Cryo‐EM structure of native human uromodulin, a zona pellucida module polymer
2020; Springer Nature; Volume: 39; Issue: 24 Linguagem: Inglês
10.15252/embj.2020106807
ISSN1460-2075
AutoresAlena Stsiapanava, Chenrui Xu, Martina Brunati, Sara Zamora‐Caballero, Céline Schaeffer, Marcel Bokhove, Ling Han, Hans Hebert, Marta Carroni, Shigeki Yasumasu, Luca Rampoldi, Bin Wu, Luca Jovine,
Tópico(s)RNA modifications and cancer
ResumoArticle16 November 2020Open Access Source DataTransparent process Cryo-EM structure of native human uromodulin, a zona pellucida module polymer Alena Stsiapanava Alena Stsiapanava orcid.org/0000-0001-6560-011X Department of Biosciences and Nutrition, Karolinska Institutet, Huddinge, Sweden These authors contributed equally to this work Search for more papers by this author Chenrui Xu Chenrui Xu orcid.org/0000-0002-5605-728X School of Biological Sciences, Nanyang Technological University, Singapore, Singapore NTU Institute of Structural Biology, Nanyang Technological University, Singapore, Singapore These authors contributed equally to this work Search for more papers by this author Martina Brunati Martina Brunati orcid.org/0000-0002-1010-0836 Molecular Genetics of Renal Disorders, Division of Genetics and Cell Biology, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Sara Zamora-Caballero Sara Zamora-Caballero orcid.org/0000-0003-4717-8845 Department of Biosciences and Nutrition, Karolinska Institutet, Huddinge, Sweden Search for more papers by this author Céline Schaeffer Céline Schaeffer orcid.org/0000-0001-5883-3951 Molecular Genetics of Renal Disorders, Division of Genetics and Cell Biology, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Marcel Bokhove Marcel Bokhove orcid.org/0000-0001-7241-5967 Department of Biosciences and Nutrition, Karolinska Institutet, Huddinge, Sweden Search for more papers by this author Ling Han Ling Han orcid.org/0000-0001-9310-4789 Department of Biosciences and Nutrition, Karolinska Institutet, Huddinge, Sweden Search for more papers by this author Hans Hebert Hans Hebert orcid.org/0000-0002-3220-9402 Department of Biosciences and Nutrition, Karolinska Institutet, Huddinge, Sweden Department of Biomedical Engineering and Health Systems, KTH Royal Institute of Technology, Huddinge, Sweden Search for more papers by this author Marta Carroni Marta Carroni orcid.org/0000-0002-7697-6427 Department of Biochemistry and Biophysics, Science for Life Laboratory, Stockholm University, Stockholm, Sweden Search for more papers by this author Shigeki Yasumasu Shigeki Yasumasu orcid.org/0000-0003-4295-477X Department of Materials and Life Sciences, Faculty of Science and Technology, Sophia University, Tokyo, Japan Search for more papers by this author Luca Rampoldi Luca Rampoldi orcid.org/0000-0002-0544-7042 Molecular Genetics of Renal Disorders, Division of Genetics and Cell Biology, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Bin Wu Corresponding Author Bin Wu [email protected] orcid.org/0000-0002-0883-8006 School of Biological Sciences, Nanyang Technological University, Singapore, Singapore NTU Institute of Structural Biology, Nanyang Technological University, Singapore, Singapore Search for more papers by this author Luca Jovine Corresponding Author Luca Jovine [email protected] orcid.org/0000-0002-2679-6946 Department of Biosciences and Nutrition, Karolinska Institutet, Huddinge, Sweden School of Biological Sciences, Nanyang Technological University, Singapore, Singapore Search for more papers by this author Alena Stsiapanava Alena Stsiapanava orcid.org/0000-0001-6560-011X Department of Biosciences and Nutrition, Karolinska Institutet, Huddinge, Sweden These authors contributed equally to this work Search for more papers by this author Chenrui Xu Chenrui Xu orcid.org/0000-0002-5605-728X School of Biological Sciences, Nanyang Technological University, Singapore, Singapore NTU Institute of Structural Biology, Nanyang Technological University, Singapore, Singapore These authors contributed equally to this work Search for more papers by this author Martina Brunati Martina Brunati orcid.org/0000-0002-1010-0836 Molecular Genetics of Renal Disorders, Division of Genetics and Cell Biology, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Sara Zamora-Caballero Sara Zamora-Caballero orcid.org/0000-0003-4717-8845 Department of Biosciences and Nutrition, Karolinska Institutet, Huddinge, Sweden Search for more papers by this author Céline Schaeffer Céline Schaeffer orcid.org/0000-0001-5883-3951 Molecular Genetics of Renal Disorders, Division of Genetics and Cell Biology, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Marcel Bokhove Marcel Bokhove orcid.org/0000-0001-7241-5967 Department of Biosciences and Nutrition, Karolinska Institutet, Huddinge, Sweden Search for more papers by this author Ling Han Ling Han orcid.org/0000-0001-9310-4789 Department of Biosciences and Nutrition, Karolinska Institutet, Huddinge, Sweden Search for more papers by this author Hans Hebert Hans Hebert orcid.org/0000-0002-3220-9402 Department of Biosciences and Nutrition, Karolinska Institutet, Huddinge, Sweden Department of Biomedical Engineering and Health Systems, KTH Royal Institute of Technology, Huddinge, Sweden Search for more papers by this author Marta Carroni Marta Carroni orcid.org/0000-0002-7697-6427 Department of Biochemistry and Biophysics, Science for Life Laboratory, Stockholm University, Stockholm, Sweden Search for more papers by this author Shigeki Yasumasu Shigeki Yasumasu orcid.org/0000-0003-4295-477X Department of Materials and Life Sciences, Faculty of Science and Technology, Sophia University, Tokyo, Japan Search for more papers by this author Luca Rampoldi Luca Rampoldi orcid.org/0000-0002-0544-7042 Molecular Genetics of Renal Disorders, Division of Genetics and Cell Biology, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Bin Wu Corresponding Author Bin Wu [email protected] orcid.org/0000-0002-0883-8006 School of Biological Sciences, Nanyang Technological University, Singapore, Singapore NTU Institute of Structural Biology, Nanyang Technological University, Singapore, Singapore Search for more papers by this author Luca Jovine Corresponding Author Luca Jovine [email protected] orcid.org/0000-0002-2679-6946 Department of Biosciences and Nutrition, Karolinska Institutet, Huddinge, Sweden School of Biological Sciences, Nanyang Technological University, Singapore, Singapore Search for more papers by this author Author Information Alena Stsiapanava1, Chenrui Xu2,3, Martina Brunati4, Sara Zamora-Caballero1, Céline Schaeffer4, Marcel Bokhove1, Ling Han1, Hans Hebert1,5, Marta Carroni6, Shigeki Yasumasu7, Luca Rampoldi4, Bin Wu *,2,3 and Luca Jovine *,1,2 1Department of Biosciences and Nutrition, Karolinska Institutet, Huddinge, Sweden 2School of Biological Sciences, Nanyang Technological University, Singapore, Singapore 3NTU Institute of Structural Biology, Nanyang Technological University, Singapore, Singapore 4Molecular Genetics of Renal Disorders, Division of Genetics and Cell Biology, IRCCS San Raffaele Scientific Institute, Milan, Italy 5Department of Biomedical Engineering and Health Systems, KTH Royal Institute of Technology, Huddinge, Sweden 6Department of Biochemistry and Biophysics, Science for Life Laboratory, Stockholm University, Stockholm, Sweden 7Department of Materials and Life Sciences, Faculty of Science and Technology, Sophia University, Tokyo, Japan *Corresponding author. Tel: +65 69082207; E-mail: [email protected] *Corresponding author. Tel: +46 852480000; E-mail: [email protected] The EMBO Journal (2020)39:e106807https://doi.org/10.15252/embj.2020106807 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Assembly of extracellular filaments and matrices mediating fundamental biological processes such as morphogenesis, hearing, fertilization, and antibacterial defense is driven by a ubiquitous polymerization module known as zona pellucida (ZP) "domain". Despite the conservation of this element from hydra to humans, no detailed information is available on the filamentous conformation of any ZP module protein. Here, we report a cryo-electron microscopy study of uromodulin (UMOD)/Tamm–Horsfall protein, the most abundant protein in human urine and an archetypal ZP module-containing molecule, in its mature homopolymeric state. UMOD forms a one-start helix with an unprecedented 180-degree twist between subunits enfolded by interdomain linkers that have completely reorganized as a result of propeptide dissociation. Lateral interaction between filaments in the urine generates sheets exposing a checkerboard of binding sites to capture uropathogenic bacteria, and UMOD-based models of heteromeric vertebrate egg coat filaments identify a common sperm-binding region at the interface between subunits. SYNOPSIS Urinary glycoprotein uromodulin (UMOD) forms filaments via its C-terminal zona pellucida (ZP) module, a conserved building block of many polymeric extracellular proteins. Cryo-EM of native human UMOD filaments and structurally-related vertebrate egg coat material sheds light on the atomic architecture of ZP module polymers and how it may contribute to their many biological functions. Comparison of the filament structure with that of the UMOD precursor explains how propeptide dissociation starts polymerization by triggering intermolecular interaction between ZP modules. The linker between the ZP-N and ZP-C moieties of the ZP module undergoes a dramatic conformational change during polymerization. In the filament, each UMOD subunit embraces the ZP-C and ZP-N domains of the neighboring molecules, giving rise to a highly stable helix made up of interlocked subunits with a twist angle of 180 degrees. UMOD filaments can assemble into a multivalent molecular "Velcro" that facilitates the capture of uropathogenic bacteria by the N-terminal region of the protein. By adopting an architecture similar to the UMOD homopolymer, heteromeric egg coat filaments present sperm-binding regions at the interface between subunits. Introduction The ZP "domain" is a conserved sequence of ~ 260 amino acids that was first recognized in UMOD as well as ZP2 and ZP3 components of the mammalian egg coat, the zona pellucida (Bork & Sander, 1992). Functional, biochemical, and structural studies showed that this element is a protein polymerization module that consists of two distinct but topologically related immunoglobulin-like domains, ZP-N and ZP-C (Jovine et al, 2002, 2004; Bokhove & Jovine, 2018). These are characterized by different intramolecular disulfide bond patterns and separated by a linker that, in crystal structures of non-polymeric precursor forms of ZP module proteins, can be either flexible or rigid, giving rise to different relative arrangements of ZP-N and ZP-C (Bokhove & Jovine, 2018). For example, the precursor of ZP3 is secreted as an antiparallel homodimer where the two moieties of the ZP module are connected by a largely disordered linker, with each ZP-N domain both lying against the ZP-C domain of the same subunit and interacting with the ZP-C of the other (Han et al, 2010). On the other hand, the interdomain linker of the UMOD precursor is entirely structured by forming an α-helix (α1) and a β-strand (β1) that pack against ZP-C; this orients the ZP-N domain so that it homodimerizes with ZP-N from another molecule (Bokhove et al, 2016a, 2016b). Despite these differences, in both ZP3 and UMOD the last β-strand of ZP-C (βG)—generally referred to as the external hydrophobic patch (EHP)—is part of a polymerization-blocking C-terminal propeptide (CTP) whose protease-dependent release is required for protein incorporation into filaments (Jovine et al, 2004; Schaeffer et al, 2009). Notably, in both mammalian egg coat proteins and UMOD, this process is dependent on membrane anchoring of the precursors (Jovine et al, 2002; Brunati et al, 2015); however, it is unclear how propeptide dissociation triggers polymerization, and the molecular basis of ZP module-mediated protein assembly remains essentially unknown. To address these questions, we exploited the natural abundance of UMOD (Tamm & Horsfall, 1950; Serafini-Cessi et al, 2003) to study ZP module filaments by cryo-electron microscopy (cryo-EM). First recognized as a major component of hyaline casts in 1873 (Rovida, 1873) and then described as an inhibitor of viral hemagglutination (Tamm & Horsfall, 1950; Serafini-Cessi et al, 2003), UMOD is expressed by cells of the thick ascending limb of Henle's loop as a highly glycosylated, intramolecularly disulfide-bonded, and glycosylphosphatidylinositol (GPI)-anchored precursor. This consists of three epidermal growth factor-like domains (EGF I-III), a cysteine-rich domain (D8C), a fourth EGF domain (EGF IV), and the ZP module, followed by a consensus cleavage site (CCS; often referred to as CFCS in other ZP module proteins) and the EHP-including CTP (Fig EV1A) (Serafini-Cessi et al, 2003; Bokhove et al, 2016a). Hepsin protease-mediated cleavage of the CCS leads to dissociation of mature UMOD from the CTP and triggers its incorporation into homopolymeric filaments (Schaeffer et al, 2009; Brunati et al, 2015). These protect against urinary tract infections by binding to uropathogenic E. coli (UPEC), reduce nephrolithiasis, and are involved in the regulation of water/electrolyte balance and kidney innate immunity (Serafini-Cessi et al, 2003; Devuyst et al, 2017; Weiss et al, 2020). While common variants of UMOD are strongly associated with risk of chronic kidney disease, higher levels of a monomeric form of UMOD that circulates in the serum and regulates renal and systemic oxidative stress were recently linked to a lower risk for mortality and cardiovascular disease in older adults (LaFavers et al, 2019; Steubl et al, 2020). Thus, elucidating how UMOD polymerization is regulated is not only important for ZP module proteins in general, but also crucial to understand the diverse biological functions of this key urinary molecule. Click here to expand this figure. Figure EV1. Full-length UMOD filaments: comparison with elastase-treated material and stability in 6 M urea A. Domain organization of the secreted human UMOD precursor. Magenta, EGF I-III; salmon, D8C domain; orange, EGF IV; light blue and dark blue, ZP-N and ZP-C domains; red, ZP-N/ZP-C linker; gray, internal hydrophobic patch (IHP); black, CCS; yellow, EHP. A thick black horizontal line marks the CTP, with a brown circle depicting the GPI anchor attachment. Inverted tripods show N-glycans, with the high-mannose chain attached to D8C N275 colored cyan. Black and orange arrows indicate the position of the hepsin (F587|R588) and elastase (S291|S292) cleavage sites, respectively, with thin horizontal bars indicating the extent of UMODfl and UMODe. B. Representative Volta phase plate micrographs of native UMODfl filaments. Although tree/front views are predominant, a number of zig-zag/side views can be seen in the right-most micrograph. The yellow arrows show examples of how twisting of individual UMOD filaments generates both views. Scale bars: 50 nm. C. Reducing Coomassie-stained SDS–PAGE analysis of the UMODfl (6 µg; lane 1) and UMODe (3 and 5 µg; lanes 2, 3) material used for structure determination. D. Representative micrograph of UMODe filaments, showing the absence of branches. Scale bar: 50 nm. E. Superposition of the UMODfl (salmon) and UMODe (cyan) cryo-EM maps shows that only the former shows density for a globular domain protruding from the core of the filaments. This reveals the approximate location of the elastase cleavage site, corresponding to the N-terminus of UMODe, within the structure of UMODfl (orange arrows). F. Coomassie-stained SDS–PAGE analysis of supernatant and pellet fractions of purified native UMOD filaments, incubated with increasing amounts of urea. No significant breakdown of the polymers is observed at urea concentrations below 7 M. Source data are available online for this figure. Download figure Download PowerPoint Results Structure of the UMOD filament To obtain information on the supramolecular structure of UMOD, we first imaged human urine samples by cryo-EM. This showed that the protein forms semi-regular sheets through lateral interaction of micrometer-long filaments, whose pairing generates features that were previously interpreted as the projection of a double-helical structure (Jovine et al, 2002) (Fig 1A). Imaging of purified samples of full-length native UMOD (UMODfl) showed that the majority of filaments had a tree-like structure, resulting from the regular alternation of ~12 nm-long branches protruding at an angle of 50 to 60 degrees from either side of the polymeric core (Fig 1B); other filaments instead adopted a zig-zag shape consistent with early negative stain EM studies of UMOD (Bayer, 1964) (Fig 1C). In agreement with the observation that the two types of structures occasionally interconvert within individual filaments (Fig EV1B), helical reconstruction of UMODfl showed that these apparently distinct conformations in fact correspond to different views of a single type of filament with 62.5 Å axial rise and 180° twist. The latter parameter, which is even more extreme than the −166.6° helical twist of F-actin (Dominguez & Holmes, 2011), severely complicated structure determination together with the thinness (~35 Å) and flexibility of the filament core. By averaging 288,403 helical segments, we were, however, able to obtain a cryo-EM map of UMODfl with an estimated average resolution of 3.8 Å, as well as a 3.4 Å map of the corresponding filament core (Figs 1F–J and EV2, Appendix Figs S1 and S2 and Table S1). To inform model building, we also studied native UMOD digested with elastase (UMODe), a protease that removes the entire N-terminal region of the protein (EGF I-III + D8C) by cutting at a single site just before EGF IV (Jovine et al, 2002) (Fig EV1A and C and Appendix Table S1). This leads to loss of UMOD filament branches (Figs 1D and E, and EV1D), and comparison of the resulting UMODe density with that of UMODfl allowed us to identify the location of the EGF IV N-terminus in the maps (Figs 1F and K, and EV1E). Using this information, we could unambiguously dock the crystallographic model of UMOD EGF IV and ZP-N (Bokhove et al, 2016a) into the UMODfl map and then fit the crystal structure of ZP-C (Bokhove et al, 2016a). These placements were validated by the presence of density for the N-glycans attached to ZP-N N396 (Fig 1G and Movie EV1) and ZP-C N513 (Fig EV2B, right panel). Subsequently, a continuous stretch of unexplained density contacting both domains was identified as the ZP-N/ZP-C linker of a third molecule (UMOD 3) that embraces the previously placed ZP-C and ZP-N, which belong to adjacent protein subunits (UMOD 2 and UMOD 4, respectively; Figs 1H and I and 2, Movies EV2 and EV3). This revealed that the relative arrangement of the ZP module moieties of filamentous UMOD is completely different from that of its homodimeric precursor (Bokhove et al, 2016a), so that the distance between the centers of mass of the ZP-N and ZP-C β-sandwiches increases from 41 to 91 Å upon polymerization (Fig 3A). Consistent with a ~ 120 Å axial periodicity (Jovine et al, 2002), this ZP module conformation allows UMOD monomers to interact head-to-tail (ZP-N-to-ZP-C), with one and two-half subunits per turn (Figs 1F and 2). Figure 1. Overall structure of human UMOD filaments A. Electron micrograph of unstained UMOD filament sheets in human urine. The inset highlights a double helix-like structure resulting from juxtaposition of two individual filaments. Scale bars: 50 nm and 10 nm (inset). B, C. "Tree" front view and "zig-zag" side view of purified native UMOD filaments, imaged using a Volta phase plate. Scale bars: 10 nm. D, E. Front and side views of UMODe filaments, showing the absence of branches. Scale bars: 10 nm. F. Orthogonal views of the sharpened cryo-EM map of UMODfl (3.8 Å resolution), oriented as in panels (B and C), respectively. The map is fitted with an atomic model that consists of a complete EGF IV + ZP module (chain A; blue), the ZP-C domain of a second molecule (chain B; teal), and the EGF IV + ZP-N domain of a third one (chain C; magenta). G–J. Details of the UMODfl map shown in panel (F): ZP-N N396 glycan (G); interdomain linker α1β (H) and β1 (I); ZP-C αEFβ/ZP-N βF′ intermolecular interface (J). K. Sharpened cryo-EM map of UMODe (4.0 Å resolution) in two orthogonal views oriented as in panels (D and E). Comparison of this map with that of UMODfl identifies density belonging to the N-terminal half of UMOD (salmon contour in the front view of panel F), which is lost upon site-specific cleavage by elastase (orange arrow). L. Goodsell-style depiction of a complete UMODfl filament model, with protein subunits shown in different colors. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Sharpened cryo-EM map of the UMODfl filament core (3.4 Å resolution) A. Overall view of the entire polymerization region of a UMOD molecule (blue), wrapped around the ZP-C domain (teal) and EGF IV + ZP-N domains (magenta) of the preceding and following subunits, respectively. B. Details of different parts of the map, highlighting the separation of β-strands (left panel) and the quality of side chain density (middle and right panels). The map is fitted with an atomic model of UMOD where carbon atoms of different chains are colored according to panel A. Download figure Download PowerPoint Figure 2. Interaction between each copy of UMOD and the ZP modules of four other subunits generates a unique filament architecture A. A section of a UMOD filament is shown that consists of 5 consecutive subunits (UMOD 1-5) related by the helical symmetry operation indicated in the top panel. In the middle panel, where the helical axis is represented by a large black arrow, subunits are depicted in cartoon (UMOD 1, 3, and 5) and surface (UMOD 2, 3, and 4) representation to highlight protein–protein interfaces (with UMOD 1 ZP-N and UMOD 5 ZP-C omitted for clarity). In the filament, the ZP-N/ZP-C linker of each molecule (e.g., UMOD 3) wraps around the ZP-C domain of the subunit that precedes it (UMOD 2) and the ZP-N domain of the subunit that follows it (UMOD 4); additionally, the ZP-N and ZP-C domains of the same molecule are engaged in interactions with the ZP-C domain of the subunit that in turn precedes UMOD 2 (UMOD 1) and the ZP-N domain of the subunit that follows UMOD 4 (UMOD 5), respectively. As summarized in the bottom panel, every UMOD subunit is thus interacting with another four by being engaged in six interfaces that belong to three different types (ZP-N/ZP-C, black arrow; ZP-N/β1, dark red arrow; E′FG, α1β/ZP-C, light red arrow). Subunits 3, 2, and 4 in this figure correspond to Fig 3 chains A, B, and C, respectively. B. Path of the interdomain linkers of UMOD 1-5, whose domains are outlined in the background. The view is rotated by ~ 40° around the Y-axis, compared to panel (A). Download figure Download PowerPoint Figure 3. Conformational changes and protein–protein interactions underlying UMOD polymerization A. Comparison of the precursor and polymeric structures of UMOD shows how dissociation of the EHP triggers a major conformational change in the ZP module. This involves a significant rearrangement of the interdomain linker, which not only completely dissociates from ZP-C but also changes secondary structure upon conversion of α1 in the precursor to α1β in the polymer. Molecules are depicted in cartoon representation, with only one subunit of the UMOD precursor homodimer shown; structural elements are colored as in Fig EV1A, with the N- and C-terminal halves of the ZP-N/ZP-C linker colored bright and dark red, respectively. B. In the ZP-C domain of the precursor form of UMOD (left panel, teal), the polymerization-blocking EHP β-strand interacts hydrophobically with a short α-helix (αEF) encompassing the FXF motif. Hepsin-mediated cleavage of the CCS of this molecule (chain B/UMOD 2) triggers release of its EHP, which is replaced by α1β from the interdomain linker of a second UMOD subunit (chain A/UMOD 3) (right panel). This allows the FXF motif of molecule B to form an intermolecular β-sheet (αEFβ/βF′; upper dashed box) with the ZP-N fg loop of a third, incoming subunit (chain C/UMOD 4, magenta). Another result of the CCS cleavage is that the C-terminus of mature UMOD 2 is freed for interaction with the D8C domain of the same molecule (not shown). Elements are shown as in panel (A), with disulfide bonds and glycan residues represented by thick dark gray and thin black sticks, respectively; β-strands are labeled as in the UMOD precursor (Bokhove et al, 2016a). C. Hidden Markov model logos, highlighting the conservation of selected residues shown in panels (B, D, and E). D. Hydrophobic interactions stabilize the interface between the E′FG extension of the ZP-N domain of chain A and the ZP-C domain of chain B, corresponding to the dashed box in the lower right part of panel (B). E. Details of the interface between the ZP-C domain of chain B and the ZP-N domain of chain C, showing a different view of the area boxed in the upper right part of panel (B). Download figure Download PowerPoint Lower map resolution outside the filament core and lack of close structural homologues precluded accurate modeling of D8C. However, different ab initio prediction programs suggested that—consistent with the expected presence of multiple intramolecular disulfides (Hamlin & Fish, 1977; Yang et al, 2004)—this domain adopts a compact fold with average dimensions that closely match the globular density protruding from EGF IV (Fig EV3A and B). Notably, the density for the short C-terminal tail of hepsin-processed UMOD, whose flexibility is restricted by the last disulfide of the protein (C6527-C8582), merges with that of D8C (Fig EV3B). This suggests that cleavage of UMOD not only activates its ZP-C domain for polymerization, but also allows it to interact with D8C and, in turn, orient the N-terminal region of the protein relative to the core of the polymer. Despite this additional conformational constraint, the latter appears to be highly mobile and swings relative to the filament core, as suggested by the blurred densities seen in 2D class images (Appendix Fig S2C). A multi-body refinement that focused on the branch was then performed to refine the density of the whole N-terminal region of UMOD (Appendix Fig S1 and Fig EV3C). Considering the previous placement of D8C, this local map at moderate resolution agrees with the dimensions of a homology model of EGF I-III (Fig EV3C and D). By combining this information with the refined coordinates of EGF IV + ZP, we could assemble a complete filament model consistent with both the tree and zig-zag views of UMODfl (Figs 1L and EV3D). Click here to expand this figure. Figure EV3. Docking of D8C and EGF I-III domain models into the density map of UMODfl A. D8C domain models created using I-TASSER (blue shades) or Robetta (magenta shades) have approximately the same overall dimensions. B. Consistent with the location of the elastase cleavage site (orange arrow) that immediately precedes the EGF IV domain (orange), the top model of D8C generated by Robetta (salmon) can be straightforwardly docked into the globular density protruding from the core of the filament. The unsharpened cryo-EM map of UMODfl is shown, and a black arrow indicates the C6527-C8582 disulfide, which orients the C-terminal tail of mature UMOD (thick lemon tube) toward D8C. The latter also packs against the loop that connects C6527 to βD (thin lemon tube). C. Using multi-body refinement in RELION, we performed a focused refinement of the density corresponding to the complete N-terminal branch of UMOD. This locally refined map suggested the location of EGF I-III, which served as a guideline to build a model of full-length UMOD. D. The gray density depicts the N-terminal branch, treated as body 1, whereas the cyan density shows the rest of the filament segment, treated as body 2. After being separately refined, these two local maps were merged to produce a composite map of UMODfl, which was used as a reference for model building and as a starting point for Fig 6B. UMOD branch domains are indicated and colored as in Fig S1A. Download figure Download PowerPoint Filament formation involves a major conformational change in the ZP module's interdomain linker A dramatic rearrangement of the ZP-N/ZP-C linker region during polymerization underlies the highly different ZP module conformations of the precursor and filamentous forms of UMOD (Fig 3A). In the former, the interdomain linker consists of an α-helix (α1) and a β-strand (β1) that pack against ZP-C β-strand A—an element implicated in polymerization and known as internal hydrophobic patch (IHP; Jovine et al, 2004)—and, in the case of β1, also interact with the EHP (Bokhove et al, 2016a). In polymeric UMOD, α1 and the amino acids that follow it transform into a 13-residue long twisted β-strand (α1β; alternatively described as two distinct β strands, α1β′ (D430-S434) and α1β" (A438-M442), separated by a three-residue linker) (Fig 1H and Movie EV2). This substitutes the EHP of the previous subunit by hydrogen bonding to its F and A" strands as well as facing βA/IHP (Figs 2A and 3B), with linker residue L435 inserted into a conserved hydrophobic pocket formed by IHP M460 and L462, βB V487, and βF L564 (Fig EV4A and B). Interestingly, an identical copy of the DM
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