactions with other proteins, whose activities are thereby enhanced or repressed. In this work we describe another example of such mechanism, by which p8 binds and negatively regulates MSL1, a histone acetyl transferase (HAT)-associated protein, which in turn binds the DNA-damage-associated 53BP1 protein to facilitate DNA repair following DNA gamma-irradiation. Contrary to the HAT-associated activity, MSL1-dependent DNA-repair activity is almost completely dependent on 53BP1 expression. The picture that has emerged from our findings is that 53BP1 could be a scaffold that gets the HAT MSL1-dependent DNA-repair activity to the sites of DNA damage. Finally, we also found that, although p8 expression is transiently activated after gamma-irradiation, it is eventually submitted to sustained down-regulation, presumably to allow development of MSL1-associated DNA-repair activity. We conclude that interaction of MSL1 with 53BP1 brings MSL1-dependent HAT activity to the vicinity of damaged DNA. MSL1-dependent HAT activity, which is negatively regulated by the stress protein p8, induces chromatin remodeling and relaxation allowing access to DNA of the repair machinery.

Thermodynamics of zinc binding to hepatitis C virus NS3 protease: a folding by binding event.

The hepatitis C virus (HCV) nonstructural protein 3 (NS3) protease is responsible for the processing of the non-structural region of the viral precursor polyprotein in infected hepatic cells. HCV NS3 is a zinc-dependent serine protease. The zinc ion, which is bound far away from the active site and considered to have a structural role, is essential for the structural integrity of the protein; furthermore, the ion is required for the hydrolytic activity. Consequently, the NS3 zinc binding site has been considered for a long time as a possible target for drug discovery. As a first step towards this goal, the energetics of the NS3-zinc interaction and its effect on the NS3 conformation must be established and discussed. The thermodynamic characterization of zinc binding to NS3 protease by isothermal titration calorimetry and spectroscopy is presented here. Spectroscopic and calorimetric results suggest that a considerable conformational change in the protein is coupled to zinc binding. The energetics of the conformational change is comparable to that of the folding of a protein of similar size. Therefore, zinc binding to NS3 protease can be considered as a "folding by binding" event.