skip to main content

Title: Structural basis for ligand modulation of the CCR2 conformational landscape

CC chemokine receptor 2 (CCR2) is a part of the chemokine receptor family, an important class of therapeutic targets. These class A G-protein coupled receptors (GPCRs) are involved in mammalian signaling pathways and control cell migration toward endogenous CC chemokine ligands, named for the adjacent cysteine motif on their N terminus. Chemokine receptors and their associated ligands are involved in a wide range of diseases and thus have become important drug targets. CCR2, in particular, promotes the metastasis of cancer cells and is also implicated in autoimmunity-driven type-1 diabetes, diabetic nephropathy, multiple sclerosis, asthma, atherosclerosis, neuropathic pain, and rheumatoid arthritis. Although promising, CCR2 antagonists have been largely unsuccessful to date. Here, we investigate the effect of an orthosteric and an allosteric antagonist on CCR2 dynamics by coupling long-timescale molecular dynamics simulations with Markov-state model theory. We find that the antagonists shift CCR2 into several stable inactive conformations that are distinct from the crystal structure conformation and disrupt a continuous internal water and sodium ion pathway, preventing transitions to an active-like state. Several metastable conformations present a cryptic drug-binding pocket near the allosteric site that may be amenable to targeting with small molecules. Without antagonists, the apo dynamics reveal intermediate conformations more » along the activation pathway that provide insight into the basal dynamics of CCR2 and may also be useful for future drug design.

« less
Authors:
; ;
Publication Date:
NSF-PAR ID:
10091524
Journal Name:
Proceedings of the National Academy of Sciences
Page Range or eLocation-ID:
Article No. 201814131
ISSN:
0027-8424
Publisher:
Proceedings of the National Academy of Sciences
Sponsoring Org:
National Science Foundation
More Like this
  1. The lack of biologically relevant protein structures can hinder rational design of small molecules to target G protein-coupled receptors (GPCRs). While ensemble docking using multiple models of the protein target is a promising technique for structure-based drug discovery, model clustering and selection still need further investigations to achieve both high accuracy and efficiency. In this work, we have developed an original ensemble docking approach, which identifies the most relevant conformations based on the essential dynamics of the protein pocket. This approach is applied to the study of small-molecule antagonists for the PAC1 receptor, a class B GPCR and a regulator of stress. As few as four representative PAC1 models are selected from simulations of a homology model and then used to screen three million compounds from the ZINC database and 23 experimentally validated compounds for PAC1 targeting. Our essential dynamics ensemble docking (EDED) approach can effectively reduce the number of false negatives in virtual screening and improve the accuracy to seek potent compounds. Given the cost and difficulties to determine membrane protein structures for all the relevant states, our methodology can be useful for future discovery of small molecules to target more other GPCRs, either with or without experimental structures.
  2. The PDZ family has drawn attention as possible drug targets because of the domains’ wide ranges of function and highly conserved binding pockets. The PICK1 PDZ domain has been proposed as a possible drug target because the interactions between the PICK1 PDZ domain and the GluA2 subunit of the AMPA receptor have been shown to progress neurodegenerative diseases. BIO124 has been identified as a sub µM inhibitor of the PICK1–GluA2 interaction. Here, we use all-atom molecular dynamics simulations to reveal the atomic-level interaction pattern between the PICK1 PDZ domain and BIO124. Our simulations reveal three unique binding conformations of BIO124 in the PICK1 PDZ binding pocket, referred to here as state 0, state 1, and state 2. Each conformation is defined by a unique hydrogen bonding network and a unique pattern of hydrophobic interactions between BIO124 and the PICK1 PDZ domain. Interestingly, each conformation of BIO124 results in different dynamic changes to the PICK1 PDZ domain. Unlike states 1 and 2, state 0 induces dynamic coupling between BIO124 and the αA helix. Notably, this dynamic coupling with the αA helix is similar to what has been observed in other PDZ–ligand complexes. Our analysis indicates that the interactions formed between BIO124more »and I35 may be the key to inducing dynamic coupling with the αA helix. Lastly, we suspect that the conformational shifts observed in our simulations may affect the stability and thus the overall effectiveness of BIO124. We propose that a physically larger inhibitor may be necessary to ensure sufficient interactions that permit stable binding between a drug and the PICK1 PDZ domain.« less
  3. Abstract Background

    Emerging RNA viruses that target the central nervous system (CNS) lead to cognitive sequelae in survivors. Studies in humans and mice infected with West Nile virus (WNV), a re-emerging RNA virus associated with learning and memory deficits, revealed microglial-mediated synapse elimination within the hippocampus. Moreover, CNS-resident memory T (TRM) cells activate microglia, limiting synapse recovery and inducing spatial learning defects in WNV-recovered mice. The signals involved in T cell-microglia interactions are unknown.

    Methods

    Here, we examined immune cells within the murine WNV-recovered forebrain using single-cell RNA sequencing to identify putative ligand-receptor pairs involved in intercellular communication between T cells and microglia. Clustering and differential gene analyses were followed by protein validation and genetic and antibody-based approaches utilizing an established murine model of WNV recovery in which microglia and complement promote ongoing hippocampal synaptic loss.

    Results

    Profiling of host transcriptome immune cells at 25 days post-infection in mice revealed a shift in forebrain homeostatic microglia to activated subpopulations with transcriptional signatures that have previously been observed in studies of neurodegenerative diseases. Importantly, CXCL16/CXCR6, a chemokine signaling pathway involved in TRM cell biology, was identified as critically regulating CXCR6 expressing CD8+TRM cell numbers within the WNV-recovered forebrain. We demonstrate that CXCL16 is highlymore »expressed by all myeloid cells, and its unique receptor, CXCR6, is highly expressed on all CD8+T cells. Using genetic and pharmacological approaches, we demonstrate that CXCL16/CXCR6 not only is required for the maintenance of WNV-specific CD8 TRM cells in the post-infectious CNS, but also contributes to their expression of TRM cell markers. Moreover, CXCR6+CD8+T cells are required for glial activation and ongoing synapse elimination.

    Conclusions

    We provide a comprehensive assessment of the role of CXCL16/CXCR6 as an interaction link between microglia and CD8+T cells that maintains forebrain TRM cells, microglial and astrocyte activation, and ongoing synapse elimination in virally recovered animals. We also show that therapeutic targeting of CXCL16 in mice during recovery may reduce CNS CD8+TRM cells.

    « less
  4. Abstract Background

    Wnt genes code for ligands that activate signaling pathways during development in Metazoa. Through the canonical Wnt (cWnt) signaling pathway, these genes regulate important processes in bilaterian development, such as establishing the anteroposterior axis and posterior growth. In Arthropoda, Wnt ligands also regulate segment polarity, and outgrowth and patterning of developing appendages. Arthropods are part of a lineage called Panarthropoda that includes Onychophora and Tardigrada. Previous studies revealed potential roles of Wnt genes in regulating posterior growth, segment polarity, and growth and patterning of legs in Onychophora. Unlike most other panarthropods, tardigrades lack posterior growth, but retain segmentation and appendages. Here, we investigated Wnt genes in tardigrades to gain insight into potential roles that these genes play during development of the highly compact and miniaturized tardigrade body plan.

    Results

    We analyzed published genomes for two representatives of Tardigrada,Hypsibius exemplarisandRamazzottius varieornatus. We identified single orthologs ofWnt4,Wnt5,Wnt9,Wnt11, andWntA, as well as twoWnt16paralogs in both tardigrade genomes. We only found aWnt2ortholog inH. exemplaris. We could not identify orthologs ofWnt1,Wnt6,Wnt7,Wnt8, orWnt10. We identified most other components of cWnt signaling in both tardigrade genomes. However, we were unable to identify an ortholog ofarrow/Lrp5/6, a gene that codes for a Frizzled co-receptor of Wnt ligands. Additionally,more »we found that some other animals that have lost several Wnt genes and are secondarily miniaturized, like tardigrades, are also missing an ortholog ofarrow/Lrp5/6. We analyzed the embryonic expression patterns of Wnt genes inH. exemplarisduring developmental stages that span the establishment of the AP axis through segmentation and leg development. We detected expression of all Wnt genes inH. exemplarisbesides one of theWnt16paralogs. During embryo elongation, expression of several Wnt genes was restricted to the posterior pole or a region between the anterior and posterior poles. Wnt genes were expressed in distinct patterns during segmentation and development of legs inH. exemplaris, rather than in broadly overlapping patterns.

    Conclusions

    Our results indicate that Wnt signaling has been highly modified in Tardigrada. While most components of cWnt signaling are conserved in tardigrades, we conclude that tardigrades have lostWnt1,Wnt6,Wnt7,Wnt8, andWnt10, along witharrow/Lrp5/6. Our expression data may indicate a conserved role of Wnt genes in specifying posterior identities during establishment of the AP axis. However, the loss of several Wnt genes and the distinct expression patterns of Wnt genes during segmentation and leg development may indicate that combinatorial interactions among Wnt genes are less important during tardigrade development compared to many other animals. Based on our results, and comparisons to previous studies, we speculate that the loss of several Wnt genes in Tardigrada may be related to a reduced number of cells and simplified development that accompanied miniaturization and anatomical simplification in this lineage.

    « less
  5. Infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) involves the attachment of the receptor-binding domain (RBD) of its spike proteins to the ACE2 receptors on the peripheral membrane of host cells. Binding is initiated by a down-to-up conformational change in the spike protein, the change that presents the RBD to the receptor. To date, computational and experimental studies that search for therapeutics have concentrated, for good reason, on the RBD. However, the RBD region is highly prone to mutations, and is therefore a hotspot for drug resistance. In contrast, we here focus on the correlations between the RBD and residues distant to it in the spike protein. This allows for a deeper understanding of the underlying molecular recognition events and prediction of the highest-effect key mutations in distant, allosteric sites, with implications for therapeutics. Also, these sites can appear in emerging mutants with possibly higher transmissibility and virulence, and preidentifying them can give clues for designing pan-coronavirus vaccines against future outbreaks. Our model, based on time-lagged independent component analysis (tICA) and protein graph connectivity network, is able to identify multiple residues that exhibit long-distance coupling with the RBD opening. Residues involved in the most ubiquitous D614G mutation and themore »A570D mutation of the highly contagious UK SARS-CoV-2 variant are predicted ab initio from our model. Conversely, broad-spectrum therapeutics like drugs and monoclonal antibodies can target these key distant-but-conserved regions of the spike protein.« less