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Structural Basis of Highly Specific Interaction Between Nephrin And BASIC RESEARCH www.jasn.org Structural Basis of Highly Specific Interaction between Nephrin and MAGI1 in Slit Diaphragm Assembly and Signaling Zhuangfeng Weng,1,2 Yuan Shang,3 Zeyang Ji,3 Fei Ye,3,4 Lin Lin,1 Rongguang Zhang,1,2 and Jinwei Zhu1 Due to the number of contributing authors, the affiliations are listed at the end of this article. ABSTRACT Background The slit diaphragm is a specialized adhesion junction between opposing podocytes, establishing the final filtration barrier that prevents passage of proteins from the capillary lumen into the urinary space. Nephrin, the key structural and signaling adhesion molecule expressed in the slit diaphragm, contains an evolutionally conserved, atypical PDZ-binding motif (PBM) reported to bind to a variety of proteins in the slit diaphragm. Several mutations in NPHS1 (the gene encoding nephrin) that result in nephrin lacking an intact PBM are associated with glomerular diseases. However, the molecular basis of nephrin-PBM–mediated pro- tein complexes is still unclear. Methods Using a combination of biochemic, biophysic, and cell biologic approaches, we systematically investigated the interactions between nephrin-PBM and PDZ domain–containing proteins in the slit diaphragm. Results We found that nephrin-PBM specifically binds to one member of the membrane-associated gua- nylate kinase family of scaffolding proteins, MAGI1, but not to another, MAGI2. The complex structure of MAGI1-PDZ3/nephrin-PBM reveals that the Gly at the 23 position of nephrin-PBM is the determining feature for MAGI1-PDZ3 recognition, which sharply contrasts with the typical PDZ/PBM binding mode. A single gain-of-function mutation within MAGI2 enabled nephrin-PBM binding. In addition, using our structural analysis, we developed a highly efficient inhibitory peptide capable of specifically blocking the nephrin/ MAGI1 interaction. Conclusions MAGI1 interacts with nephrin-PBM with exquisite specificity. A newly developed, potent inhibitory peptide that blocks this interaction may be useful for future functional investigations in vivo. Our findings also provide possible explanations for the diseases caused by NPHS1 mutations. J Am Soc Nephrol 29: ccc–ccc, 2018. doi: https://doi.org/10.1681/ASN.2017121275 Slit diaphragm is a highly specialized cell-cell junction formed by the neighboring podocytes and functions Received December 11, 2017. Accepted June 19, 2018. as the final filtration barrier that prevents passage of macromolecules from the capillary lumen into the Published online ahead of print. Publication date available at www.jasn.org. urinary space.1,2 Disruption of the architecture of slit diaphragm can lead to severe nephrotic syn- Present address: Dr. Yuan Shang, Center for Biomedical In- formatics and Biostatistics, University of Arizona, Tucson, drome, with symptoms including massive protein- Arizona. uria, hypoalbuminemia, and edema.3,4 fi Correspondence: Dr. Rongguang Zhang or Dr. Jinwei Zhu, NPHS1,encodingthede ning adhesion molecule Shanghai Institute of Biochemistry and Cell Biology, Chinese inNephrinintheslitdiaphragm,wasidentified as the Academy of Sciences, 333 Haike Road, Shanghai 201203, China. major causative gene for congenital nephrotic syn- E-mail: [email protected] or [email protected] drome of the Finnish type (CNF).5 To date, over 160 Copyright © 2018 by the American Society of Nephrology J Am Soc Nephrol 29: ccc–ccc, 2018 ISSN : 1046-6673/2909-ccc 1 BASIC RESEARCH www.jasn.org mutations in NPHS1 have been discovered in the patients with Significance Statement CNF.6 NPHS1 knockout mice showed defective assembly of the slit diaphragm and impaired barrier function of podocytes.7 Although nephrin plays crucial roles in formation of the podocyte slit Nephrin consists of extracellular eight Ig-like domains, a fibro- diaphragmand in dynamic regulationof signaling pathways intheslit nectin type III-like domain, and an unstructured intracellular diaphragm, the mechanisms at molecular level are poorly un- derstood. In addition, several mutations in NPHS1 (the gene en- domain (Figure 1A). Nephrins form the unique zipper-like struc- coding nephrin) that affect nephrin’s atypical PDZ-binding motif ture via the homotypic interactions between the Ig domains from (PBM) have been associated with glomerular diseases. Using a va- the adjacent podocytes.8 The intracellular domain of Nephrin riety of approaches, the authors demonstrate that nephrin’sPBM (Nephrin-CT) contains an evolutionarily conserved atypical specifically interacts with the PDZ3 domain of MAGI1, a member of fi PDZ domain-binding motif (PBM; “2LPFELRGHLV”)atthe the membrane-associated guanylate kinase family. The ndings provide a molecular basis of nephrin/MAGI1 interaction in slit di- C-terminus, not consistent with the canonical type I, type II, or aphragm assembly and signaling, as well as possible insights at the type III PBMs (Figure 1A).9 Mutations of NPHS1 lacking partial molecular level for diseases caused by alterations in NPHS1. intracellular domain including the C-terminal PBM lead to con- genital nephrotic syndrome.6 Specifically, a truncating mutation of Nephrin (p.L1240fs12863) that lacks the very C-terminal Va- insights into Nephrin-mediated protein complex in slit line of PBM and instead bears an additional 45 amino acids was diaphragm assembly and signaling. The potent inhibitory peptide identified in patients with steroid-resistant nephrotic syndrome could serve as a useful manipulating tool to dissect differential (SRNS)(Figure1A,SupplementalFigure1).10 These genetic data rolesofMAGIisoformsinfutureinvestigations. implied that Nephrin, in addition to playing key roles in the assembly of the slit diaphragm, also serves as an intracellular signaling hub to orchestrate the dynamic regulation of the slit METHODS diaphragm. There is much evidence indicating that Nephrin forms Protein Expression and Purification multicomponent complexes with a variety of PDZ domain– The coding sequences of MAGI1-PDZ3 (residues 820–920) and containing proteins in the slit diaphragm, including membrane- Nephrin-PBM (residues 1247–1256) were amplified from associated guanylate kinase family proteins (e.g.,MAGI1/2,ZO-1, mouse brain complementary DNA. For the isothermal titration and CASK),11,12 the Par6/Par3 complex,13 scribble,14 etc. Nephrin- calorimetry (ITC) assay, wild-type or various mutants of PBM–mediated protein complexes play essential roles in mainte- MAGI1-PDZ3 and Nephrin-PBM were cloned into a modified nance of glomerular filtration barrier integrity.11 However, little version of pET15b vector and pET32a vector, respectively. His6- 2+ is known about whether the atypical PBM of Nephrin directly tagged proteins were purified with Ni -NTA agarose affinity interacts with these PDZ-containing proteins. If it does, how is it chromatography, followed by size-exclusion chromatography specifically recognized by its target(s)? We believe that a better (SEC). For the glutathione-S-transferase (GST) pull-down assay, understanding of the molecular basis underlying the Nephrin- MAGI1-PDZ3 or Nephrin-PBM was fused to the C-terminus of PBM–mediated complex formation can provide valuable insights GSTusing the pGEX-4T-1 vector and purified by GSH-Sepharose into the physiologic roles of Nephrin in the assembly and dynamic affinity chromatography, followed by another round of SEC. All of regulation of the slit diaphragm, as well as the pathogenesis of the constructs were expressed in Escherichia coli BL21 (DE3) host nephrotic syndrome caused by alterations of NPHS1. cells at 16°C for 16–18 hours. In this study, we systematically studied the interactions between For the reconstitution of the MAGI1-PDZ3/Nephrin-PBM fi fi Nephrin-PBM and PDZ domain–containing proteins in complex, His6-MAGI1-PDZ3 was rstly puri ed as described slit diaphragm and found that only MAGI1 bound directly to above, and the His6-tag was then removed by incubation with Nephrin-PBM. We further discovered that MAGI1-PDZ3 binds human rhinovirus 3C protease overnight, followed by another to Nephrin-PBM with high specificity and affinity. The precise round of SEC. The commercial synthetic Nephrin-PBM peptide molecular basis underlying this interaction was elucidated by was mixed with MAGI1-PDZ3 in a molar excess of 3:1 (approx- solving the complex structure. Unexpectedly, the atypical PBM imately 15 mg/ml total complex protein in PBS buffer; pH 7.4). of Nephrin binds to MAGI1-PDZ3 with a mode distinct from the All of the peptides used in this study were commercial syn- canonical PDZ/PBM interactions. In addition to the canonical thesized by ChinaPeptide Co., Ltd. binding interface at the aB/bB groove, the N-terminus of Neph- rin-PBM adopts a short helix structure to engage the hydrophobic GST Pull-Down Assay surface formed by bB/bC of PDZ3. Interestingly, the highly con- GST-tagged MAGI1-PDZ3 was incubated with thioredoxin- served residue Gly(23) of Nephrin-PBM plays a critical role in the tagged Nephrin-PBM in the assay buffer (50 mM Tris [pH 8.0], formation of the MAGI1/Nephrin complex, which is different 100mMNaCl,1mMDTT,and1mMEDTA)for1hourat4°C.For from the typical PDZ/PBM binding modes. We further inhibitory peptide competition experiments, GST-tagged Neph- developed a potent inhibitory peptide that can specifically block rin-PBMwasincubatedwithHis-taggedMAI1-PDZ3inthepres- the MAGI1/Nephrin interaction in vitro and in vivo. Our bioche- ence of the PBM_H-2T peptide or the PBM_G-3A peptide in the mic, biophysic, and cell biologic studies could provide valuable assay buffer. After incubation, the mixture was centrifuged at 2 Journal of the American Society of Nephrology J Am Soc Nephrol 29: ccc–ccc,2018 www.jasn.org BASIC RESEARCH individually for 30 minutes. After washing three times, the bound proteins were eluted by boiling with 20 ml23 SDS- PAGE loading dye and detected by Western blotting with anti-Flag antibody (Sigma). ITC Assay The ITC assay was performed on a MicroCal ITC-200 (Malvern Panalytical, UK) at 25°C. All of the protein samples were in the buffer containing 50 mM Tris (pH 8.0), 100 mM NaCl, 1 mM EDTA, and 1 mM DTT. Wild- type and various mutants of Nephrin-PBM (approximately 0.6 mM) and MAGI1-PDZ3 (approximately 0.04 mM) were placed in a syringe and the sample cell, respectively.
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