1. Mehler, M.F. and Purpura, D.P., Autism, fever, epigenetics and the locus coeruleus, Brain Research Reviews 59:383-392 (2009).
2. Witter et al., In utero beta2 adrenergic agonist exposure and adverse neurophysiologic and behavioral outcomes. American J. of Obstetrics & Gynecology December 2009: 553-559 (2009).
3. Rasmussen et at., Crystal structure of the human β2-adrenergic G-protein-coupled receptor .Nature 450:383-388 (2007).
4. Warne et al., Structure of a β1-adrenergic G-protein-coupled receptor. Nature 454:486-491 (2008).
5. Scheerer et al., Crystal structure of Opsin in its G-protein-interacting conformation, Nature 455: 497-502 (2009).
6. Cherezov et al., High-resolution crystal structure of an engineered human of the human β2-adrenergic G-protein-coupled receptor. Science 318:1258-1265 (2007).
7. Hanson et al., A specific cholesterol binding site is established by the 2.8 Angstrom structure of the human β2-adrenergic receptor Structure 16:897-905 (2008).
www.rcsb.org Rutgers Center for Structural Biology. Database of biological structures.
Concise answers to the Study Guide questions and molecular images resulting from the modeling exercises are available on the Proteopedia website. Simply type in ‘autism’ in the search bar.
1. Autism & the Catecholamine System. Mehlers & Purpura draw attention to parent reports of the ability to communicate sometimes improving for autistic individuals suffering from fever or having an epileptic seizure. This raises the question of how such complex neurobiological activities as speech and language production are affected by these events. They hypothesize that the locus coeruleus-noradrenergc system (LC-NR) is restored to normality during these episodes. Where is the locus coeruleus? What is the structure of norephinephrine (noradrenaline)? How many neurons are in the LC? To what regions of the brain do they project (i.e. where are the NR varicosities?)? What brain regions project (afferents) to the LC? Explain why prenatal stress exposure can affect post-natal mental life of the child (HINT: cortisol synthesis might be epigenetically marked during pregnancy). Do data collected on stressed MeCP2-mutated mice support this hypothesis? Stress in modern life is increasing; stress influences the LC-NR system; autism is on the rise. Is this a significant correlation?
Witter et al. discuss the possibility that prenatal exposure to the bronchodilator terbutaline used to prevent premature uterine contractions can give rise to autism. Terbutaline, a substance banned by the world anti-doping agency for use by Olympic athletes, is a β2-adrenergic receptor agonist. Compare the structures of terbutaline, noradrenaline, and adrenaline. How do Witter et al. suppose that abnormal β2-adrenergic receptor activation causes permanent changes in the brain and peripheral tissues? (HINT: consider the targets of the second messenger system). What is the normal physiological mechanism that controls β2-adrenergic receptor activity in response to over-stimulation? (HINT: acetylcholine system can counteract the catecholamine system in the cardiovascular system). Why doesn’t this regulatory system work for individuals with autism according to Witter et al. (HINT: what is parasympathetic tone? )? What are the most convincing clinical studies cited by Witter et al. to support their theory? What are the most convincing animal studies cited? What polymorphisms in the β2-adrenergic receptor gene are risk factors for terbutaline sensitivity? Asthma is on the increase; pregnant women take anti-asthma drugs; autism is on the increase. Is this a significant correlation?
2. The structure of β2-adrenergic receptor with β-blockers. What is the mechanism by which β-blockers reduce blood pressure? (HINT: the renin-angiotensin system). GPCR’s are the largest class of membrane proteins in the human genome and are the pharmacological targets of drugs accounting for 50% of dollar sales. There are 670 GPCR’s in the human genome. They are notoriously difficult to crystallize even in detergent-containing solutions. How did Rassmussen et al. get around this problem? (HINT: use β-blocker to stabilize and FAB to form crystal contacts).
We have seen repeatedly that the key to interpreting crystal structures is to compare members of a homologous class. Rhodopsin transduces light into conformational changes that trigger G-protein coupled metabolic changes. It is the paradigm member of this class. Compare the binding interactions involving carazolol with β2AR and 11-cis-retinal with rhodopsin (see also paper by Cherezov et al).
One shortcoming of the original β2AR:carazolol structure is that the extracellular loops are disordered on β2AR. The Cherezov et al. paper and a recent one by Warne et al. (β1AR with a bound β-blocker) report ordered electron density for this region. Using SPDBV, prepare illustrations of the differences between ECL2 of the β1AR and β2AR structures with the same region of rhodopsin (figure 2). What notable interaction occurs between the E(D)RY motif and the ECL2 loop of β1AR? Adrenaline and noradrenaline bind to β1AR. Describe the binding site on β1AR for the antagonist (β-blocker) cyanapindolol and compare it with the binding of adrenaline. Is it apparent why one is an antagonist while the other is able to initiate changes felt 30 Angstroms away at the Gα binding surface? An important aim of pharmaceutical development is to find selective β-blockers. Does the paper by Warne et al. offer any clues suggesting how this specificity might be achieved?
Warne et al. dispute the importance of the D(E)RY ‘ionic lock’ for maintaining the inactive state. What is their justification?
3. Rhodopsin Activation of Heterotrimeric G-proteins. Describe what happens to the bound 11-cis-retinal moiety following the absorption of a photon by rhodopsin. How does opsin (the protein part) react to retinal isomerization? (HINT: consider what happens to the conserved E(D)RY motif in forming the ‘ionic lock’ --- see fig 3 of Rasmussen et al.). When a GPCR is activated (by agonist binding in the case of β-adrenergic receptors or light in the case of rhodopsin), a site is opened up on the cytoplasmic surface for the binding of G-proteins. At low pH opsin can adopt a retinal-free, but still activated state (ops*) that can bind G-proteins.
Scheerer et al. succeeded in obtaining a crystal structure of ops* with a bound peptide derived from the the COOH-terminal 20 amino acid residues of Gα (GαCT). How were these crystals prepared? All CPCR’s are based on the seven helix trans-membrane fold. A notable difference amongst the β-adrenergic receptors and rhodopsin is a swinging out of the cytoplasmic half of TM-6, a conformational change associated with G-protein activation. What amino acid is at the pivot point of this conformational change (it is not a glycine!)? The binding of GαCT is enabled by changes in two conserved regions of the rhodopsin family: the previously mentioned E(D)RY motif and NPxxY(x)5,6F, a region encompassing the end of TM7 and the beginning of helix 8. Essentially, R135 breaks its two salt-bridges, and forms new interactions with the bound peptide and the TM7-helix 8 kink. Using SPDBV model the interactions that R135 makes with Y223 on TM5 and C347 on the GαCT.
The GαCT has a COOH-terminal ‘cap’. Using SPDBV, model the cap – that is, show the sidechain-mainchain interaction that stabilizes the end of the helix. Where do the ‘cap’ residues fall on a Ramachandran plot?
4. Structural Analysis of Cholesterol Binding to β2AR. The crystal structure of the engineered β2AR-4TL:carazolol complex (Cherezov et al.) revealed an unusual mode of dimerization in which the principal crystallographic interaction between complexes was mediated by cholesterol molecules. The question thus presented itself: were these physiologically relevant? This was resolved with the determination of a mutated structure engineered along similar lines, but with a different bound β2AR-blocker (timolol). In this structure the cholesterol molecules were not part of an interface, yet still bound at the same location on the surface of the protein. This enabled the definition of GPCR class A cholesterol consensus motif (CCM). Using SPDBV produce several different images showing how cholesterol is bound by this motif. What is the role of arginine residues? To get rid of the pesky lysozyme structure – delete residues with numbers over 1000. What biophysical experiment validates the role of the CCM in binding cholesterol? (HINT: calorimetry)
- Return to A Structural Biologist Looks at Autism: A Course of Study -