Structural basis for CD97 recognition of the decay-accelerating factor CD55 suggests mechanosensitive activation of adhesion GPCRs

Abstract

The adhesion G protein–coupled receptor CD97 and its ligand complement decay-accelerating factor CD55 are important binding partners in the human immune system. Dysfunction in this binding has been linked to immune disorders such as multiple sclerosis and rheumatoid arthritis, as well as various cancers. Previous literatures have indicated that the CD97 includes 3 to 5 epidermal growth factor (EGF) domains at its N terminus and these EGF domains can bind to the N-terminal short consensus repeat (SCR) domains of CD55. However, the details of this interaction remain elusive, especially why the CD55 binds with the highest affinity to the shortest isoform of CD97 (EGF1,2,5). Herein, we designed a chimeric expression construct with the EGF1,2,5 domains of CD97 and the SCR1–4 domains of CD55 connected by a flexible linker and determined the complex structure by crystallography. Our data reveal that the two proteins adopt an overall antiparallel binding mode involving the SCR1–3 domains of CD55 and all three EGF domains of CD97. Mutagenesis data confirmed the importance of EGF5 in the interaction and explained the binding specificity between CD55 and CD97. The architecture of CD55–CD97 binding mode together with kinetics suggests a force-resisting shearing stretch geometry when forces applied to the C termini of both proteins in the circulating environment. The potential of the CD55–CD97 complex to withstand tensile force may provide a basis for the mechanosensing mechanism for activation of adhesion G protein–coupled receptors. The adhesion G protein–coupled receptor CD97 and its ligand complement decay-accelerating factor CD55 are important binding partners in the human immune system. Dysfunction in this binding has been linked to immune disorders such as multiple sclerosis and rheumatoid arthritis, as well as various cancers. Previous literatures have indicated that the CD97 includes 3 to 5 epidermal growth factor (EGF) domains at its N terminus and these EGF domains can bind to the N-terminal short consensus repeat (SCR) domains of CD55. However, the details of this interaction remain elusive, especially why the CD55 binds with the highest affinity to the shortest isoform of CD97 (EGF1,2,5). Herein, we designed a chimeric expression construct with the EGF1,2,5 domains of CD97 and the SCR1–4 domains of CD55 connected by a flexible linker and determined the complex structure by crystallography. Our data reveal that the two proteins adopt an overall antiparallel binding mode involving the SCR1–3 domains of CD55 and all three EGF domains of CD97. Mutagenesis data confirmed the importance of EGF5 in the interaction and explained the binding specificity between CD55 and CD97. The architecture of CD55–CD97 binding mode together with kinetics suggests a force-resisting shearing stretch geometry when forces applied to the C termini of both proteins in the circulating environment. The potential of the CD55–CD97 complex to withstand tensile force may provide a basis for the mechanosensing mechanism for activation of adhesion G protein–coupled receptors. Adhesion G protein–coupled receptors (GPCRs) are a subfamily of GPCRs that participate in a wide variety of functions from cell adhesion to immune defense and development, and, consequently, their dysfunction is linked to a myriad of negative health effects including inflammation, neurological disease, and cancer (1Langenhan T. Adhesion G protein-coupled receptors-candidate metabotropic mechanosensors and novel drug targets.Basic Clin. Pharmacol. Toxicol. 2020; 126 Suppl 6: 5-16Crossref PubMed Scopus (15) Google Scholar). Adhesion GPCRs are characterized by variable tandem adhesion domains followed by a common GPCR autoproteolysis-inducing (GAIN) domain at the extracellular region and a canonical seven-transmembrane (7TM) domain at the C-terminal region (2Langenhan T. Aust G. Hamann J. Sticky signaling--adhesion class G protein-coupled receptors take the stage.Sci. Signal. 2013; 6re3Crossref PubMed Scopus (168) Google Scholar). The epidermal growth factor (EGF) subgroup of the adhesion GPCR includes five members, CD97, and EGF module–containing mucin-like hormone receptor (EMR) numbers 1 through 4. Each of these members exhibits similar structural patterns but with variant numbers of EGF-like domains. Uniquely among the members, CD97 is widely expressed on granulocytes, monocytes, macrophage, dendritic cells, and smooth muscle cells (3Jaspars L.H. Vos W. Aust G. Van Lier R.A. Hamann J. Tissue distribution of the human CD97 EGF-TM7 receptor.Tissue Antigens. 2001; 57: 325-331Crossref PubMed Scopus (66) Google Scholar). Recent in vivo studies using mAbs have indicated that CD97 plays a prominent role in neutrophil migration and antibacterial immunity (4Leemans J.C. te Velde A.A. Florquin S. Bennink R.J. de Bruin K. van Lier R.A. van der Poll T. Hamann J. The epidermal growth factor-seven transmembrane (EGF-TM7) receptor CD97 is required for neutrophil migration and host defense.J. Immunol. 2004; 172: 1125-1131Crossref PubMed Google Scholar). Furthermore, CD97 has also been identified as a tumor-associated receptor and it is significantly upregulated in many carcinomas, including gastric, colorectal, and pancreatic (5Aust G. Eichler W. Laue S. Lehmann I. Heldin N.E. Lotz O. Scherbaum W.A. Dralle H. Hoang-Vu C. CD97: A dedifferentiation marker in human thyroid carcinomas.Cancer Res. 1997; 57: 1798-1806PubMed Google Scholar, 6Steinert M. Wobus M. Boltze C. Schutz A. Wahlbuhl M. Hamann J. Aust G. Expression and regulation of CD97 in colorectal carcinoma cell lines and tumor tissues.Am. J. Pathol. 2002; 161: 1657-1667Abstract Full Text Full Text PDF PubMed Google Scholar, 7Aust G. Steinert M. Schutz A. Boltze C. Wahlbuhl M. Hamann J. Wobus M. CD97, but not its closely related EGF-TM7 family member EMR2, is expressed on gastric, pancreatic, and esophageal carcinomas.Am. J. Clin. Pathol. 2002; 118: 699-707Crossref PubMed Scopus (69) Google Scholar). CD97 has three alternative gene-spliced isoforms containing between three and five EGF domains: EGF1,2,5, EGF1,2,3,5, and EGF1–5 (8Gray J.X. Haino M. Roth M.J. Maguire J.E. Jensen P.N. Yarme A. Stetler-Stevenson M.A. Siebenlist U. Kelly K. CD97 is a processed, seven-transmembrane, heterodimeric receptor associated with inflammation.J. Immunol. 1996; 157: 5438-5447PubMed Google Scholar). These isoforms have been linked to distinct functions, an aspect believed to derive from the different ligands they accommodate (9Liu D. Trojanowicz B. Ye L. Li C. Zhang L. Li X. Li G. Zheng Y. Chen L. The invasion and metastasis promotion role of CD97 small isoform in gastric carcinoma.PLoS One. 2012; 7e39989Crossref PubMed Scopus (27) Google Scholar, 10Eichler W. CD97 isoform expression in leukocytes.J. Leukoc. Biol. 2000; 68: 561-567PubMed Google Scholar). CD97 was first reported to bind CD55 (or decay-accelerating factor), a regulator of the complement system (11Hamann J. Vogel B. van Schijndel G.M. van Lier R.A. The seven-span transmembrane receptor CD97 has a cellular ligand (CD55, DAF).J. Exp. Med. 1996; 184: 1185-1189Crossref PubMed Google Scholar). Antibody blocking, domain deletion, and swapping experiments have verified the critical role of the first two EGF domains of CD97 in binding CD55 (12Hamann J. Stortelers C. Kiss-Toth E. Vogel B. Eichler W. van Lier R.A. Characterization of the CD55 (DAF)-binding site on the seven-span transmembrane receptor CD97.Eur. J. Immunol. 1998; 28: 1701-1707Crossref PubMed Scopus (90) Google Scholar, 13Lin H.H. Stacey M. Saxby C. Knott V. Chaudhry Y. Evans D. Gordon S. McKnight A.J. Handford P. Lea S. Molecular analysis of the epidermal growth factor-like short consensus repeat domain-mediated protein-protein interactions: Dissection of the CD97-CD55 complex.J. Biol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). the of has been to CD97 binding affinity with CD55. to CD97 can also bind to a of ligands including E. A. D. C. Aust G. is an for CD97 on Immunol. 2012; PubMed Scopus Google and T. Y. L. L. Stetler-Stevenson Kelly K. CD97, an adhesion receptor on cells, through binding on PubMed Scopus Google and bind to the as well as M.J. W. M. Stacey M. H.H. Gordon S. van Lier R.A. Hamann J. Expression of the CD97 and isoforms on a interaction with on Leukoc. Biol. PubMed Scopus Google binds to the These binding together with the different isoforms of CD97, with distinct CD55 is a with short consensus repeat (SCR) domains at the N CD55 the complement by the and plays a critical role in and defense and Immunol. PubMed Google Scholar). The binding of CD55 to CD97 can cell from and the CD55–CD97 are in the for multiple sclerosis L. de Vos Hamann J. M.J. van M. van Lier R.A. Expression of the EGF-TM7 receptor CD97 and its ligand CD55 in multiple 2002; Full Text Full Text PDF PubMed Scopus Google inflammation, and rheumatoid Vogel G. J. L. K. Hamann J. van H. E. of CD97 in The role of and on the of Immunol. PubMed Scopus Google Scholar). CD55 is also linked to immunity cells with CD97, in cell activation and an in cell and M. Stacey M. Gordon S. J. I. CD55 on human cells by Immunol. PubMed Google Scholar). CD97 the highest with in the with three in EGF domains and However, has been to bind to CD55 with a that of CD97 H.H. Stacey M. Saxby C. Knott V. Chaudhry Y. Evans D. Gordon S. McKnight A.J. Handford P. Lea S. Molecular analysis of the epidermal growth factor-like short consensus repeat domain-mediated protein-protein interactions: Dissection of the CD97-CD55 complex.J. Biol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, H.H. Stacey M. Hamann J. Gordon S. McKnight A.J. EMR2, a novel EGF-TM7 on is closely related to 2000; PubMed Scopus Google Scholar). of the domains of CD55 P. P. J. D. J. R.A. Evans regulation at the The structure of decay-accelerating U. S. A. 2004; PubMed Scopus Google and the EGF1,2,5 domains of R.J. I. P. H. Knott V. P. Handford Lea and of a novel cell receptor Biol. Full Text Full Text PDF PubMed Scopus Google reveal an However, to the of structure for CD55 in complex with CD97 EMR2, the binding mode and as well as the by the remain Herein, we the structure of the domains of the complex as determined at by crystallography. of the complex structure not an overall antiparallel binding mode but also the specificity for CD97 by CD55. The CD55 SCR1–4 domains and CD97 EGF1,2,5 domains from However, the of the two not a complex in of the affinity between the two we designed a linker including a site to the C terminus of EGF1,2,5 and the N terminus of by a mode on H.H. Stacey M. Saxby C. Knott V. Chaudhry Y. Evans D. Gordon S. McKnight A.J. Handford P. Lea S. Molecular analysis of the epidermal growth factor-like short consensus repeat domain-mediated protein-protein interactions: Dissection of the CD97-CD55 complex.J. Biol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, R.J. I. P. H. Knott V. P. Handford Lea and of a novel cell receptor Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). The chimeric construct was expressed and to and of the linker with a of two to the CD55 and CD97 the construct was well The and with of the complex and indicated the of a CD55–CD97 by the flexible linker and the chimeric complex and the data to the structure with using of CD55 and as and the structure was to and of and and for the highest are in and are the and of of between of data for for the highest are in and are the and of of between of data for in a of the in the complex can the linker region is with its two with of and A and Previous binding studies of CD55 and CD97 have the importance of the N-terminal domains of both proteins in and designed linker was to the between the C terminus of EGF1,2,5 and the N terminus of SCR1–4 in A but not to the in B. Furthermore, A binding mode can a with a structural of this is the of and the from R.J. I. P. H. Knott V. P. Handford Lea and of a novel cell receptor Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). of the from we of an G. M. of flexible using PubMed Scopus Google for the chimeric complex in the the binding studies have been to the A binding mode these of the binding mode in A and we the A binding mode The CD55 and CD97 bind antiparallel to involving N-terminal tandem domains of CD55 and CD97 in the complex closely with with a of to and to is Previous of CD55 domains have a of and in the of and complex the of is and the are similar of as the CD55 of studies in CD97, in the is when with of its CD97 EMR2, and EGF5 bind a in the N-terminal to the of These together with of the linker between the two indicated the binding mode between CD55 and CD97 is not by the The by the was an that is the for interaction J. L. interactions: in the between binding affinity and 2013; PubMed Scopus Google Scholar). are in the and this is the that with domains in the Our of the domain that of its are by in to to for the domains. Our complex structure two in CD97 and and site in CD55 but of these are in the The can three the the and the the we that of a with of also with and and a of The of from the in the a of and of the of this and of to and of the of in a to of the of the the are by three of between and and and the of of EGF5 a by and and These are with the and domains as critical for CD55–CD97 structure by EGF5 of CD97 as well as of CD55. the of EGF5 in the CD55–CD97 may why the EGF1,2,5 isoform of CD97 a binding affinity to CD55 the two and The in the CD55–CD97 complex to with the characterized affinity and binding H.H. Stacey M. Saxby C. Knott V. Chaudhry Y. Evans D. Gordon S. McKnight A.J. Handford P. Lea S. Molecular analysis of the epidermal growth factor-like short consensus repeat domain-mediated protein-protein interactions: Dissection of the CD97-CD55 complex.J. Biol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, J. L. interactions: in the between binding affinity and 2013; PubMed Scopus Google Scholar). this we the binding kinetics of CD55 and CD97 by CD55 a and of CD97 Our that CD97 binds to CD55 with a of the reported affinity and in with a between the and binding affinity J. L. interactions: in the between binding affinity and 2013; PubMed Scopus Google Scholar). Furthermore, with the determined H.H. Stacey M. Saxby C. Knott V. Chaudhry Y. Evans D. Gordon S. McKnight A.J. Handford P. Lea S. Molecular analysis of the epidermal growth factor-like short consensus repeat domain-mediated protein-protein interactions: Dissection of the CD97-CD55 complex.J. Biol. 2001; Full Text Full Text PDF PubMed Scopus Google the two proteins to with a and These are in with structural of the antiparallel binding that by tandem domains at both The in binding kinetics between and to the the CD55 was to the may a in and by CD97 H.H. Stacey M. Saxby C. Knott V. Chaudhry Y. Evans D. Gordon S. McKnight A.J. Handford P. Lea S. Molecular analysis of the epidermal growth factor-like short consensus repeat domain-mediated protein-protein interactions: Dissection of the CD97-CD55 complex.J. Biol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). proteins both expressed and from cells, in the the CD97 and CD55 proteins expressed from H.H. Stacey M. Saxby C. Knott V. Chaudhry Y. Evans D. Gordon S. McKnight A.J. Handford P. Lea S. Molecular analysis of the epidermal growth factor-like short consensus repeat domain-mediated protein-protein interactions: Dissection of the CD97-CD55 complex.J. Biol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). affinity of CD97 to the of the binding the and of CD97 to a in CD55 with this the binding affinity of and by and The CD55–CD97 in structure not with the from R.J. I. P. H. Knott V. P. Handford Lea and of a novel cell receptor Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). the the and N-terminal of in CD55 bind to the of CD97 with a Furthermore, that not such a between CD97 and the also why the isoform of CD97 has the highest binding affinity with CD55. a that the is it all three in the binding the for the are as they from E. structure that the for CD55 by different isoforms of CD97 as well as its the by EGF5 is is this EGF domain may the binding affinity and specificity of the different CD97 isoforms with CD55. the and EGF1–5 the domain the of EGF5 and the domain of CD55. The EGF5 is to a in the The of this suggests that in the two the domain may in the binding of CD55 with similar the importance of a in the with we the to an in the EGF1,2,5 and the that the an in binding the of EGF5 in the binding with CD55 The in the together with a potential of its with the first two EGF may to binding affinity of the two isoforms and the EGF1,2,5 The EGF1,2,5 of CD97 and is by three and affinity of CD97 H.H. Stacey M. Saxby C. Knott V. Chaudhry Y. Evans D. Gordon S. McKnight A.J. Handford P. Lea S. Molecular analysis of the epidermal growth factor-like short consensus repeat domain-mediated protein-protein interactions: Dissection of the CD97-CD55 complex.J. Biol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). with different of EGF1,2,5 indicated similar of in binding affinity the of these variant in the binding of three variant are in the binding with and their can in the complex The first is in the to in of the CD97 domain the and of CD55. The of is from and and this a interaction in the similar interaction in the A and The is in a region in CD97, the and its adopt a the to its and a to in the CD55 domain A and A is not in EMR2, as the of is in EMR2, the the of CD97 and is in this region when it binds to CD55. The is not in the binding it may not the binding of with CD55. are as well as in the between different The of CD97, and are in all is but by a in the and are and and are in they are by and including of CD97 and and of are among these in in the in the with this have that the interaction is and is between human and Haino M. Kelly K. of CD97 and of its interaction with the decay-accelerating factor PubMed Scopus Google Scholar, J. van C. A. C. K. van Lier R.A. Molecular and of Immunol. 2000; PubMed Google Scholar). to human CD97, CD97 also includes three isoforms but with different and to a of that to J. van C. A. C. K. van Lier R.A. Molecular and of Immunol. 2000; PubMed Google Scholar). to the may the domain and binding with CD55. these the of the interaction was to between and Vogel G. J. L. K. Hamann J. van H. E. of CD97 in The role of and on the of Immunol. PubMed Scopus Google Scholar, de D. Hamann J. of CD55 CD97 in PubMed Scopus Google Scholar, J. van Lier R.A. Expression of the activation CD97 and its ligand CD55 in rheumatoid PubMed Scopus Google Scholar). the three EGF domains of CD97, is different from the two domains of its A and The and are a and a van de interaction between a in the of the EGF5 domain and a in the domain The and the are also in the and domains similar domain in the and EGF1–5 CD55 also similar van de between the and domains and their and domains the van de in the and are by that in the and the are and the of domain Furthermore, the is not in and its by a a in the an similar to and the the is to with this is in to between the domains. 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CD97 are and to the site through multiple including the CD55–CD97 a CD97 is in to CD55 cell adhesion and of a between these two is as a of The overall architecture of the CD55–CD97 binding mode together with structural details to a shearing stretch is to force applied to the S. M. H. S. Chen H. J. of the of PubMed Google Scholar). the first of with this the is to the force through the C termini of CD55 and CD97, can is to that the of the CD55–CD97 complex by is a force The potential of a of force suggests that the CD55–CD97 may force to the is believed to important for the of adhesion many adhesion the domain is associated with the extracellular region through its N-terminal that a of the extracellular domain (2Langenhan T. Aust G. Hamann J. Sticky signaling--adhesion class G protein-coupled receptors take the stage.Sci. Signal. 2013; 6re3Crossref PubMed Scopus (168) Google Scholar, D. A.A. J. A novel domain of GPCRs J. 2012; PubMed Scopus Google Scholar). The force may a of the domain and it from the when CD97 its extracellular region from the cell this of can in the H. D. van E. van Lier R.A. Hamann J. of the protein-coupled receptor CD97 on circulating with its ligand Immunol. 2013; PubMed Scopus Google Scholar). the has been to and bind the I. J. L. A. M. S. T. A the the adhesion G protein-coupled receptors and Full Text Full Text PDF PubMed Scopus Google Scholar, C. L. V. J. S. T. I. The of the adhesion GPCR is by its J. PubMed Scopus Google Scholar, S. T. I. of the of the adhesion G protein-coupled receptor Res. PubMed Scopus Google Scholar, L. Adhesion G protein-coupled receptors are by of a U. S. A. PubMed Scopus Google Scholar). complement factor 1 was reported to bind and the C S. S. S. E. H. B. T. C. binding to cells and in PubMed Scopus Google Scholar). an for CD97 has to its antiparallel binding with CD55 may provide a for potential of the tensile force to the and domains for force was reported to in the CD97 a G D. D. S. E. M. J. L. I. L. Aust G. of the of cellular Full Text Full Text PDF PubMed Scopus (15) Google Scholar). The of mechanosensing mechanism for adhesion GPCRs has been V. G protein-coupled receptor muscle U. S. A. PubMed Google Scholar, I. S. A. M. T. The adhesion GPCR has functions in cell by interaction with Full Text Full Text PDF PubMed Scopus Google Scholar, J. C. D. V. R.J. T. The adhesion GPCR Full Text Full Text PDF PubMed Scopus Google Scholar, A. of adhesion G protein-coupled receptor Biol. 2020; Full Text Full Text PDF PubMed Scopus Google the is in of that an force-resisting the of the CD55–CD97 complex in immune disorders and carcinomas, this complex structure may provide for and a of the of the human The CD97 EGF CD55 and chimeric complex expressed and as Y. Zhang M. M.J. W. of the adhesion G protein-coupled receptor CD97 related to its and cell Pharmacol. PubMed Scopus Google Scholar). the EGF1,2,5 isoform of CD97 CD55 chimeric complex was a W. A and system for in Biol. 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