Structural insights into the extracellular recognition of the human serotonin 2B receptor by an antibody

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Abstract

Monoclonal antibodies provide an attractive alternative to small-molecule therapies for a wide range of diseases. Given the importance of G protein-coupled receptors (GPCRs) as pharmaceutical targets, there has been an immense interest in developing therapeutic monoclonal antibodies that act on GPCRs. Here we present the 3.0-Å resolution structure of a complex between the human 5-hydroxytryptamine 2B (5-HT2B) receptor and an antibody Fab fragment bound to the extracellular side of the receptor, determined by serial femtosecond crystallography with an X-ray free-electron laser. The antibody binds to a 3D epitope of the receptor that includes all three extracellular loops. The 5-HT2B receptor is captured in a well-defined active-like state, most likely stabilized by the crystal lattice. The structure of the complex sheds light on the mechanism of selectivity in extracellular recognition of GPCRs by monoclonal antibodies.

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Crystal structure of the human beta2 adrenergic G-protein-coupled receptor.

Søren G F Rasmussen, Hee-Jung Choi, Daniel M Rosenbaum … (2007)

Structural analysis of G-protein-coupled receptors (GPCRs) for hormones and neurotransmitters has been hindered by their low natural abundance, inherent structural flexibility, and instability in detergent solutions. Here we report a structure of the human beta2 adrenoceptor (beta2AR), which was crystallized in a lipid environment when bound to an inverse agonist and in complex with a Fab that binds to the third intracellular loop. Diffraction data were obtained by high-brilliance microcrystallography and the structure determined at 3.4 A/3.7 A resolution. The cytoplasmic ends of the beta2AR transmembrane segments and the connecting loops are well resolved, whereas the extracellular regions of the beta2AR are not seen. The beta2AR structure differs from rhodopsin in having weaker interactions between the cytoplasmic ends of transmembrane (TM)3 and TM6, involving the conserved E/DRY sequences. These differences may be responsible for the relatively high basal activity and structural instability of the beta2AR, and contribute to the challenges in obtaining diffraction-quality crystals of non-rhodopsin GPCRs.

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Structure-function of the G protein-coupled receptor superfamily.

Vsevolod Katritch, Vadim Cherezov, Raymond C. Stevens (2013)

During the past few years, crystallography of G protein-coupled receptors (GPCRs) has experienced exponential growth, resulting in the determination of the structures of 16 distinct receptors-9 of them in 2012 alone. Including closely related subtype homology models, this coverage amounts to approximately 12% of the human GPCR superfamily. The adrenergic, rhodopsin, and adenosine receptor systems are also described by agonist-bound active-state structures, including a structure of the receptor-G protein complex for the β(2)-adrenergic receptor. Biochemical and biophysical techniques, such as nuclear magnetic resonance and hydrogen-deuterium exchange coupled with mass spectrometry, are providing complementary insights into ligand-dependent dynamic equilibrium between different functional states. Additional details revealed by high-resolution structures illustrate the receptors as allosteric machines that are controlled not only by ligands but also by ions, lipids, cholesterol, and water. This wealth of data is helping redefine our knowledge of how GPCRs recognize such a diverse array of ligands and how they transmit signals 30 angstroms across the cell membrane; it also is shedding light on a structural basis of GPCR allosteric modulation and biased signaling.

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Activation and allosteric modulation of a muscarinic acetylcholine receptor

Andrew C. Kruse, Aaron Ring, Aashish Manglik … (2013)

Despite recent advances in crystallography of G protein-coupled receptors (GPCRs), little is known about the mechanism of their activation process, as only the β2 adrenergic receptor (β2AR) and rhodopsin have been crystallized in fully active conformations. Here, we report the structure of an agonist-bound, active state of the human M2 muscarinic acetylcholine receptor stabilized by a G-protein mimetic camelid antibody fragment isolated by conformational selection using yeast surface display. In addition to the expected changes in the intracellular surface, the structure reveals larger conformational changes in the extracellular region and orthosteric binding site than observed in the active states of the β2AR and rhodopsin. We also report the structure of the M2 receptor simultaneously binding the orthosteric agonist iperoxo and the positive allosteric modulator LY2119620. This structure reveals that LY2119620 recognizes a largely pre-formed binding site in the extracellular vestibule of the iperoxo-bound receptor, inducing a slight contraction of this outer binding pocket. These structures offer important insights into activation mechanism and allosteric modulation of muscarinic receptors.