Absolute binding free energy calculations: On the accuracy of computational scoring of protein–ligand interactions

Calculating the absolute binding free energies is a challenging task. Reliable estimates of binding free energies should provide a guide for rational drug design. It should also provide us with deeper understanding of the correlation between protein structure and its function. Further applications may include identifying novel molecular scaffolds and optimizing lead compounds in computer‐aided drug design. Available options to evaluate the absolute binding free energies range from the rigorous but expensive free energy perturbation to the microscopic linear response approximation (LRA/β version) and related approaches including the linear interaction energy (LIE) to the more approximated and considerably faster scaled protein dipoles Langevin dipoles (PDLD/S‐LRA version) as well as the less rigorous molecular mechanics Poisson–Boltzmann/surface area (MM/PBSA) and generalized born/surface area (MM/GBSA) to the less accurate scoring functions. There is a need for an assessment of the performance of different approaches in terms of computer time and reliability. We present a comparative study of the LRA/β, the LIE, the PDLD/S‐LRA/β, and the more widely used MM/PBSA and assess their abilities to estimate the absolute binding energies. The LRA and LIE methods perform reasonably well but require specialized parameterization for the nonelectrostatic term. The PDLD/S‐LRA/β performs effectively without the need of reparameterization. Our assessment of the MM/PBSA is less optimistic. This approach appears to provide erroneous estimates of the absolute binding energies because of its incorrect entropies and the problematic treatment of electrostatic energies. Overall, the PDLD/S‐LRA/β appears to offer an appealing option for the final stages of massive screening approaches. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Filamin A Binds to CCR2B and Regulates Its Internalization

The chemokine (C-C motif) receptor 2B (CCR2B) is one of the two isoforms of the receptor for monocyte chemoattractant protein-1 (CCL2), the major chemoattractant for monocytes, involved in an array of chronic inflammatory diseases. Employing the yeast two-hybrid system, we identified the actin-binding protein filamin A (FLNa) as a protein that associates with the carboxyl-terminal tail of CCR2B. Co-immunoprecipitation experiments and in vitro pull down assays demonstrated that FLNa binds constitutively to CCR2B. The colocalization of endogenous CCR2B and filamin A was detected at the surface and in internalized vesicles of THP-1 cells. In addition, CCR2B and FLNa were colocalized in lamellipodia structures of CCR2B-expressing A7 cells. Expression of the receptor in filamin-deficient M2 cells together with siRNA experiments knocking down FLNa in HEK293 cells, demonstrated that lack of FLNa delays the internalization of the receptor. Furthermore, depletion of FLNa in THP-1 monocytes by RNA interference reduced the migration of cells in response to MCP-1. Therefore, FLNa emerges as an important protein for controlling the internalization and spatial localization of the CCR2B receptor in different dynamic membrane structures.     

On the origin of the electrostatic barrier for proton transport in aquaporin

The nature of the electrostatic barrier for proton transport in aquaporins is analyzed by semimacroscopic and microscopic models. It is found that the barrier is associated with the loss of the generalized solvation energy upon moving from the bulk solvent to the center of the channel. It is clarified that our solvation concept includes the effect of the protein polar groups and ionized residues. The nature of the contributions to the solvation barrier is examined by using the linear response approximation. It is found that the residues in the NPA region contribute much less than what would be deduced from calculations that do not consider the protein reorganization. It is clarified that the contributions of different structural or electrostatic elements to the solvation barrier can be established by removing these elements and examining the corresponding effect on the barrier height. Using this definition and “mutating” the NPA residues to their non-polar analogues establishes that these residues do not provide the major contribution to the solvation barrier.     

Synergistic action of growth hormone and insulin-like growth factor I (IGF-I) on proliferation and estradiol secretion in porcine granulosa and theca cells cultured alone or in coculture

The effect of growth hormone (GH) on insulin-like growth factor I (IGF-I) secretion and the effects of GH and IGF-I on [(3)H] thymidine incorporation and estradiol (E2) secretion by theca interna (Tc) and granulosa cells (Gc) cultured alone and in coculture were studied in cultured porcine follicular cells, prepared from small (SF), medium (MF) and large (LF) preovulatory follicles. We demonstrated that both Tc and Gc secrete IGF-I and that GH had no effect on IGF-I secretion by Tc but, increased IGF-I secretion by Gc isolated from SF and cultured alone or in coculture. IGF-I stimulated secretion of E2 by all cells, except in Tc derived from SF in which the effect was not statistically significant. The only stimulatory effect of concurrent treatment with GH on E2 secretion was noted in Tc derived from MF. IGF-I increased the [(3)H] thymidine incorporation in all Tc cells but GH did not augment this effect. In Gc, IGF-I stimulated [(3)H] thymidine incorporation in cells derived from SF and MF but not from LF. GH had no stimulatory effect except on Gc derived from LF and grown alone. The highest stimulatory effect was observed in SF. This was smaller in MF and no effect was noted in LF. In conclusion, our work shows that both Gc and Tc are sites of IGF-I production and for the first time shows the stimulatory role of IGF-I in proliferation of Tc cells derived from all types of follicles and augmentation of E2 secretion in Tc derived from MF and LF. The promotion of the mitogenic activity in Tc by IGF-I during all stages of follicular development suggests an important role for theca cells in follicular growth.     

Enduring toxicity of transgenic Anabaena PCC 7120 expressing mosquito larvicidal genes from Bacillus thuringiensis ssp. israelensis

Persistence of biological control agents against mosquito larvae was tested under simulated field conditions. Mosquito larvicidal activity of transgenic Anabaena PCC 7120 expressing cry4Aa, cry11Aa and p20 from Bacillus thuringiensis ssp. israelensis was greater than B. thuringiensis ssp. israelensis primary powder (fun 89C06D) or wettable powder (WP) (Bactimos products) when either mixed with silt or exposed to sunlight outdoors. Reduction of Bactimos primary powder toxicity was at least 10-fold higher than Anabaena's after mixing with silt. In outdoors experiments, Bactimos WP remained toxic (over 30% mortality of 3rd instar Aedes aegypti larvae) for 2-4 days only, while transgenic Anabaena's toxicity endured 8-21 days.     

Complete sequence and organization of pBtoxis,the toxin-coding plasmid of Bacillus thuringiensis subsp. israelensis

The entire 127,923-bp sequence of the toxin-encoding plasmid pBtoxis from Bacillus thuringiensis subsp. israelensis is presented and analyzed. In addition to the four known Cry and two known Cyt toxins, a third Cyt-type sequence was found with an additional C-terminal domain previously unseen in such proteins. Many plasmid-encoded genes could be involved in several functions other than toxin production. The most striking of these are several genes potentially affecting host sporulation and germination and a set of genes for the production and export of a peptide antibiotic.     

Simulating redox coupled proton transfer in cytochrome c oxidase: looking for the proton bottleneck

Gaining a detailed understanding of the molecular nature of the redox coupled proton transfer in cytochrome c oxidase (COX) is one of the challenges of modern biophysics. The present work addresses this by integrating approaches for simulations of proton transport (PTR) and electron transfer (ET). The resulting method converts the electrostatic energies of different charge configurations and reorganization energies to free-energy profiles for different PTR and ET pathways. This approach provides for the first time a tool to study the actual activation barriers and kinetics of different feasible PTR processes in the cycle of COX. Using this tool, we explore the PTR through the bottleneck water molecules. It is found that a stepwise PTR along this commonly assumed path leads to far too high barriers and is, thus, inconsistent with the observed kinetics. Furthermore, the simulated free-energy profile does not provide a simple gating mechanism. Fortunately, we obtain reasonable kinetics when we consider a PTR that involves a concerted transfer of protons to and from E286. Finally, semi-qualitative considerations of the forward and backward barriers point toward open questions about the actual gating process and offer a feasible pumping mechanism. Although further studies are clearly needed, we believe that our approach offers a general and effective tool for correlating the structure of COX with its function.     

Comparative Sensitivity to UV-B Radiation of Two Bacillus thuringiensis Subspecies and Other Bacillus sp.

Susceptibility of Bacillus thuringiensis spores and toxins to the UV-B range (280–330 nm) of the solar spectrum reaching Earth's surface may be responsible for its inactivation and low persistence in nature. Spores of the mosquito larvicidal B. thuringiensis subsp. israelensis were significantly more resistant to UV-B than spores of the lepidopteran-active subsp. kurstaki. Spores of subsp. israelensis were as resistant to UV-B as spores of B. subtilis and more resistant than spores of the closely related B. cereus and another mosquito larvicidal species B. sphaericus. Sensitivity of B. thuringiensis subsp. israelensis spores to UV-B radiation depended upon their culture age; 24-h cultures, approaching maximal larvicidal activity, were still sensitive. Maximal resistance to UV-B was achieved only at 48 h. Received: 13 December 2000/Accepted: 19 January 2001