CD1b-restricted GEM T cell responses are modulated by Mycobacterium tuberculosis mycolic acid meromycolate chains

Tuberculosis, caused by Mycobacterium tuberculosis, remains a major human pandemic. Germline-encoded mycolyl lipid-reactive (GEM) T cells are donor-unrestricted and recognize CD1b-presented mycobacterial mycolates. However, the molecular requirements governing mycolate antigenicity for the GEM T cell receptor (TCR) remain poorly understood. Here, we demonstrate CD1b expression in tuberculosis granulomas and reveal a central role for meromycolate chains in influencing GEM-TCR activity. Meromycolate fine structure influences T cell responses in TB-exposed individuals, and meromycolate alterations modulate functional responses by GEM-TCRs. Computational simulations suggest that meromycolate chain dynamics regulate mycolate head group movement, thereby modulating GEM-TCR activity. Our findings have significant implications for the design of future vaccines that target GEM T cells

Direct validation of the single step classical to quantum free energy perturbation

The use of the Zwanzig equation in the calculation of single-step perturbations to provide first-principles (ab initio) quantum mechanics (QM) correction terms to molecular mechanics (MM) free energy cycles is well established. A rigorous test of the ability to converge such calculations would be very useful in this context. In this work, we perform a direct assessment of the convergence of the MM to QM perturbation, by attempting the reverse QM to MM perturbation. This required the generation of extensive QM molecular dynamics trajectories, using density functional theory (DFT), within the representative biological system of a DNA adenosine–thymidine dimer. Over 100 ps of dynamics with the PBE functional and 6.25 ps with the LDA functional were generated. We demonstrate that calculations with total potential energies are very poorly convergent due to a lack of overlap of phase space distributions between ensembles. While not theoretically rigorous, the use of interaction energies provides far superior convergence, despite the presence of nonclassical charge transfer effects within the DFT trajectories. The source of poor phase space overlap for total energies is diagnosed, the approximate quantification of overlaps suggesting that even for the comparatively simple system considered here convergence of total energy calculations within a reasonable simulation time is unfeasible

A Monte Carlo Resampling approach for the calculation of hybrid classical and quantum free energies

Hybrid free energy methods allow estimation of free energy differences at the quantum mechanics (QM) level with high efficiency by performing sampling at the classical mechanics (MM) level. Various approaches to allow the calculation of QM corrections to classical free energies have been proposed. The single step free energy perturbation approach starts with a classically generated ensemble, a subset of structures of which are post-processed to obtain QM energies for use with the Zwanzig equation. This gives an estimate of the free energy difference associated with the change from an MM to a QM Hamiltonian. Owing to the poor numerical properties of the Zwanzig equation however, recent developments have produced alternative methods which aim to provide access to the properties of the true QM ensemble. Here we propose an approach based on the resampling of MM structural ensembles and application of a Monte Carlo acceptance test which in principle, can generate the exact QM ensemble or intermediate ensembles between the MM and QM states. We carry out a detailed comparison against the Zwanzig equation and recently proposed non-Boltzmann methods. As a test system we use a set of small molecule hydration free energies for which hybrid free energy calculations are performed at the semi-empirical Density Functional Tight Binding level. Equivalent ensembles at this level of theory have also been generated allowing the reverse QM to MM perturbations to be performed along with a detailed analysis of the results. Additionally, a previously published nucleotide base pair data set simulated at the QM level using ab initio molecular dynamics is also considered. We provide a strong rationale for the use of the Monte Carlo Resampling and non-Boltzmann approaches by showing that configuration space overlaps can be estimated which provide useful diagnostic information regarding the accuracy of these hybrid approaches

A Monte Carlo Resampling Approach for the Calculation of Hybrid Classical and Quantum Free Energies

Hybrid free energy methods allow estimation of free energy differences at the quantum mechanics (QM) level with high efficiency by performing sampling at the classical mechanics (MM) level. Various approaches to allow the calculation of QM corrections to classical free energies have been proposed. The single step free energy perturbation approach starts with a classically generated ensemble, a subset of structures of which are postprocessed to obtain QM energies for use with the Zwanzig equation. This gives an estimate of the free energy difference associated with the change from an MM to a QM Hamiltonian. Owing to the poor numerical properties of the Zwanzig equation, however, recent developments have produced alternative methods which aim to provide access to the properties of the true QM ensemble. Here we propose an approach based on the resampling of MM structural ensembles and application of a Monte Carlo acceptance test which in principle, can generate the exact QM ensemble or intermediate ensembles between the MM and QM states. We carry out a detailed comparison against the Zwanzig equation and recently proposed non-Boltzmann methods. As a test system we use a set of small molecule hydration free energies for which hybrid free energy calculations are performed at the semiempirical Density Functional Tight Binding level. Equivalent ensembles at this level of theory have also been generated allowing the reverse QM to MM perturbations to be performed along with a detailed analysis of the results. Additionally, a previously published nucleotide base pair data set simulated at the QM level using <i>ab initio</i> molecular dynamics is also considered. We provide a strong rationale for the use of the Monte Carlo Resampling and non-Boltzmann approaches by showing that configuration space overlaps can be estimated which provide useful diagnostic information regarding the accuracy of these hybrid approaches

CD1c antigen presentation

CD1c-dependent self-reactive T cells are abundant in human blood, but self-antigens presented by CD1c to the T cell receptors of these cells are unknown. Here we present a crystal structure of CD1c determined at 2.4 Å revealing an extended ligand binding potential of the antigen groove and a substantially different conformation compared to known CD1c structures. Computational simulations exploring different occupancy states of the groove re-enacted these different CD1c conformations, and suggested cholesteryl esters (CE) and acylated steryl glycosides (ASG) as new ligand classes for CD1c. Confirming this, we show that binding of CE and ASG to CD1c enables the binding of human CD1c self-reactive T cell receptors. Hence, human CD1c adopts different conformations dependent on ligand occupancy of its groove, with CE and ASG stabilising CD1c conformations that provide a footprint for binding of CD1c self-reactive T cell receptors

Cholesteryl esters stabilize human CD1c conformations for recognition by self-reactive T cells

Cluster of differentiation 1c (CD1c)-dependent self-reactive T cells are abundant in human blood, but self-antigens presented by CD1c to the T-cell receptors of these cells are poorly understood. Here we present a crystal structure of CD1c determined at 2.4 Å revealing an extended ligand binding potential of the antigen groove and a substantially different conformation compared with known CD1c structures. Computational simulations exploring different occupancy states of the groove reenacted these different CD1c conformations and suggested cholesteryl esters (CE) and acylated steryl glycosides (ASG) as new ligand classes for CD1c. Confirming this, we show that binding of CE and ASG to CD1c enables the binding of human CD1c self-reactive T-cell receptors. Hence, human CD1c adopts different conformations dependent on ligand occupancy of its groove, with CE and ASG stabilizing CD1c conformations that provide a footprint for binding of CD1c self-reactive T-cell receptors