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Oct 21, 2007 - recognizes the third intracellular loop (IL3) of the native .... Biology, Stanford University School of Medicine, 299 Campus Drive, Stanford,.
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A monoclonal antibody for G protein–coupled receptor crystallography Peter W Day1,4, Søren G F Rasmussen1,4, Charles Parnot1, Juan Jose´ Fung1, Asna Masood2, Tong Sun Kobilka1, Xiao-Jie Yao1, Hee-Jung Choi1,3, William I Weis1,3, Daniel K Rohrer2 & Brian K Kobilka1 G protein–coupled receptors (GPCRs) constitute the largest family of signaling proteins in mammals, mediating responses to hormones, neurotransmitters, and senses of sight, smell and taste. Mechanistic insight into GPCR signal transduction is limited by a paucity of high-resolution structural information. We describe the generation of a monoclonal antibody that recognizes the third intracellular loop (IL3) of the native human b2 adrenergic (b2AR) receptor; this antibody was critical for acquiring diffraction-quality crystals.

Efforts to crystallize membrane proteins in general, and GPCRs in particular, have been hampered by intrinsic characteristics of integral membrane proteins. Bovine rhodopsin is the only GPCR for which a high-resolution structure has been determined by X-ray crystallography1–3; this is in part due to its natural abundance and atypical stability. The seven hydrophobic transmembrane helices of GPCRs make poor surfaces for crystal contacts, and the extracellular and intracellular domains are often relatively short and/or poorly structured. Antibody fragments (Fab, Fv) that recognize native protein conformations have been shown to facilitate crystallization of other membrane proteins by increasing the polar surface area for protein-protein contacts and by restricting the flexibility of mobile domains4. Our goal was to generate monoclonal antibodies to facilitate b2AR crystallization. The b2AR belongs to the rhodopsin family of GPCRs, and is one of nine adrenergic receptor subtypes that respond to adrenaline and noradrenaline. Unlike rhodopsin, the b2AR must be expressed in recombinant systems to obtain sufficient quantities of protein for crystallography, and it is considerably less stable in detergent solutions compared to rhodopsin. The b2AR contains relatively unstructured domains that may be involved in functionally important protein-protein interactions. Based on protease susceptibility and intramolecular fluorescence resonance energy transfer experiments5, the carboxyl terminus and IL3 are the most unstructured domains. Removal of these unstructured

domains by deletion and truncation should improve the overall order of the receptor, but this would be at the expense of reducing the polar surface area and potential crystal lattice contacts. We were interested in generating monoclonal antibodies that bind and stabilize IL3 because this sequence links two independent folding domains: the amino terminus through transmembrane helix 5 (TM5) and TM6 through the C terminus. These two domains can be expressed as separate proteins and associate noncovalently to form a functional receptor6. An antibody that binds to IL3 has the potential for stabilizing noncovalent interactions between the transmembrane segments while providing polar surface for crystal lattice contacts. We prepared antigen by reconstituting purified, functional b2AR at high density into phospholipid vesicles (1 mg of receptor per 1 mg of phospholipids; Supplementary Methods online). The phospholipid environment ensures the functional integrity of the protein after injection into mice. To provide additional stability, the b2AR was bound with carazolol, a high-affinity inverse agonist. To facilitate the immune response, phospholipid vesicles consisted of a 10:1 mixture (by weight) of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and the adjuvant Lipid A. These vesicles contained randomly oriented b2AR, so that both cytoplasmic and extracellular domains were presented to immune cells. All experiments involving animals were overseen and approved by the Medarex Animal Care and Use Committee, in compliance with all regulations as set forth by the Public Health Service policy on Humane Care and Use of Laboratory Animals and the guidelines established by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALACI). We generated monoclonal antibodies using a conventional fusion protocol (Supplementary Methods). Fusions from two mice yielded 17 hybridoma clones that produced antibody to b2AR as determined by an enzyme-linked immunosorbent assay on immobilized phospholipid vesicles containing purified b2AR. Nine of these hybridomas produced sufficient quantities of b2ARspecific antibody for characterization. The antibodies bound the intracellular or extracellular surface of the receptor in immunofluorescence experiments with HEK-293 cells stably expressing an N-terminally Flag-tagged version of the b2AR. Antibodies that recognize an intracellular epitope only stain permeabilized cells. Staining of cells that were either fixed, or fixed and permeabilized indicated that five of the antibodies bound to the intracellular face and four bound to the extracellular surface (Supplementary Fig. 1 online). To facilitate crystallization of the b2AR, antibodies should bind to a three-dimensional surface rather than a flexible linear epitope, such as the distal N or C termini7. We therefore screened the 9 monoclonal antibodies, along with positive (M1 antibody) and


of Molecular and Cellular Physiology, Stanford University School of Medicine, 279 Campus Drive, Stanford, Palo Alto, California 94305, USA. 2Medarex, Inc., 521 Cottonwood Drive, Milpitas, California 95035, USA. 3Department of Structural Biology, Stanford University School of Medicine, 299 Campus Drive, Stanford, Palo Alto, California 94305, USA. 4These authors contributed equally to this work. Correspondence should be addressed to B.K. ([email protected]).




negative (myc antibody; 9E10) controls, for binding to b2AR denatured with sodium dodecyl sulfate (SDS) and spotted on nitrocellulose (Fig. 1a). Antibodies 5 and 9 showed the weakest binding to denatured protein, even though they showed immunostaining comparable to M1 on binding to native receptor in fixed cells (Supplementary Fig. 1). The reduced binding of antibodies 5 and 9 to SDS-denatured b2AR suggested that these antibodies bind to an epitope that is present only in the native form of the protein. As discussed above, our goal was to identify an antibody that recognized IL3 of the native b2AR. Antibodies 2, 5, 6, 7 and 9 all reacted with intracellular epitopes. To select for antibodies that may interact with IL3, we examined the effect of antibody binding on the fluorescence of b2AR labeled at Cys265 (at the cytoplasmic end of TM6) with tetramethylrhodamine. Tetramethylrhodamine bound to Cys265 is predicted to be at the interface between TM5 and TM6. Previous studies have shown that tetramethylrhodamine bound to Cys265 is sensitive to ligand-induced conformational changes in the b2AR8. We reasoned that antibodies binding to IL3

a MW (kDa) 106.9



β2AR + Fab 5



5 30 0


Fab 5 30






37.2 28.2

N L I R T D N Q Q A Y I V H A I A S V I N V TM6 S I V I F F S F V V P L WC P L V L T F TM5 I M V F T G M I V Y S I G L R A L K T V E H K F K Q Exposed L C F K S S R IL3 E R L G A H K 28 kDa R G 30 kDa V Q Q H T 27 kDa N L F R L Q R G S K D G I Q V E Q D K S E Protected C




could stabilize a specific conformation of TM5 relative to TM6 that would be detected by a change in tetramethylrhodamine fluorescence. Antibody 5 induced the greatest fluorescence response and had the highest affinity for the b2AR (Fig. 1b). The fluorescence change induced by antibody 5 was considerably greater than the response to antibody 9, the other intracellular binder that reacted weakly with the SDS-denatured receptor. Notably, there was a response to antibodies 8 and 4, two antibodies that bind to an extracellular epitope. This suggested that binding of these antibodies to the extracellular surface of the b2AR influences the structure around the cytoplasmic end of TM6. Based on these results, we chose antibody 5 for crystallization experiments. Antibody 5 bound to an intracellular epitope exhibited relatively high affinity for native b2AR (Fig. 1b) but bound weakly to SDS-denatured protein (Fig. 1a). Antibody 5 also induced the largest fluorescence response in b2AR labeled at Cys265 with tetramethylrhodamine. These results suggested that antibody 5 was likely to bind to the native IL3.

b Fluorescence intensity change (%)

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Percentage change in fluorescence

Figure 1 | Binding characteristics of b2AR-specific 4 a 150 b 12 5 antibodies. (a) Representative dot blots showing 125 6 10 binding of nine b2AR-specific antibodies to 7 denatured receptor (bottom). Equal amounts 100 8 8 of SDS-denatured b2AR in the presence of 9 75 6 b-mercaptoethanol were spotted in triplicate on 50 4 nitrocellulose strips. The strips were blocked with 5% nonfat dry milk in phosphate-buffered saline 2 25 (pH 7.4) with 0.05% Tween-20 and then probed 0 with 1 mg/ml of the indicated antibodies diluted –9 –8 –7 –6 9E10 M1 1 2 3 4 5 6 7 8 9 10 10 10 10 in blocking buffer. Binding of the primary antibody Antibody concentration (M) to the denatured b2AR was detected with an Alexa-688 labeled mouse secondary antibody. The average dot intensity from three independent experiments is shown in the graph (top). Error bars, s.d.; n ¼ 3. The binding of all 9 antibodies to denatured b2AR was reduced compared to M1 binding to the linear Flag epitope (M1). (b) Dose-response curves showing the effect of increasing amounts of antibodies on the fluorescence intensity of b2AR labeled at Cys265 with tetramethylrhodamine b2AR-TMR8. b2AR-TMR was diluted to 4 nM in 500 ml of buffer consisting of 0.1% dodecylmaltoside, 100 mM sodium chloride and 20 mM HEPES buffer (pH 7.5). Error bars, s.d.; n ¼ 3.


Iso + Fab 5

Fab 5






Figure 2 | Fab 5 binds IL3 of the b2AR but does not effect structural changes associated with G-protein activation. (a) Western blot analysis of the b2AR digested with trypsin for the indicated amounts of time in the absence and presence of Fab 5 (left). The fragmented b2AR was visualized using an Alexa-680– labeled M1 antibody against the N-terminal Flag epitope. In the absence of Fab 5, two fragments of B27 and 29 kDa appear, corresponding to cleavage in IL3. In contrast, the presence of Fab 5 protects the N-terminal end of the loop. The diagram shows IL3 connecting TM5 and TM6. Molecular weights of corresponding N-terminal fragments containing the Flag epitope are marked. Residues sensitive to trypsin digest are shown in red. (b) The change in the bimane fluorescence of b2AR labeled with monobromobimane at H271C at the cytoplasmic end of TM6. Bimane fluorescence is quenched by Trp135 at the cytoplasmic end of TM3 upon agonist binding9. The response to the full agonist isoproterenol (Iso), isoproterenol plus Fab 5 and Fab 5 alone are shown. Fluorescence intensity was corrected for background fluorescence from buffer and ligands in all experiments. The data are the mean ± s.e.m. of two independent experiments performed in triplicate. 2 | ADVANCE ONLINE PUBLICATION | NATURE METHODS

© 2007 Nature Publishing Group

BRIEF COMMUNICATIONS For crystallization, Fab fragments are preferred over full-length monoclonal antibodies because they lack the flexible hinge separating constant domains 1 and 2 of the heavy chain. We generated Fab 5 fragments from purified antibody 5 by papain cleavage and purified them by ion exchange chromatography. The dissociation constant for Fab 5 binding to purified b2AR was 150 nM, as determined by isothermal titration calorimetry (Supplementary Fig. 2 online). As expected, this value is higher than the halfmaximal effective concentration (23 nM) observed for the intact antibody in the fluorescence experiments (Fig. 1b). To localize the intracellular epitope of the b2AR that interacts with Fab 5, we performed limited tryptic digestion of purified b2AR. Cleavage of purified b2AR with trypsin yielded two bands with molecular weights of approximately 27 and 29 kDa on a western blot probed with M1 anti-Flag (Fig. 2a). The sizes of these fragments indicate trypsin digestion at two of ten potential sites in IL3 (Fig. 2a). When preincubated with Fab 5, the 27-kDa band disappeared. suggesting that the antibody binds to one of the N-terminal trypsin-cleavage sites in IL3 (Fig. 2a). One concern about the use of Fabs to facilitate crystal formation is the possibility that Fabs stabilize a minor conformation of the receptor that does not reflect the native structure. We therefore determined the effect of Fab binding on ligand-binding properties and on agonist-induced conformational changes. Fab 5 had no significant effect on antagonist or agonist binding affinity (Supplementary Table 1 online; P ¼ 0.653 for antagonist and P ¼ 0.335 for agonist, two-tailed paired t-test). IL3 is known to be important for G-protein activation. Fab 5 prevented coupling of purified b2AR to purified G protein (data not shown), most likely because of steric competition. However, it is possible that Fab 5 restricts the conformational changes associated with receptor activation. We determined the effect of Fab binding on agonist-induced conformational changes using a fluorescence-based assay9. Agonist binding induces a change that brings the fluorophore monobromobimane bound to Cys271 at the cytoplasmic end of TM6 closer to Trp135 at the cytoplasmic end of TM3, resulting in a decrease in bimane fluorescence. This fluorescence change was not affected by binding of antibody 5 (Fig. 2b). In conclusion, although Fab 5 required a native b2AR to bind, it did not restrict the movement of transmembrane segments involved in ligand binding and agonist activation. Fab fragments crystallize much more readily than membrane proteins, and Fab crystals are likely to be a source of false positives in crystallization screens. To more easily identify crystals of the Fab 5–b2AR complex, we labeled purified b2AR at Cys265 with tetramethylrhodamine (b2AR-TMR). We formed the Fab 5–b2ARTMR complex by mixing b2AR-TMR with a stoichiometric excess of Fab 5 and isolating the complex by size-exclusion chromatography. Small fluorescent crystals formed by vapor phase diffusion using ammonium sulfate as the precipitant (Supplementary Fig. 3

online). No crystals formed from b2AR alone or from Fab 5 alone under these conditions, indicating that the additional protein interactions and the stabilizing effects of the antibody were critical for successful crystallization of the b2AR. Refinement of the crystallization conditions has since produced diffractionquality crystals and an anisotropic 3.4 A˚/3.7 A˚ resolution structure of the Fab 5–b2AR complex10. The crystal structure confirmed that Fab 5 binds to a tertiarystructure epitope consisting of nine amino acids at the N-terminal end of IL3 (Ile233–Val242) and two amino acids at the C-terminal end (Leu266 and Lys270)10. The structure resolved by this approach will be an invaluable tool for understanding GPCR function at the molecular level. Moreover, as IL3 of the b2AR can be exchanged with that of other GPCRs, it is possible that this Fab can be used to obtain crystal structures of other GPCRs. Note: Supplementary information is available on the Nature Methods website. ACKNOWLEDGMENTS This study was supported by a US National Institutes of Health Vascular Biology Training Grant 5 T32 HL007708 (to P.W.D.), the Lundbeck Foundation (to S.G.F.R.), National Institute of General Medical Sciences grant GM56169 (to W.I.W.), National Institute of Neurological Disorders and Stroke grant NS28471 (to B.K.K.), the Mather Charitable Foundation (to B.K.K.) and a generous gift from 7TM Pharma (to B.K.K.). B.K.K. and W.I.W. are grateful for the advice of R. MacKinnon in the preparation and use of antibody fragments for crystallography. AUTHOR CONTRIBUTIONS P.W.D. characterized antibody binding to the b2AR on HEK-293 cells and spotted on nitrocellulose. S.G.F.R. purified antibody 5, prepared Fab 5, determined the receptor binding surface of Fab 5 by tryptic digestion, determined ligand binding affinities for the b2AR in the presence of Fab 5 and crystallized the b2AR-TMR-Fab 5 complex. C.P. and J.J.F. characterized antibody-induced fluorescence changes of b2AR-TMR. A.M. and D.K.R. generated the antibodies and performed initial antibody screening. T.S.K. prepared monoclonal antibody 5. X.-J.Y. performed bimane fluorescence experiments. H.-J.C. and W.I.W. performed isothermal titration calorimetry experiments on b2AR and Fab 5. P.W.D. and B.K.K. prepared the manuscript. COMPETING INTERESTS STATEMENT The authors declare competing financial interests: details accompany the full-text HTML version of the paper at Published online at Reprints and permissions information is available online at 1. Li, J., Edwards, P.C., Burghammer, M., Villa, C. & Schertler, G.F. J. Mol. Biol. 343, 1409–1438 (2004). 2. Okada, T. et al. J. Mol. Biol. 342, 571–583 (2004). 3. Palczewski, K. et al. Science 289, 739–745 (2000). 4. Hunte, C. & Michel, H. Curr. Opin. Struct. Biol. 12, 503–508 (2002). 5. Granier, S. et al. J. Biol. Chem. (2007). 6. Kobilka, B.K. et al. Science 238, 650–656 (1987). 7. Mancia, F. et al. Proc. Natl. Acad. Sci. USA 104, 4303–4308 (2007). 8. Swaminath, G. et al. J. Biol. Chem. 279, 686–691 (2004). 9. Yao, X. et al. Nat. Chem. Biol. 2, 417–422 (2006). 10. Rasmussen, S.G.F. et al. Nature (in the press).


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