Ab initio molecular dynamics-based assignment of the protonation state of pepstatin A/HIV-1 protease cleavage site

A recent 13C NMR experiment (Smith et al. Nature Struct. Biol. 1996, 3, 946-950) on the Asp 25-Asp25' dyad in pepstatin A/HIV-1 protease measured two separate resonance lines, which were interpreted as being a singly protonated dyad. We address this issue by performing ab initio molecular dynamics calculations on models for this site accompanied by calculations of 13C NMR chemical shifts and isotopic shifts. We find that already on the picosecond time-scale the model proposed by Smith et al. is not stable and evolves toward a different monoprotonated form whose NMR pattern differs from the experimental one. We suggest, instead, a different protonation state in which both aspartic groups are protonated. Despite the symmetric protonation state, the calculated 13C NMR properties are in good agreement with the experiment. We rationalize this result using a simple valence bond model, which explains the chemical inequality of the two C sites. The model calculations, together with our calculations on the complex, allow also the rationalization of 13C NMR properties on other HIV-1 PR/inhibitor complexes. Both putative binding of the substrate to the free enzyme, which has the dyad singly protonated (Piana, S.; Carloni, P. Proteins: Struct., Funct., Genet. 2000, 39, 26-36), and pepstatin A binding to the diprotonated form are consistent with the inverse solvent isotope effect on the onset of inhibition of pepsin by pepstatin and the kinetic iso-mechanism proposed for aspartic proteases (Cho, T.-K.; Rebholz, K.; Northrop, D.B. Biochemistry 1994, 33, 9637-9642).     

Evolutionarily conserved functional mechanics across pepsin-like and retroviral aspartic proteases

The biological function of the aspartic protease from HIV-1 has recently been related to the conformational flexibility of its structural scaffold. Here, we use a multistep strategy to investigate whether the same mechanism affects the functionality in the pepsin-like fold. (i) We identify the set of conserved residues by using sequence-alignment techniques. These residues cluster in three distinct regions: near the cleavage-site cavity, in the four beta-sheets cross-linking the two lobes, and in a solvent-exposed region below the long beta-hairpin in the N-terminal lobe. (ii) We elucidate the role played by the conserved residues for the enzymatic functionality of one representative member of the fold family, the human beta-secretase, by means of classical molecular dynamics (MD). The conserved regions exhibit little overall mobility and yet are involved into the most important modes of structural fluctuations. These modes influence the substrate-catalytic aspartates distance through a relative rotation of the N- and C-terminal lobes. (iii) We investigate the effects of this modulation by estimating the reaction free energy at different representative substrate/enzyme conformations. The activation free energy is strongly affected by large-scale protein motions, similarly to what has been observed in the HIV-1 enzyme. (iv) We extend our findings to all other members of the two eukaryotic and retroviral fold families by recurring to a simple, topology-based, energy functional. This analysis reveals a sophisticated mechanism of enzymatic activity modulation common to all aspartic proteases. We suggest that aspartic proteases have been evolutionarily selected to possess similar functional motions despite the observed fold variations.     

Role and Perspective of Molecular Simulation-Based Investigation of RNA–Ligand Interaction: From Small Molecules and Peptides to Photoswitchable RNA Binding

Aberrant RNA–protein complexes are formed in a variety of diseases. Identifying the ligands that interfere with their formation is a valuable therapeutic strategy. Molecular simulation, validated against experimental data, has recently emerged as a powerful tool to predict both the pose and energetics of such ligands. Thus, the use of molecular simulation may provide insight into aberrant molecular interactions in diseases and, from a drug design perspective, may allow for the employment of less wet lab resources than traditional in vitro compound screening approaches. With regard to basic research questions, molecular simulation can support the understanding of the exact molecular interaction and binding mode. Here, we focus on examples targeting RNA–protein complexes in neurodegenerative diseases and viral infections. These examples illustrate that the strategy is rather general and could be applied to different pharmacologically relevant approaches. We close this study by outlining one of these approaches, namely the light-controllable association of small molecules with RNA, as an emerging approach in RNA-targeting therapy.     

Radiative events as a probe of dark forces at GeV-scale <Emphasis Type="Italic">e</Emphasis><Superscript>+</Superscript><Emphasis Type="Italic">e</Emphasis><Superscript>−</Superscript> colliders

High-luminosity e ⁺ e ⁻ colliders at the GeV scale (flavor factories) have been recently recognized to be an ideal environment to search for a light weakly coupled vector boson U (dark photon) emerging in several new physics models. At flavor factories a particularly clean channel is the production of the U boson in association with a photon, followed by the decay of the U boson into lepton pairs. Beyond the approximations addressed in previous works, we perform an exact lowest order calculation of the signal and background processes of this channel. We also include the effect of initial- and final-state QED corrections neglected so far, to show how they affect the distributions of experimental interest. We present new results for the expected statistical significance to a dark photon signal at KLOE/KLOE-2 and future super-B factories. The calculation is implemented in a new release of the event generator BabaYaga@NLO, which is available for full event simulations and data analysis.     

Structure and Dynamics of Oligonucleotides in the Gas Phase

By combining ion‐mobility mass spectrometry experiments with sub‐millisecond classical and ab initio molecular dynamics we fully characterized, for the first time, the dynamic ensemble of a model nucleic acid in the gas phase under electrospray ionization conditions. The studied oligonucleotide unfolds upon vaporization, loses memory of the solution structure, and explores true gas‐phase conformational space. Contrary to our original expectations, the oligonucleotide shows very rich dynamics in three different timescales (multi‐picosecond, nanosecond, and sub‐millisecond). The shorter timescale dynamics has a quantum mechanical nature and leads to changes in the covalent structure, whereas the other two are of classical origin. Overall, this study suggests that a re‐evaluation on our view of the physics of nucleic acids upon vaporization is needed.     

Status and accuracy of the Monte Carlo generators for luminosity measurements

The status and accuracy of the precision Monte Carlo generators used for luminosity measurements at flavour factories is reviewed.It is shown that,thanks to a considerable,long-term effort in tuned comparisons between the predictions of independent programs,as well as in the validation of the generators against the presently available calculations of the next-to-next-to-leading order QED corrections to Bhabha scattering,the theoretical accuracy reached by the most precise tools is of about one per mille.This error estimate is valid for realistic experimental cuts,appears to be quite robust and is already sufficient for very accurate luminosity measurements.However,recent progress and possible advances to further improve it are also discussed.     

Binding of novel azole-bridged dinuclear platinum(II) anticancer drugs to DNA: insights from hybrid QM/MM molecular dynamics simulations

Dinuclear Pt-containing compounds might be used to overcome the intrinsic and acquired cell resistance of widely used anticancer drugs such as cisplatin. Recently, the complexes [[cis-Pt(NH3)2]2(mu-OH)(mu-pz)](NO3)2 (with pz = pyrazolate) (1), [[cis-Pt(NH3)2]2(mu-OH)(mu-1,2,3-ta-N(1),N(2))](NO3)2 (with ta = 1,2,3-triazolate) (2), and the binding of 1 to d(CpTpCpTpG*pG*pTpCpTpCp) have been characterized. Here we provide the structural and electronic properties of the free drugs, of the intermediates of binding to guanine bases, and of the products, by performing DFT calculations. Our results show that in 2 an isomerization of the Pt-coordination sphere from N(2) to N(3) of the triazolate unit determines a thermodynamic stabilization of approximately 20 kcal/mol as a consequence of the formation of an allylic structure. In addition, hybrid quantum-classical molecular dynamics simulations of 1 and 2 DNA adducts have shed light on the structural distortions that the drugs induce to the DNA duplex. Our calculations show that the rise and the tilt of the two adjacent guanines are identical in the presence of 1 and 2, but they markedly increase when 2 binds in the N(1),N(3) fashion. In addition, the drugs do not provoke any kink upon binding to the double-stranded DNA, suggesting that they may act with a mechanism different than that of cisplatin. The accuracy of our calculations is established by a comparison with the NMR data for the corresponding complex with 1.     

Structure and Raman spectrum of clavulanic acid in aqueous solution

The calculation of the vibrational Raman spectrum of enzyme-bound beta-lactamase inhibitors may be of help to understand the mechanisms responsible for bacterial drug resistance. Here, we present a study of the solvation structure and the vibrational properties of clavulanate, an important beta-lactamase inhibitor, in aqueous solution as obtained from full quantum and hybrid empirical/quantum molecular dynamics simulations at ambient conditions. The analysis of the vibrational density of states indicates that hybrid empirical/quantum mechanical simulations are able to properly describe the vibrational levels of clavulanate in solution. In addition, we propose a computationally efficient protocol to calculate the vibrational Raman effect for large solute molecules in water, which is able to faithfully reproduce the experimentally recorded clavulanate Raman spectrum and discloses the possibility to employ hybrid simulations to assign the experimental Raman spectra of inhibitors bound to beta-lactamases.