Beta2-Adrenergic receptor crystal structure

Good news from The Scripps Research Institute. The human beta2-adrenergic receptor (beta2-AR) has been crystallized ! !

From Crystal structure of beta2-AR

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.

From Crystal structure of beta2-AR

Figure 5 shows carazolol (yellow) bound to the beta2-AR.

From Crystal structure of 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.

From Crystal structure of beta2-AR

GPCRs have amazing structural plasticity to accommodate many different binding ligands. In particular, 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 other than rhodopsin has been challenging because of their inherent structural flexibility and instability. The flexibility of the ICL3 region probably leads to the receptor's conformational heterogeneity and crystallization problems.

The authors developed a clever 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.

From Crystal structure of beta2-AR

The beta2-AR-T4L did not couple to G-proteins, so it was not a functional G-protein, 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." (D.M. Rosenbaum, 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 an antibody (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 beta2-AR-T4L (blue/grey) and the beta2-AR-Fab (yellow/grey).

From Crystal structure of beta2-AR

In the previous 7 years, rhodopsin was the only GPCR structure that was available. 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.


References

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

Dense habits

Rupert Sheldrake said,

"An on-going system of habits, built up through the past, and what's happened in the past. And habits have a certain density, I mean, matter is, in a sense, dense because it is so deeply habitual. There's a sense in which habits are the basis of the kind of density of the sheer materiality of the natural world, its sheer resistance to the imagination, the fact that everything is so deeply embedded in habit. And then, left to themselves of course, habits would just fossilize, and the whole world would just become intensely, repetitively habitual. But they can't be left to themselves because there is another active process going on, which is the cosmological expansion, associated with the continued presence of chaos within the universe, which means that habits are permanently, or all the time, or at least intermissibly being interrupted by asteroids hitting the earth, Or, as we see in our own lives, our habits are being permanently being disrupted by unexpected accidents. This creates new conditions, new possibility, new vacuums where things can happen."

Intensification of LSD effects by reserpine, human studies

Antipsychotic drugs including reserpine and chlorpromazine do not always block LSD intoxication. In some cases, LSD responses are heightened after acute reserpine treatment. Three independent studies from 1957 to 1965 showed this was the case.

In 1965, Resnick studied drug-drug interactions between reserpine and LSD in 3 male volunteers. Reserpine (Serpasil) 500 mg/day was administered for two weeks, then each participant had a session with 75 ug LSD.

"All three subjects volunteered the information that, following reserpine treatment for two weeks, the experiences produced by LSD-25 were very markedly enhanced." (O. Resnick, 1965)

An article from the American Journal of Psychiatry discussed LSD and reserpine interactions in patients with chronic schizophrenia. Freedman wrote,

"In collaboration with Benton, 14 chronically schizophrenic women were tested. Two days after 10 mg of reserpine the patients felt recovered from the reserpine; the only sign of an altered brain milieu was the miosis. They then received 120 ug LSD-25 and showed prolonged and toxic reactions: marked tremor and akathisia in the majority and in 1 an occulogyric crisis. Each felt the drug was less pleasant than her control LSD-25 and that the effects lasted longer." (D.X. Freedman, 1963)

Isbell and Logan studied twelve criminals who were serving sentences for violations of the Harrison Narcotic Act. They reported that reserpine treatment does not block the LSD psychosis, and may make it worse.

“The combinations of reserpine and LSD were so disagreeable that the patients were persuaded to complete all the experiments only with the greatest difficulty. In addition to the usual symptoms experienced after LSD, the patients reported other symptoms, which seemed to be of two sorts: first, the usual side-effects of reserpine such as nasal stuffiness, nausea, diarrhea, vomiting, lethargy, weakness, and dizziness on standing, second, severer mental effects. The latter included nervousness and confusion, which exceeded that experienced after LSD alone." (H. Isbell, 1957)

Table 3 below shows the "patients worse" result, when given LSD plus reserpine.

From Reserpine

Most of the human participants had a significant change in perception with reserpine plus LSD, and the evidence here shows that reserpine gave no particular alleviation of the LSD effect. The unpleasant intensification of effects produced by reserpine plus LSD suggest that reserpine should be avoided before or during LSD use.

Reserpine releases 5-HT from bound form, and it is often cited for the monoaminergic depletion and depression theory, since it is known that normal people taking reserpine become clinically depressed.


References

Resnick, O., D.M. Krus and M. Raskin (1965). Accentuation of the psychological effects of LSD-25 in normal subjects treated with reserpine. Life Sci. 4, 1433-1437. DOI:10.1016/0024-3205(65)90022-6

FREEDMAN, D. X. (1963). Psychotomimetic drugs and brain biogenic amines. Am. J. Psychiatry. 119, 843-850.

ISBELL, H. and C.R. LOGAN (1957). Studies on the diethylamide of lysergic acid (LSD-25). II. effects of chlorpromazine, azacyclonol, and reserpine on the intensity of the LSD-reaction. AMA Arch. Neurol. Psychiatry. 77, 350-358.

Reserpine and chlorpromazine, two antipsychotic drugs

From Reserpine

In the late fifties, Dr. Nathan Kline treated schizophrenia patients with the drug reserpine, an alkaloid isolated from Rauwolfia serpentina. The antipsychotic drug chlorpromazine appeared around the same time. In 1955, Bleurer and Stoll wrote,

"...reserpine and chlorpromazine soothe and relax patients to an extent formerly unknown to the doctors writing this report. . . after 2-3 days of excessive sleeping, from which the patient can be awakened, the patient's mood is more indifferent, less impulsive, quieter, and more relaxed."

Some antipsychotic drugs including chlorpromazine and reserpine have affinity for both dopamine and serotonin receptors.


References

BLEULER, M. and W.A. STOLL 1955. Clinical use of reserpine in psychiatry: Comparison with chlorpromazine. Ann. N. Y. Acad. Sci. 61, 167-173. DOI:10.1111/j.1749-6632.1955.tb42463.x

Effect of temperature on free radicals

Electron spin resonance (ESR) signals are commonly observed in surviving mammalian tissue, especially brain and liver tissue.

Heat destroys free radicals in tissues. If a sample of liver tissue is boiled for 5 minutes, the ESR signal is lost irreversibly.

From ESR free radical signals

If temperatures are reduced, free radical content increases, and persists in frozen tissues. Long-lived solvated electrons have been observed by ESR in frozen concentrated sugar solutions. When temperatures are elevated again, the ESR signal and free radical content falls.

Color centers can be induced in crystals of simple organic compounds by irradiating at low temperature with x-rays or electrons. The visible color centers are temperature-dependent and can be bleached by warming the sample.

F-centers are electrons that are trapped within a crystal. It has been shown that F-center fluorescence is temperature-dependent. With decreasing temperature the F-center band becomes narrower and its peak position shifts to shorter wavelengths. Thus at low temperature, quantum efficiency is high, and there is more energy within the F-center band (Schulman,J.H. 1962).

In summary, the lifetime of a radical is several minutes at room temperature and several hours at -78 C. This occurs at low temperature because quantum efficiency is high, and a radical is stabilized.

As with free radicals, there is a corresponding temperature effect on the appearance of charge-transfer complexes, which are assemblies of atoms that are associated with one or more free radicals. Complexing is greatly enhanced by freezing, thus many experiments with charge-transfer complexes store the sample at -80 C or below.



Reference

Commoner B. and J. L. Ternberg. (1961). Free radicals in surviving tissues. Proc.Natl.Acad.Sci.U.S.A. 47, 1374-1384. 10.1073/pnas.47.9.1374

Schulman, J. H. and Compton, W. D. Color centers in solids. Pergamon Press: New York, 1962.

Free radicals in surviving tissues

The leaves of plants and germinating seeds contain unpaired electrons, or free radicals, which can be detected from their interaction with a magnetic field in an electron spin resonance (ESR) spectrometer. The ESR spectrometer applies an external magnetic field to the sample, and the "ESR signal" is a measurement of the absorption of microwave energy by these unpaired electrons, as the magnetic field is varied. In 1961, Commoner and Ternberg used the ESR technique to detect free radicals in mammalian tissues.

The ESR signal from the surviving tissue of the body remains for up to 100 minutes after death. In the research described below, rats, mice, and guinea pigs were sacrificed, and the surviving tissue was quickly removed and placed into an ESR spectrometer. The signals obtained from surviving samples of guinea pig tissue are shown (below). Liver, intestine, and kidneys had the largest amount of free radicals, whereas skeletal muscle had an undetectable ESR signal.

From ESR free radical signals

Liver tissue from guinea pig, rat, and mouse had a high ESR signal, as shown in the bar graphs (below).

From ESR free radical signals

The authors reasoned that the ESR signal observed in mammalian tissues might be due to the enzymatic redox activity of mitochondria. Liver and kidney organs are relatively rich in mitochondria, and gave the most robust ESR signal, whereas skeletal muscle has almost no signal and is known to contain relatively few mitochondria.


Reference

COMMONER B. and J. L. TERNBERG. (1961). Free radicals in surviving tissues. Proc. Natl. Acad. Sci. U. S. A. 47, 1374-1384. DOI:10.1073/pnas.47.9.1374