Using Steady-State Kinetics to Quantitate Substrate Selectivity and Specificity: A Case Study with Two Human Transaminases.

We examined the ability of two human cytosolic transaminases, aspartate aminotransferase (GOT1) and alanine aminotransferase (GPT), to transform their preferred substrates whilst discriminating against similar metabolites. This offers an opportunity to survey our current understanding of enzyme selectivity and specificity in a biological context. Substrate selectivity can be quantitated based on the ratio of the kcat/KM values for two alternative substrates (the 'discrimination index'). After assessing the advantages, implications and limits of this index, we analyzed the reactions of GOT1 and GPT with alternative substrates that are metabolically available and show limited structural differences with respect to the preferred substrates. The transaminases' observed selectivities were remarkably high. In particular, GOT1 reacted ~106-fold less efficiently when the side-chain carboxylate of the 'physiological' substrates (aspartate and glutamate) was replaced by an amido group (asparagine and glutamine). This represents a current empirical limit of discrimination associated with this chemical difference. The structural basis of GOT1 selectivity was addressed through substrate docking simulations, which highlighted the importance of electrostatic interactions and proper substrate positioning in the active site. We briefly discuss the biological implications of these results and the possibility of using kcat/KM values to derive a global measure of enzyme specificity.

Reversible processes in collagen dehydration: A molecular dynamics study.

Collagen dehydration is an unavoidable damaging process that causes the lack of fibers' physical properties and it is usually irreversible. However, the identification of low hydration conditions that permit a recovering of initial collagen features after a rehydration treatment is particularly of interest. Monitoring structural changes by means of MD simulations, we investigated the hydration-dehydration-rehydration cycle of two microfibril models built on different fragments of the sequence of rat tail collagen type I. The microfibrils have different hydropathic features, to investigate the influence of amino acid composition on the whole process. We showed that with low hydration at a level corresponding to the first shell, microfibril gains in compactness and tubularity. Crucially, some water molecules remain trapped inside the fibrils, allowing, by rehydrating, a recovery of the initial collagen structural features. Water rearranges in cluster around the protein, and its first layer is more anchored to the surface. However, these changes in distribution and mobility in low hydration conditions get back with rehydration.

Interplay among the "flipping" glutamine, a conserved phenylalanine, water and hydrogen bonds within a blue-light sensing LOV domain.

In this work we exploited time-resolved photoacoustics (PA) and molecular dynamics (MD) simulations to investigate the function of a conserved phenylalanine residue in blue sensing (BL) LOV domains. The LOV photocycle involves reversible formation of a photoproduct (LOV390) where the flavin mononucleotide (FMN) chromophore is covalently bound to a cysteine. LOV390 thermally returns to the dark adapted state (LOV447) with a lifetime τrec (s-to-h). In the LOV domain of Bacillus subtilis BsYtvA, the conserved F46 is one of the few residues undergoing a pronounced light-driven conformational change. PA and spectroscopic data show that in the YtvA variants F46A and F46Y light-induced structural changes are much smaller than those in the wild type (wt) protein, τrec is strongly accelerated and the energy content of LOV390 is lower for F46Y. MD simulations for each variant in the LOV447 and LOV390 states revealed an overall very stable structure of the BsYtvA-LOV domain. The largest variations emerged for the conserved HB network that includes FMN, Q123 (the "flipping" glutamine of LOV domains), and the conserved N104 and N94, with strong dependence on the presence of water. The lateral chain of Q123 in wt-LOV447 can adopt three alternative conformations, and movements act in concert with F46 flexibility. In LOV390, Q123 remains instead fixed in the orientation adopted in the crystal structure. Interestingly, in F46A, Q123 is locked in a LOV447-like conformation (pseudo-dark-adapted state), in both LOV447 and LOV390. In LOV447 of F46Y the tyrosine hydroxyl group fixes a water molecule, which induces a Q123 conformation similar to wt-LOV390, i.e. a pseudo-photoproduct state. These pseudo-dark-adapted and photoproduct-like conformations of the Q123 sidechain may account for the strong acceleration of the photocycle in the two variants. Given the importance of the "flipping" glutamine in light-to-signal propagation in LOV proteins, the results presented here underscore a crucial structural and functional role of the conserved F46. MD results also indicate that F46 is not directly engaged in permeability of the FMN pocket, but is involved in solvent ordering and the formation of water bridges.

Deciphering protein dynamics changes along the pathway of retinol uptake by cellular retinol-binding proteins 1 and 2.

Four Cellular Retinol-binding Proteins (CRBP 1, 2, 3, 4) are encoded in the human genome. CRBP 1 and 2, sharing a 56% amino acid sequence identity, exhibit the highest binding affinities for retinol. Previous NMR studies provided some insights into the mechanism of retinol uptake, but details of such mechanism remain to be elucidated. Herein, the results of molecular dynamics simulations for the uptake of retinol by CRBP 1 and 2 are consistent with the presence of two different retinol entry points, both involving the 'cap region' (α-helices I and II and neighboring loops). We observed that a hydrophobic patch at the surface of the 'portal region' (α-helix II, CD and EF loops) of CRBP 1 attracts retinol, which accesses the binding cavity through an opening generated by the concerted movements of Arg58 and Phe57, present in the CD loop. In CRBP 2 a different distribution of the surface residues of the 'cap region' allows retinol to access the binding cavity by sinking in a hydrophobic matrix between the two α-helices. Polar interactions mainly affect retinol movements inside the ß-barrel cavities of both CRBPs. The interaction energy profiles are in agreement with the different behavior of the two protein systems.

Structural and molecular determinants affecting the interaction of retinol with human CRBP1.

Four cellular retinol-binding protein (CRBP) types (CRBP1,2,3,4) are encoded in the human genome. Here, we report on X-ray analyses of human apo- and holo-CRBP1, showing nearly identical structures, at variance with the results of a recent study on the same proteins containing a His-Tag, which appears to be responsible for a destabilizing effect on the apoprotein. The analysis of crystallographic B-factors for our structures indicates that the putative portal region, in particular α-helix-II, along with Arg58 and the E-F loop, is the most flexible part of both apo- and holoprotein, consistent with its role in ligand uptake and release. Fluorometric titrations of wild type and mutant forms of apo-CRBP1, coupled with X-ray analyses, provided insight into structural and molecular determinants for the interaction of retinol with CRBP1. An approximately stoichiometric binding of retinol to wild type apo-CRBP1 (Kd∼4.5nM), significantly lower binding affinity for both mutants Q108L (Kd∼65nM) and K40L (Kd∼70nM) and very low binding affinity for the double mutant Q108L/K40L (Kd∼250nM) were determined, respectively. Overall, our data indicate that the extensive apolar interactions between the ligand and hydrophobic residues lining the retinol binding cavity are sufficient to keep it in its position bound to CRBP1. However, polar interactions of the retinol hydroxyl end group with Gln108 and Lys40 play a key role to induce a high binding affinity and specificity for the interaction.

The DNA binding mechanism of a SSB protein from Lactococcus lactis siphophage p2.

Virulent lactococcal phages of the Siphoviridae family are responsible for the industrial milk fermentation failures worldwide. Lactococcus lactis, a Gram-positive bacterium widely used for the manufacture of fermented dairy products, is subjected to infections by virulent phages, predominantly those of the 936 group, including phage p2. Among the proteins coded by lactococcal phage genomes, of special interest are those expressed early, which are crucial to efficiently carry out the phage lytic cycle. We previously identified and solved the 3D structure of lactococcal phage p2 ORF34, a single stranded DNA binding protein (SSBp2). Here we investigated the molecular basis of ORF34 binding mechanism to DNA. DNA docking on SSBp2 and Molecular Dynamics simulations of the resulting complex identified R15 as a crucial residue for ssDNA binding. Electrophoretic Mobility Shift Assays (EMSA) and Atomic Force Microscopy (AFM) imaging revealed the inability of the Arg15Ala mutant to bind ssDNA, as compared to the native protein. Since R15 is highly conserved among lactococcal SSBs, we propose that its role in the SSBp2/DNA complex stabilization might be extended to all the members of this protein family.

One substrate many enzymes virtual screening uncovers missing genes of carnitine biosynthesis in human and mouse.

The increasing availability of experimental and computational protein structures entices their use for function prediction. Here we develop an automated procedure to identify enzymes involved in metabolic reactions by assessing substrate conformations docked to a library of protein structures. By screening AlphaFold-modeled vitamin B6-dependent enzymes, we find that a metric based on catalytically favorable conformations at the enzyme active site performs best (AUROC Score=0.84) in identifying genes associated with known reactions. Applying this procedure, we identify the mammalian gene encoding hydroxytrimethyllysine aldolase (HTMLA), the second enzyme of carnitine biosynthesis. Upon experimental validation, we find that the top-ranked candidates, serine hydroxymethyl transferase (SHMT) 1 and 2, catalyze the HTMLA reaction. However, a mouse protein absent in humans (threonine aldolase; Tha1) catalyzes the reaction more efficiently. Tha1 did not rank highest based on the AlphaFold model, but its rank improved to second place using the experimental crystal structure we determined at 2.26 Å resolution. Our findings suggest that humans have lost a gene involved in carnitine biosynthesis, with HTMLA activity of SHMT partially compensating for its function.

New Insights into the Enhancement of Adventitious Root Formation Using N,N'-Bis(2,3-methylenedioxyphenyl)urea.

Adventitious rooting is a process of postembryonic organogenesis strongly affected by endogenous and exogenous factors. Although adventitious rooting has been exploited in vegetative propagation programs for many plant species, it is a bottleneck for vegetative multiplication of difficult-to-root species, such as many woody species. The purpose of this research was to understand how N,N'-bis-(2,3-methylenedioxyphenyl)urea could exert its already reported adventitious rooting adjuvant activity, starting from the widely accepted knowledge that adventitious rooting is a hormonally tuned progressive process. Here, by using specific in vitro bioassays, histological analyses, molecular docking simulations and in vitro enzymatic bioassays, we have demonstrated that this urea derivative does not interfere with polar auxin transport; it inhibits cytokinin oxidase/dehydrogenase (CKX); and, possibly, it interacts with the apoplastic portion of the auxin receptor ABP1. As a consequence of this dual binding capacity, the lifespan of endogenous cytokinins could be locally increased and, at the same time, auxin signaling could be favored. This combination of effects could lead to a cell fate transition, which, in turn, could result in increased adventitious rooting.

Solution nuclear magnetic resonance structure and molecular dynamics simulations of a murine 18.5 kDa myelin basic protein segment (S72-S107) in association with dodecylphosphocholine micelles.

The 18.5 kDa myelin basic protein (MBP), the most abundant splice isoform in adult mammalian myelin, is a multifunctional, intrinsically disordered protein involved in the development and compaction of the myelin sheath in the central nervous system. A highly conserved central segment comprises a membrane-anchoring amphipathic α-helix followed by a proline-rich segment that represents a ligand for SH3 domain-containing proteins. Here, we have determined using solution nuclear magnetic resonance spectroscopy the structure of a 36-residue peptide fragment of MBP (murine 18.5 kDa residues S72-S107, denoted the α2-peptide) comprising these two structural motifs, in association with dodecylphosphocholine (DPC) micelles. The structure was calculated using CS-ROSETTA (version 1.01) because the nuclear Overhauser effect restraints were insufficient for this protein. The experimental studies were complemented by molecular dynamics simulations of a corresponding 24-residue peptide fragment (murine 18.5 kDa residues E80-G103, denoted the MD-peptide), also in association with a DPC micelle in silico. The experimental and theoretical results agreed well with one another, despite the independence of the starting structures and analyses, both showing membrane association via the amphipathic α-helix, and a sharp bend in the vicinity of the Pro93 residue (murine 18.5 kDa sequence numbering). Overall, the conformations elucidated here show how the SH3 ligand is presented to the cytoplasm for interaction with SH3 domain-containing proteins such as Fyn and contribute to our understanding of myelin architecture at the molecular level.

Looking over toxin-K(+) channel interactions. Clues from the structural and functional characterization of α-KTx toxin Tc32, a Kv1.3 channel blocker.

α-KTx toxin Tc32, from the Amazonian scorpion Tityus cambridgei, lacks the dyad motif, including Lys27, characteristic of the family and generally associated with channel blockage. The toxin has been cloned and expressed for the first time. Electrophysiological experiments, by showing that the recombinant form blocks Kv1.3 channels of olfactory bulb periglomerular cells like the natural Tc32 toxin, when tested on the Kv1.3 channel of human T lymphocytes, confirmed it is in an active fold. The nuclear magnetic resonance-derived structure revealed it exhibits an α/ß scaffold typical of the members of the α-KTx family. TdK2 and TdK3, all belonging to the same α-KTx 18 subfamily, share significant sequence identity with Tc32 but diverse selectivity and affinity for Kv1.3 and Kv1.1 channels. To gain insight into the structural features that may justify those differences, we used the recombinant Tc32 nuclear magnetic resonance-derived structure to model the other two toxins, for which no experimental structure is available. Their interaction with Kv1.3 and Kv1.1 has been investigated by means of docking simulations. The results suggest that differences in the electrostatic features of the toxins and channels, in their contact surfaces, and in their total dipole moment orientations govern the affinity and selectivity of toxins. In addition, we found that, regardless of whether the dyad motif is present, it is always a Lys side chain that physically blocks the channels, irrespective of its position in the toxin sequence.

Conformational choreography of a molecular switch region in myelin basic protein--molecular dynamics shows induced folding and secondary structure type conversion upon threonyl phosphorylation in both aqueous and membrane-associated environments.

The 18.5 kDa isoform of myelin basic protein is essential to maintaining the close apposition of myelin membranes in central nervous system myelin, but its intrinsic disorder (conformational dependence on environment), a variety of post-translational modifications, and a diversity of protein ligands (e.g., actin and tubulin) all indicate it to be multifunctional. We have performed molecular dynamics simulations of a conserved central segment of 18.5 kDa myelin basic protein (residues Glu80-Gly103, murine sequence numbering) in aqueous and membrane-associated environments to ascertain the stability of constituent secondary structure elements (α-helix from Glu80-Val91 and extended poly-proline type II from Thr92-Gly103) and the effects of phosphorylation of residues Thr92 and Thr95, individually and together. In aqueous solution, all four forms of the peptide bent in the middle to form a hydrophobic cluster. The phosphorylated variants were stabilized further by electrostatic interactions and formation of ß-structures, in agreement with previous spectroscopic data. In simulations performed with the peptide in association with a dimyristoylphosphatidylcholine bilayer, the amphipathic α-helical segment remained stable and membrane-associated, although the degree of penetration was less in the phosphorylated variants, and the tilt of the α-helix with respect to the plane of the membrane also changed significantly with the modifications. The extended segment adjacent to this α-helix represents a putative SH3-ligand and remained exposed to the cytoplasm (and thus accessible to binding partners). The results of these simulations demonstrate how this segment of the protein can act as a molecular switch: an amphipathic α-helical segment of the protein is membrane-associated and presents a subsequent proline-rich segment to the cytoplasm for interaction with other proteins. Phosphorylation of threonyl residues alters the degree of membrane penetration of the α-helix and the accessibility of the proline-rich ligand and can stabilize a ß-bend. A bend in this region of 18.5 kDa myelin basic protein suggests that the N- and C-termini of the proteins can interact with different leaflets of the myelin membrane and explain how a single protein can bring them close together.

Oxygen diffusion pathways in mutated forms of a LOV photoreceptor from Methylobacterium radiotolerans: A molecular dynamics study.

Mr4511 from Methylobacterium radiotolerans is a photoreceptor of the light, oxygen voltage (LOV) family, binding flavin mononucleotide (FMN) as a chromophore. It exhibits the prototypical LOV photocycle, with the reversible formation of an FMN-Cys71 adduct via fast decay of the FMN triplet state. Mr4511 has high potential as a photosensitiser for singlet oxygen (SO) upon mutation of C71. Mr4511-C71S shows a triplet lifetime (τ T) of several hundreds of microseconds, ensuring efficient energy transfer to dioxygen to form SO. In this work, we have explored the potential diffusion pathways for dioxygen within Mr4511 using molecular dynamics (MD) simulations. The structural model of wild-type (wt) Mr4511 showed a dimeric structure stabilised by a strong leucine zipper at the two C-terminal helical ends. We then introduced in silico the C71S mutation and analysed transient and persistent oxygen channels. MD simulations indicate that the chromophore binding site is highly accessible to dioxygen. Mutations that might favour SO generation were designed based on their position with respect to FMN and the oxygen channels. In particular, the C71S-Y61T and C71S-Y61S variants showed an increased diffusion and persistence of oxygen molecules inside the binding cavity.

Characterization of a deswapped triple mutant bovine odorant binding protein.

The stability and functionality of GCC-bOBP, a monomeric triple mutant of bovine odorant binding protein, was investigated, in the presence of denaturant and in acidic pH conditions, by both protein and 1-aminoanthracene ligand fluorescence measurements, and compared to that of both bovine and porcine wild type homologues. Complete reversibility of unfolding was observed, though refolding was characterized by hysteresis. Molecular dynamics simulations, performed to detect possible structural changes of the monomeric scaffold related to the presence of the ligand, pointed out the stability of the ß-barrel lipocalin scaffold.

Influence of membrane surface charge and post-translational modifications to myelin basic protein on its ability to tether the Fyn-SH3 domain to a membrane in vitro.

Myelin basic protein (MBP) is a highly post-translationally modified, multifunctional structural component of central nervous system myelin, adhering to phospholipid membranes and assembling cytoskeletal proteins, and has previously been shown to bind SH3 domains in vitro and tether them to a membrane surface [Polverini, E., et al. (2008) Biochemistry 47, 267-282]. Since molecular modeling shows that the Fyn-SH3 domain has a negative surface charge density even after binding the MBP ligand, we have investigated the influence of negative membrane surface charge and the effects of post-translational modifications to MBP on the interaction of the Fyn-SH3 domain with membrane-associated MBP. Using a sedimentation assay with multilamellar vesicles consisting of neutral phosphatidylcholine (PC) and negatively charged phosphatidylinositol (PI), we demonstrate that increasing the negative surface charge of the membrane by increasing the proportion of PI reduces the amount of Fyn-SH3 domain that binds to membrane-associated MBP, due to electrostatic repulsion. When one of the phosphoinositides, PI(4)P or PI(4,5)P(2) was substituted for PI in equal proportion, none of the Fyn-SH3 domain bound to MBP under the conditions that were used. Post-translational modifications of MBP which reduced its net positive charge, i.e., phosphorylation or arginine deimination, increased the degree of repulsion of Fyn-SH3 from the membrane surface, an effect further modulated by the lipid charge. This study suggests that changes in membrane negative surface charge due to protein or lipid modifications, which could occur during cell signaling, can regulate the binding of the Fyn-SH3 domain to membrane-associated MBP and thus could regulate the activity of Fyn at the oligodendrocyte membrane surface.

Insight on collagen self-assembly mechanisms by coupling molecular dynamics and UV spectroscopy techniques.

Self-assembly of rat tail collagen type I was investigated by means of turbidity measurements and molecular dynamics simulations. Turbidity curves collected at different pH values show that the rate of aggregation was not linear in dependence from pH, with the fastest kinetics at pH 5.0 and the lowest at neutral pH. MD simulations were carried out on two regions with different hydropathicity, monitoring the aggregation of up to four staggered tropocollagen fragments at different ionic strength. At physiological conditions, association of lowly charged regions occurs more easily than for highly charged ones, the latter seeming to aggregate in a sequential way. The first contacts indicate for both regions that the driving force is hydrophobic, the electrostatic contribution becoming relevant at short distance. The direct inter-tropocollagen H-bonds confirm that fibrillogenesis is driven by loss of surface water from the monomers and involves in large percentage hydroxyproline residues. Low ionic strength dynamics leads to the formation of incorrect assemblies, driven by not shielded pairwise charge interactions.

A monoclonal antibody inhibits gelatinase B/MMP-9 by selective binding to part of the catalytic domain and not to the fibronectin or zinc binding domains.

Gelatinase B/matrix metalloproteinase-9 (MMP-9) is a multidomain enzyme functioning in acute and chronic inflammatory and neoplastic diseases. It belongs to a family of more than 20 related zinc proteinases. Therefore, the discovery and the definition of the action mechanism of selective MMP inhibitors form the basis for future therapeutics. The monoclonal antibody REGA-3G12 is a most selective inhibitor of human gelatinase B. REGA-3G12 was found to recognize the aminoterminal part and not the carboxyterminal O-glycosylated and hemopexin protein domains. A variant of gelatinase B, lacking the two carboxyterminal domains, was expressed in insect cells and fragmented with purified proteinases. The fragments were probed by one- and two-dimensional Western blot and immunoprecipitation experiments with REGA-3G12 to map the interactions between the antibody and the enzyme. The interaction unit was identified by Edman degradation analysis as the glycosylated segment from Trp(116) to Lys(214) of gelatinase B. The sequence of this segment was analysed by hydrophobicity/hydrophilicity, accessibility and flexibility profiling. Four hydrophilic peptides were chemically synthesized and used in binding and competition assays. The peptide Gly(171)-Leu(187) in molar excess inhibited partially the binding of MMP-9 to REGA-3G12 and thus refines the structure of the conformational binding site. These results define part of the catalytic domain of gelatinase B/MMP-9, and not the zinc-binding or fibronectin domains, as target for the development of selective inhibitors.

Novel Naja atra cardiotoxin 1 (CTX-1) derived antimicrobial peptides with broad spectrum activity.

Naja atra subsp. atra cardiotoxin 1 (CTX-1), produced by Chinese cobra snakes, belonging to Elapidae family, is included in the three-finger toxin family and exerts high cytotoxicity and antimicrobial activity too. Using as template mainly the tip and the subsequent ß-strand of the first "finger" of this toxin, different sequences of 20 amino acids linear peptides have been designed in order to avoid toxic effects but to maintain or even strengthen the partial antimicrobial activity already seen for the complete toxin. As a result, the sequence NCP-0 (Naja Cardiotoxin Peptide-0) was designed as ancestor and subsequently 4 other variant sequences of NCP-0 were developed. These synthesized variant sequences have shown microbicidal activity towards a panel of reference and field strains of Gram-positive and Gram-negative bacteria. The sequence named NCP-3, and its variants NCP-3a and NCP-3b, have shown the best antimicrobial activity, together with low cytotoxicity against eukaryotic cells and low hemolytic activity. Bactericidal activity has been demonstrated by minimum bactericidal concentration (MBC) assay at values below 10 µg/ml for most of the tested bacterial strains. This potent antimicrobial activity was confirmed even for unicellular fungi Candida albicans, Candida glabrata and Malassezia pachydermatis (MBC 50-6.3 µg/ml), and against the fast-growing mycobacteria Mycobacterium smegmatis and Mycobacterium fortuitum. Moreover, NCP-3 has shown virucidal activity on Bovine Herpesvirus 1 (BoHV1) belonging to Herpesviridae family. The bactericidal activity is maintained even in a high salt concentration medium (125 and 250 mM NaCl) and phosphate buffer with 20% Mueller Hinton (MH) medium against E. coli, methicillin resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa reference strains. Considering these in vitro obtained data, the search for active sequences within proteins presenting an intrinsic microbicidal activity could provide a new way for discovering a large number of novel and promising antimicrobial peptides families.

A killer mimotope with therapeutic activity against AIDS-related opportunistic micro-organisms inhibits ex-vivo HIV-1 replication.

OBJECTIVE: To verify whether a synthetic therapeutic killer decapeptide (KP), a functional mimotope of a yeast killer toxin with wide-spectrum microbicidal activity, inclusive of AIDS-related opportunistic micro-organisms, through interaction with beta-glucan receptors, which has been found to possess sequence homology with critical segments in gp160 V1/V2 and V3 loops, may also be inhibiting HIV-1 replication. METHODS: Primary peripheral blood mononuclear cells (PBMCs) cultures established from HIV-1-infected patients were treated with KP in comparison with zidovudine and supernatants and cells were harvested for analysis of HIV RNA and proviral contents, respectively. Virus production in exogenous in-vitro PBMCs infection with lymphocytotropic and monocytotropic HIV-1 strains was also assessed in presence of KP by enzyme-linked immunosorbent assay HIV p24 gag antigen detection. The binding affinity of KP to CD4, CCR5 and CXCR4 was evaluated on CD4-CCR5 or CD4-CXCR4 transfected astroglioma cell lines. RESULTS: KP was shown to be devoid of cytotoxicity on PBMCs and to inhibit HIV-1 replication in PBMCs of a patient in the acute phase of infection. The antiretroviral activity of KP, which proved to be more potent than zidovudine at micromolar concentrations, is abolished by beta 1,3-glucan but not by beta 1,6-glucan. Down-regulation of CCR5 co-receptor, and/or physical block of the gp120-receptor interaction are possible mechanisms of KP activity. CONCLUSION: KP appears to be the first antibody-derived short peptide displaying an inhibitory activity against HIV-1 and related opportunistic micro-organisms by different mechanisms of action.

Blue news: NTP binding properties of the blue-light sensitive YtvA protein from Bacillus subtilis.

The blue-light sensitive protein YtvA from Bacillus subtilis is built of a photoactive, flavin-binding LOV (Light, Oxygen and Voltage) domain and a STAS domain with unknown function. Here we show that YtvA binds a fluorescent derivative of guanosine triphosphate (GTPTR) that can be displaced by both GTP or ATP. Unspecific NTP (N=G or A) binding is supported by the molecular model of YtvA-STAS. Blue-light activation of YtvA results in small and dark-reversible spectroscopic changes for GTPTR, suggesting that light-driven conformational changes are transmitted from the LOV core to the GTPTR binding site. These results support the idea that STAS domains may have a general NTP binding role and open a way to investigate the molecular functionality of YtvA-STAS.

Quinoline-2-carboxaldehyde thiosemicarbazones and their Cu(II) and Ni(II) complexes as topoisomerase IIa inhibitors.

A series of quinoline-2-carboxaldehyde thiosemicarbazones and their copper(II) and nickel(II) complexes were synthesized and characterized. In all complexes the ligands are in the E configuration with respect to the imino bond and behave as terdentate. The copper(II) complexes form square planar derivatives with one molecule of terdentate ligand and chloride ion. A further non-coordinated chloride ion compensates the overall charge. Nickel(II) ions form instead octahedral complexes with two ligands for each metal ion, independently from the stoichiometric metal:ligand ratio used in the synthesis. Ligands and complexes were tested for their antiproliferative properties on histiocytic lymphoma cell line U937. Copper(II) derivatives are systematically more active than the ligands and the nickel complexes. All copper derivatives result in inhibiting topoisomerase IIa in vitro. Computational methods were used to propose a model to explain the different extent of inhibition presented by these compounds. The positive charge of the dissociated form of the copper complexes may play a key role in their action.