Hallucinogenic molecules bind to G-protein coupled receptors (GPCRs), a large family that includes the beta2-AR and serotonin 2A receptor (5-HT2AR). A popular theory is that hallucinogens such as psilocybin and DMT affect constitutive signaling at the 5-HT2AR. Hallucinogenic drugs probably bind to dopamine GPCRs too. The results with the carazolol-bound beta2-AR structure can be extended to psychedelic research since the 5-HT2AR is highly conserved with the beta2-AR, and the beta2-AR was crystallized with the inverse agonist carazolol, which closely resembles psilocin.
Figure 5 below shows carazolol (yellow) bound to the beta2-AR.
The beta2-AR has seven transmembrane helices (Figure 1 below). Intracellular loop 3 (ICL3) is found on the intracellular side of the cell membrane, where G-proteins bind to the receptor. The 5-HT2AR couples to Gq/11, via interactions involving ICL3. The ICL3 domains of various GPCRs have been shown to provide docking sites for beta/gamma G-proteins subunits, as well as arrestins.
GPCRs have amazing structural plasticity to accommodate many different binding ligands. In particular, the ICL3 has been identified as a highly unstructured region using protease susceptibility and intramolecular fluorescence resonance energy transfer experiments. Obtaining high-resolution structures of GPCRs has been challenging because of the unstructured regions. The inherent flexibility of the ICL3 region probably leads to the receptor's conformational heterogeneity and crystallization problems.
The authors developed a strategy to obtain the beta2-AR crystals. They replaced ICL3 with T4 lysozyme (T4L), a well-folded protein that restricts the movement of helices. The final construct used for crystallization trials had residues 231 to 262 of the beta2-AR replaced by amino acids 2 to 161 of T4L. The authors called the fusion protein "beta2-adrenergic receptor-T4L," or beta2-AR-T4L.
The beta2-AR-T4L did not couple to G-proteins, so it was not a functional receptor, and the presence of T4L at the ICL3 region could potentially affect the arrangement of the normal protein, but there didn't seem to be another option. The researchers set up over 2,000 conditions at 4 C and 20 C to attempt to grow wild-type crystals of beta2-AR.
"Despite substantial efforts, we were unable to grow diffraction-quality crystals from purified, homogeneous wild-type (WT) beta2-adrenergic receptor." (Rosenbaum,D.M. 2007)This effort is a testimony to the flexibility of the beta2-AR and perhaps GPCRs in general. A tightly packed protein such as T4 lysozyme can be crystallized easily, but a native GPCR cannot be crystallized because it vibrates too much to "freeze out". By attaching T4L as a seed for the crystallization process, the researchers sufficiently minimized the molecular motions of the beta2-AR protein.
Another strategy for obtaining beta2-AR crystals involved making monoclonal antibodies to the ICL3, and then fragments of these antibodies (Fab). The researchers were able to crystallize a beta2-AR-Fab5 construct, and remarkably, binding of Fab5 to beta2-AR did not alter agonist or antagonist binding affinities, so presumably the native structure of the beta2-AR was unaffected.
The figure below shows the constructs that were crystallized: beta2-AR-T4L (blue/grey) and the beta2-AR-Fab5 (yellow/grey).
In the previous 7 years, rhodopsin was the only GPCR structure that was available. It was possible to infer structure-function relationships in GPCRs by referring to rhodopsin, but in the next few months, the beta2-AR crystal structure may be utilized to generate better theoretical models of the 5-HT2AR, which has not yet been crystallized, and make predictions about the orientation of hallucinogens in the binding pocket. The beta2-AR crystal structure may be utilized in the next months to generate better theoretical models of the 5-HT2AR, which has not yet been crystallized, and make predictions about the orientation of hallucinogens in the binding pocket.
Cherezov V., D. M. Rosenbaum, M. A. Hanson, S. G. Rasmussen, F. S. Thian, T. S. Kobilka, H. J. Choi, P. Kuhn, W. I. Weis, B. K. Kobilka and R. C. Stevens (2007). High-resolution crystal structure of an engineered human beta2-adrenergic G protein-coupled receptor. Science 318, 1258-1265. 10.1126/science.1150577
Rosenbaum D. M., V. Cherezov, M. A. Hanson, S. G. Rasmussen, F. S. Thian, T. S. Kobilka, H. J. Choi, X. J. Yao, W. I. Weis, R. C. Stevens and B. K. Kobilka (2007). GPCR engineering yields high-resolution structural insights into beta2-adrenergic receptor function. Science 318, 1266-1273. 10.1126/science.1150609
Rasmussen S. G., H. J. Choi, D. M. Rosenbaum, T. S. Kobilka, F. S. Thian, P. C. Edwards, M. Burghammer, V. R. Ratnala, R. Sanishvili, R. F. Fischetti, G. F. Schertler, W. I. Weis and B. K. Kobilka (2007). Crystal structure of the human beta2 adrenergic G-protein-coupled receptor. Nature 450, 383-387. 10.1038/nature06325