Effect of Ions and Sequence Variants on the Antagonist Binding Properties of the Histamine H1 Receptor.

The histamine H1 receptor (H1R) is a G protein-coupled receptor (GPCR) and represents a main target in the treatment of allergic reactions as well as inflammatory reactions and depressions. Although the overall effect of antagonists on H1 function has been extensively investigated, rather little is known about the potential modulatory effect of ions or sequence variants on antagonist binding. We investigated the dynamics of a phosphate ion present in the crystal structure and of a sodium ion, for which we determined the position in the allosteric pocket by metadynamics simulations. Both types of ions exhibit significant dynamics within their binding site; however, some key contacts remain stable over the simulation time, which might be exploited to develop more potent drugs targeting these sites. The dynamics of the ions is almost unaffected by the presence or absence of doxepin, as also reflected in their small effect (less than 1 kcal·mol-1) on doxepin binding affinity. We also examined the effect of four H1R sequence variants observed in the human population on doxepin binding. These variants cause a reduction in doxepin affinity of up to 2.5 kcal·mol-1, indicating that personalized medical treatments that take into account individual mutation patterns could increase precision in the dosage of GPCR-targeting drugs.

Specific Engineered G Protein Coupling to Histamine Receptors Revealed from Cellular Assay Experiments and Accelerated Molecular Dynamics Simulations.

G protein-coupled receptors (GPCRs) are targets of extracellular stimuli and hence occupy a key position in drug discovery. By specific and not yet fully elucidated coupling profiles with α subunits of distinct G protein families, they regulate cellular responses. The histamine H2 and H4 receptors (H2R and H4R) are prominent members of Gs- and Gi-coupled GPCRs. Nevertheless, promiscuous G protein and selective Gi signaling have been reported for the H2R and H4R, respectively, the molecular mechanism of which remained unclear. Using a combination of cellular experimental assays and Gaussian accelerated molecular dynamics (GaMD) simulations, we investigated the coupling profiles of the H2R and H4R to engineered mini-G proteins (mG). We obtained coupling profiles of the mGs, mGsi, or mGsq proteins to the H2R and H4R from the mini-G protein recruitment assays using HEK293T cells. Compared to H2R-mGs expressing cells, histamine responses were weaker (pEC50, Emax) for H2R-mGsi and -mGsq. By contrast, the H4R selectively bound to mGsi. Similarly, in all-atom GaMD simulations, we observed a preferential binding of H2R to mGs and H4R to mGsi revealed by the structural flexibility and free energy landscapes of the complexes. Although the mG α5 helices were consistently located within the HR binding cavity, alternative binding orientations were detected in the complexes. Due to the specific residue interactions, all mG α5 helices of the H2R complexes adopted the Gs-like orientation toward the receptor transmembrane (TM) 6 domain, whereas in H4R complexes, only mGsi was in the Gi-like orientation toward TM2, which was in agreement with Gs- and Gi-coupled GPCRs structures resolved by X-ray/cryo-EM. These cellular and molecular insights support (patho)physiological profiles of the histamine receptors, especially the hitherto little studied H2R function in the brain, as well as of the pharmacological potential of H4R selective drugs.

Agonist Binding and G Protein Coupling in Histamine H2 Receptor: A Molecular Dynamics Study.

The histamine H2 receptor (H2R) plays an important role in the regulation of gastric acid secretion. Therefore, it is a main drug target for the treatment of gastroesophageal reflux or peptic ulcer disease. However, there is as of yet no 3D-structural information available hampering a mechanistic understanding of H2R. Therefore, we created a model of the histamine-H2R-Gs complex based on the structure of the ternary complex of the ß2-adrenoceptor and investigated the conformational stability of this active GPCR conformation. Since the physiologically relevant motions with respect to ligand binding and conformational changes of GPCRs can only partly be assessed on the timescale of conventional MD (cMD) simulations, we also applied metadynamics and Gaussian accelerated molecular dynamics (GaMD) simulations. A multiple walker metadynamics simulation in combination with cMD was applied for the determination of the histamine binding mode. The preferential binding pose detected is in good agreement with previous data from site directed mutagenesis and provides a basis for rational ligand design. Inspection of the H2R-Gs interface reveals a network of polar interactions that may contribute to H2R coupling selectivity. The cMD and GaMD simulations demonstrate that the active conformation is retained on a µs-timescale in the ternary histamine-H2R-Gs complex and in a truncated complex that contains only Gs helix α5 instead of the entire G protein. In contrast, histamine alone is unable to stabilize the active conformation, which is in line with previous studies of other GPCRs.

A Metadynamics-Based Protocol for the Determination of GPCR-Ligand Binding Modes.

G protein-coupled receptors (GPCRs) are a main drug target and therefore a hot topic in pharmaceutical research. One important prerequisite to understand how a certain ligand affects a GPCR is precise knowledge about its binding mode and the specific underlying interactions. If no crystal structure of the respective complex is available, computational methods can be used to deduce the binding site. One of them are metadynamics simulations which have the advantage of an enhanced sampling compared to conventional molecular dynamics simulations. However, the enhanced sampling of higher-energy states hampers identification of the preferred binding mode. Here, we present a novel protocol based on clustering of multiple walker metadynamics simulations which allows identifying the preferential binding mode from such conformational ensembles. We tested this strategy for three different model systems namely the histamine H1 receptor in combination with its physiological ligand histamine, as well as the ß 2 adrenoceptor with its agonist adrenaline and its antagonist alprenolol. For all three systems, the proposed protocol was able to reproduce the correct binding mode known from the literature suggesting that the approach can more generally be applied to the prediction of GPCR ligand binding in future.

Conformational Dynamics of Herpesviral NEC Proteins in Different Oligomerization States.

All herpesviruses use a heterodimeric nuclear egress complex (NEC) to transport capsids out of host cell nuclei. Despite their overall similar structure, NECs may differ significantly in sequence between different viruses. Up to now, structural information is limited to isolated NEC heterodimers and to large hexagonal lattices made up of hexagonal ring-like structures ("Hexagons"). The present study aimed to expand the existing structural knowledge with information on the dynamics of NECs from different viruses and in different oligomerization states. For this task, comparative molecular dynamics simulations were performed of the free NEC heterodimers from three different viruses (HCMV (human cytomegalovirus), HSV-1 (herpes simplex virus 1), and PRV (pseudorabies virus)). In addition, higher oligomerization states comprising two or six NEC heterodimers were characterized for HCMV and HSV-1. The study revealed that the isolated NEC heterodimers from α- (HSV-1, PRV) and ß-herpesviruses (HCMV) differ significantly in their dynamics, which can be attributed to a poorly conserved interface region between the NEC subdomains. These differences become smaller for higher oligomerization states, and both HCMV and HSV-1 individual Hexagons exhibit a common region of enhanced dynamics, which might be of functional relevance for the formation of curved vesicle structures or the recognition of hexameric capsid proteins.

Binding of histamine to the H1 receptor-a molecular dynamics study.

Binding of histamine to the G-protein coupled histamine H1 receptor plays an important role in the context of allergic reactions; however, no crystal structure of the resulting complex is available yet. To deduce the histamine binding site, we performed unbiased molecular dynamics (MD) simulations on a microsecond time scale, which allowed to monitor one binding event, in which particularly the residues of the extracellular loop 2 were involved in the initial recognition process. The final histamine binding pose in the orthosteric pocket is characterized by interactions with Asp1073.32, Tyr1083.33, Thr1945.43, Asn1985.46, Trp4286.48, Tyr4316.51, Phe4326.52, and Phe4356.55, which is in agreement with existing mutational data. The conformational stability of the obtained complex structure was subsequently confirmed in 2 µs equilibrium MD simulations, and a metadynamics simulation proved that the detected binding site represents an energy minimum. A complementary investigation of a D107A mutant, which has experimentally been shown to abolish ligand binding, revealed that this exchange results in a significantly weaker interaction and enhanced ligand dynamics. This finding underlines the importance of the electrostatic interaction between the histamine ammonium group and the side chain of Asp1073.32 for histamine binding.

Interaction of Glycolipids with the Macrophage Surface Receptor Mincle - a Systematic Molecular Dynamics Study.

Synthetic analogues of mycobacterial trehalose-dimycolate such as trehalose acyl esters have been proposed as novel adjuvants for vaccination. They induce an immune response by binding to the macrophage C-type lectin receptor Mincle. The binding site of trehalose is known, but there is yet only very limited structural information about the binding mode of the acyl esters. Here, we performed a systematic molecular dynamics study of trehalose mono-and diesters with different chain lengths. All acyl chains investigated exhibited a high flexibility and interacted almost exclusively with a hydrophobic groove on Mincle. Despite the limited length of this hydrophobic groove, the distal parts of the longer monoesters can still form additional interactions with this surface region due to their conformational flexibility. In diesters, a certain length of the second acyl chain is required to contact the hydrophobic groove. However, a stable concomitant accommodation of both acyl chains in the groove is hampered by the conformational rigidity of Mincle. Instead, multiple dynamic interaction modes are observed, in which the second acyl chain contributes to binding. This detailed structural information is considered helpful for the future design of more affine ligands that may foster the development of novel adjuvants.

Role of the N-terminus for the stability of an amyloid-ß fibril with three-fold symmetry.

A key player in Alzheimer's disease is the peptide amyloid-beta (Aß), whose aggregation into small soluble oligomers, protofilaments, and fibrils finally leads to plaque deposits in human brains. The aggregation behavior of Aß is strongly modulated by the nature and composition of the peptide's environment and by its primary sequence properties. The N-terminal residues of Aß play an important role, because they are known to change the peptide's aggregation propensity. Since these residues are for the first time completely resolved at the molecular level in a three-fold symmetric fibril structure derived from a patient, we chose that system as template for a systematic investigation of the influence of the N-terminus upon structural stability. Using atomistic molecular dynamics simulations, we examined several fibrillar systems comprising three, six, twelve and an infinite number of layers, both with and without the first eight residues. First, we found that three layers are not sufficient to stabilize the respective Aß topology. Second, we observed a clear stabilizing effect of the N-terminal residues upon the overall fibril fold: truncated Aß systems were less stable than their full-length counterparts. The N-terminal residues Arg5, Asp7, and Ser8 were found to form important interfilament contacts stabilizing the overall fibril structure of three-fold symmetry. Finally, similar structural rearrangements of the truncated Aß species in different simulations prompted us to suggest a potential mechanism involved in the formation of amyloid fibrils with three-fold symmetry.