Influence of substrate/inoculum ratio, inoculum source and ammonia inhibition on anaerobic digestion of poultry waste.

Poultry wastes are rich in organic matter, allowing their use as substrates for biogas production by anaerobic digestion (AD). The major difficulty in the anaerobic digestion of this protein-rich waste is ammonia inhibition. Different results of biochemical methane potential (BMP) were obtained after the mesophilic anaerobic digestion of different avian waste in batch mode. It was shown that using two different inoculum (Liger and Saint-Brieuc) sources and different substrate-to-inoculum (S/I) ratios does not have a significant effect on the biochemical methane potential of organic laying hen droppings (OLHD); an average of 0.272 Nm3 CH4·kg-1·VS was obtained with both inocula. Otherwise, it affects the hydrolysis constant KH, and it decreases when the substrate-to-inoculum ratio increases. Furthermore, Liger is the most suitable inoculum for our substrate because it shows stability during the process even with different organic loads. Comparing the biochemical methane potential of multiple avian wastes such as organic laying hen droppings and different slaughterhouse waste highlights the importance of slaughterhouse waste in the anaerobic digestion process because of the high methane yield observed especially with the viscera (0.779 Nm3 CH4·kg-1 VS, SD = 0.027 Nm3 CH4·kg-1 VS). Moreover, methane production was affected by increasing the ammonia concentrations; when [N-NH3] > 9.8 g·N-NH3·L-1, the biochemical methane potential decreases and the lag phase increases (λ > 30 days); a total inhibition of the process was observed when ammonia concentration is above 21.8 g·L-1.

Multi-Step Biomass Fractionation of Grape Seeds from Pomace, a Zero-Waste Approach.

Grape seeds are the wineries' main by-products, and their disposal causes ecological and environmental problems. In this study seeds from the pomace waste of autochthonous grape varieties from Lebanon, Obeidi (white variety) and Asswad Karech (red variety) were used for a multi-step biomass fractionation. For the first step, a lipid extraction was performed, and the obtained yield was 12.33% (w/w) for Obeidi and 13.04% (w/w) for Asswad Karech. For the second step, polyphenols' recovery from the defatted seeds was carried out, resulting in 12.0% (w/w) for Obeidi and 6.6% (w/w) for Asswad Karech, with Obeidi's extract having the highest total phenolic content (333.1 ± 1.6 mg GAE/g dry matter) and antioxidant activity (662.17 ± 0.01 µg/mL of Trolox equivalent). In the third step, the defatted and dephenolized seeds were subsequently extracted under alkaline conditions and the proteins were isoelectric precipitated. The recovered protein extract was 3.90% (w/w) for Obeidi and 4.11% (w/w) for Asswad Karech seeds, with Asswad Karech's extract having the highest protein content (64 ± 0.2 mg protein/g dry matter). The remaining exhausted residue can be valorized in cosmetic scrubs formulations as a replacement for plastic microbeads. The designed zero-waste approach multi-step biomass fractionation has the potential to improve the valorization of the side products (grape seeds) of these two Lebanese autochthonous grape varieties.

Interfacial Water Many-Body Effects Drive Structural Dynamics and Allosteric Interactions in SARS-CoV-2 Main Protease Dimerization Interface.

Following our previous work ( Chem. Sci. 2021, 12, 4889-4907), we study the structural dynamics of the SARS-CoV-2 Main Protease dimerization interface (apo dimer) by means of microsecond adaptive sampling molecular dynamics simulations (50 µs) using the AMOEBA polarizable force field (PFF). This interface is structured by a complex H-bond network that is stable only at physiological pH. Structural correlations analysis between its residues and the catalytic site confirms the presence of a buried allosteric site. However, noticeable differences in allosteric connectivity are observed between PFFs and non-PFFs. Interfacial polarizable water molecules are shown to appear at the heart of this discrepancy because they are connected to the global interface H-bond network and able to adapt their dipole moment (and dynamics) to their diverse local physicochemical microenvironments. The water-interface many-body interactions appear to drive the interface volume fluctuations and to therefore mediate the allosteric interactions with the catalytic cavity.

Quantum-Chemistry Based Design of Halobenzene Derivatives With Augmented Affinities for the HIV-1 Viral G4/C16 Base-Pair.

The HIV-1 integrase (IN) is a major target for the design of novel anti-HIV inhibitors. Among these, three inhibitors which embody a halobenzene ring derivative (HR) in their structures are presently used in clinics. High-resolution X-ray crystallography of the complexes of the IN-viral DNA transient complex bound to each of the three inhibitors showed in all cases the HR ring to interact within a confined zone of the viral DNA, limited to the highly conserved 5'CpA 3'/5'TpG 3' step. The extension of its extracyclic CX bond is electron-depleted, owing to the existence of the "sigma-hole." It interacts favorably with the electron-rich rings of base G4. We have sought to increase the affinity of HR derivatives for the G4/C16 base pair. We thus designed thirteen novel derivatives and computed their Quantum Chemistry (QC) intermolecular interaction energies (ΔE) with this base-pair. Most compounds had ΔE values significantly more favorable than those of the HR of the most potent halobenzene drug presently used in clinics, Dolutegravir. This should enable the improvement in a modular piece-wise fashion, the affinities of halogenated inhibitors for viral DNA (vDNA). In view of large scale polarizable molecular dynamics simulations on the entirety of the IN-vDNA-inhibitor complexes, validations of the SIBFA polarizable method are also reported, in which the evolution of each ΔE(SIBFA) contribution is compared to its QC counterpart along this series of derivatives.

Reconciling NMR Structures of the HIV-1 Nucleocapsid Protein NCp7 Using Extensive Polarizable Force Field Free-Energy Simulations.

Using polarizable (AMOEBA) and nonpolarizable (CHARMM) force fields, we compare the relative free energy stability of two extreme conformations of the HIV-1 nucleocapsid protein NCp7 that had been previously experimentally advocated to prevail in solution. Using accelerated sampling techniques, we show that they differ in stability by no more than 0.75-1.9 kcal/mol depending on the reference protein sequence. While the extended form appears to be the most probable structure, both forms should thus coexist in water explaining the differing NMR findings.

Influence of pretreatment conditions on lignocellulosic fractions and methane production from grape pomace.

The lignocellulosic structure of grape pomace requires the use of pretreatments facilitating microbial decomposition of the matter and enhancing methane production. In this study, the effects of various pretreatments (freezing, alkaline treatment using NaOH and NH3, acid treatment using HCl, ultrasounds and pulsed electric fields) were examined in batch mode. The highest methane production (0.178Nm3kg-1 of COD) was attained after alkaline treatment with 10% NaOH w/w dry basis, at 20°C and for 24h. This result is due to the degradation of more than 50% of lignin and about 22% of cellulose present in grape pomace. The coupling of this pretreatment with freezing at -20°C exhibited the highest methane production of 0.2194±0.0007Nm3kg-1 of COD. When applied to a larger scale continuous digester, this coupled pretreatment increased methane production by about 27%, compared to the untreated samples, promoting the green valorization of the biomass.

Anaerobic digestion of grape pomace: Effect of the hydraulic retention time on process performance and fibers degradability.

To optimize the anaerobic digestion of grape pomace under mesophilic conditions, continuous digesters were operated at different hydraulic retention times (HRT) (30, 20, 15 and 10 days) equivalent to organic loading rates (OLR) of 2.5, 3.7, 5.7 and 7.3 kg COD m-3 d-1, respectively. At HRTs of 30 and 20 days, steady state conditions were observed with methane yields of 0.984 ±â€¯0.013 NL d-1 and 1.362 ±â€¯0.018 NL d-1, respectively. The HRT of 15 days was found critical because of acids accumulation through the experiments. When the OLR of 5.7 kg COD m-3 d-1 was reached, methane production was found to be instable. Finally, at HRT of 10 days, a failure of the system was observed due to the washing of the methanogenic microorganisms. Regarding the degradability of the lignocellulosic fractions, the maximum reduction yields for hemicellulose and cellulose were noted for HRTs of 30 and 20 days, while lignin was not degraded throughout the different experiments. For an optimization of the process, HRT of 20 days can therefore be recommended for productive use in large-scale applications.

Calibration of 1,2,4-Triazole-3-Thione, an Original Zn-Binding Group of Metallo-ß-Lactamase Inhibitors. Validation of a Polarizable MM/MD Potential by Quantum Chemistry.

In the context of the SIBFA polarizable molecular mechanics/dynamics (PMM/PMD) procedure, we report the calibration and a series of validation tests for the 1,2,4-triazole-3-thione (TZT) heterocycle. TZT acts as the chelating group of inhibitors of dizinc metallo-ß-lactamases (MBL), an emerging class of Zn-dependent bacterial enzymes, which by cleaving the ß-lactam bond of most ß-lactam antibiotics are responsible for the acquired resistance of bacteria to these drugs. Such a study is indispensable prior to performing PMD simulations of complexes of TZT-based inhibitors with MBL's, on account of the anchoring role of TZT in the dizinc MBL recognition site. Calibration was done by comparisons to energy decomposition analyses (EDA) of high-level ab initio QC computations of the TZT complexes with two probes: Zn(II), representative of "soft" dications, and water, representative of dipolar molecules. We performed distance variations of the approach of each probe to each of the two TZT atoms involved in Zn ligation, the S atom and the N atom ortho to it, so that each SIBFA contribution matches its QC counterpart. Validations were obtained by performing in- and out-of-plane angular variations of Zn(II) binding in monoligated Zn(II)-TZT complexes. The most demanding part of this study was then addressed. How well does ΔE(SIBFA) and its individual contributions compare to their QC counterparts in the dizinc binding site of one MBL, L1, whose structure is known from high-resolution X-ray crystallography? Six distinct complexes were considered, namely each separate monozinc site, and the dizinc site, whether ligated or unligated by TZT. Despite the large magnitude of the interaction energies, in all six complexes ΔE(SIBFA) can match ΔE(QC) with relative errors <2% and the proper balance of individual energy contributions. The computations were extended to the dizinc site of another MBL, VIM-2, and its complexes with two other TZT analogues. ΔE(SIBFA) faithfully reproduced ΔE(QC) in terms of magnitude, ranking of the three ligands, and trends of the separate energy contributions. A preliminary extension to correlated calculations is finally presented. All these validations should enable a secure design of a diversity of TZT-containing MBL inhibitors: a structurally and energetically correct anchoring of TZT should enable all other inhibitor groups to in turn optimize their interactions with the other target MBL residues.

The inhibition process of HIV-1 integrase by diketoacids molecules: Understanding the factors governing the better efficiency of dolutegravir.

The Human Immunodeficiency Virus-1 integrase is responsible for the covalent insertion of a newly synthesized double-stranded viral DNA into the host cells, and is an emerging target for antivirus drug design. Raltegravir (RAL) and elvitegravir (EVG) are the first two integrase strand transfer inhibitors used in therapy. However, treated patients eventually develop detrimental resistance mutations. By contrast, a recently approved drug, dolutegravir (DTG), presents a high barrier to resistance. This study aims to understand the increased efficiency of DTG upon focusing on its interaction properties with viral DNA. The results showed DTG to be involved in more extended interactions with viral DNA than EVG. Such interactions involve the halobenzene and scaffold of DTG and EVG and bases 5'G-43', 3'A35'and 3'C45'.

Importance of explicit smeared lone-pairs in anisotropic polarizable molecular mechanics. Torture track angular tests for exchange-repulsion and charge transfer contributions.

A correct representation of the short-range contributions such as exchange-repulsion (Erep ) and charge-transfer (Ect ) is essential for the soundness of separable, anisotropic polarizable molecular mechanics potentials. Within the context of the SIBFA procedure, this is aimed at by explicit representations of lone pairs in their expressions. It is necessary to account for their anisotropic behaviors upon performing not only in-plane, but also out-of-plane, variations of a probe molecule or cation interacting with a target molecule or molecular fragment. Thus, Erep and Ect have to reproduce satisfactorily the corresponding anisotropies of their quantum chemical (QC) counterparts. A significant improvement of the out-of-plane dependencies was enabled when the sp2 and sp localized lone-pairs are, even though to a limited extent, delocalized on both sides of the plane, above and below the atom bearer but at the closely similar angles as the in-plane lone pair. We report calibration and validation tests on a series of monoligated complexes of a probe Zn(II) cation with several biochemically relevant ligands. Validations are then performed on several polyligated Zn(II) complexes found in the recognition sites of Zn-metalloproteins. Such calibrations and validations are extended to representative monoligated and polyligated complexes of Mg(II) and Ca(II). It is emphasized that the calibration of all three cations was for each ΔE contribution done on a small training set bearing on a limited number of representative N, O, and S monoligated complexes. Owing to the separable nature of ΔE, a secure transferability is enabled to a diversity of polyligated complexes. For these the relative errors with respect to the target ΔE(QC) values are generally < 3%. Overall, the article proposes a full set of benchmarks that could be useful for force field developers. © 2017 Wiley Periodicals, Inc.

A Comparative Study of the Phenolic and Technological Maturities of Red Grapes Grown in Lebanon.

Grape harvest date is determined according to the technological and phenolic maturities. These parameters were calculated for different red grape (Vitis vinifera L.) varieties (Cabernet Sauvignon, Merlot, Syrah, Cabernet Franc) over four years (2008, 2009, 2010, and 2011) (642 samples). Titratable acidity and sugar content of the grapes were used to determine the technological maturity, whereas Glories (1 and 2) and ITV (Institut Technique de la Vigne et du Vin) methods were used to monitor their phenolic maturity. The ITV method allows the monitoring of phenolic maturity by the quantification of total polyphenol index and anthocyanins, while the Glories method enables the quantitative evolution of extractable anthocyanins and tannins of the grapes. A correlation was shown between the harvest dates obtained by both ITV and Glories (R2 = 0.7 - 0.93). Phenolic maturity of grapes can, therefore, be optimized by the application of both ITV and Glories. Similarly, a correlation was observed between technological and phenolic harvest dates. The effect of climate on the phenolic content of grapes was also studied. The highest temperatures(up to 25◦C)accompanied by the lowest rainfall (null value), induced the maximal concentration of polyphenols in grapes. Thermal and water stresses were also shown to enhance the grapes' polyphenolic production.

A targeted DNA substrate mechanism for the inhibition of HIV-1 integrase by inhibitors with antiretroviral activity.

We recently reported that viral DNA could be the primary target of raltegravir (RAL), an efficient anti-HIV-1 drug, which acts by inhibiting integrase. To elucidate this mechanism, we conducted a comparative analysis of RAL and TB11, a diketoacid abandoned as an anti-HIV-1 drug for its weak efficiency and marked toxicity, and tested the effects of the catalytic cofactor Mg(2+) (5 mm) on drug-binding properties. We used circular dichroism and fluorescence to determine drug affinities for viral DNA long terminal repeats (LTRs) and peptides derived from the integrase active site and DNA retardation assays to assess drug intercalation into DNA base pairs. We found that RAL bound more tightly to LTR ends than did TB11 (a diketo acid bearing an azido group) and that Mg(2+) significantly increased the affinity of both RAL and TB11. We also observed a good relationship between drug binding with processed LTR and strand transfer inhibition. This unusual type of inhibition was caused by Mg(2+)-assisted binding of drugs to DNA substrate, rather than to enzyme. Notably, while RAL bound exclusively to the cleavable/cleaved site, TB11 further intercalated into DNA base pairs and interacted with the integrase-derived peptides. These unwanted binding sites explain the weaker bioavailability and higher toxicity of TB11 compared with the more effective RAL.

Anaerobic digestion of grape pomace: Biochemical characterization of the fractions and methane production in batch and continuous digesters.

In this study, we have estimated the biogas and methane production from grape pomace (variety Cabernet Franc). The physical and chemical characteristics of the raw material were determined, and the structural polysaccharides were identified and analyzed by the Van Soest method. Batch anaerobic digestions were carried out to assess the methane production of the grape pomace, pulp and seeds. The obtained cumulative methane productions are 0.125, 0.165 and 0.052 Nm(3) kg COD(-1) for grape pomace, pulps and seeds, respectively. The effect of grinding on the methane potential of the substrates, as a mechanical pretreatment, was evaluated. We found that it increased the anaerobic biodegradability for grape pomace, pulp and seeds by 13.1%, 4.8% and 22.2%, respectively. On the other hand, the methane potential of the grape pomace was determined in a laboratory pilot plant (12L) continuously mixed with an organic loading rate of 2.5 kg COD m(3) d(-1) and a hydraulic retention time of 30 days. The corresponding biogas production was 6.43 × 10(-3) Nm(3) d(-1), with a methane content of 62.3%. Thus, the pilot plant's efficiency compared to that achieved in the batch process was 81.2%. Finally, a significant correlation was found between the biochemical content and methane production.

Could the "Janus-like" properties of the halobenzene CX bond (X=Cl, Br) be leveraged to enhance molecular recognition?

The CX bond in halobenzenes (XCl, Br) exhibits a dual character, being electron-deficient along the CX direction, and electron-rich on its flanks. We sought to amplify both features by resorting to electron-withdrawing and electron-donating substituents, respectively. This was done by quantum chemistry (QC) computations in the recognition sites of three protein targets: farnesyl transferase, coagulation factor Xa, and the HIV-1 integrase. In this context, some substituents, notably fluorine, CF3 , and NHCH3 , afforded significant overall gains in the binding energies as compared to the parent halobenzene, in the 2-5 kcal/mol range. In fact, we found that some di- and up to tetra-substitutions enabled even larger gains than those they contribute separately owing to many-body effects. Moreover, desolvation was also found to be a key contributor to the energy balances. As a consequence, some particular substituents, contributing to reduce the halobenzene dipole moment, accordingly reduced solvation: this factor acted in synergy with their enhancement of the intermolecular interaction energies along and around the CX bond. We could thus leverage the "Janus-like" properties of such a bond and the fact that it can be tuned and possibly amplified by well-chosen substituents. We propose a simple yet rigorous computational strategy resorting to QC to prescreen novel substituted halobenzenes. The QC results on the recognition sites then set benchmarks to validate polarizable molecular mechanics/dynamics approaches used to handle the entirety of the inhibitor-protein complex.

Effect of the drying process on the intensification of phenolic compounds recovery from grape pomace using accelerated solvent extraction.

In light of their environmental and economic interests, food byproducts have been increasingly exploited and valorized for their richness in dietary fibers and antioxidants. Phenolic compounds are antioxidant bioactive molecules highly present in grape byproducts. Herein, the accelerated solvent extraction (ASE) of phenolic compounds from wet and dried grape pomace, at 45 °C, was conducted and the highest phenolic compounds yield (PCY) for wet (16.2 g GAE/100 g DM) and dry (7.28 g GAE/100 g DM) grape pomace extracts were obtained with 70% ethanol/water solvent at 140 °C. The PCY obtained from wet pomace was up to two times better compared to the dry byproduct and up to 15 times better compared to the same food matrices treated with conventional methods. With regard to Resveratrol, the corresponding dry pomace extract had a better free radical scavenging activity (49.12%) than the wet extract (39.8%). The drying pretreatment process seems to ameliorate the antiradical activity, especially when the extraction by ASE is performed at temperatures above 100 °C. HPLC-DAD analysis showed that the diversity of the flavonoid and the non-flavonoid compounds found in the extracts was seriously affected by the extraction temperature and the pretreatment of the raw material. This diversity seems to play a key role in the scavenging activity demonstrated by the extracts. Our results emphasize on ASE usage as a promising method for the preparation of highly concentrated and bioactive phenolic extracts that could be used in several industrial applications.

Substituent-modulated affinities of halobenzene derivatives to the HIV-1 integrase recognition site. Analyses of the interaction energies by parallel quantum chemical and polarizable molecular mechanics.

The C-X bond of halobenzenes (X = Cl, Br) has a dual character, its electron density being depleted in its prolongation and built-up on its sides. We have recently considered three protein or nucleic acid recognition sites of halobenzenes and quantified the energy gains that either electron-attracting substituents or electron-donating ones contribute due to such a character (El Hage et al., paper in revision). Nonadditivity was found to impact the total interaction energies. We focus here on one recognition site, that of the HIV-1 integrase, in which the halobenzene ring of the drug elvitegravir is sandwiched between a guanine and a cytosine base. We perform energy-decomposition analyses of the ab initio quantum-chemistry (QC) binding energies of the parent halobenzene ring and its derivatives with this G-C base pair. In these complexes, the nonadditivity of ΔE could be traced back mostly to the polarization contribution Epol. In view of large-scale applications to the entirety of the complex formed between the integrase, the viral DNA, and the whole drug, the analyses were performed in parallel with a polarizable molecular mechanics method, SIBFA. This method could faithfully reproduce most features of the QC energies. This is due to its use of QC-derived distributed multipoles and polarizabilities, which enable us to account for both nonisotropy and nonadditivity.

Could an anisotropic molecular mechanics/dynamics potential account for sigma hole effects in the complexes of halogenated compounds?

Halogenated compounds are gaining an increasing importance in medicinal chemistry and materials science. Ab initio quantum chemistry (QC) has unraveled the existence of a "sigma hole" along the C-X (X = F, Cl, Br, I) bond, namely, a depletion of electronic density prolonging the bond, concomitant with a build-up on its sides, both of which are enhanced along the F < Cl < Br < I series. We have evaluated whether these features were intrinsically built-in in an anisotropic, polarizable molecular mechanics (APMM) procedure such as SIBFA (sum of interactions between fragments ab initio computed). For that purpose, we have computed the interaction energies of fluoro-, chloro-, and bromobenzene with two probes: a divalent cation, Mg(II), and water approaching X through either one H or its O atom. This was done by parallel QC energy-decomposition analyses (EDA) and SIBFA computations. With both probes, the leading QC contribution responsible for the existence of the sigma hole is the Coulomb contribution E(c). For all three halogenated compounds, and with both probes, the in- and out-of-plane angular features of E(c) were closely mirrored by the SIBFA electrostatic multipolar contribution (E(MTP)). Resorting to such a contribution thus dispenses with empirically-fitted "extra", off-centered partial atomic charges as in classical molecular mechanics/dynamics.

Unprocessed viral DNA could be the primary target of the HIV-1 integrase inhibitor raltegravir.

Integration of HIV DNA into host chromosome requires a 3'-processing (3'-P) and a strand transfer (ST) reactions catalyzed by virus integrase (IN). Raltegravir (RAL), commonly used in AIDS therapy, belongs to the family of IN ST inhibitors (INSTIs) acting on IN-viral DNA complexes (intasomes). However, studies show that RAL fails to bind IN alone, but nothing has been reported on the behaviour of RAL toward free viral DNA. Here, we assessed whether free viral DNA could be a primary target for RAL, assuming that the DNA molecule is a receptor for a huge number of pharmacological agents. Optical spectroscopy, molecular dynamics and free energy calculations, showed that RAL is a tight binder of both processed and unprocessed LTR (long terminal repeat) ends. Complex formation involved mainly van der Waals forces and was enthalpy driven. Dissociation constants (Kds) revealed that RAL affinity for unbound LTRs was stronger than for bound LTRs. Moreover, Kd value for binding of RAL to LTRs and IC50 value (half concentration for inhibition) were in same range, suggesting that RAL binding to DNA and ST inhibition are correlated events. Accommodation of RAL into terminal base-pairs of unprocessed LTR is facilitated by an extensive end fraying that lowers the RAL binding energy barrier. The RAL binding entails a weak damping of fraying and correlatively of 3'-P inhibition. Noteworthy, present calculated RAL structures bound to free viral DNA resemble those found in RAL-intasome crystals, especially concerning the contacts between the fluorobenzyl group and the conserved 5'C(4)pA(3)3' step. We propose that RAL inhibits IN, in binding first unprocessed DNA. Similarly to anticancer drug poisons acting on topoisomerases, its interaction with DNA does not alter the cut, but blocks the subsequent joining reaction. We also speculate that INSTIs having viral DNA rather IN as main target could induce less resistance.

Specificity of LTR DNA recognition by a peptide mimicking the HIV-1 integrase {alpha}4 helix.

HIV-1 integrase integrates retroviral DNA through 3'-processing and strand transfer reactions in the presence of a divalent cation (Mg(2+) or Mn(2+)). The alpha4 helix exposed at the catalytic core surface is essential to the specific recognition of viral DNA. To define group determinants of recognition, we used a model composed of a peptide analogue of the alpha4 helix, oligonucleotides mimicking processed and unprocessed U5 LTR end and 5 mM Mg(2+). Circular dichroism, fluorescence and NMR experiments confirmed the implication of the alpha4 helix polar/charged face in specific and non-specific bindings to LTR ends. The specific binding requires unprocessed LTR ends-i.e. an unaltered 3'-processing site CA downward arrowGT3'-and is reinforced by Mg(2+) (K(d) decreases from 2 to 0.8 nM). The latter likely interacts with the ApG and GpT3' steps of the 3'-processing site. With deletion of GT3', only persists non-specific binding (K(d) of 100 microM). Proton chemical shift deviations showed that specific binding need conserved amino acids in the alpha4 helix and conserved nucleotide bases and backbone groups at LTR ends. We suggest a conserved recognition mechanism based on both direct and indirect readout and which is subject to evolutionary pressure.

An unusual helix turn helix motif in the catalytic core of HIV-1 integrase binds viral DNA and LEDGF.

BACKGROUND: Integrase (IN) of the type 1 human immunodeficiency virus (HIV-1) catalyzes the integration of viral DNA into host cellular DNA. We identified a bi-helix motif (residues 149-186) in the crystal structure of the catalytic core (CC) of the IN-Phe185Lys variant that consists of the alpha(4) and alpha(5) helices connected by a 3 to 5-residue turn. The motif is embedded in a large array of interactions that stabilize the monomer and the dimer. PRINCIPAL FINDINGS: We describe the conformational and binding properties of the corresponding synthetic peptide. This displays features of the protein motif structure thanks to the mutual intramolecular interactions of the alpha(4) and alpha(5) helices that maintain the fold. The main properties are the binding to: 1- the processing-attachment site at the LTR (long terminal repeat) ends of virus DNA with a K(d) (dissociation constant) in the sub-micromolar range; 2- the whole IN enzyme; and 3- the IN binding domain (IBD) but not the IBD-Asp366Asn variant of LEDGF (lens epidermal derived growth factor) lacking the essential Asp366 residue. In our motif, in contrast to the conventional HTH (helix-turn-helix), it is the N terminal helix (alpha(4)) which has the role of DNA recognition helix, while the C terminal helix (alpha(5)) would rather contribute to the motif stabilization by interactions with the alpha(4) helix. CONCLUSION: The motif, termed HTHi (i, for inverted) emerges as a central piece of the IN structure and function. It could therefore represent an attractive target in the search for inhibitors working at the DNA-IN, IN-IN and IN-LEDGF interfaces.