HT-SuMD: making molecular dynamics simulations suitable for fragment-based screening. A comparative study with NMR

Fragment-based lead discovery (FBLD) is one of the most efficient methods to develop new drugs. We present here a new computational protocol called High-Throughput Supervised Molecular Dynamics (HT-SuMD), which makes it possible to automatically screen up to thousands of fragments, representing therefore a new valuable resource to prioritise fragments in FBLD campaigns. The protocol was applied to Bcl-XL, an oncological protein target involved in the regulation of apoptosis through protein-protein interactions. Initially, HT-SuMD performances were validated against a robust NMR-based screening, using the same set of 100 fragments. These independent results showed a remarkable agreement between the two methods. Then, a virtual screening on a larger library of additional 300 fragments was carried out and the best hits were validated by NMR. Remarkably, all the in silico selected fragments were confirmed as Bcl-XL binders. This represents, to date, the largest computational fragments screening entirely based on MD

Calmodulin Enhances Cryptochrome Binding to INAD in Drosophila Photoreceptors

Light is the main environmental stimulus that synchronizes the endogenous timekeeping systems in most terrestrial organisms. Drosophila cryptochrome (dCRY) is a light-responsive flavoprotein that detects changes in light intensity and wavelength around dawn and dusk. We have previously shown that dCRY acts through Inactivation No Afterpotential D (INAD) in a light-dependent manner on the Signalplex, a multiprotein complex that includes visual-signaling molecules, suggesting a role for dCRY in fly vision. Here, we predict and demonstrate a novel Ca2+-dependent interaction between dCRY and calmodulin (CaM). Through yeast two hybrid, coimmunoprecipitation (Co-IP), nuclear magnetic resonance (NMR) and calorimetric analyses we were able to identify and characterize a CaM binding motif in the dCRY C-terminus. Similarly, we also detailed the CaM binding site of the scaffold protein INAD and demonstrated that CaM bridges dCRY and INAD to form a ternary complex in vivo. Our results suggest a process whereby a rapid dCRY light response stimulates an interaction with INAD, which can be further consolidated by a novel mechanism regulated by CaM

The mitochondrial inhibitor IF1 binds to the ATP synthase OSCP subunit and protects cancer cells from apoptosis

: The mitochondrial protein IF1 binds to the catalytic domain of the ATP synthase and inhibits ATP hydrolysis in ischemic tissues. Moreover, IF1 is overexpressed in many tumors and has been shown to act as a pro-oncogenic protein, although its mechanism of action is still debated. Here, we show that ATP5IF1 gene disruption in HeLa cells decreases colony formation in soft agar and tumor mass development in xenografts, underlining the role of IF1 in cancer. Notably, the lack of IF1 does not affect proliferation or oligomycin-sensitive mitochondrial respiration, but it sensitizes the cells to the opening of the permeability transition pore (PTP). Immunoprecipitation and proximity ligation analysis show that IF1 binds to the ATP synthase OSCP subunit in HeLa cells under oxidative phosphorylation conditions. The IF1-OSCP interaction is confirmed by NMR spectroscopy analysis of the recombinant soluble proteins. Overall, our results suggest that the IF1-OSCP interaction protects cancer cells from PTP-dependent apoptosis under normoxic conditions

Exploring iron-binding to human frataxin and to selected Friedreich ataxia mutants by means of NMR and EPR spectroscopies

The neurodegenerative disease Friedreich ataxia results from a deficiency of frataxin, a mitochondrial protein. Most patients have a GAA expansion in the first intron of both alleles of frataxin gene, whereas a minority of them are heterozygous for the expansion and contain a mutation in the other allele. Frataxin has been claimed to participate in iron homeostasis and biosynthesis of FeS clusters, however its role in both pathways is not unequivocally defined. In this work we combined different advanced spectroscopic analyses to explore the iron-binding properties of human frataxin, as isolated and at the FeS clusters assembly machinery. For the first time we used EPR spectroscopy to address this key issue providing clear evidence of the formation of a complex with a low symmetry coordination of the metal ion. By 2D NMR, we confirmed that iron can be bound in both oxidation states, a controversial issue, and, in addition, we were able to point out a transient interaction of frataxin with a N-terminal 6his-tagged variant of ISCU, the scaffold protein of the FeS clusters assembly machinery. To obtain insights on structure/function relationships relevant to understand the disease molecular mechanism(s), we extended our studies to four clinical frataxin mutants. All variants showed a moderate to strong impairment in their ability to activate the FeS cluster assembly machinery in vitro, while keeping the same iron-binding features of the wild type protein. This supports the multifunctional nature of frataxin and the complex biochemical consequences of its mutations.Fil: Bellanda, Massimo. Università di Padova; ItaliaFil: Maso, Lorenzo. Università di Padova; ItaliaFil: Doni, Davide. Università di Padova; ItaliaFil: Bortolus, M.. Università di Padova; ItaliaFil: De Rosa, E.. Università di Padova; ItaliaFil: Lunardi, Federica. Università di Padova; ItaliaFil: Alfonsi, Arianna. Università di Padova; ItaliaFil: Noguera, Martín Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Herrera, Maria Georgina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Santos, Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Carbonera, Donatella. Università di Padova; ItaliaFil: Costantini, Paola. Università di Padova; Itali

Calmodulin enhances cryptochrome binding to INAD in Drosophila photoreceptors

Light is the main environmental stimulus that synchronizes the endogenous timekeeping systems in most terrestrial organisms. Drosophila cryptochrome (dCRY) is a light-responsive flavoprotein that detects changes in light intensity and wavelength around dawn and dusk. We have previously shown that dCRY acts through Inactivation No Afterpotential D (INAD) in a light-dependent manner on the Signalplex, a multiprotein complex that includes visual-signaling molecules, suggesting a role for dCRY in fly vision. Here, we predict and demonstrate a novel Ca2+-dependent interaction between dCRY and calmodulin (CaM). Through yeast two hybrid, coimmunoprecipitation (Co-IP), nuclear magnetic resonance (NMR) and calorimetric analyses we were able to identify and characterize a CaM binding motif in the dCRY C-terminus. Similarly, we also detailed the CaM binding site of the scaffold protein INAD and demonstrated that CaM bridges dCRY and INAD to form a ternary complex in vivo. Our results suggest a process whereby a rapid dCRY light response stimulates an interaction with INAD, which can be further consolidated by a novel mechanism regulated by CaM

A molecular dynamics strategy for CS\u3b1\u3b2 peptides disulfide-assisted model refinement

Many cysteine-stabilized antimicrobial peptides from a variety of living organisms could be good candidates for the development of anti-infective agents. In the absence of experimentally obtained structural data, peptide modeling is an essential tool for understanding structure\u2013activity relationships and for optimizing the bioactive moieties. Focusing on cysteine-rich peptide structures, we reproduced the case of structure predictions in the so-called midnight zone. We developed our protocol on a training set derived by clustering the available cysteine-stabilized \u3b1\u3b2 (CS\u3b1\u3b2) structures in nine different representative families and tested it on peptides randomly selected from each family. Starting from draft models, we tested a structure-based disulfide predictor and we used cysteine distances as constraints during molecular dynamics. Finally, we proposed an analysis for final structure selection. Accordingly, we obtained a mean root mean square deviation improvement of 21% for the test set. Our findings demonstrate that it is possible to predict the network of disulfide bridges in cysteine-stabilized peptides and to use this result to improve the accuracy of structural predictions. Finally, we applied the methods to predict the structure of royalisin, a cysteine-rich peptide with unknown structure

Mono- and Dithiol Glutaredoxins in the Trypanothione-Based Redox Metabolism of Pathogenic Trypanosomes

SIGNIFICANCE: Glutaredoxins are ubiquitous small thiol proteins of the thioredoxin-fold superfamily. Two major groups are distinguished based on their active sites: the dithiol (2-C-Grxs) and the monothiol (1-C-Grxs) glutaredoxins with a CXXC and a CXXS active site motif, respectively. Glutaredoxins are involved in cellular redox and/or iron sulfur metabolism. Usually their functions are closely linked to the glutathione system. Trypanosomatids, the causative agents of several tropical diseases, rely on trypanothione as principal low molecular mass thiol, and their glutaredoxins readily react with the unique bis(glutathionyl) spermidine conjugate. RECENT ADVANCES: Two 2-C-Grxs and three 1-C-Grxs have been identified in pathogenic trypanosomatids. The 2-C-Grxs catalyze the reduction of glutathione disulfide by trypanothione and display reductase activity towards protein disulfides, as well as protein-glutathione mixed disulfides. In vitro, all three 1-C-Grxs as well as the cytosolic 2-C-Grx of Trypanosoma brucei can complex an iron-sulfur cluster. Recently the structure of the 1-C-Grx1 has been solved by NMR spectroscopy. The structure is very similar to those of other 1-C-Grxs, with some differences in the loop containing the conserved cis-Pro and the surface charge distribution. CRITICAL ISSUES: Although four of the five trypanosomal glutaredoxins proved to coordinate an iron-sulfur cluster in vitro, the physiological role of the mitochondrial and cytosolic proteins, respectively, has only started to be unraveled. FUTURE DIRECTIONS: The use of trypanothione by the glutaredoxins has established a novel role for this parasite-specific dithiol. Future work should reveal if these differences can be exploited for the development of novel antiparasitic drugs

Structure of the Cytosolic Portion of the Motor Protein Prestin and Functional Role of the STAS Domain in SLC26/SulP Anion Transporters

Prestin is the motor protein responsible for the somatic electromotility of cochlear outer hair cells and is essential for normal hearing sensitivity and frequency selectivity of mammals. Prestin is a member of mammalian solute-linked carrier 26 (SLC26) anion exchangers, a family of membrane proteins capable of transporting a wide variety of monovalent and divalent anions. SLC26 transporters play important roles in normal human physiology in different tissues, and many of them are involved in genetic diseases. SLC26 and related SulP transporters carry a hydrophobic membrane core and a C-terminal cytosolic portion that is essential in plasma membrane targeting and protein function. This C-terminal portion is mainly composed of a STAS (sulfate transporters and anti- sigma factor antagonist) domain, whose name is due to a remote but significant sequence similarity with bacterial ASA (anti- sigma factor antagonist) proteins. Here we present the crystal structure at 1.57 Å resolution of the cytosolic portion of prestin, the first structure of a SulP transporter STAS domain, and its characterization in solution by heteronuclear multidimensional NMR spectroscopy. Prestin STAS significantly deviates from the related bacterial ASA proteins, especially in the N-terminal region, which-although previously considered merely as a generic linker between the domain and the last transmembrane helix-is indeed fully part of the domain. Hence, unexpectedly, our data reveal that the STAS domain starts immediately after the last transmembrane segment and lies beneath the lipid bilayer. A structure-function analysis suggests that this model can be a general template for most SLC26 and SulP anion transporters and supports the notion that STAS domains are involved in functionally important intramolecular and intermolecular interactions. Mapping of disease-associated or functionally harmful mutations on STAS structure indicates that they can be divided into two categories: those causing significant misfolding of the domain and those altering its interaction properties