Clustering molecular dynamics conformations of the CC'-loop of the PD-1 immuno-checkpoint receptor.

Molecular mechanisms within the checkpoint receptor PD-1 are essential for its activation by PD-L1 as well as for blocking such an activation via checkpoint inhibitors. We use molecular dynamics to scrutinize patterns of atomic motion in PD-1 without a ligand. Molecular dynamics is performed for the whole extracellular domain of PD-1, and the analysis focuses on its CC'-loop and some adjacent Cα-atoms. We extend previous work by applying common nearest neighbor clustering (Cnn) and compare the performance of this method with Daura clustering as well as UMAP dimension reduction and subsequent agglomerative linkage clustering. As compared to Daura clustering, we found Cnn less sensitive to cutoff selection and better able to return representative clusters for sets of different 3D atomic conformations. Interestingly, Cnn yields results quite similar to UMAP plus linkage clustering.

Accelerated Enveloping Distribution Sampling to Probe the Presence of Water Molecules.

Determining the presence of water molecules at protein-ligand interfaces is still a challenging task in free-energy calculations. The inappropriate placement of water molecules results in the stabilization of wrong conformational orientations of the ligand. With classical alchemical perturbation methods, such as thermodynamic integration (TI), it is essential to know the amount of water molecules in the active site of the respective ligands. However, the resolution of the crystal structure and the correct assignment of the electron density do not always lead to a clear placement of water molecules. In this work, we apply the one-step perturbation method named accelerated enveloping distribution sampling (AEDS) to determine the presence of water molecules in the active site by probing them in a fast and straightforward way. Based on these results, we combined the AEDS method with standard TI to calculate accurate binding free energies in the presence of buried water molecules. The main idea is to perturb the water molecules with AEDS such that they are allowed to alternate between regular water molecules and non-interacting dummy particles while treating the ligand with TI over an alchemical pathway. We demonstrate the use of AEDS to probe the presence of water molecules for six different test systems. For one of these, previous calculations showed difficulties to reproduce the experimental binding free energies, and here, we use the combined TI-AEDS approach to tackle these issues.

Molecular dynamics identifies semi-rigid domains in the PD-1 checkpoint receptor bound to its natural ligand PD-L1.

Cells in danger of being erroneously attacked by leucocytes express PD-L1 on their surface. These cells activate PD-1 on attacking leucocytes and send them to death, thus curbing erroneous, autoimmune attack. Unfortunately, cancer cells exploit this mechanism: By expressing PD-L1, they guard themselves against leucocyte attack and thereby evade immune clearance. Checkpoint inhibitors are drugs which re-enable immune clearance of cancer cells by blocking the binding of PD-L1 to PD-1 receptors. It is therefore of utmost interest to investigate these binding mechanisms. We use three 600 ns all-atom molecular dynamics simulations to scrutinize molecular motions of PD-1 with its binding partner, the natural ligand PD-L1. Usually, atomic motion patterns are evaluated against whole molecules as a reference, disregarding that such a reference is a dynamic entity by itself, thus degrading stability of the reference. As a remedy, we identify semi-rigid domains, lending themselves as more stable and reliable reference frames against which even minute differences in molecular motion can be quantified precisely. We propose an unsupervised three-step procedure. In previous work of our group and others, minute differences in motion patterns proved decisive for differences in function. Here, several highly reliable frames of reference are established for future investigations based on molecular motion.

Conformational flexibility of a free and TCR-bound pMHC-I protein investigated by long-term molecular dynamics simulations.

BACKGROUND: Major histocompatibility complexes (MHCs) play a crucial role in the cell-mediated adaptive immune response as they present antigenic peptides (p) which are recognized by host T cells through a complex formation of the T cell receptor (TCR) with pMHC. In the present study, we report on changes in conformational flexibility within a pMHC molecule upon TCR binding by looking at molecular dynamics (MD) simulations of the free and the TCR-bound pMHC-I protein of the LC13-HLA-B*44:05-pEEYLQAFTY complex. RESULTS: We performed long-term MD simulations with a total simulation time of 8 µs, employing 10 independent 400 ns replicas for the free and the TCR-bound pMHC system. Upon TCR ligation, we observed a reduced dynamic flexibility in the central residues of the peptide and the MHC α1-helix, altered occurrences of hydrogen bonds between the peptide and the MHC, a reduced conformational entropy of the peptide-binding groove, as well as a decreased solvent accessible surface area. CONCLUSIONS: In summary, our results from 8 µs MD simulations indicate a restricted conformational space of the MHC peptide-binding groove upon TCR ligation and suggest a minimum simulation time of approximately 100 ns for biomolecules of comparable complexity to draw meaningful conclusions. Given the relatively long total simulation time, our results contribute to a more detailed view on conformational flexibility properties of the investigated free and TCR-bound pMHC-I system.