Probing allosteric interactions in homo-oligomeric molecular machines using solution NMR spectroscopy

Developments in solution NMR spectroscopy have significantly impacted the biological questions that can now be addressed by this methodology. By means of illustration, we present here a perspective focusing on studies of a number of molecular machines that are critical for cellular homeostasis. The role of NMR in elucidating the structural dynamics of these important molecules is emphasized, focusing specifically on intersubunit allosteric communication in homo-oligomers. In many biophysical studies of oligomers, allostery is inferred by showing that models specifically including intersubunit communication best fit the data of interest. Ideally, however, experimental studies focusing on one subunit of a multisubunit system would be performed as an important complement to the more traditional bulk measurements in which signals from all components are measured simultaneously. Using an approach whereby asymmetric molecules are prepared in concert with NMR experiments focusing on the structural dynamics of individual protomers, we present examples of how intersubunit allostery can be directly observed in high-molecular-weight protein systems. These examples highlight some of the unique roles of solution NMR spectroscopy in studies of complex biomolecules and emphasize the important synergy between NMR and other atomic resolution biophysical methods.

Molecular modelling approaches predicted 1,2,3-triazolyl ester of ketorolac (15K) to be a novel allosteric modulator of the oncogenic kinase PAK1

P21-activated kinases (PAKs) are serine/threonine protein kinase which have six different isoforms (PAK1–6). Of those, PAK1 is overexpressed in many cancers and considered to be a major chemotherapeutic target. Most of the developed PAK1 inhibitor drugs work as pan-PAK inhibitors and show undesirable toxicity due to having untargeted kinase inhibition activities. Selective PAK1 inhibitors are therefore highly desired and oncogenic drug hunters are trying to develop allosteric PAK1 inhibitors. We previously synthesized 1,2,3-triazolyl ester of ketorolac (15K) through click chemistry technique, which exhibits significant anti-cancer effects via inhibiting PAK1. Based on the selective anticancer effects of 15K against PAK1-dependent cancer cells, we hypothesize that it may act as an allosteric PAK1 inhibitor. In this study, computational analysis was done with 15K to explore its quantum chemical and thermodynamic properties, molecular interactions and binding stability with PAK1, physicochemical properties, ADMET, bioactivities, and druglikeness features. Molecular docking analysis demonstrates 15K as a potent allosteric ligand that strongly binds to a novel allosteric site of PAK1 (binding energy ranges – 8.6 to – 9.2 kcal/mol) and does not target other PAK isoforms; even 15K shows better interactions than another synthesized PAK1 inhibitor. Molecular dynamics simulation clearly supports the stable binding properties of 15K with PAK1 crystal. Density functional theory-based calculations reveal that it can be an active drug with high softness and moderate polarity, and ADMET predictions categorize it as a non-toxic drug as evidenced by in vitro studies with brine shrimp and fibroblast cells. Structure–activity relationship clarifies the role of ester bond and triazol moiety of 15K in establishing novel allosteric interactions. Our results summarize that 15K selectively inhibits PAK1 as an allosteric inhibitor and in turn shows anticancer effects without toxicity.

Identification of the Ghrelin and Cannabinoid CB2 Receptor Heteromer Functionality and Marked Upregulation in Striatal Neurons from Offspring of Mice under a High-Fat Diet

Cannabinoids have been reported as orexigenic, i.e., as promoting food intake that, among others, is controlled by the so-called “hunger” hormone, ghrelin. The aim of this paper was to look for functional and/or molecular interactions between ghrelin GHSR1a and cannabinoid CB2 receptors at the central nervous system (CNS) level. In a heterologous system we identified CB2-GHSR1a receptor complexes with a particular heteromer print consisting of impairment of CB2 receptor/Gi-mediated signaling. The blockade was due to allosteric interactions within the heteromeric complex as it was reverted by antagonists of the GHSR1a receptor. Cannabinoids acting on the CB2 receptor did not affect cytosolic increases of calcium ions induced by ghrelin acting on the GHSR1a receptor. In situ proximity ligation imaging assays confirmed the expression of CB2-GHSR1a receptor complexes in both heterologous cells and primary striatal neurons. We tested heteromer expression in neurons from offspring of high-fat-diet mouse mothers as they have more risk to be obese. Interestingly, there was a marked upregulation of those complexes in striatal neurons from siblings of pregnant female mice under a high-fat diet.

Elastic Network Modeling of Cellular Networks Unveils Sensor and Effector Genes that Control Information Flow

The high-level organization of the cell is embedded in long-range interactions that connect distinct cellular processes. Existing approaches for detecting long-range interactions consist of propagating information from source nodes through cellular networks, but the selection of source nodes is inherently biased by prior knowledge. Here, we sought to derive an unbiased view of long-range interactions by adapting a perturbation-response scanning strategy initially developed for identifying allosteric interactions within proteins. We deployed this strategy onto an elastic network model of the yeast genetic network. The genetic network revealed a superior propensity for long-range interactions relative to simulated networks with similar topology. Long-range interactions were detected systematically throughout the network and found to be enriched in specific biological processes. Furthermore, perturbation-response scanning identified the major sources and receivers of information in the network, named effector and sensor genes, respectively. Effectors formed dense clusters centrally integrated into the network, whereas sensors formed loosely connected antenna-shaped clusters. Long-range interactions between effector and sensor clusters represent the major paths of information in the network. Our results demonstrate that elastic network modeling of cellular networks constitutes a promising strategy to probe the high-level organization of the cell.

Interfacial Water Many-body Effects Drive Structural Dynamics and Allosteric interactions in SARS-CoV-2 Main Protease Dimerization Interface

Following our previous work (Chem. Sci., 2021, 12, 4889 – 4907), we study the structural dynamics of the SARS-CoV-2 Main Protease dimerization interface (apo dimer) by means of microsecond adaptive sampling molecular dynamics simulations (50 microseconds) using the AMOEBA polarizable force field (PFF). This interface is structured by a complex H-bond network that is only stable at physiological pH. Structural correlations analysis between its residues and the catalytic site confirms the presence of a buried allosteric site. However, noticeable differences in allosteric connectivity are observed between PFFs and non-PFFs. Interfacial polarizable water molecules are shown to appear at the heart of this discrepancy, since they are connected to the global interface H-bond network and able to adapt their dipole moment (and dynamics) to their diverse local physico-chemical micro-environments. The water-interface many-body interactions appear to drive the interface volume fluctuations and to therefore mediate the allosteric interactions with the catalytic cavity.

Interfacial Water Many-body Effects Drive Structural Dynamics and Allosteric interactions in SARS-CoV-2 Main Protease Dimerization Interface

Following our previous work (Chem. Sci., 2021, 12, 4889 – 4907), we study the structural dynamics of the SARS-CoV-2 Main Protease dimerization interface (apo dimer) by means of microsecond adaptive sampling molecular dynamics simulations (50 microseconds) using the AMOEBA polarizable force field (PFF). This interface is structured by a complex H-bond network that is only stable at physiological pH. Structural correlations analysis between its residues and the catalytic site confirms the presence of a buried allosteric site. However, noticeable differences in allosteric connectivity are observed between PFFs and non-PFFs. Interfacial polarizable water molecules are shown to appear at the heart of this discrepancy, since they are connected to the global interface H-bond network and able to adapt their dipole moment (and dynamics) to their diverse local physico-chemical micro-environments. The water-interface many-body interactions appear to drive the interface volume fluctuations and to therefore mediate the allosteric interactions with the catalytic cavity.

Interfacial Water Many-body Effects Drive Structural Dynamics and Allosteric interactions in SARS-CoV-2 Main Protease Dimerization Interface

Following our previous work (Chem. Sci., 2021, 12, 4889 – 4907), we study the structural dynamics of the SARS-CoV-2 Main Protease dimerization interface (apo dimer) by means of microsecond adaptive sampling molecular dynamics simulations (50 microseconds) using the AMOEBA polarizable force field (PFF). This interface is structured by a complex H-bond network that is only stable at physiological pH. Structural correlations analysis between its residues and the catalytic site confirms the presence of a buried allosteric site. However, noticeable differences in allosteric connectivity are observed between PFFs and non-PFFs. Interfacial polarizable water molecules are shown to appear at the heart of this discrepancy, since they are connected to the global interface H-bond network and able to adapt their dipole moment (and dynamics) to their diverse local physico-chemical micro-environments. The water-interface many-body interactions appear to drive the interface volume fluctuations and to therefore mediate the allosteric interactions with the catalytic cavity.