Tianyi Yang

The folded structure of a protein is understood to be an optimal energy state. However, previous studies have shown that certain amino acid residue positions that play a critical role in protein function are often in a suboptimal energy state or “frustrated”. Here, we leverage over 1200 three-dimensional structures of G protein-coupled receptors (GPCRs) to demonstrate that residues at the interface between GPCR and its ligand or G protein contain a higher density of frustrated residues compared to other structural regions in the receptor. Likewise, the Gα subunit of the trimeric G proteins shows multiple clusters of highly frustrated residues on its surface that overlap with their effector protein (Gbγ, RGS, Adenylyl cyclase, Ric8) binding interfaces. Our study highlights the use of protein frustration as one of the multiple structural properties to identify protein–protein interfaces and for prospective prediction of potential ligand binding sites.

The structurally disordered intracellular loops (ICLs) of G protein-coupled receptors (GPCRs) play a critical role in G protein coupling. In our previous work, we used a combination of FRET-based and computational methodologies to show that the third intracellular loop (ICL3) modulates the activity and G protein coupling selectivity in GPCRs. In the current study, we have uncovered the role of several lipid components in modulating the conformational ensemble of ICL3 of the β2-adrenergic receptor (β2AR). Our findings indicate that phosphatidylinositol 4,5-bisphosphate (PIP2) in the inner leaflet of the membrane bilayer acts as a stabilizing anchor for ICL3, opening the intracellular cavity to facilitate G protein coupling. This interaction between PIP2 and ICL3 causes tilting of β2AR within the cellular membrane. Notably, this tilting of the receptor is supported by ganglioside GM3 stabilizing the extracellular loops on the outer leaflet of the bilayer, thereby exerting an allosteric effect on the orthosteric ligand binding pocket. Our results underscore the significance of lipids in modulating GPCR activity, proposing an allosteric mechanism that occurs through the receptor’s orientation within the membrane.

Human c-Cbl is a RING-type ligase and plays a central role in the protein degradation cascade. To elucidate its conformational changes related to substrate binding, we performed molecular dynamics simulations of different variants/states of c-Cbl for a cumulative time of 68 its. Our simulations demonstrate that before the substrate binds, the RING domain samples a broad set of conformational states at a biologically relevant salt concentration, including the closed, partially open, and fully open states, whereas substrate binding leads to a restricted conformational sampling. Phe378 and the C-terminal region play an essential role in stabilizing the partially open state. To visualize the allosteric signal transmission pathways from the substrate-binding site to the 40 A apart RING domain and identify the critical residues for allostery, we have created a subgraph from the optimal and suboptimal paths. Redundant paths are seen in the SH2 domain where the substrate binds, while the major bottlenecks are found at the junction between the SH2 domain and the linker helix region as well as that between the SH2 domain and the 4H bundle. These bottlenecks separate the paths into two overall routes. The nodes/residues at the bottlenecks on the subgraph are considered allosteric hot spots. This subgraph approach provides a general tool for network visualization and determination of critical residues for allostery. The structurally and allosterically critical residues identified in our work are testable and would provide valuable insights into the emerging strategies for drug discovery, such as targeted protein degradation.