G protein-coupled receptors (GPCRs) in the G protein-coupled active state have higher affinity for agonists as compared with when they are in the inactive state, but the molecular basis for this is ...unclear. We have determined four active-state structures of the β
-adrenoceptor (β
AR) bound to conformation-specific nanobodies in the presence of agonists of varying efficacy. Comparison with inactive-state structures of β
AR bound to the identical ligands showed a 24 to 42% reduction in the volume of the orthosteric binding site. Potential hydrogen bonds were also shorter, and there was up to a 30% increase in the number of atomic contacts between the receptor and ligand. This explains the increase in agonist affinity of GPCRs in the active state for a wide range of structurally distinct agonists.
Adenosine receptors and β-adrenoceptors are G-protein-coupled receptors (GPCRs) that activate intracellular G proteins on binding the agonists adenosine or noradrenaline, respectively. GPCRs have ...similar structures consisting of seven transmembrane helices that contain well-conserved sequence motifs, indicating that they are probably activated by a common mechanism. Recent structures of β-adrenoceptors highlight residues in transmembrane region 5 that initially bind specifically to agonists rather than to antagonists, indicating that these residues have an important role in agonist-induced activation of receptors. Here we present two crystal structures of the thermostabilized human adenosine A(2A) receptor (A(2A)R-GL31) bound to its endogenous agonist adenosine and the synthetic agonist NECA. The structures represent an intermediate conformation between the inactive and active states, because they share all the features of GPCRs that are thought to be in a fully activated state, except that the cytoplasmic end of transmembrane helix 6 partially occludes the G-protein-binding site. The adenine substituent of the agonists binds in a similar fashion to the chemically related region of the inverse agonist ZM241385 (ref. 8). Both agonists contain a ribose group, not found in ZM241385, which extends deep into the ligand-binding pocket where it makes polar interactions with conserved residues in H7 (Ser 277(7.42) and His 278(7.43); superscripts refer to Ballesteros-Weinstein numbering) and non-polar interactions with residues in H3. In contrast, the inverse agonist ZM241385 does not interact with any of these residues and comparison with the agonist-bound structures indicates that ZM241385 sterically prevents the conformational change in H5 and therefore it acts as an inverse agonist. Comparison of the agonist-bound structures of A(2A)R with the agonist-bound structures of β-adrenoceptors indicates that the contraction of the ligand-binding pocket caused by the inward motion of helices 3, 5 and 7 may be a common feature in the activation of all GPCRs.
The catalytic domain of the F-ATPase in mitochondria protrudes into the matrix of the organelle, and is attached to the membrane domain by central and peripheral stalks. Energy for the synthesis of ...ATP from ADP and phosphate is provided by the transmembrane proton-motive-force across the inner membrane, generated by respiration. The proton-motive force is coupled mechanically to ATP synthesis by the rotation at about 100 times per second of the central stalk and an attached ring of c-subunits in the membrane domain. Each c-subunit carries a glutamate exposed around the midpoint of the membrane on the external surface of the ring. The rotation is generated by protonation and deprotonation successively of each glutamate. Each 360° rotation produces three ATP molecules, and requires the translocation of one proton per glutamate by each c-subunit in the ring. In fungi, eubacteria, and plant chloroplasts, ring sizes of c₁₀—c₁₅ subunits have been observed, implying that these enzymes need 3.3—5 protons to make each ATP, but until now no higher eukaryote has been examined. As shown here in the structure of the bovine F₁-c-ring complex, the c-ring has eight c-subunits. As the sequences of c-subunits are identical throughout almost all vertebrates and are highly conserved in invertebrates, their F-ATPases probably contain c₈-rings also. Therefore, in about 50,000 vertebrate species, and probably in many or all of the two million invertebrate species, 2.7 protons are required by the F-ATPase to make each ATP molecule.
The structure of the intact ATP synthase from the α-proteobacterium Paracoccus denitrificans, inhibited by its natural regulatory ζ-protein, has been solved by X-ray crystallography at 4.0 Å ...resolution. The ζ-protein is bound via its N-terminal α-helix in a catalytic interface in the F1 domain. The bacterial F1 domain is attached to the membrane domain by peripheral and central stalks. The δ-subunit component of the peripheral stalk binds to the N-terminal regions of two α-subunits. The stalk extends via two parallel long α-helices, one in each of the related b and b' subunits, down a noncatalytic interface of the F1 domain and interacts in an unspecified way with the a-subunit in the membrane domain. The a-subunit lies close to a ring of 12 c-subunits attached to the central stalk in the F1 domain, and, together, the central stalk and c-ring form the enzyme's rotor. Rotation is driven by the transmembrane proton-motive force, by a mechanism where protons pass through the interface between the a-subunit and c-ring via two half-channels in the a-subunit. These half-channels are probably located in a bundle of four α-helices in the a-subunit that are tilted at ∼30° to the plane of the membrane. Conserved polar residues in the two α-helices closest to the c-ring probably line the proton inlet path to an essential carboxyl group in the c-subunit in the proton uptake site and a proton exit path from the proton release site. The structure has provided deep insights into the workings of this extraordinary molecular machine.
MicroED uses very small three-dimensional protein crystals and electron diffraction for structure determination. We present an improved data collection protocol for MicroED called 'continuous ...rotation'. Microcrystals are continuously rotated during data collection, yielding more accurate data. The method enables data processing with the crystallographic software tool MOSFLM, which resulted in improved resolution for the model protein lysozyme. These improvements are paving the way for the broad implementation and application of MicroED in structural biology.
G-protein-coupled receptors (GPCRs) are essential components of the signalling network throughout the body. To understand the molecular mechanism of G-protein-mediated signalling, solved structures ...of receptors in inactive conformations and in the active conformation coupled to a G protein are necessary. Here we present the structure of the adenosine A(2A) receptor (A(2A)R) bound to an engineered G protein, mini-Gs, at 3.4 Å resolution. Mini-Gs binds to A(2A)R through an extensive interface (1,048 Å2) that is similar, but not identical, to the interface between Gs and the β2-adrenergic receptor. The transition of the receptor from an agonist-bound active-intermediate state to an active G-protein-bound state is characterized by a 14 Å shift of the cytoplasmic end of transmembrane helix 6 (H6) away from the receptor core, slight changes in the positions of the cytoplasmic ends of H5 and H7 and rotamer changes of the amino acid side chains Arg3.50, Tyr5.58 and Tyr7.53. There are no substantial differences in the extracellular half of the receptor around the ligand binding pocket. The A(2A)R-mini-Gs structure highlights both the diversity and similarity in G-protein coupling to GPCRs and hints at the potential complexity of the molecular basis for G-protein specificity.
The objective of any modern data‐processing program is to produce from a set of diffraction images a set of indices (hkls) with their associated intensities (and estimates of their uncertainties), ...together with an accurate estimate of the crystal unit‐cell parameters. This procedure should not only be reliable, but should involve an absolute minimum of user intervention. The process can be conveniently divided into three stages. The first (autoindexing) determines the unit‐cell parameters and the orientation of the crystal. The unit‐cell parameters may indicate the likely Laue group of the crystal. The second step is to refine the initial estimate of the unit‐cell parameters and also the crystal mosaicity using a procedure known as post‐refinement. The third step is to integrate the images, which consists of predicting the positions of the Bragg reflections on each image and obtaining an estimate of the intensity of each reflection and its uncertainty. This is carried out while simultaneously refining various detector and crystal parameters. Basic features of the algorithms employed for each of these three separate steps are described, principally with reference to the program MOSFLM.
Adenosine triphosphate (ATP) synthase contains a rotary motor involved in biological energy conversion. Its membrane-embedded FO sector has a rotation generator fueled by the proton-motive force, ...which provides the energy required for the synthesis of ATP by the F$_1$ domain. An electron density map obtained from crystals Of a subcomplex of yeast mitochondrial ATP synthase shows a ring of 10 c subunits. Each c subunit forms an α-helical hairpin. The interhelical loops of six to seven of the c subunits are in dose contact with the γ and δ subunits of the central stalk. The extensive contact between the c ring and the stalk suggests that they may rotate as an ensemble during catalysis.
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