Structural insights into the extracellular recognition of the human serotonin 2B receptor by an antibody Andrii Ishchenkoa, Daniel Wackerb, Mili Kapoorc,1, Ai Zhangc, Gye Won Hana, Shibom Basud,2, Nilkanth Patele, Marc Messerschmidtf, Uwe Weierstallg, Wei Liuh, Vsevolod Katritche, Bryan L. Rothb,i,j, Raymond C. Stevensa,e, and Vadim Cherezova,3 aDepartment of Chemistry, Bridge Institute, University of Southern California, Los Angeles, CA 90089; bDepartment of Pharmacology, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, NC 27599-7365; cDepartment of Integrative Structural, and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037; dDepartment of Chemistry and Biochemistry, Arizona State University, Tempe, AZ 85281; eDepartment of Biological Sciences, Bridge Institute, University of Southern California, Los Angeles, CA 90089; fBioXFEL Science and Technology Center, Buffalo, NY 14203; gDepartment of Physics, Arizona State University, Tempe, AZ 85281; hSchool of Molecular Sciences, Biodesign Center for Applied Structural Discovery, Biodesign Institute, Arizona State University, Tempe, AZ 85287; iDivision of Chemical Biology and Medicinal Chemistry, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7365; and jNational Institute of Mental Health Psychoactive Drug Screening Program, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7365 Edited by K. Christopher Garcia, Stanford University, Stanford, CA, and approved June 21, 2017 (received for review January 17, 2017) Monoclonal antibodies provide an attractive alternative to small- the agonist ergotamine (ERG) and a selective antibody Fab molecule therapies for a wide range of diseases. Given the impor- fragment, and solved its structure by serial femtosecond crystallog- tance of G protein-coupled receptors (GPCRs) as pharmaceutical raphy (SFX) with an X-ray free-electron laser (XFEL). Given that targets, there has been an immense interest in developing therapeutic all previous structural information on GPCR recognition is limited monoclonal antibodies that act on GPCRs. Here we present the 3.0-Å to Fabs/nanobodies bound to the intracellular side of the receptors resolution structure of a complex between the human 5-hydroxy- (13–17), this work provides unprecedented molecular insights into tryptamine 2B (5-HT2B) receptor and an antibody Fab fragment bound the 3D antibody-binding extracellular epitope of the receptor. to the extracellular side of the receptor, determined by serial femto- Moreover, whereas previous 5-HT2B/ERG structures revealed con- second crystallography with an X-ray free-electron laser. The anti- formational characteristics of an intermediate receptor activation BIOCHEMISTRY body binds to a 3D epitope of the receptor that includes all three state (18, 19), the 5-HT2B/ERG-Fab structure captures the receptor extracellular loops. The 5-HT2B receptor is captured in a well-defined in a distinct active-like state, which shows extensive activation-related active-like state, most likely stabilized by the crystal lattice. The struc- changes throughout the receptor manifested in triggered “micro- ture of the complex sheds light on the mechanism of selectivity in switches” and concerted movements of helices VI and VII. extracellular recognition of GPCRs by monoclonal antibodies. Results GPCR | active state | antibody recognition | X-ray free-electron laser | Antibody Binding Characterization. The monoclonal antibody serial femtosecond crystallography P2C2-IgG raised against human 5-HT2B receptor bound to ERG was received as a gift from Bird Rock Bio, San Diego, CA. ntibodies comprise hypervariable domains that evolve in re- P2C2 heavy and light chains were subcloned to enable expression – sponse to antigenic stimuli, making them capable of selectively of a P2C2 Fab fragment in insect cells. ELISA experiments A – binding virtually any macromolecule. Thanks to their potentially confirmed that the purified P2C2 Fab binds selectively to the high affinity, selectivity, long duration of action, and engineered extracellular epitope of the human 5-HT2B receptor, both in the ability to penetrate the blood–brain barrier, monoclonal antibodies (mAbs) provide an attractive alternative to small-molecule thera- Significance pies (1). G protein-coupled receptors (GPCRs) represent highly attractive targets for therapeutic mAbs because of their involvement Highly selective monoclonal antibodies recognizing the extra- in signal transduction, associated with many important diseases, and cellular 3D epitope of G protein-coupled receptors represent their localization in the cell plasma membrane (2). Production of valuable tools for elucidating receptor function and localiza- mAbs against GPCRs, however, is a challenging task because of tion in the cell and show promise for a range of therapeutic difficulties in obtaining sufficient quantities of solubilized, purified, applications. Here we present the structure of a complex be- and functional antigen. The most abundant class A GPCRs are tween the human serotonin 2B receptor, captured in an active- characterized by very small solvent-exposed domains, making pro- like state, and an antibody Fab fragment, bound to the extra- duction of high affinity, selective mAbs even more difficult (3–7). cellular side of the receptor. The structure uncovers the Furthermore, antibodies can recognize GPCRs in different func- mechanisms of receptor activation and of extracellular receptor tional states, depending on the presence of agonists or antagonists recognition by antibodies. (8). Some mAbs can also recognize specific receptor conformations independent of ligand presence, and there is a potential for iden- Author contributions: A.I., D.W., B.L.R., R.C.S., and V.C. designed research; A.I., D.W., M.K., “ ” A.Z., M.M., U.W., and W.L. performed research; B.L.R. contributed new reagents/analytic tification of biased mAbs that can selectively guide receptor ac- tools; A.I., D.W., G.W.H., S.B., N.P., V.K., R.C.S., and V.C. analyzed data; and A.I., V.K., and tivity to specific signaling pathways (3, 9). The first anti-GPCR V.C. wrote the paper. therapeutic mAb, mogamulizumab (KW-0761, POTELIGEO), The authors declare no conflict of interest. targeting the CCR4 receptor, was approved in 2012 in Japan for the This article is a PNAS Direct Submission. treatment of relapsed or refractory adult T-cell leukemia-lymphoma Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, (10). Currently, there are more than a dozen mAbs targeting ex- www.pdb.org (PDB ID code 5TUD). tracellular sites of GPCRs in different stages of clinical trials for the 1Present address: Amplyx Pharamaceuticals Inc., San Diego, CA 92121. treatment of HIV, inflammation and immune disorders, athero- 2Present address: Department of Synchrotron Radiation and Nanotechnology, sclerosis, cancer, and other major chronic diseases (11, 12). Paul-Scherer Institute, Swiss Light Source, 5232 Villigen, Switzerland. To gain insight into the molecular basis of extracellular rec- 3To whom correspondence should be addressed. Email: [email protected]. ognition of GPCRs by mAbs, we crystallized a complex between This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. the human 5-hydroxytryptamine 2B (5-HT2B) receptor bound to 1073/pnas.1700891114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1700891114 PNAS | August 1, 2017 | vol. 114 | no. 31 | 8223–8228 Downloaded by guest on September 29, 2021 presence (pKd = −8.50 ± 0.26) and absence (pKd = −8.86 ± 0.28) of ECL2, together forming a three-stranded β-sheet (Fig. 1A). In- of ERG (Fig. S1A). terestingly, the tip of CDR-H3 extends toward the lipid membrane, likely anchoring its hydrophobic residues Ile106 and Leu107 in the Structure of the 5-HT2B/ERG-Fab Complex. For structural studies, we membrane near helices III and IV of the receptor. CDR- used the same engineered construct of the human 5-HT2B receptor as H2 interacts mostly with ECL2 and ECL3. Asn50 (CDR-H2) forms ECL2 the one previously used to obtain 5-HT2B/ERG structures (18, 19). ahydrogenbondwithAsp198 , Tyr52 (CDR-H2) with Size-exclusion chromatography (SEC) and pull-down assays con- Glu212ECL2, and Ser54 (CDR-H2) with Gln3557.28 (Fig. 1B). CDR- firmed formation of a complex between purified receptor and anti- H1 interacts only with ECL2 and has the least number of contacts. ECL2 body (Fig. S2). The 5-HT2B/ERG-Fab structure was determined at Thr30(CDR-H1)makesahydrogenbondwithGlu212 ,and 3.0-Å resolution (Fig. 1, Fig. S3,andTable S1). The complex was Trp33 (CDR-H1) makes a nonpolar contact with Asp198ECL2 (Fig. crystallized in lipidic cubic phase (LCP) (Fig. S3 A and B)inaP21 1C). The Fab light chain, specifically CDR-L3, also forms multiple space group, with two complexes (chains ABC and DEF) per hydrogen bonds and salt bridges with the receptor (Fig. 1D). On the asymmetric unit (Fig. S4). Because both structures are nearly identical receptor side, one of the key residues for Fab recognition is ECL2 B–D (rmsdCα ∼0.3 Å for the receptor and ∼1.3 Å for the whole complex) Asp198 (Fig. 1 ). Its acidic side chain protrudes into the (Fig. S5), we hereafter focus on the description of the ABC complex. interface between the light and heavy chains of Fab, interacting with both of them. Asp198ECL2 forms a salt bridge with Arg90 (CDR- Specific Interactions Between Fab and 5-HT2B. The Fab forms extensive L3), and hydrogen bonds with Tyr93 (CDR-L3), Asn50 (CDR-H2), interactions with all three extracellular loops (ECLs) of the and His35 (CDR-H1), as well as makes a hydrophobic contact with receptor with a total buried surface of 945 Å2 (Figs. 1 and 2 and Trp33 (CDR-H1). Mutational studies using ELISA further con- Tables S2 and S3). On the Fab side, interactions involve all three firmed the importance of Asp198ECL2 for binding of P2C2–Fab, complementarity-determining regions (CDR) of the heavy chain which showed at least three orders-of-magnitude decreased affinity (CDR-H1, CDR-H2, and CDR-H3), as well as CDR-L1 and CDR- of Fab binding to the D198N mutant (Fig.