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Biophysical and Functional Characterization of Norrin Signaling Through Frizzled4,” by Injin Bang, Hee Ryung Kim, Andrew H

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Biophysical and Functional Characterization of Norrin Signaling Through Frizzled4,” by Injin Bang, Hee Ryung Kim, Andrew H Correction BIOPHYSICS AND COMPUTATIONAL BIOLOGY Correction for “Biophysical and functional characterization of Norrin signaling through Frizzled4,” by Injin Bang, Hee Ryung Kim, Andrew H. Beaven, Jinuk Kim, Seung-Bum Ko, Gyu Rie Lee, Hasup Lee, Wonpil Im, Chaok Seok, Ka Young Chung, and Hee-Jung Choi, which was first published August 13, 2018; 10.1073/ pnas.1805901115 (Proc Natl Acad Sci USA 115:8787–8792). The authors note that Wei Kan should be added to the author list between Gyu Rie Lee and Hasup Lee. Wei Kan should be credited with contributing new reagents/analytic tools and analyz- ing data. The corrected author line, affiliation line, and author contributions appear below. The online version has been corrected. Injin Banga,1, Hee Ryung Kimb,1, Andrew H. Beavenc,2, Jinuk Kima, Seung-Bum Koa, Gyu Rie Leed,3, Wei Kane, Hasup Leed,4, Wonpil Imf,g, Chaok Seokd, Ka Young Chungb,5, and Hee-Jung Choia,5 aDepartment of Biological Sciences, Seoul National University, 08826 Seoul, Republic of Korea; bSchool of Pharmacy, Sungkyunkwan University, 16419 Suwon, Republic of Korea; cDepartment of Chemistry, The University of Kansas, Lawrence, KS 66047; dDepartment of Chemistry, Seoul National University, 08826 Seoul, Republic of Korea; eDepartment of Structural Biology, Stanford University School of Medicine, Stanford, CA 94305; fDepartment of Biological Sciences, Lehigh University, Bethlehem, PA 18015; and gDepartment of Bioengineering, Lehigh University, Bethlehem, PA 18015 Author contributions: H.-J.C. designed research; I.B., H.R.K., J.K., and S.-B.K. performed research; W.K. and K.Y.C. contributed new reagents/analytic tools; I.B., H.R.K., A.H.B., G.R.L., W.K., H.L., W.I., C.S., and K.Y.C. analyzed data; and I.B. and H.-J.C. wrote the paper. Published under the PNAS license. Published online October 29, 2018. www.pnas.org/cgi/doi/10.1073/pnas.1817333115 CORRECTION www.pnas.org PNAS | November 6, 2018 | vol. 115 | no. 45 | E10807 Downloaded by guest on October 4, 2021 Biophysical and functional characterization of Norrin signaling through Frizzled4 Injin Banga,1, Hee Ryung Kimb,1, Andrew H. Beavenc,2, Jinuk Kima, Seung-Bum Koa, Gyu Rie Leed,3, Hasup Leed,4, Wonpil Ime,f, Chaok Seokd, Ka Young Chungb,5, and Hee-Jung Choia,5 aDepartment of Biological Sciences, Seoul National University, 08826 Seoul, Republic of Korea; bSchool of Pharmacy, Sungkyunkwan University, 16419 Suwon, Republic of Korea; cDepartment of Chemistry, The University of Kansas, Lawrence, KS 66047; dDepartment of Chemistry, Seoul National University, 08826 Seoul, Republic of Korea; eDepartment of Biological Sciences, Lehigh University, Bethlehem, PA 18015; and fDepartment of Bioengineering, Lehigh University, Bethlehem, PA 18015 Edited by Jeremy Nathans, Johns Hopkins University, Baltimore, MD, and approved July 17, 2018 (received for review April 5, 2018) Wnt signaling is initiated by Wnt ligand binding to the extracel- showed that the binding surface on the CRD for Norrin overlaps lular ligand binding domain, called the cysteine-rich domain (CRD), with that for the Wnt finger loop, but the lipid-mediated in- of a Frizzled (Fzd) receptor. Norrin, an atypical Fzd ligand, specif- teraction of XWnt8 is missing in Norrin (9, 10). ically interacts with Fzd4 to activate β-catenin–dependent canon- Most structural studies on the Fzd family have focused on the ical Wnt signaling. Much of the molecular basis that confers Norrin extracellular domain to understand how Fzd achieves its ligand selectivity in binding to Fzd4 was revealed through the structural specificity. To our knowledge, no structural information about – study of the Fzd4CRD Norrin complex. However, how the ligand the Fzd transmembrane domain (TMD) or the linker domain interaction, seemingly localized at the CRD, is transmitted across full- between the CRD and the TMD is available yet. The linker length Fzd4 to the cytoplasm remains largely unknown. Here, we domain is highly divergent among Fzd subtypes, varying in length show that a flexible linker domain, which connects the CRD to the from approximately 40 aa in Fzd4 to 100 aa in Fzd8, and in transmembrane domain, plays an important role in Norrin signaling. sequence as well. The functional role of the linker domain has The linker domain directly contributes to the high-affinity interac- not yet been studied to our awareness. tion between Fzd4 and Norrin as shown by ∼10-fold higher binding Although X-ray crystallography remains the favorite tool for affinity of Fzd4CRD to Norrin in the presence of the linker. Swapping structural evaluation of membrane proteins, it is, in general, a BIOPHYSICS AND the Fzd4 linker with the Fzd5 linker resulted in the loss of Norrin challenging and time-consuming approach, and the crystal struc- COMPUTATIONAL BIOLOGY signaling, suggesting the importance of the linker in ligand-specific ture provides only a thermodynamically static view of the protein. cellular response. In addition, structural dynamics of Fzd4 associated The structural dynamics of a protein are as important as its static with Norrin binding investigated by hydrogen/deuterium exchange structure in understanding its function. Hydrogen/deuterium ex- MS revealed Norrin-induced conformational changes on the linker change MS (HDX-MS) has been successfully used to probe the domain and the intracellular loop 3 (ICL3) region of Fzd4. Cell-based functional assays showed that linker deletion, L430A and L433A Significance mutations at ICL3, and C-terminal tail truncation displayed reduced β-catenin–dependent signaling activity, indicating the functional sig- nificance of these sites. Together, our results provide functional and Wnt signaling has a broad spectrum of effects on cellular biochemical dissection of Fzd4 in Norrin signaling. physiology and diseases like cancer. Much of the specificity and regulation mechanism of the Frizzled (Fzd) family, the main receptor of the Wnt pathway, remains elusive, but exploiting Frizzled 4 | linker domain | Norrin | Dishevelled | signaling the activation mechanism of Fzd is pivotal to understanding the Wnt signaling pathway. Here, by using biophysical and nt signaling plays an important role in embryonic develop- biochemical techniques, we identify the key conformational Wment, tissue homeostasis, and cell proliferation in adult tissues. changes that occur as the Norrin ligand binds to Fzd4, and we Aberrant Wnt signaling causes various human diseases such as cancer show the functional implications of the involved regions. We and neurodegenerative disease (1). Frizzled (Fzd) receptors on the also reveal that the linker region plays an important role in cell surface recognize Wnt ligands by their extracellular cysteine-rich communicating ligand binding with cytoplasmic signaling. Our domain (CRD) (2) and transmit their signal to the cytoplasm. Al- observations offer insight into the activation mechanism of the though Wnt is the main ligand for the Fzd family, Fzd4 has an Fzd family and the regulation of the Wnt signaling pathway. atypical and highly specific ligand called Norrin. Norrin functions as a growth factor that is especially required for angiogenesis in the eye Author contributions: H.-J.C. designed research; I.B., H.R.K., J.K., and S.-B.K. performed (3), and mutations in the gene encoding human Norrin are associated research; K.Y.C. contributed new reagents/analytic tools; I.B., H.R.K., A.H.B., G.R.L., H.L., with Norrie disease, which impairs vision (4). Specific Norrin binding W.I., C.S., and K.Y.C. analyzed data; and I.B. and H.-J.C. wrote the paper. to Fzd4 (5) activates the β-catenin–dependent canonical Wnt signal- The authors declare no conflict of interest. ing pathway, in which Dishevelled (Dvl), as a cytoplasmic downstream This article is a PNAS Direct Submission. molecule, is shown to interact with intracellular loop 3 (ICL3) and Published under the PNAS license. the highly conserved KTXXXW motif of Fzd (6, 7). 1I.B. and H.R.K. contributed equally to this work. From a structural point of view, Norrin is completely different 2Present address: Department of Biomedical Engineering, University of Minnesota, from Wnt. Norrin has a cystine-knot motif and forms a homo- Minneapolis, MN 55455. 3 dimer through intermolecular disulfide bonds (8, 9). Unlike Wnt, Present address: Department of Biochemistry, University of Washington, Seattle, WA 98195. in which a palmitoleic acid lipid group is attached at a conserved 4Present address: Organic Material Lab, Samsung Advanced Institute of Technology, serine (10), Norrin is not lipid-modified. The molecular basis of Gyeonggi-do, 443-803 Suwon, Republic of Korea. the specific interaction of Norrin with Fzd4 was shown by the 5To whom correspondence may be addressed. Email: [email protected] or choihj@snu. crystal structures of the Fzd4CRD–Norrin complex, in which two ac.kr. CRD molecules interact with a head-to-tail Norrin dimer in a 2:2 This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. stoichiometry (9). A structural comparison of the Fzd4CRD–Norrin 1073/pnas.1805901115/-/DCSupplemental. complex with the Fzd8CRD–Xenopus Wnt8 (XWnt8) complex www.pnas.org/cgi/doi/10.1073/pnas.1805901115 PNAS Latest Articles | 1of6 not show any response to Norrin treatment (Fig. 1B). This im- plies that further oligomerization of Fzd4 above the basal level can be enhanced by Norrin in vivo, probably through its dimeric form. Recently, the Wnt5a ligand was shown to induce Fzd4 oligomerization through its lipid moiety (12). Although Fzd4 oligomerization appears to be the common response to Norrin and Wnt, the molecular mechanism of Fzd4 oligomerization by these ligands likely differs because of their structural differences. Structural Modeling of Fzd4–Norrin Complex. To better visualize how Fzd4 interacts with Norrin, nine candidate model structures were generated by computational modeling that used template- Fig. 1. Fzd4 oligomerization in vivo observed with BRET. All measurements based modeling (TBM), domain docking, linker modeling, and were done with at least four repeats, and the error bars are drawn with SEM.
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