1. GPCR activation: a mutagenic spotlight on crystal structures
Edward C. Hulme 
Trends in Pharmacological Sciences 
Volume 34, Issue 1, Pages (January 2013)
DOI: /j.tips
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2. Figure 1
Interactions between the residues of the G-protein-binding microdomain in the (a) inactive and (b) active states of the β2 adrenoceptor (β2AR) and (c–e) three views of the structure of the agonist–β2AR–Gs complex. Transmembrane (TM) domains are labelled. Ala substitution of the homologues of the G-protein-binding microdomain residues D2.50, I3.46, R3.50, N7.49, and Y7.53 (cyan) caused > 100-fold reductions in the M1 muscarinic acetylcholine receptor (mAChR) signalling efficacy. Side chains coloured grey are in the first shell of these core residues and include 1.50, 2.43, 2.46, 2.49, 2.53, 3.39, and 7.46, where Ala substitution caused 10- to 100-fold reductions in signalling efficacy (Table 1). (a) Inactive state (PDB 2RH1) and (b) active state (PDB 3SN6). The side chains of L3.43 and I6.40 are surrounded by dotted surfaces. Y391 (magenta) is four residues from the C terminus of the Gsα subunit. Inter-residue distances (yellow dotted lines) are given in Å. (c) R3.50 (cyan) and F3.58 (cyan) mark the upper and lower limits of Gsα contact (backbone brown, Y391 magenta) with the agonist (yellow)–receptor (rainbow) complex. There may be a contact between intracellular loop 1 (E1.61, R1.62) and D312 (grey dots) in Gβ (green). (d) TM3 (green) and ICL2 (cyan) provide the main conduit for signal transduction from ligand (yellow) to Gsα. R3.50 in the G-protein-binding microdomain of the receptor (cyan, inter-residue contacts emphasised by yellow dots) anchors the α5 helix (magenta) of Gsα at Y391. F3.58 (cyan) inserts into a pocket between H41 (αN-β1 strand, magenta), V217 (β2-β3 strand) and F376 (α5 helix). Destabilisation induces GDP (yellow spheres) dissociation, leading to opening of the Ras-α helical (αH) domain boundary and rupture of the contact between R201 (red dots) in ground-state αH (grey) (superposition of central parts of Ras domains of PDB 1AZT and 3SN6) and E50 (green dots) in the phosphate binding loop of the Ras domain. GDP contact residues are green in open-state Gsα. αH residues that swing out from the αH-Ras domain boundary are coloured red in closed ground-state Gsα. (e) The C-terminal three residues of Gsα form a reverse turn contacting residues 6.32/6.33 and 6.36/6.37 in TM6 (orange spheres) and in TM5 (yellow spheres) of the β2AR.
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3. Figure 2
Inactive (2RH1) and active (3SN6) states of the β2 adrenoceptor (β2AR) superposed using transmembrane (TM) domain 2 (V2.38–I2.65) and the proposed division of the structure into functional domains. The receptor backbone is shown as strands or ribbons. Colours denote functional domains defined as follows: anchor domain, V2.38–W99 (ECL1), blue; signal transduction domain, T100 (ECL1)–W4.50, cyan; ligand-binding domain L1, I4.51–P5.50, green; restriction domain, L5.51–W6.48, yellow; ligand-binding domain L2, L6.49–V7.44, orange; and actuator domain, G1.49–T2.37/N7.45–C348 (H8), red. The bound antagonist (carazolol) is magenta and the agonist (BI ) is yellow. Important amino acids are shown as sticks. TM domains, extracellular (EC) and intracellular (IC) loops and H8 are labelled. Elements of the structure have been removed to increase clarity. (a,b) Opposite views parallel to the bilayer showing the superposition of Trp residues. Both inactive and active states are coloured by functional domain and shown as strands. W99 in ECL1 is resolved only in the inactive state but the α-carbon positions are superposed. Ribbons show Pro-induced disruption of intra-helical hydrogen bonds. (c–e) Conformational changes in functional domains during activation. The ground state is coloured grey. (c) Signal transduction (cyan) and restriction (yellow) domains viewed parallel to the bilayer. (d) L1 (green, wide turn in TM5 labelled) and L2 (orange) ligand-binding domains viewed from the extracellular side. (e) Restriction (yellow) and actuator (red) domains viewed from the intracellular side.
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4. Figure 3
Motifs in the signal transduction domain of the β2 adrenoceptor (β2AR): transmembrane domain (TM) 3 and intracellular loop 2. (a) Stabilising motifs in the ground state of the β2AR (2RH1). Contacts between conserved side chains create an arc of contacts linking W4.50 to W6.48, channelling TM3. The conserved W in the WxxG motif in ECL1 contacts the conserved disulfide linkage. Amino acids are shown as spheres and cholesterol molecules as sticks. (b,c) A hydrogen bond (yellow dashes) between the side chain of T3.42 and the backbone CO of A3.38 reinforces a turn of helix in TM3 in the inactive (yellow, 2RH1) and active (cyan, 3SN6) states of the β2AR. The bound antagonist is magenta and the agonist is yellow. Key residues are surrounded by dotted surfaces to show contacts. (d,e) A hydrogen bond (yellow dashes) between D3.49 and Y3.60 stabilises a short helix in ICL2 in the inactive state of the β1AR (2VT4, yellow) and the active state of the β2AR (3SN6, cyan). F3.58 is in a different conformation in the two structures.
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5. Figure 4
Prolines functionalise the transmembrane (TM) domain of G-protein-coupled receptors (GPCRs) to bind ligands. A wide turn (cyan) N-terminal to P5.50 (i=0) in TM5 of the β2 adrenoceptor (β2AR) in (a) the inactive state (2RH1) and (b) the active state (3SN6). i–6–i–1 and i–7–i–2 CO–NH hydrogen bonds are shown by yellow dashes. Contacts between the extrahelical CO group of S5.46 and I3.40 (also marked by yellow dashes) are emphasised by dotted surfaces. The bound antagonist is magenta and the agonist yellow, with hydrogen bonds as yellow dots. (c) A kink (cyan) at P4.59 in TM4 of the M3 muscarinic acetylcholine receptor (mAChR) (4DAJ) positions W4.57 to contact both the bound antagonist (tiotropium, magenta) and T5.42. Hydrogen bonds and van der Waals contacts are shown as yellow dashes. (d) A hydrogen bond between the side chain of N2.59 and the backbone CO of V2.55 creates a wide turn (cyan) in TM2 of the M2 mAChR (3UON). This bond, the i–5–i hydrogen bonds, and key side-chain–ligand contacts are shown by yellow dashes. (e) Disruption (cyan) by P2.58 of helical hydrogen bonds (yellow dashes) functionalises TM2 of the κ-opioid receptor (4DJH) to bind the antagonist JDTic (magenta) at V2.53, T2.56, and Q2.60.
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6. Figure 5
The transmembrane (TM) domain 6 clamp and the activation-dependent conformational rearrangement of TM domains 5 and 6. (a,b) Orthogonal views of interactions of TM6 (orange) of the M3 mAChR (4DAJ) with residues homologous to Ala-substitutions that increase ACh affinity in the M1 muscarinic acetylcholine receptor (mAChR; Table 1). Interacting residues are shown as spheres. (a) TM3 (green); a hydrogen bond between Y5.58 and S6.38 is indicated by yellow dots. (b) TM7 (cyan). (c,d) The conformations of TM5 (yellow ribbons) and TM6 (orange ribbons) in the (c) inactive (2RH1) and (d) active (3SN6) states of the β2 adrenoceptor (β2AR). Key residues are shown as sticks. The Pro kink (cyan) in TM6 creates positions at 6.51, 6.52, and 6.55 that favour binding of (c) antagonist (magenta) and (d) agonist (yellow). (d) On activation, the Pro kink angle closes but the hydrogen bond distances from 6.40 to 6.44 and 6.44 to 6.48 are extended and F6.44 acquires a contact with M5.54. (c) Steric obstructions (dots) of TM5 by TM6 are resolved and residues (spheres) that contact the G protein, including A6.33, T6.36, and L6.37, form a coherent surface.
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7. Figure 6
The transmembrane domain 7 (TM7) anchored-knee motif and helix 8 of the β2 adrenoceptor (β2AR), and movements of the extracellular domain and TM7 that accompany activation. (a,b) Helical sections of TM7 are shown as yellow ribbons. Hydrogen bonds are indicated by yellow dashes. The H8 helix is coloured grey. Key residues and the palmitate group are shown as sticks. (a) Inactive state of β2AR (2RH1); the free loop is indicated by a cyan strand. The conserved GNxxV motif in TM1 and P7.50 are shown by green transparent spheres. (b) Activated state of β2AR (3SN6); the free loop (cyan ribbon) is reincorporated into the N-terminal domain. The C-terminal helix is partly unwound. (c) Conformation of the L1 and L2 ligand-binding domains in superpositions of the ground state (2RH1; grey), covalent agonist-ligated state (3PDS; cyan), and activated state (3SN6; orange) of the β2AR. Key side chains are indicated by sticks or lines. A hydrogen bond between TM7 and ECL2 is shown by yellow dashes. Dots around L7.37 indicate a position whose dynamics may be important for signalling. The bound agonist is indicated by yellow sticks. (d) Relationships between the cationic amine nitrogen (N+) of the ligand, the binding-site aspartate (D3.32), and key residues in TM7 in the ground (grey), covalent agonist-ligated (cyan), and active (orange) states of the β2AR. The CO group of Y7.43 is hydrogen-bonded (yellow dashes) to the NH of S7.46. Inward movements of Y7.35 are correlated with upward movements of N7.49, leading to the formation of a hydrogen bond (yellow dashes) with D2.50.
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8. Figure 7
The ligand-binding site and extracellular vestibule of the M2 and M3 muscarinic acetylcholine receptors (mAChRs). Superposition of M2 (3UON, grey) and M3 (4DAJ, cyan, rainbow) mAChRs. (a) Ligand binding residues. The antagonist QNB (3UON) is shown as yellow lines. Amino acid residues are shown as sticks. A water molecule is indicated by a green sphere. Key hydrogen bonds are shown as yellow dashed lines. (b) A view into the extracellular vestibule of the M3 mAChR (4DAJ). The Tyr residues forming the roof of the orthosteric binding pocket are shown as cyan sticks. The ligand, tiotropium, is shown as green lines. A hydrogen bond (yellow dashes) between ECL3 and ECL2 is shown. Positions of constitutively active mutations at the tops of the transmembrane (TM) helices (4.60, 5.40, 6.58, 6.59, 7.40) are indicated by magenta spheres. They interdigitate with residues involved in ligand binding (W4.57, T5.42) or in the second shell of the binding site (W7.35). (c) Location in the M3 mAChR (4DAJ) of residues (green sticks) homologous to those involved in positive allosteric modulation of acetylcholine binding in M1 and M4 mAChRs. The floor of the allosteric binding pocket is formed by tyrosine residues (cyan sticks) that form the roof of the orthosteric binding site. The residues marked in purple form a microdomain that may modulate the dynamics of the orthosteric site. An apparent cavity is shown by white mesh.
Trends in Pharmacological Sciences  , 67-84DOI: ( /j.tips )
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9. Figure 8
Intramolecular cavities in the β2 adrenoceptor (β2AR) and the M2 muscarinic acetylcholine receptor (mAChR). The receptor backbone is indicated by rainbow strands. Side chains that contribute to the active state are green, and those that form intramolecular contacts that restrict the receptor to the ground state are magenta. Key side chains are labelled. Cavities are shown as mesh surfaces. Water molecules are indicated by yellow spheres. (a) β2AR ground state (2RH1); the central cavity is surrounded by residues beneath the binding site (V3.37, W6.48; green) and in the TM6 clamp (L3.43, I6.40, F6.44; magenta) and the G-protein-binding domain (D2.50, N7.49; green) and its first shell (L2.46, A2.49, M2.53, S3.39, N7.45, S7.46; green) and contains three resolved water molecules. (b) M2 mAChR antagonist state (3UON); the key contact between L3.43 and I6.40 is shown as magenta spheres. (c) β2AR active state (3SN6) showing closure of the central cavity. (d) Superposition of β2AR ground (yellow) and active (cyan) states focusing on I3.40 and L3.43; Cδ1 of the side chain of I3.40 moves upwards by 4.9Å and L3.43 moves by 2.0Å, as indicated by white dashes.
Trends in Pharmacological Sciences  , 67-84DOI: ( /j.tips )
Copyright © 2012 Elsevier Ltd Terms and Conditions