Shivani Ahuja

Structural conversion of neurotoxic amyloid-[beta]1-42 oligomers to fibrils

The amyloid-β1–42 (Aβ42) peptide rapidly aggregates to form oligomers, protofibils and fibrils en route to the deposition of amyloid plaques associated with Alzheimers disease. We show that low-temperature and low-salt conditions can stabilize disc-shaped oligomers (pentamers) that are substantially more toxic to mouse cortical neurons than protofibrils and fibrils. We find that these neurotoxic oligomers do not have the β-sheet structure characteristic of fibrils. Rather, the oligomers are composed of loosely aggregated strands whose C termini are protected from solvent exchange and which have a turn conformation, placing Phe19 in contact with Leu34. On the basis of NMR spectroscopy, we show that the structural conversion of Aβ42 oligomers to fibrils involves the association of these loosely aggregated strands into β-sheets whose individual β-strands polymerize in a parallel, in-register orientation and are staggered at an intermonomer contact between Gln15 and Gly37.

Helix movement is coupled to displacement of the second extracellular loop in rhodopsin activation

The second extracellular loop (EL2) of rhodopsin forms a cap over the binding site of its photoreactive 11-cis retinylidene chromophore. A crucial question has been whether EL2 forms a reversible gate that opens upon activation or acts as a rigid barrier. Distance measurements using solid-state 13C NMR spectroscopy between the retinal chromophore and the β4 strand of EL2 show that the loop is displaced from the retinal binding site upon activation, and there is a rearrangement in the hydrogen-bonding networks connecting EL2 with the extracellular ends of transmembrane helices H4, H5 and H6. NMR measurements further reveal that structural changes in EL2 are coupled to the motion of helix H5 and breaking of the ionic lock that regulates activation. These results provide a comprehensive view of how retinal isomerization triggers helix motion and activation in this prototypical G protein–coupled receptor.

Location of the Retinal Chromophore in the Activated State of Rhodopsin

Rhodopsin is a highly specialized G protein-coupled receptor (GPCR) that is activated by the rapid photochemical isomerization of its covalently bound 11-cis-retinal chromophore. Using two-dimensional solid-state NMR spectroscopy, we defined the position of the retinal in the active metarhodopsin II intermediate. Distance constraints were obtained between amino acids in the retinal binding site and specific 13C-labeled sites located on the β-ionone ring, polyene chain, and Schiff base end of the retinal. We show that the retinal C20 methyl group rotates toward the second extracellular loop (EL2), which forms a cap on the retinal binding site in the inactive receptor. Despite the trajectory of the methyl group, we observed an increase in the C20-Gly188 (EL2) distance consistent with an increase in separation between the retinal and EL2 upon activation. NMR distance constraints showed that the β-ionone ring moves to a position between Met207 and Phe208 on transmembrane helix H5. Movement of the ring toward H5 was also reflected in increased separation between the Cϵ carbons of Lys296 (H7) and Met44 (H1) and between Gly121 (H3) and the retinal C18 methyl group. Helix-helix interactions involving the H3-H5 and H4-H5 interfaces were also found to change in the formation of metarhodopsin II reflecting increased retinal-protein interactions in the region of Glu122 (H3) and His211 (H5). We discuss the location of the retinal in metarhodopsin II and its interaction with sequence motifs, which are highly conserved across the pharmaceutically important class A GPCR family, with respect to the mechanism of receptor activation.

Highly conserved tyrosine stabilizes the active state of rhodopsin

Light-induced isomerization of the 11-cis-retinal chromophore in the visual pigment rhodopsin triggers displacement of the second extracellular loop (EL2) and motion of transmembrane helices H5, H6, and H7 leading to the active intermediate metarhodopsin II (Meta II). We describe solid-state NMR measurements of rhodopsin and Meta II that target the molecular contacts in the region of the ionic lock involving these three helices. We show that a contact between Arg1353.50 and Met2576.40 forms in Meta II, consistent with the outward rotation of H6 and breaking of the dark-state Glu1343.49-Arg1353.50-Glu2476.30 ionic lock. We also show that Tyr2235.58 and Tyr3067.53 form molecular contacts with Met2576.40. Together these results reveal that the crystal structure of opsin in the region of the ionic lock reflects the active state of the receptor. We further demonstrate that Tyr2235.58 and Ala1323.47 in Meta II stabilize helix H5 in an active orientation. Mutation of Tyr2235.58 to phenylalanine or mutation of Ala1323.47 to leucine decreases the lifetime of the Meta II intermediate. Furthermore, the Y223F mutation is coupled to structural changes in EL2. In contrast, mutation of Tyr3067.53 to phenylalanine shows only a moderate influence on the Meta II lifetime and is not coupled to EL2.

Light activation of rhodopsin: insights from molecular dynamics simulations guided by solid-state NMR distance restraints

Structural restraints provided by solid-state NMR measurements of the metarhodopsin II intermediate are combined with molecular dynamics simulations to help visualize structural changes in the light activation of rhodopsin. Since the timescale for the formation of the metarhodopsin II intermediate (>1 ms) is beyond that readily accessible by molecular dynamics, we use NMR distance restraints derived from 13C dipolar recoupling measurements to guide the simulations. The simulations yield a working model for how photoisomerization of the 11-cis retinylidene chromophore bound within the interior of rhodopsin is coupled to transmembrane helix motion and receptor activation. The mechanism of activation that emerges is that multiple switches on the extracellular (or intradiscal) side of rhodopsin trigger structural changes that converge to disrupt the ionic lock between helices H3 and H6 on the intracellular side of the receptor.

Structure and function of G protein-coupled receptors using NMR spectroscopy.

Nathans and Hogness [1] cloned the gene for the visual pigment rhodopsin in 1983, and found that the receptor contained seven hydrophobic membrane-spanning helices. At roughly the same time, the paradigm in the visual receptor field that rhodopsin functions as a light-activated Ca2+ channel was being overturned in favor of a competing model that rhodopsin is responsible for activating a heterotrimeric G-protein [2]. In 1986, a landmark paper by Strader, Lefkowitz and co-workers [3] reported the sequence of the β2-adrenergic receptor. They recognized that the β2-adrenergic receptor shares significant sequence homology with rhodopsin, including seven transmembrane (TM) segments. They proposed that the amine ligands of the adrenergic receptors intercalate into the TM helices and trigger receptor activation in the same manner as the retinal in rhodopsin. Because the receptors of the adenylate cyclase system were known to activate G-proteins [2,4,5], the similarity between the β2-adrenergic receptor and rhodopsin provided the first hint of the existence of a large G protein-coupled receptor (GPCR) family. Early structural work in the emerging GPCR field was guided by two pioneering studies. In 1993, Schertler, Villa and Henderson [6] reported an electron density projection map based on cryo-EM images of rhodopsin that showed the seven-helix architecture is distinctly different from that of bacteriorhodopsin, which had been a favored template for modeling GPCRs. In 1996, Hubbell and Khorana [7] used site-directed spin labeling in combination with EPR spectroscopy to reveal that activation is mediated by rigid body rotation of the TM helices. A breakthrough in the GPCR field came in 2000 with the determination of the rhodopsin crystal structure [8] (Fig. 1). The structure was solved of the dark, inactive conformation of the protein. The structure of the active state, however, has been more elusive. When illuminated, the crystals of rhodopsin crack and dissolve [9], presumably due to rigid body helix motions that accompany receptor activation. In 2007, the crystal structure of the β2-adrenergic receptor was solved with the inverse agonist carazolol bound in the active site [10]. The structure of this first ligand-activated GPCR confirmed that the overall seven-transmembrane helix architecture is similar to that of rhodopsin. Together the rhodopsin and β2-adrenergic receptor structures provide a high-resolution framework for understanding the structural features that are responsible for locking these receptors in the off conformation, and a starting point for investigating the mechanism of receptor activation. Fig. 1 Crystal structure of rhodopsin. The first crystal structure of a GPCR was obtained in 2000 by Palczewski and Okada [8]. Views of rhodopsin spanning the membrane bilayer (a) and from the extracellular (or intradiscal) surface of the membrane (b) show the ... In this review, we describe the progress that has been made using NMR spectroscopy to characterize the structure and dynamics of GPCRs in membrane environments. NMR methods have been used extensively to establish the structural changes occurring upon the activation of rhodopsin, as well as several ligand-activated receptors. We describe below the importance of these receptors in terms of their cell biology and pharmacology, and then outline the role that NMR can play in answering questions of structure and function. 1.1 Cell biology The 7-transmembrane helix GPCRs have evolved to recognize and transduce signals as diverse as light, Ca2+, small organic molecules and proteins. These receptors are found in both vertebrates and invertebrates, and are typically divided into six classes (Class A–F) based on sequence homology and functional similarity [11–13]. However, the classification is still open to debate. For example, on the basis of phylogeny, the human GPCRs have been divided into five families (Rhodopsin-like, Secretin, Adhesion, Glutamate, and Frizzled/Taste2) [14]. In this scheme, the Class A receptors correspond to the Rhodopsin-like family, but the Class B receptors are divided into the Secretin and Adhesion families. Nevertheless, in all classification schemes proposed to date, the lack of homology between classes or families suggests that nature has converged on the same seven transmembrane helix framework multiple times. The Class A (Rhodopsin-like family) receptors respond to the presence of diverse stimuli ranging from light absorption to the binding of various ligands, which include small molecule amines and hormones. Class B (Secretin and Adhesion families) receptors are activated by peptides of the glucagon hormone family [15,16]. The Class C (Glutamate family) GPCRs are comprised of the metabotropic glutamate receptors. These receptors are characterized by a large N-terminal ligand binding domain [17], which appears to be structurally homologous to the amino terminal domain of the ligand-gated ionotropic glutamate receptors in postsynaptic neuronal membranes [18]. Pheromones (e.g. α-factor) secreted by Saccharomyces cerevisiae bind to Class D GPCRs (e.g. STE2) during the mating process. Similar mechanisms are involved in the mating of several fungi [19]. Class E receptors have been implicated in the chemotactic migration of slime mold and can potentially be exploited as antifungal targets [20,21]. Class F (Frizzled/smoothened/taste2 family) contains receptors in the Wnt signaling pathway [14], which perform indispensable roles in embryonic development [22]. The Class A receptors are by far the most populated class of GPCRs. In the GPCR database there are over 20,000 Class A sequences (http://www.gpcr.org/). In humans, 952 of 1061 GPCRs identified in the human genome are in Class A. Of the 952 human Class A receptors, most (509) are olfactory receptors. The remaining Class A GPCRs are subdivided into 18 subfamilies including the well studied visual and small molecule amine receptors, as well as hormone and peptide receptors. Despite the breadth of this group, there exists a degree of sequence conservation among these receptors. Furthermore, the Class A receptors share similar intracellular proteins (e.g. protein kinases, arrestins) that mediate receptor desensitization.

6-s-cis Conformation and polar binding pocket of the retinal chromophore in the photoactivated state of rhodopsin.

The visual pigment rhodopsin is unique among the G protein-coupled receptors in having an 11-cis retinal chromophore covalently bound to the protein through a protonated Schiff base linkage. The chromophore locks the visual receptor in an inactive conformation through specific steric and electrostatic interactions. This efficient inverse agonist is rapidly converted to an agonist, the unprotonated Schiff base of all-trans retinal, upon light activation. Here, we use magic angle spinning NMR spectroscopy to obtain the (13)C chemical shifts (C5-C20) of the all-trans retinylidene chromophore and the (15)N chemical shift of the Schiff base nitrogen in the active metarhodopsin II intermediate. The retinal chemical shifts are sensitive to the conformation of the chromophore and its molecular interactions within the protein-binding site. Comparison of the retinal chemical shifts in metarhodopsin II with those of retinal model compounds reveals that the Schiff base environment is polar. In particular, the (13)C15 and (15)Nepsilon chemical shifts indicate that the C horizontal lineN bond is highly polarized in a manner that would facilitate Schiff base hydrolysis. We show that a strong perturbation of the retinal (13)C12 chemical shift observed in rhodopsin is reduced in wild-type metarhodopsin II and in the E181Q mutant of rhodopsin. On the basis of the T(1) relaxation time of the retinal (13)C18 methyl group and the conjugated retinal (13)C5 and (13)C8 chemical shifts, we have determined that the conformation of the retinal C6-C7 single bond connecting the beta-ionone ring and the retinylidene chain is 6-s-cis in both the inactive and the active states of rhodopsin. These results are discussed within the general framework of ligand-activated G protein-coupled receptors.

Structural Transitions of Transmembrane Helix 6 in the Formation of Metarhodopsin I

Absorption of light by the visual pigment rhodopsin triggers a rapid cis-trans photoisomerization of its retinal chromophore and a series of conformational changes in both the retinal and protein. The largest structural change is an outward tilt of transmembrane helix H6 that increases the separation of the intracellular ends of H6 and H3 and opens up the G-protein binding site. In the dark state of rhodopsin, Glu247 at the intracellular end of H6 forms a salt bridge with Arg135 on H3 to tether H6 in an inactive conformation. The Arg135-Glu247 interaction is broken in the active state of the receptor, and Arg135 is then stabilized by interactions with Tyr223, Met257, and Tyr306 on helices H5, H6, and H7, respectively. To address the mechanism of H6 motion, solid-state NMR measurements are undertaken of Metarhodopsin I (Meta I), the intermediate preceding the active Metarhodopsin II (Meta II) state of the receptor. (13)C NMR dipolar recoupling measurements reveal an interhelical contact of (13)Cζ-Arg135 with (13)Cε-Met257 in Meta I but not with (13)Cζ-Tyr223 or (13)Cζ-Tyr306. These observations suggest that helix H6 has rotated in the formation of Meta I but that structural changes involving helices H5 and H7 have not yet occurred. Together, our results provide insights into the sequence of events leading up to the outward motion of H6, a hallmark of G protein-coupled receptor activation.