Structural transitions in TCTP tumor protein upon binding to the anti-apoptotic protein family member Mcl-1.

Translationally Controlled Tumor Protein (TCTP) serves as a pro-survival factor in tumor cells, inhibiting the mitochondrial apoptosis pathway by enhancing the function of anti-apoptotic Bcl-2 family members Mcl-1 and Bcl-xL. TCTP specifically binds to Bcl-xL, preventing Bax-dependent Bcl-xL-induced cytochrome c release, and it reduces Mcl-1 turnover by inhibiting its ubiquitination, thereby decreasing Mcl-1-mediated apoptosis. TCTP harbors a BH3-like motif that forms a ß-strand buried in the globular domain of the protein. In contrast, the crystal structure of the TCTP BH3-like peptide in complex with the Bcl-2 family member Bcl-xL reveals an α-helical conformation for the BH3-like motif, suggesting significant structural changes upon complex formation. Employing biochemical and biophysical methods, including limited proteolysis, circular dichroism, NMR, and SAXS, we describe the TCTP complex with the Bcl-2 homolog Mcl-1. Our findings demonstrate that full-length TCTP binds to the BH3 binding groove of Mcl-1 via its BH3-like motif, experiencing conformational exchange at the interface on a micro- to milli-second timescale. Concurrently, the TCTP globular domain becomes destabilized, transitioning into a molten-globule state. Furthermore, we establish that the non-canonical residue D16 within the TCTP BH3-like motif reduces stability while enhancing the dynamics of the intermolecular interface. In conclusion, we detail the structural plasticity of TCTP and discuss its implications for partner interactions and future anticancer drug design strategies aimed at targeting TCTP complexes.

Time-Resolved Protein Side-Chain Motions Unraveled by High-Resolution Relaxometry and Molecular Dynamics Simulations.

Motions of proteins are essential for the performance of their functions. Aliphatic protein side chains and their motions play critical roles in protein interactions: for recognition and binding of partner molecules at the surface or serving as an entropy reservoir within the hydrophobic core. Here, we present a new NMR method based on high-resolution relaxometry and high-field relaxation to determine quantitatively both motional amplitudes and time scales of methyl-bearing side chains in the picosecond-to-nanosecond range. We detect a wide variety of motions in isoleucine side chains in the protein ubiquitin. We unambiguously identify slow motions in the low nanosecond range, which, in conjunction with molecular dynamics computer simulations, could be assigned to transitions between rotamers. Our approach provides unmatched detailed insight into the motions of aliphatic side chains in proteins and provides a better understanding of the nature and functional role of protein side-chain motions.

Time dependence of NMR observables reveals salient differences in the accumulation of early aggregated species between human islet amyloid polypeptide and amyloid-ß.

Type 2 diabetes mellitus and Alzheimer's disease are characterized by the accumulation of fibrillar amyloid deposits consisting mainly of islet amyloid polypeptide (IAPP) and amyloid-ß (Aß), respectively. Fibril formation is a multi-step nucleation process that involves the transient build-up of oligomeric species that are thought to be the most toxic components. To gain more insight into the molecular mechanism of early IAPP aggregated species formation, we performed a combination of direct and indirect biophysical approaches on IAPP and also on Aß42 for the sake of comparison. Thioflavin T fluorescence kinetics measurements revealed a stronger autocatalytic behaviour of IAPP and a weaker concentration dependence of fibrillization half-time t1/2, as compared to Aß42. Our NMR experiments highlight the absence of micelle reservoir or supercritical regime in the studied concentration range, indicating that the low concentration dependence of IAPP fibril formation can be ascribed to saturable pathways. IAPP and Aß42 displayed marked differences in formation of oligomeric species, as observed by 1D 1H, pulsed-field gradient (PFG) diffusion and saturation transfer difference (STD) NMR experiments. A fast equilibrium between monomer and oligomeric species was detected in the case of Aß42 but not IAPP, with a significant build-up of aggregated species, as shown by the time dependence of diffusion coefficient and STD magnetization transfer efficiency during the aggregation process. Altogether our data show significant differences between IAPP and Aß42 regarding the microscopic events of amyloid species formation.

Structure and Dynamics of an Intrinsically Disordered Protein Region That Partially Folds upon Binding by Chemical-Exchange NMR.

Many intrinsically disordered proteins (IDPs) and protein regions (IDRs) engage in transient, yet specific, interactions with a variety of protein partners. Often, if not always, interactions with a protein partner lead to partial folding of the IDR. Characterizing the conformational space of such complexes is challenging: in solution-state NMR, signals of the IDR in the interacting region become broad, weak, and often invisible, while X-ray crystallography only provides information on fully ordered regions. There is thus a need for a simple method to characterize both fully and partially ordered regions in the bound state of IDPs. Here, we introduce an approach based on monitoring chemical exchange by NMR to investigate the state of an IDR that folds upon binding through the observation of the free state of the protein. Structural constraints for the bound state are obtained from chemical shifts, and site-specific dynamics of the bound state are characterized by relaxation rates. The conformation of the interacting part of the IDR was determined and subsequently docked onto the structure of the folded partner. We apply the method to investigate the interaction between the disordered C-terminal region of Artemis and the DNA binding domain of Ligase IV. We show that we can accurately reproduce the structure of the core of the complex determined by X-ray crystallography and identify a broader interface. The method is widely applicable to the biophysical investigation of complexes of disordered proteins and folded proteins.

ECL1i, d(LGTFLKC), a novel, small peptide that specifically inhibits CCL2-dependent migration.

CC chemokine receptor type 2 (CCR2) is a key molecule in inflammatory diseases and is an obvious drug target for the treatment of inflammation. A number of nonpeptidic, competitive CCR2 antagonists have been developed, but none has yet been approved for clinical use. Our aim was to identify a short peptide that showed allosteric antagonism against human and mouse CCR2. On the basis of sequence analysis and 3-dimensional modeling, we identified an original 7-d-amino acid peptidic CCR2 inhibitor that we have called extracellular loop 1 inverso (ECL1i), d(LGTFLKC). In vitro, ECL1i selectively and potently inhibits CC chemokine ligand type 2 (CCL2)-triggered chemotaxis (IC50, 2 µM) but no other conventional CCL2-associated events. We used the classic competitive CCR2 antagonist, BMS22 {2-[(isopropylaminocarbonyl)amino]-N-[2-[[cis-2-[[4-(methylthio)benzoyl]amino]cyclohexyl]amino]-2-oxoethyl]-5-(trifluoromethyl)benzamide}, as positive control and inhibited CCL2-dependent chemotaxis with an IC50 of 18 nM. As negative control, we used a peptide with the same composition as ECL1i, but in a different sequence, d(FKLTLCG). In vivo, ECL1i (4 mg/kg) interfered with CCR2-positive cell recruitment and attenuated disease progression in experimental autoimmune encephalomyelitis, a mouse model of multiple sclerosis. This study establishes ECL1i as the first allosteric inhibitor of CCR2 with functional selectivity. ECL1i is a promising new agent in therapeutic development, and it may, by its selective effect, increase our understanding of CCR2 signaling pathways and functions.-Auvynet, C., Baudesson de Chanville, C., Hermand, P., Dorgham, K., Piesse, C., Pouchy, C., Carlier, L., Poupel, L., Barthélémy, S., Felouzis, V., Lacombe, C., Sagan, S., Salomon, B., Deterre, P., Sennlaub, F., Combadière, C. ECL1i, d(LGTFLKC), a novel, small peptide that specifically inhibits CCL2-dependent migration.

Multivalent interactions of the disordered regions of XLF and XRCC4 foster robust cellular NHEJ and drive the formation of ligation-boosting condensates in vitro.

In mammalian cells, DNA double-strand breaks are predominantly repaired by non-homologous end joining (NHEJ). During repair, the Ku70-Ku80 heterodimer (Ku), X-ray repair cross complementing 4 (XRCC4) in complex with DNA ligase 4 (X4L4) and XRCC4-like factor (XLF) form a flexible scaffold that holds the broken DNA ends together. Insights into the architectural organization of the NHEJ scaffold and its regulation by the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) were recently obtained by single-particle cryo-electron microscopy analysis. However, several regions, especially the C-terminal regions (CTRs) of the XRCC4 and XLF scaffolding proteins, have largely remained unresolved in experimental structures, which hampers the understanding of their functions. Here we used magnetic resonance techniques and biochemical assays to comprehensively characterize the interactions and dynamics of the XRCC4 and XLF CTRs at residue resolution. We show that the CTRs of XRCC4 and XLF are intrinsically disordered and form a network of multivalent heterotypic and homotypic interactions that promotes robust cellular NHEJ activity. Importantly, we demonstrate that the multivalent interactions of these CTRs lead to the formation of XLF and X4L4 condensates in vitro, which can recruit relevant effectors and critically stimulate DNA end ligation. Our work highlights the role of disordered regions in the mechanism and dynamics of NHEJ and lays the groundwork for the investigation of NHEJ protein disorder and its associated condensates inside cells with implications in cancer biology, immunology and the development of genome-editing strategies.

Characterization of a New Immunosuppressive and Antimicrobial Peptide, DRS-DA2, Isolated from the Mexican Frog, Pachymedusa dacnicolor.

Inflammatory and antimicrobial diseases constitute a major burden for society, and fighting them is a WHO strategic priority. Most of the treatments available to fight inflammatory diseases are anti-inflammatory drugs, such as corticosteroids or immunomodulators that lack cellular specificity and lead to numerous side effects. In addition to suppressing undesired inflammation and reducing disease progression, these drugs lessen the immune system protective functions. Furthermore, treating infectious diseases is more and more challenging due to the rise of microbial resistance to antimicrobial drugs. Thus, controlling the inflammatory process locally without compromising the ability to combat infections is an essential feature in the treatment of inflammatory diseases. We isolated three forms (DRS-DA2N, DRS-DA2NE, and DRS-DA2NEQ) of the same peptide, DRS-DA2, which belongs to the dermaseptin family, from the Mexican tree frog Pachymedusa dacnicolor. Interestingly, DRS-DA2N and DRS-DA2NEQ exhibit a dual activity by inducing the death of leukocytes as well as that of Gram-negative and Gram-positive bacteria, including multiresistant strains, without affecting other cells such as epithelial cells or erythrocytes. We showed that the death of both immune cells and bacteria is induced rapidly by DRS-DA2 and that the membrane is permeabilized, leading to the loss of membrane integrity. We also validated the capacity of DRS-DA2 to regulate the pool of inflammatory cells in vivo in a mouse model of noninfectious peritonitis. After the induction of peritonitis, a local injection of DRS-DA2N could decrease the number of inflammatory cells locally in the peritoneal cavity without inducing a systemic effect, as no changes in the number of inflammatory cells could be detected in blood or in the bone marrow. Collectively, these data suggest that this peptide could be a promising tool in the treatment of inflammatory diseases, such as inflammatory skin diseases, as it could reduce the number of inflammatory cells locally without suppressing the ability to combat infections.

Investigation of homeodomain membrane translocation properties: insights from the structure determination of engrailed-2 homeodomain in aqueous and membrane-mimetic environments.

In addition to their well-known DNA-binding properties, homeodomains have the ability to efficiently translocate across biological membranes through still poorly-characterized mechanisms. To date, most biophysical studies addressing the mechanisms of internalization have focused on small synthetic peptides rather than full-length globular homeodomains. In this work, we characterized the conformational properties of chicken Engrailed 2 homeodomain (En2HD) in aqueous solution and in membrane mimetic environments using circular dichroism, Trp fluorescence, and NMR spectroscopy. En2HD adopts a well-defined three-helical bundle fold in aqueous solution. The Trp-48 residue, which is critical for internalization, is fully buried in the hydrophobic core. Circular dichroism and fluorescence reveal that a conformational transition occurs in anionic lipid vesicles and in micelles. En2HD loses its native three-dimensional structure in micellar environments but, remarkably, near-native helical secondary structures are maintained. Long-range interactions could be detected using site-directed spin labels, indicating that the three helices do not adopt extended orientations. Noncovalent paramagnetic probes yielded information about helix positioning and unveiled the burial of critical aromatic and basic residues within the micelles. Our results suggest that electrostatic interactions with membranes may be determinant in inducing a conformational change enabling Trp-48 to insert into membranes.

Multivalent interactions of the disordered regions of XLF and XRCC4 foster robust cellular NHEJ and drive the formation of ligation-boosting condensates in vitro.

In mammalian cells, DNA double-strand breaks are predominantly repaired by non-homologous end joining (NHEJ). During repair, the Ku70/80 heterodimer (Ku), XRCC4 in complex with DNA Ligase 4 (X4L4), and XLF form a flexible scaffold that holds the broken DNA ends together. Insights into the architectural organization of the NHEJ scaffold and its regulation by the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) have recently been obtained by single-particle cryo-electron microscopy analysis. However, several regions, especially the C-terminal regions (CTRs) of the XRCC4 and XLF scaffolding proteins, have largely remained unresolved in experimental structures, which hampers the understanding of their functions. Here, we used magnetic resonance techniques and biochemical assays to comprehensively characterize the interactions and dynamics of the XRCC4 and XLF CTRs at atomic resolution. We show that the CTRs of XRCC4 and XLF are intrinsically disordered and form a network of multivalent heterotypic and homotypic interactions that promotes robust cellular NHEJ activity. Importantly, we demonstrate that the multivalent interactions of these CTRs led to the formation of XLF and X4L4 condensates in vitro which can recruit relevant effectors and critically stimulate DNA end ligation. Our work highlights the role of disordered regions in the mechanism and dynamics of NHEJ and lays the groundwork for the investigation of NHEJ protein disorder and its associated condensates inside cells with implications in cancer biology, immunology and the development of genome editing strategies.

A cationic motif upstream Engrailed2 homeodomain controls cell internalization through selective interaction with heparan sulfates.

Engrailed2 (En2) is a transcription factor that transfers from cell to cell through unconventional pathways. The poorly understood internalization mechanism of this cationic protein is proposed to require an initial interaction with cell-surface glycosaminoglycans (GAGs). To decipher the role of GAGs in En2 internalization, we have quantified the entry of its homeodomain region in model cells that differ in their content in cell-surface GAGs. The binding specificity to GAGs and the influence of this interaction on the structure and dynamics of En2 was also investigated at the amino acid level. Our results show that a high-affinity GAG-binding sequence (RKPKKKNPNKEDKRPR), upstream of the homeodomain, controls En2 internalization through selective interactions with highly-sulfated heparan sulfate GAGs. Our data underline the functional importance of the intrinsically disordered basic region upstream of En2 internalization domain, and demonstrate the critical role of GAGs as an entry gate, finely tuning homeoprotein capacity to internalize into cells.

The C-terminal domain of the Uup protein is a DNA-binding coiled coil motif.

The bacterial Uup protein belongs to the REG subfamily of soluble ATP-binding cassette (ABC) ATPases, and is implicated in precise excision of transposons. In Escherichia coli, the uup gene encodes a 72 kDa polypeptide that comprises two ABC domains, separated by a linker region, and a 12kDa C-terminal domain (CTD). Uup binds double-stranded DNA with no sequence specificity, and we previously demonstrated that the CTD domain is a crucial region that participates in DNA-binding activity. We report herein the NMR structure of Uup CTD, consisting of an intramolecular antiparallel two-stranded coiled coil motif. Structural comparison with analogous coiled coil domains reveals that Uup CTD contains an atypical 3(10)-helix in the α-hairpin region that contributes to the hydrophobic core. Using NMR titration experiments, we identified residues of the CTD domain involved in the binding to double-stranded DNA. These residues are located on two opposite surfaces at the base of the coiled coil, formed by the N- and C-terminal extremities, where a strictly conserved proline residue induces an overwinding of the coiled coil. Finally, preliminary analysis of NMR spectra recorded on distinct Uup constructs precludes a fully flexible positioning of the CTD domain in full-length Uup. These structural data are the first reported for a non-ATPase domain within ABC REG subfamily.

Structural and biochemical characterization of NarE, an iron-containing ADP-ribosyltransferase from Neisseria meningitidis.

NarE is a 16 kDa protein identified from Neisseria meningitidis, one of the bacterial pathogens responsible for meningitis. NarE belongs to the family of ADP-ribosyltransferases (ADPRT) and catalyzes the transfer of ADP-ribose moieties to arginine residues in target protein acceptors. Many pathogenic bacteria utilize ADP-ribosylating toxins to modify and alter essential functions of eukaryotic cells. NarE is further the first ADPRT which could be shown to bind iron through a Fe-S center, which is crucial for the catalytic activity. Here we present the NMR solution structure of NarE, which shows structural homology to other ADPRTs. Using NMR titration experiments we could identify from Chemical Shift Perturbation data both the NAD binding site, which is in perfect agreement with a consensus sequence analysis between different ADPRTs, as well as the iron coordination site, which consists of 2 cysteines and 2 histidines. This atypical iron coordination is also capable to bind zinc. These results could be fortified by site-directed mutagenesis of the catalytic region, which identified two functionally crucial residues. We could further identify a main interaction region of NarE with antibodies using two complementary methods based on antibody immobilization, proteolytic digestion, and mass spectrometry. This study combines structural and functional features of NarE providing for the first time a characterization of an iron-dependent ADPRT.

Biophysical studies of the interaction between calmodulin and the R²87-T³¹¹ region of human estrogen receptor α reveals an atypical binding process.

The transcriptional activity of human estrogen receptor ERα is modulated by a number of coregulatory proteins among which calmodulin (CaM). Segment 295-311 in the hinge region of ERα has previously been proposed to be the CaM binding site. In this work, we investigate the molecular mechanism of the interaction of CaM with peptides derived from the hinge region of ERα, using a biophysical approach combining isothermal titration calorimetry, fluorescence, CD and NMR. The ERα17p peptide, corresponding to the previously identified 295-311 region of ERα, recruits mainly the C-terminal domain of Ca(4)CaM, as shown by NMR spectroscopy. In contrast, a longer peptide, ERα25p, extended on the N-terminal side (residues 287-311) interacts with both N- and C-terminal domains of Ca(4)CaM. These results lead to a new delineation of the CaM binding site, encompassing residues 287-294. In particular, fluorescence spectroscopy reveals that the conserved W(292) residue is engaged within hydrophobic pockets on Ca(4)CaM. ITC results show that ERα25p binds Ca(4)CaM with an atypical 2:1 stoichiometry and a dissociation constant in the micromolar range. Based on the NMR titration of Ca(4)CaM by ERα25p showing a biphasic behavior for several residues, we suggest that concerted conformational changes of CaM domains may be required to accommodate the binding of a second peptide. CD spectra indicate that ERα25p partially folds into an α-helix upon binding to Ca(4)CaM. Hence, ERα25p is a new CaM-binding ligand that could be appropriate for the synthesis of derivatives able to control ER-dependent transcription, particularly in the context of hormone-dependent breast tumors.

Anionic lipids induce a fold-unfold transition in the membrane-translocating Engrailed homeodomain.

Homeoprotein transcription factors have the property of interacting with membranes through their DNA-binding homeodomain, which is involved in unconventional internalization and secretion. Both processes depend on membrane-translocating events but their detailed molecular mechanisms are still poorly understood. We have previously characterized the conformational properties of Engrailed 2 homeodomain (EnHD) in aqueous solution and in micelles as membrane-mimetic environments. In the present study, we used small isotropic lipid bicelles as a more relevant membrane-mimetic model to characterize the membrane-bound state of EnHD. We show that lipid bicelles, in contrast to micelles, adequately reproduce the requirement of anionic lipids in the membrane binding and conformational transition of EnHD. The fold-unfold transition of EnHD induced by anionic lipids was characterized by NMR using 1H, 13C, 15N chemical shifts, nuclear Overhauser effects, residual dipolar couplings, intramolecular and intermolecular paramagnetic relaxation enhancements induced by site-directed spin-label or paramagnetic lipid probe, respectively. A global unpacking of EnHD helices is observed leading to a loss of the native fold. However, near-native propensities of EnHD backbone conformation are maintained in membrane environment, including not only the three helices but also the turn connecting helices H2 and H3. NMR and coarse-grained molecular dynamics simulations reveal that the EnHD adopts a shallow insertion in the membrane, with the three helices oriented parallel to the membrane. EnHD explores extended conformations and closed U-shaped conformations, which are stabilized by anionic lipid recruitment.

Structural Bases for the Involvement of Phosphatidylinositol-4,5-bisphosphate in the Internalization of the Cell-Penetrating Peptide Penetratin.

Cell-penetrating peptides cross cell membranes through various parallel internalization pathways. Herein, we analyze the role of the negatively charged lipid phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) in the internalization of Penetratin. Contributions of both inner leaflet and outer leaflet pools of PI(4,5)P2 were revealed by quantifying the internalization of Penetratin in cells treated with PI(4,5)P2 binders. Studies on model systems showed that Penetratin has a strong affinity for PI(4,5)P2 and interacts selectively with this lipid, even in the presence of other negatively charged lipids, as demonstrated by affinity photo-crosslinking experiments. Differential scanning calorimetry experiments showed that Penetratin induces lateral segregation in PI(4,5)P2-containing liposomes, which was confirmed by coarse-grained molecular dynamics simulations. NMR experiments indicated that Penetratin adopts a stabilized helical conformation in the presence of PI(4,5)P2-containing membranes, with an orientation parallel to the bilayer plane, which was also confirmed by all-atom simulations. NMR and photo-crosslinking experiments also suggest a rather shallow insertion of the peptide in the membrane. Put together, our findings suggest that PI(4,5)P2 is a privileged interaction partner for Penetratin and that it plays an important role in Penetratin internalization.

Drug Repurposing: Deferasirox Inhibits the Anti-Apoptotic Activity of Mcl-1.

INTRODUCTION: With the aim of repositioning commercially available drugs for the inhibition of the anti-apoptotic myeloid cell leukemia protein, Mcl-1, implied in various cancers, five molecules, highlighted from a published theoretical screening, were selected to experimentally validate their affinity toward Mcl-1. RESULTS: A detailed NMR study revealed that only two of the five tested drugs, Torsemide and Deferasirox, interacted with Mcl-1. NMR data analysis allowed the complete characterization of the binding mode of both drugs to Mcl-1, including the estimation of their affinity for Mcl-1. Biological assays evidenced that the biological activity of Torsemide was lower as compared to the Deferasirox, which was able to efficiently and selectively inhibit the anti-apoptotic activity of Mcl-1. Finally, docking and molecular dynamics led to a 3D model for the Deferasirox:Mcl-1 complex and revealed the positioning of the drug in the Mcl-1 P2/P3 pockets as well as almost all synthetic Mcl-1 inhibitors. Interestingly, contrary to known synthetic Mcl-1 inhibitors which interact through Arg263, Deferasirox, establishes a salt bridge with Lys234. CONCLUSION: Deferasirox could be a potential candidate for drug repositioning as Mcl-1 inhibitor.

Peptidoglycan potentiates the membrane disrupting effect of the carboxyamidated form of DMS-DA6, a Gram-positive selective antimicrobial peptide isolated from Pachymedusa dacnicolor skin.

The occurrence of nosocomial infections has been on the rise for the past twenty years. Notably, infections caused by the Gram-positive bacteria Staphylococcus aureus represent a major clinical problem, as an increase in antibiotic multi-resistant strains has accompanied this rise. There is thus a crucial need to find and characterize new antibiotics against Gram-positive bacteria, and against antibiotic-resistant strains in general. We identified a new dermaseptin, DMS-DA6, produced by the skin of the Mexican frog Pachymedusa dacnicolor, with specific antibacterial activity against Gram-positive bacteria. This peptide is particularly effective against two multiple drug-resistant strains Enterococcus faecium BM4147 and Staphylococcus aureus DAR5829, and has no hemolytic activity. DMS-DA6 is naturally produced with the C-terminal carboxyl group in either the free or amide forms. By using Gram-positive model membranes and different experimental approaches, we showed that both forms of the peptide adopt an α-helical fold and have the same ability to insert into, and to disorganize a membrane composed of anionic lipids. However, the bactericidal capacity of DMS-DA6-NH2 was consistently more potent than that of DMS-DA6-OH. Remarkably, rather than resulting from the interaction with the negatively charged lipids of the membrane, or from a more stable conformation towards proteolysis, the increased capacity to permeabilize the membrane of Gram-positive bacteria of the carboxyamidated form of DMS-DA6 was found to result from its enhanced ability to interact with peptidoglycan.

Structure-guided design of pyridoclax derivatives based on Noxa / Mcl-1 interaction mode.

Protein-protein interactions are attractive targets because they control numerous cellular processes. In oncology, apoptosis regulating Bcl-2 family proteins are of particular interest. Apoptotic cell death is controlled via PPIs between the anti-apoptotic proteins hydrophobic groove and the pro-apoptotic proteins BH3 domain. In ovarian carcinoma, it has been previously demonstrated that Bcl-xL and Mcl-1 cooperate to protect tumor cells against apoptosis. Moreover, Mcl-1 is a key regulator of cancer cell survival and is a known resistance factor to Bcl-2/Bcl-xL pharmacological inhibitors making it an attractive therapeutic target. Here, using a structure-guided design from the oligopyridine lead Pyridoclax based on Noxa/Mcl-1 interaction we identified a new derivative, active at lower concentration as compared to Pyridoclax. This new derivative selectively binds to the Mcl-1 hydrophobic groove and releases Bak and Bim from Mcl-1 to induce cell death and sensitize cancer cells to Bcl-2/Bcl-xL targeting strategies.

Solution structure of the region 51-160 of human KIN17 reveals an atypical winged helix domain.

Human KIN17 is a 45-kDa eukaryotic DNA- and RNA-binding protein that plays an important role in nuclear metabolism and in particular in the general response to genotoxics. Its amino acids sequence contains a zinc finger motif (residues 28-50) within a 30-kDa N-terminal region conserved from yeast to human, and a 15-kDa C-terminal tandem of SH3-like subdomains (residues 268-393) only found in higher eukaryotes. Here we report the solution structure of the region 51-160 of human KIN17. We show that this fragment folds into a three-alpha-helix bundle packed against a three-stranded beta-sheet. It belongs to the winged helix (WH) family. Structural comparison with analogous WH domains reveals that KIN17 WH module presents an additional and highly conserved 3(10)-helix. Moreover, KIN17 WH helix H3 is not positively charged as in classical DNA-binding WH domains. Thus, human KIN17 region 51-160 might rather be involved in protein-protein interaction through its conserved surface centered on the 3(10)-helix.

Investigating the role of GXXXG motifs in helical folding and self-association of plasticins, Gly/Leu-rich antimicrobial peptides.

Plasticins (PTC) are dermaseptin-related antimicrobial peptides characterized by a large number of leucine and glycine residues arranged in GXXXG motifs that are often described to promote helix association within biological membranes. We report the structure and interaction properties of two plasticins, PTC-B1 from Phyllomedusa bicolor and a cationic analog of PTC-DA1 from Pachymedusa dacnicolor, which exhibit membrane-lytic activities on a broad range of microorganisms. Despite a high number of glycine, CD and NMR spectroscopy show that the two plasticins adopt mainly alpha-helical conformations in a wide variety of environments such as trifluoroethanol, detergent micelles and lipid vesicles. In DPC and SDS, plasticins adopt well-defined helices that lie parallel to the micelle surface, all glycine residues being located on the solvent-exposed face. Spectroscopic data and cross-linking experiments indicate that the GXXXG repeats in these amphipathic helices do not provide a strong oligomerization interface, suggesting a different role from GXXXG motifs found in transmembrane helices.