Movement Of Transmembrane Helix Research Articles

Structural basis of GABAB receptor\u2013Gi protein coupling

G-protein-coupled receptors (GPCRs) have central roles in intercellular communication1,2. Structural studies have revealed how GPCRs can activate G proteins. However, whether this mechanism is conserved among all classes of GPCR remains unknown. Here we report the structure of the class-C heterodimeric GABAB receptor, which is activated by the inhibitory transmitter GABA, in its active form complexed with Gi1 protein. We found that a single G protein interacts with the GB2 subunit of the GABAB receptor at a site that mainly involves intracellular loop 2 on the side of the transmembrane domain. This is in contrast to the G protein binding in a central cavity, as has been observed with other classes of GPCR. This binding mode results from the active form of the transmembrane domain of this GABAB receptor being different from that of other GPCRs, as it shows no outside movement of transmembrane helix 6. Our work also provides details of the inter- and intra-subunit changes that link agonist binding to G-protein activation in this heterodimeric complex.

Activation of the GLP-1 receptor by a non-peptidic agonist.

Class B G-protein-coupled receptors are major targets for the treatment of chronic diseases, including diabetes and obesity1. Structures of active receptors reveal peptide agonists engage deep within the receptor core, leading to an outward movement of extracellular loop 3 and the tops of transmembrane helices 6 and 7, an inward movement of transmembrane helix 1, reorganization of extracellular loop 2 and outward movement of the intracellular side of transmembrane helix 6, resulting in G-protein interaction and activation2-6. Here we solved the structure of a non-peptide agonist, TT-OAD2, bound to the glucagon-like peptide-1 (GLP-1) receptor. Our structure identified an unpredicted non-peptide agonist-binding pocket in which reorganization of extracellular loop 3 and transmembrane helices 6 and 7 manifests independently of direct ligand interaction within the deep transmembrane domain pocket. TT-OAD2 exhibits biased agonism, and kinetics of G-protein activation and signalling that are distinct from peptide agonists. Within the structure, TT-OAD2 protrudes beyond the receptor core to interact with the lipid or detergent, providing an explanation for the distinct activation kinetics that may contribute to the clinical efficacy of this compound series. This work alters our understanding of the events that drive the activation of class B receptors.

X-ray structures of endothelin ETB receptor bound to clinical antagonist bosentan and its analog.

Endothelin receptors (ETRs) have crucial roles in vascular control and are targets for drugs designed to treat circulatory-system diseases and cancer progression. The nonpeptide dual-ETR antagonist bosentan is the first oral drug approved to treat pulmonary arterial hypertension. Here we report crystal structures of human endothelin ETB receptor bound to bosentan and to the ETB-selective analog K-8794, at 3.6-Å and 2.2-Å resolution, respectively. The K-8794-bound structure reveals the detailed water-mediated hydrogen-bonding network at the transmembrane core, which could account for the weak negative allosteric modulation of ETB by Na+ ions. The bosentan-bound structure reveals detailed interactions with ETB, which are probably conserved in the ETA receptor. A comparison of the two structures shows unexpected similarity between antagonist and agonist binding. Despite this similarity, bosentan sterically prevents the inward movement of transmembrane helix 6 (TM6), and thus exerts its antagonistic activity. These structural insights will facilitate the rational design of new ETR-targeting drugs.

The Principles of Ligand Specificity on beta-2-adrenergic receptor

G protein-coupled receptors are recognized as one of the largest families of membrane proteins. Despite sharing a characteristic seven-transmembrane topology, G protein-coupled receptors regulate a wide range of cellular signaling pathways in response to various physical and chemical stimuli, and prevail as an important target for drug discovery. Notably, the recent progress in crystallographic methods led to a breakthrough in elucidating the structures of membrane proteins. The structures of β2-adrenergic receptor bound with a variety of ligands provide atomic details of the binding modes of agonists, antagonists and inverse agonists. In this study, we selected four representative molecules from each functional class of ligands and investigated their impacts on β2-adrenergic receptor through a total of 12 × 100 ns molecular dynamics simulations. From the obtained trajectories, we generated molecular fingerprints exemplifying propensities of protein-ligand interactions. For each functional class of compounds, we characterized and compared the fluctuation of the protein backbone, the volumes in the intracellular pockets, the water densities in the receptors, the domain interaction networks as well as the movements of transmembrane helices. We discovered that each class of ligands exhibits a distinct mode of interactions with mainly TM5 and TM6, altering the shape and eventually the state of the receptor. Our findings provide insightful prospective into GPCR targeted structure-based drug discoveries.

Rescue of Na+ Affinity in Aspartate 928 Mutants of Na+,K+-ATPase by Secondary Mutation of Glutamate 314

The Na(+),K(+)-ATPase binds Na(+) at three transport sites denoted I, II, and III, of which site III is Na(+)-specific and suggested to be the first occupied in the cooperative binding process activating phosphorylation from ATP. Here we demonstrate that the asparagine substitution of the aspartate associated with site III found in patients with rapid-onset dystonia parkinsonism or alternating hemiplegia of childhood causes a dramatic reduction of Na(+) affinity in the α1-, α2-, and α3-isoforms of Na(+),K(+)-ATPase, whereas other substitutions of this aspartate are much less disruptive. This is likely due to interference by the amide function of the asparagine side chain with Na(+)-coordinating residues in site III. Remarkably, the Na(+) affinity of site III aspartate to asparagine and alanine mutants is rescued by second-site mutation of a glutamate in the extracellular part of the fourth transmembrane helix, distant to site III. This gain-of-function mutation works without recovery of the lost cooperativity and selectivity of Na(+) binding and does not affect the E1-E2 conformational equilibrium or the maximum phosphorylation rate. Hence, the rescue of Na(+) affinity is likely intrinsic to the Na(+) binding pocket, and the underlying mechanism could be a tightening of Na(+) binding at Na(+) site II, possibly via movement of transmembrane helix four. The second-site mutation also improves Na(+),K(+) pump function in intact cells. Rescue of Na(+) affinity and Na(+) and K(+) transport by second-site mutation is unique in the history of Na(+),K(+)-ATPase and points to new possibilities for treatment of neurological patients carrying Na(+),K(+)-ATPase mutations.

Obstruction of Transmembrane Helical Movements in Subunit a Blocks Proton Pumping by F1Fo ATP Synthase

Subunit a plays a key role in promoting H(+) transport-coupled rotary motion of the subunit c ring in F1Fo ATP synthase. H(+) binding and release occur at Asp-61 in the middle of the second transmembrane helix (TMH) of Fo subunit c. H(+) are thought to reach cAsp61 via aqueous half-channels formed by TMHs 2-5 of subunit a. Movements of TMH4 and TMH5 have been proposed to facilitate protonation of cAsp61 from a half channel centered in a four helix bundle at the periplasmic side of subunit a. The possible necessity of these proposed TMH movements was investigated by assaying ATP driven H(+) pumping function before and after cross-linking paired Cys substitutions at the center of TMHs within subunit a. The cross-linking of the Cys pairs aG218C/I248C in TMH4 and TMH5, and aL120C/H245C in TMH2 and TMH5, inhibited H(+) pumping by 85-90%. H(+) pumping function was largely unaffected by modification of the same Cys residues in the absence of cross-link formation. The inhibition is consistent with the proposed requirement for TMH movements during the gating of periplasmic H(+) access to cAsp61. The cytoplasmic loops of subunit a have been implicated in gating H(+) release to the cytoplasm, and previous cross-linking experiments suggest that the chemically reactive regions of the loops may pack as a single domain. Here we show that Cys substitutions in these domains can be cross-linked with retention of function and conclude that these domains need not undergo large conformational changes during enzyme function.

Recent Progress in Molecular Mechanisms of Angiotensin II Type 1 and 2 Receptors

The renin-angiotensin system hormone angiotensin II Ang II plays a central role in the pathophysiology of vasoconstriction, cardiovascular hypertrophy and hyperplasia. Two distinct subtypes of Ang II receptor, type 1 AT1 and type 2 AT2, have been identified, and both have been shown to belong to the G protein-coupled receptors (GPCRs) superfamily. AT1 and AT2 receptors may have antagonistic action. While the crystal structures of GPCRs obtained from the rhodopsin, opsin, and ß1 and ß2- adrenergic receptors have recently been described in different conformational states, the crystal structures of Ang II receptors have not been elucidated. The conformation range and dynamics of the effects of ligands on GPCRs may differ from one receptor to another. This review focuses on the structure and function of Ang II receptors, such as the movement of transmembrane helices, functional selectivity for AT1 receptor activation, the possibility of constitutive activity of wild-type Ang II receptors and the homo- and hetero-dimerization of Ang II receptors.

P-type ATPases at a glance

P-type ATPases are a large family of integral membrane transporters that are of vital importance in all kingdoms of life. In eponymous distinction from the other main classes of transport ATPase – the FF1 (F-), the vacuolar (V-) and the ATP-binding cassette (ABC-) type – the P-type ATPases form

What site-directed labeling studies tell us about the mechanism of rhodopsin activation and G-protein binding

Rhodopsin is the photoreceptor protein responsible for dim-light vision in mammals. Due to extensive biophysical, structural and computational analysis of this membrane protein, it is presently the best understood G-protein coupled receptor (GPCR). Here I briefly review one approach that has been extensively used to identify dynamic and structural changes in rhodopsin--the use of site-directed labeling methods (SDL) coupled with electron paramagnetic resonance (EPR) and fluorescence spectroscopy. These SDL studies involve introducing individual cysteine residues into the receptor, then labeling them with cysteine-reactive probes for subsequent analysis by the appropriate spectroscopy. I will give a brief overview of how SDL methods are carried out and how the data is analyzed. Then, I will discuss how SDL studies were carried out on rhodopsin, and how they were used to identify a key structural change that occurs in rhodopsin upon activation--movement of transmembrane helix 6 (TM6). I will also briefly discuss how the SDL studies of rhodopsin compare with SDL studies of other GPCRs, and compare the SDL data with early and recent crystal structures of rhodopsin. Finally, I will discuss why the TM6 movement is required for rhodopsin to couple with the G-protein transducin, and speculate how this mechanism might be a universal method used by all GPCRs to bind G-proteins and perhaps other proteins involved in visual signal transduction, such as arrestin and rhodopsin kinase.

Allosteric antagonist binding sites in class B GPCRs: corticotropin receptor 1

The 41 amino acid neuropeptide, corticotropin-releasing factor (CRF) and its associated receptors CRF(1)-R and CRF(2)-R have been targeted for treating stress related disorders. Both CRF(1)-R and CRF(2)-R belong to the class B G-protein coupled receptors for which little information is known regarding the small molecule antagonist binding characteristics. However, it has been shown recently that different non-peptide allosteric ligands stabilize different receptor conformations for CRF(1)-R and hence an understanding of the ligand induced receptor conformational changes is important in the pharmacology of ligand binding. In this study, we modeled the receptor and identified the binding sites of representative small molecule allosteric antagonists for CRF(1)-R. The predicted binding sites of the investigated compounds are located within the transmembrane (TM) domain encompassing TM helices 3, 5 and 6. The docked compounds show strong interactions with H228 on TM3 and M305 on TM5 that have also been implicated in the binding by site directed mutation studies. H228 forms a hydrogen bond of varied strengths with all the antagonists in this study and this is in agreement with the decreased binding affinity of several compounds with H228F mutation. Also mutating M305 to Ile showed a sharp decrease in the calculated binding energy whereas the binding energy loss on M305 to Leu was less significant. These results are in qualitative agreement with the decrease in binding affinities observed experimentally. We further predicted the conformational changes in CRF(1)-R induced by the allosteric antagonist NBI-27914. Movement of TM helices 3 and 5 are dominant and generates three degenerate conformational states two of which are separated by an energy barrier from the third, when bound to NBI-27914. Binding of NBI-27914 was predicted to improve the interaction of the ligand with M305 and also enhanced the aromatic stacking between the ligand and F232 on TM3. A virtual ligand screening of ~13,000 compounds seeded with ~350 CRF(1)-R specific active antagonists performed on the NBI-27914 stabilized conformation of CRF(1)-R yielded a 44% increase in enrichment compared to the initially modeled receptor conformation at a 10% cutoff. The NBI-27914 stabilized conformation also shows a high enrichment for high affinity antagonists compared to the weaker ones. Thus, the conformational changes induced by NBI-27914 improved the ligand screening efficiency of the CRF(1)-R model and demonstrate a generalized application of the method in drug discovery.

A mutational wrench in the HAMP gearbox

HAMP domains communicate between input and output signalling elements in bacterial proteins. In the Tsr chemoreceptor, they convert axial movement of transmembrane helix 2 into changes in packing of the cytoplasmic kinase-control module (KCM). Zhou et al. suggest transmembrane helix 2 'tugs' on HAMP to destabilize x-da packing of the parallel four-helix bundle of the HAMP homodimer. Attractants would inhibit tugging. HAMP stability may be inversely related to stability of the a-d packing of the anti-parallel four-helix bundle of KCM, a relationship possibly facilitated by HAMP/KCM helical mismatch. The beauty of this idea lies in its simplicity and testability.

Molecular analysis of the structure and function of the angiotensin II type 1 receptor.

The renin-angiotensin system hormone angiotensin II (Ang II) plays a central role in the pathophysiology of hypertension, cardiac hypertrophy, congestive heart failure, and coronary heart disease. Two distinct subtypes of Ang II receptor, type 1 (AT1) and type 2 (AT2), have been identified, and both have been shown to belong to the G-protein-coupled receptor superfamily (GPCRs). The recent Human Genome Project has revealed more than 1,000 transmembrane (TM) receptors that belong to this superfamily, and it has been estimated that 50% of all clinically used medicines modulate GPCRs activity. Recently, there have been many new insights regarding Ang II receptors and other GPCRs, such as on homo- and hetero-oligomerization, constitutive activation, movement of TM helices, internalization, desensitization and phosphorylation, trafficking, nuclear localization, intracellular protein-induced receptor activation, and receptor-associated proteins. Although AT1 receptor antagonists which prevent Ang II-induced signaling are already clinically available, we here summarize new findings regarding their structure and function, and the possibility of new therapeutic strategies for targeting Ang II receptors through molecular biological techniques.

Constraints of opsin structure on the ligand-binding site: studies with ring-fused retinals.

Ring-fused retinal analogs were designed to examine the hula-twist mode of the photoisomerization of the 9-cis retinylidene chromophore. Two 9-cis retinal analogs, the C11-C13 five-membered ring-fused and the C12-C14 five-membered ring-fused retinal derivatives, formed the pigments with opsin. The C11-C13 ring-fused analog was isomerized to a relaxed all-trans chromophore (lambda(max) > 400 nm) at even -269 degrees C and the Schiff base was kept protonated at 0 degrees C. The C12-C14 ring-fused analog was converted photochemically to a bathorhodopsin-like chromophore (lambda(max) = 583 nm) at -196 degrees C, which was further converted to the deprotonated Schiff base at 0 degrees C. The model-building study suggested that the analogs do not form pigments in the retinal-binding site of rhodopsin but form pigments with opsin structures, which have larger binding space generated by the movement of transmembrane helices. The molecular dynamics simulation of the isomerization of the analog chromophores provided a twisted C11-C12 double bond for the C12-C14 ring-fused analog and all relaxed double bonds with a highly twisted C10-C11 bond for the C11-C13 ring-fused analog. The structural model of the C11-C13 ring-fused analog chromophore showed a characteristic flip of the cyclohexenyl moiety toward transmembrane segments 3 and 4. The structural models suggested that hula twist is a primary process for the photoisomerization of the analog chromophores.

The C9 methyl group of retinal interacts with glycine-121 in rhodopsin.

The visual pigment rhodopsin is a prototypical G protein-coupled receptor. These receptors have seven transmembrane helices and are activated by specific receptor-ligand interactions. Rhodopsin is unusual in that its retinal prosthetic group serves as an antagonist in the dark in the 11-cis conformation but is rapidly converted to an agonist on photochemical cis to trans isomerization. Receptor-ligand interactions in rhodopsin were studied in the light and dark by regenerating site-directed opsin mutants with synthetic retinal analogues. A progressive decrease in light-dependent transducin activity was observed when a mutant opsin with a replacement of Gly121 was regenerated with 11-cis-retinal analogues bearing progressively larger R groups (methyl, ethyl, propyl) at the C9 position of the polyene chain. A progressive decrease in light activity was also observed as a function of increasing size of the residue at position 121 for both the 11-cis-9-ethyl- and the 11-cis-9-propylretinal pigments. In contrast, a striking increase of receptor activity in the dark-i.e., without chromophore isomerization-was observed when the molecular volume at either position 121 of opsin or C9 of retinal was increased. The ability of bulky replacements at either position to hinder ligand incorporation and to activate rhodopsin in the dark suggests a direct interaction between these two sites. A molecular model of the retinal-binding site of rhodopsin is proposed that illustrates the specific interaction between Gly121 and the C9 methyl group of 11-cis-retinal. Steric interactions in this region of rhodopsin are consistent with the proposal that movement of transmembrane helices 3 and 6 is concomitant with receptor activation.