Different microscopic and semimicroscopic approaches for calculations of electrostatic energies in macromolecules are examined. This includes the Protein Dipoles Langevin Dipoles (PDLD) method, the semimicroscopic PDLD (PDLD/S) method, and a free energy perturbation (FEP) method. The incorporation of these approaches in the POLARIS and ENZYME modules of the MOLARIS package is described in detail. The PDLD electrostatic calculations are augmented by estimates of the relevant hydrophobic and steric contributions, as well as the effects of the ionic strength and external pH. Determination of the hydrophobic energy involves an approach that considers the modification of the effective surface area of the solute by local field effects. The steric contributions are analyzed in terms of the corresponding reorganization energies. Ionic strength effects are studied by modeling the ionic environment around the given system using a grid of residual charges and evaluating the relevant interaction using Coulomb's law with the dielectric constant of water. The performance of the FEP calculations is significantly enhanced by using special boundary conditions and evaluating the long-range electrostatic contributions using the Local Reaction Field (LRF) model. A diverse set of electrostatic effects are examined, including the solvation energies of charges in proteins and solutions, energetics of ion pairs in proteins and solutions, interaction between surface charges in proteins, and effect of ionic strength on such interactions, as well as electrostatic contributions to binding and catalysis in solvated proteins. Encouraging results are obtained by the microscopic and semimicroscopic approaches and the problems associated with some macroscopic models are illustrated. The PDLD and PDLD/S methods appear to be much faster than the FEP approach and still give reasonable results. In particular, the speed and simplicity of the PDLD/S method make it an effective strategy for calculations of electrostatic free energies in interactive docking studies. Nevertheless, comparing the results of the three approaches can provide a useful estimate of the accuracy of the calculated energies. 0 1993 by John Wiley & Sons, Inc.

One of the major problems in molecular-dynamics simulations of polar fluids or macromolecular systems is the evaluation of electrostatic interactions. A system of N atoms demands an amount of work proportional to N2 for such calculations. Truncation procedures that neglect a significant part of the long-range effects are often necessary for computational feasibility, though such procedures may introduce serious errors in the simulations. This work introduces a simple and very effective approach for treating the long-range electrostatic forces. The present method, which is referred to as the local reaction field method, follows some of the ideas of the previously developed generalized Ewald method but then develops into a much simpler method. This is done by dividing the system into M groups of atoms and evaluating separately the short- and long-range contributions to the potential of each group. The short-range potential is evaluated explicitly as in any standard truncation method, while the long-range potential is approximated by the first four terms in a multipole expansion. In addition, the long-range potential is updated only once in every L time steps leading to a method of the order of N×M×q/L+N×P where q is related to the number of expansion terms and P is the average number of atoms inside the cutoff range. The speed, accuracy, and precision of the present method is assessed by evaluating the self-energy of a sodium ion in water and the self-energies of the acidic residues of bovine pancreatic trypsin inhibitor using an adiabatic charging free-energy perturbation approach. It is found that the present method reproduces accurately the corresponding results obtained without any cutoff but an order of magnitude faster. Furthermore, at the limit of very large systems, the speed of this method can be 2 orders of magnitude faster than that of the no-cutoff method even when each group contains only a small number of atoms. It is also found that the method gives much better results for electrostatic energies in proteins than those obtained by truncation methods. The stability and speed of the local reaction field method provides a powerful tool for the microscopic evaluation of electrostatic energies in macromolecules.

The study of antibody-antigen interactions should greatly benefit from the development of quantitative models for the evaluation of binding free energies in proteins. The present work addresses this challenge by considering the test case of the binding free energies of phosphorylcholine analogs to the murine myeloma protein McPC603. This includes the evaluation of the differential binding energy as well as the absolute binding energies and their corresponding electrostatic contributions. Four different approaches are examined: the Protein Dipoles Langevin Dipoles (PDLD) method, the semi-microscopic PDLD (PDLD/S) method, a free energy perturbation (FEP) method based on an adiabatic charging procedure and a linear response approximation that accelerates the FEP calculation. The PDLD electrostatic calculations are augmented by estimates of the relevant hydrophobic and steric contributions. The determination of the hydrophobic energy involves an approach which considers the modification of the effective surface area of the solute by local field effects. The steric contributions are analyzed in terms of the corresponding reorganization energies. This treatment, which considers the protein as a harmonic system, views the steric forces as the restoring forces for the electrostatic interactions. The FEP method is found to give unreliable results with regular cut-off radii and starts to give quantitative results only in very expensive treatment with very large cut-off radii. The PDLD and PDLD/S methods are much faster than the FEP approach and give reasonable results for both the relative and absolute binding energies. The speed and simplicity of the PDLD/S method make it an effective strategy for interactive docking studies and indeed such an option is incorporated in the program MOLARIS. A component analysis of the different energy contributions of the FEP treatment and a similar PDLD analysis indicate that electrostatic effects provide the largest contribution to the differential binding energy, while the hydrophobic and steric contributions are much smaller. This finding lends further support to the idea that electrostatic interactions play a major role in determining the antigen specificity of McPC603.

Microscopic simulation of solvated proteins are used to evaluate the relationship between the macroscopic field and macroscopic polarization, thus providing the corresponding dielectric constant, E. This parameter is evaluated by two different methods which are first examined and calibrated by calculating the dielectric constant of bulk water. These calculations indicate that the reaction field, which represents the effect of the missing solvent around the given explicit region, must be included in the simulations in order to obtain a reasonable value for E. The corresponding effect is not related to the reduction of the effective interactions between charges in the reference region but to the intrinsic value ofE in that region. This means that vacuum calculations ofE in proteins might underestimate its actual value. Or in other words, calculations of E in proteins must include the effect of the reaction field or a sufficiently large number of surrounding solvent molecules. Having included the surrounding solvent in simulations of trypsin, we find that E depends on the actual protein site. In particular, we find that E can be as large as 10 in sites of catalytic importance. It is pointed out that the dielectric constant that should be used in calculations of protein properties depends on the part of the system which is not treated explicitly and is not necessarily the macroscopic E. For example, using E in electrostatic calculations that consider the protein polar groups explicitly accounts twice for some aspects of the protein polarity. Finally, possible ways to use microscopically determined dielectric constants for electrostatic calculations in protein are considered.

The generation of an adaptive immune response against intracellular pathogens requires the recruitment of effector T cells to sites of infection. Here we show that the chemokine IP-10, a specific chemoattractant for activated T cells, controls this process in mice naturally infected with Toxoplasma gondii. Neutralization of IP-10 in infected mice inhibited the massive influx of T cells into tissues and impaired antigen-specific T cell effector functions. This resulted in >1000-fold increase in tissue parasite burden and a marked increase in mortality compared to control antibody-treated mice. These observations suggest that IP-10 may play a broader role in the localization and function of effector T cells at sites of Th1 inflammation.

The host response to intracellular pathogens requires the coordinated action of both the innate and acquired immune systems. Chemokines play a critical role in the trafficking of immune cells and transitioning an innate immune response into an acquired response. We analyzed the host response of mice deficient in the chemokine receptor CCR5 following infection with the intracellular protozoan parasite Toxoplasma gondii. We found that CCR5 controls recruitment of natural killer (NK) cells into infected tissues. Without this influx of NK cells, tissues from CCR5-deficient (CCR5−/−) mice were less able to generate an inflammatory response, had decreased chemokine and interferon γ production, and had higher parasite burden. As a result, CCR5−/− mice were more susceptible to infection with T. gondii but were less susceptible to the immune-mediated tissue injury seen in certain inbred strains. Adoptive transfer of CCR5+/+ NK cells into CCR5−/− mice restored their ability to survive lethal T. gondii infection and demonstrated that CCR5 is required for NK cell homing into infected liver and spleen. This study establishes CCR5 as a critical receptor guiding NK cell trafficking in host defense.

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CD1d-restricted T cells contribute to tumor protection, but their precise roles remain unclear. Here we show that tumor cells engineered to secrete granulocyte-macrophage colony-stimulating factor induce the expansion of CD1d-restricted T cells through a mechanism that involves CD1d and macrophage inflammatory protein 2 expression by CD8␣ ؊ , CD11c ؉ dendritic cells (DCs). The antitumor immunity stimulated by vaccination with irradiated, granulocyte-macrophage colony-stimulating factor-secreting tumor cells was abrogated in CD1d-and J␣281-deficient mice, revealing a critical role for CD1d-restricted T cells in this response. The loss of antitumor immunity was associated with impaired tumorinduced T helper 2 cytokine production, although IFN-␥ secretion and cytotoxicity were preserved. DCs from immunized CD1d-deficient mice showed compromised maturation and function. Together, these results delineate a role for CD1d-restricted T cell-DC cross talk in the shaping of antitumor immunity.