Cryo-EM Structure of Native Human Uromodulin, a Zona Pellucida Module Polymer

SUMMARYAssembly 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 human, no information is available on the filamentous conformation of any ZP module protein. Here we report the cryo-electron microscopy structure 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 mammalian and avian heteromeric egg coat filaments identify a common sperm-binding region at the interface between subunits.

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Identification of Sperm-Binding Sites in the N-Terminal Domain of Bovine Egg Coat Glycoprotein ZP4

International Journal of Molecular Sciences ◽

10.3390/ijms23020762 ◽

2022 ◽

Vol 23 (2) ◽

pp. 762

Author(s):

Kamila Dilimulati ◽

Misaki Orita ◽

Yoshiki Yonahara ◽

Fabiana Lica Imai ◽

Naoto Yonezawa

Keyword(s):

Binding Sites ◽

Binding Activity ◽

Binding Region ◽

Terminal Domain ◽

Sperm Binding ◽

Bovine Sperm ◽

Selective Interaction ◽

Egg Coat

The species-selective interaction between sperm and egg at the beginning of mammalian fertilisation is partly mediated by a transparent envelope called the zona pellucida (ZP). The ZP is composed of three or four glycoproteins (ZP1–ZP4). The functions of the three proteins present in mice (ZP1–ZP3) have been extensively studied. However, the biological role of ZP4, which was found in all other mammals studied so far, has remained largely unknown. Previously, by developing a solid support assay system, we showed that ZP4 exhibits sperm-binding activity in bovines and the N-terminal domain of bovine ZP4 (bZP4 ZP-N1 domain) is a sperm-binding region. Here, we show that bovine sperm bind to the bZP4 ZP-N1 domain in a species-selective manner and that N-glycosylation is not required for sperm-binding activity. Moreover, we identified three sites involved in sperm binding (site I: from Gln-41 to Pro-46, site II: from Leu-65 to Ser-68 and site III: from Thr-108 to Ile-123) in the bZP4 ZP-N1 domain using chimeric bovine/porcine and bovine/human ZP4 recombinant proteins. These results provide in vitro experimental evidence for the role of the bZP4 ZP-N1 domain in mediating sperm binding to the ZP.

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Egg cortical granule N-acetylglucosaminidase is required for the mouse zona block to polyspermy.

The Journal of Cell Biology ◽

10.1083/jcb.123.6.1431 ◽

1993 ◽

Vol 123 (6) ◽

pp. 1431-1440 ◽

Cited By ~ 87

Author(s):

D J Miller ◽

X Gong ◽

G Decker ◽

B D Shur

Keyword(s):

Zona Pellucida ◽

Binding Sites ◽

Binding Activity ◽

Cortical Granule ◽

Cortical Granules ◽

Sperm Binding ◽

Competitive Inhibitors ◽

Glycosidase Inhibitor ◽

Egg Coat

The mammalian egg must be fertilized by only one sperm to prevent polyploidy. In most mammals studied to date, the primary block to polyspermy occurs at the zona pellucida, the mammalian egg coat, after exocytosis of the contents of the cortical granules into the perivitelline space. The exudate acts on the zona, causing it to lose its ability to bind sperm and to be penetrated by sperm previously bound to the zona. However, the cortical granule components responsible for the zona block have not been identified. Studies described herein demonstrate that N-acetylglucosaminidase is localized in cortical granules and is responsible for the loss in sperm-binding activity leading to the zona block to polyspermy. Before fertilization, sperm initially bind to the zona by an interaction between sperm surface GalTase and terminal N-acetylglucosamine residues on specific oligosaccharides of the zona glycoprotein ZP3 (Miller, D. J., M. B. Macek, and B. D. Shur. 1992. Nature (Lond.). 357:589-593). These GalTase-binding sites are lost from ZP3 after fertilization, an effect that can be duplicated by N-acetylglucosaminidase treatment. Therefore, N-acetylglucosaminidase, or a related glycosidase, may be present in cortical granules and be responsible for ZP3's loss of sperm-binding activity at fertilization. Of eight glycosidases assayed in exudates of ionophore-activated eggs, N-acetylglucosaminidase was 10-fold higher than any other activity. The enzyme was localized to cortical granules using immunoelectron microscopy. Approximately 70 or 90% of the enzyme was released from cortical granules after ionophore activation or in vivo fertilization, respectively. The isoform of N-acetylglucosaminidase found in cortical granules was identified as beta-hexosaminidase B, the beta, beta homodimer. Inhibition of N-acetylglucosaminidase released from activated eggs, with either competitive inhibitors or with specific antibodies, resulted in polyspermic binding to the zona pellucida. Another glycosidase inhibitor or nonimmune antibodies had no effect on sperm binding to activated eggs. Therefore, egg cortical granule N-acetylglucosaminidase is released at fertilization, where it inactivates the sperm GalTase-binding site, accounting for the block in sperm binding to the zona pellucida.

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Cryo-EM reveals unique structural features of the FhuCDB Escherichia coli ferrichrome importer

Communications Biology ◽

10.1038/s42003-021-02916-2 ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Wenxin Hu ◽

Hongjin Zheng

Keyword(s):

Electron Microscopy ◽

Escherichia Coli ◽

Molecular Basis ◽

Iron Uptake ◽

Structural Features ◽

Biological Processes ◽

Type Ii ◽

Functional Studies ◽

Cryo Electron Microscopy ◽

Translocation Pathway

AbstractAs one of the most elegant biological processes developed in bacteria, the siderophore-mediated iron uptake demands the action of specific ATP-binding cassette (ABC) importers. Although extensive studies have been done on various ABC importers, the molecular basis of these iron-chelated-siderophore importers are still not fully understood. Here, we report the structure of a ferrichrome importer FhuCDB from Escherichia coli at 3.4 Å resolution determined by cryo electron microscopy. The structure revealed a monomeric membrane subunit of FhuB with a substrate translocation pathway in the middle. In the pathway, there were unique arrangements of residues, especially layers of methionines. Important residues found in the structure were interrogated by mutagenesis and functional studies. Surprisingly, the importer’s ATPase activity was decreased upon FhuD binding, which deviated from the current understanding about bacterial ABC importers. In summary, to the best of our knowledge, these studies not only reveal a new structural twist in the type II ABC importer subfamily, but also provide biological insights in the transport of iron-chelated siderophores.

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Structure of the posttranslational Sec protein-translocation channel complex from yeast

Science ◽

10.1126/science.aav6740 ◽

2018 ◽

Vol 363 (6422) ◽

pp. 84-87 ◽

Cited By ~ 32

Author(s):

Samuel Itskanov ◽

Eunyong Park

Keyword(s):

Electron Microscopy ◽

Saccharomyces Cerevisiae ◽

Endoplasmic Reticulum ◽

Membrane Proteins ◽

Binding Sites ◽

Protein Translocation ◽

Secretory Proteins ◽

Conducting Channel ◽

Cryo Electron Microscopy ◽

Lateral Gate

The Sec61 protein-conducting channel mediates transport of many proteins, such as secretory proteins, across the endoplasmic reticulum (ER) membrane during or after translation. Posttranslational transport is enabled by two additional membrane proteins associated with the channel, Sec63 and Sec62, but its mechanism is poorly understood. We determined a structure of the Sec complex (Sec61-Sec63-Sec71-Sec72) from Saccharomyces cerevisiae by cryo–electron microscopy (cryo-EM). The structure shows that Sec63 tightly associates with Sec61 through interactions in cytosolic, transmembrane, and ER-luminal domains, prying open Sec61’s lateral gate and translocation pore and thus activating the channel for substrate engagement. Furthermore, Sec63 optimally positions binding sites for cytosolic and luminal chaperones in the complex to enable efficient polypeptide translocation. Our study provides mechanistic insights into eukaryotic posttranslational protein translocation.

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Use of microbeads for the detection of binding sites on the human zona pellucida: a scanning electron microscopy (SEM) assay

Andrologia ◽

10.1111/j.1439-0272.2001.tb01494.x ◽

2001 ◽

Vol 33 (5) ◽

pp. 266-271

Author(s):

H. W. Michelmann ◽

E. Töpfer-Petersen ◽

P. Schwartz ◽

G. Gratz ◽

C. Magerkurth

Keyword(s):

Electron Microscopy ◽

Scanning Electron Microscopy ◽

Zona Pellucida ◽

Binding Sites ◽

Scanning Electron

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Rac1 GTPase activates the WAVE regulatory complex through two distinct binding sites

eLife ◽

10.7554/elife.29795 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 54

Author(s):

Baoyu Chen ◽

Hui-Ting Chou ◽

Chad A Brautigam ◽

Wenmin Xing ◽

Sheng Yang ◽

...

Keyword(s):

Electron Microscopy ◽

Binding Sites ◽

Actin Polymerization ◽

Rho Gtpase ◽

Biochemical Data ◽

Actin Assembly ◽

Cellular Processes ◽

Cryo Electron Microscopy ◽

Regulatory Complex ◽

Rac1 Gtpase

The Rho GTPase Rac1 activates the WAVE regulatory complex (WRC) to drive Arp2/3 complex-mediated actin polymerization, which underpins diverse cellular processes. Here we report the structure of a WRC-Rac1 complex determined by cryo-electron microscopy. Surprisingly, Rac1 is not located at the binding site on the Sra1 subunit of the WRC previously identified by mutagenesis and biochemical data. Rather, it binds to a distinct, conserved site on the opposite end of Sra1. Biophysical and biochemical data on WRC mutants confirm that Rac1 binds to both sites, with the newly identified site having higher affinity and both sites required for WRC activation. Our data reveal that the WRC is activated by simultaneous engagement of two Rac1 molecules, suggesting a mechanism by which cells may sense the density of active Rac1 at membranes to precisely control actin assembly.

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Cryo-EM Structures of a Ferrichrome Importer FhuCDB

10.1101/2021.06.15.448540 ◽

2021 ◽

Author(s):

Wenxin Hu ◽

Hongjin Zheng

Keyword(s):

Electron Microscopy ◽

Escherichia Coli ◽

Atpase Activity ◽

Molecular Basis ◽

Iron Uptake ◽

Biological Processes ◽

Type Ii ◽

Functional Studies ◽

Cryo Electron Microscopy ◽

Translocation Pathway

As one of the most elegant biological processes developed in bacteria, the siderophore-mediated iron uptake demands the action of specific ATP-binding cassette (ABC) importers. Although extensive studies have been done on various ABC importers, the molecular basis of these iron-chelated-siderophore importers are still not fully understood. Here, we report the structure of a ferrichrome importer FhuCDB from Escherichia coli at 3.4 angstrom resolution determined by cryo electron microscopy. The structure revealed a monomeric membrane subunit of FhuB with a substrate translocation pathway in the middle. In the pathway, there were unique arrangements of residues, especially layers of methionines. Important residues found in the structure were interrogated by mutagenesis and functional studies. Surprisingly, the ATPase activity of the importer was decreased upon FhuD binding, which deviated from the current understanding about bacterial ABC importers. In summary, our studies not only reveal a new structural twist in the type II ABC importer subfamily, but also provide biological insights in the transport of iron-chelated siderophores.

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Use of microbeads for the detection of binding sites on the human zona pellucida: a scanning electron microscopy (SEM) assay

Andrologia ◽

10.1046/j.1439-0272.2001.00412.x ◽

2001 ◽

Vol 33 (5) ◽

pp. 266-271 ◽

Cited By ~ 3

Author(s):

H. W. Michelmann ◽

E. Töpfer-Petersen ◽

P. Schwartz ◽

G. Gratz ◽

C. Magerkurth

Keyword(s):

Electron Microscopy ◽

Scanning Electron Microscopy ◽

Zona Pellucida ◽

Binding Sites ◽

Scanning Electron

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The human GID complex engages two independent modules for substrate recruitment

10.1101/2021.04.07.438752 ◽

2021 ◽

Author(s):

Matthias Peter ◽

Weaam I. Mohamed ◽

Sophia L. Park ◽

Julius Rabl ◽

Alexander Leitner ◽

...

Keyword(s):

Ubiquitin Ligase ◽

Binding Sites ◽

Cell Cycle Progression ◽

Substrate Recognition ◽

E3 Ubiquitin Ligase ◽

Biological Processes ◽

Cycle Progression ◽

Cryo Electron Microscopy ◽

Biochemical Assays

The human GID (hGID) complex is an evolutionary conserved E3 ubiquitin ligase regulating diverse biological processes including glucose metabolism and cell cycle progression. However, the biochemical function and substrate recognition of the multi-subunit complex remains poorly understood. While the yeast GID complex recognizes Pro/N-end rule substrates via yeast Gid4, the human GID complex requires a WDR26/Gid7-dependent module to trigger proteasomal degradation of mammalian HBP1. Here, using biochemical assays, crosslinking-mass spectrometry and cryo-electron microscopy, we show that hGID unexpectedly engages two distinct modules for substrate recruitment, dependent on either WDR26 or GID4. WDR26 together with RanBP9 cooperate to ubiquitinate HBP1 in vitro, while GID4 is dispensable for this reaction. In contrast, GID4 functions as an adaptor for the substrate ZMYND19, which surprisingly lacks a Pro/N-end rule degron. GID4 substrate binding and ligase activity is regulated by ARMC8 alpha, while the shorter ARMC8 beta; isoform assembles into a stable hGID complex that is unable to recruit GID4. Cryo-EM reconstructions of these hGID complexes reveal the localization of WDR26 within a ring-like, tetrameric architecture and suggest that GID4 and WDR26/Gid7 utilize different, non-overlapping binding sites. Together, these data advance our mechanistic understanding of how the hGID complex recruits cognate substrates and provide insights into the regulation of its ligase activity.

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The structures of secretory and dimeric immunoglobulin A

eLife ◽

10.7554/elife.56098 ◽

2020 ◽

Vol 9 ◽

Author(s):

Sonya Kumar Bharathkar ◽

Benjamin W Parker ◽

Andrey G Malyutin ◽

Nandan Haloi ◽

Kathryn E Huey-Tubman ◽

...

Keyword(s):

Electron Microscopy ◽

Heavy Chain ◽

Binding Sites ◽

Immunoglobulin A ◽

Secretory Component ◽

Antigen Binding ◽

Effector Functions ◽

Cryo Electron Microscopy ◽

Mucosal Antibody ◽

Steric Accessibility

Secretory (S) Immunoglobulin (Ig) A is the predominant mucosal antibody, which binds pathogens and commensal microbes. SIgA is a polymeric antibody, typically containing two copies of IgA that assemble with one joining-chain (JC) to form dimeric (d) IgA that is bound by the polymeric Ig-receptor ectodomain, called secretory component (SC). Here, we report the cryo-electron microscopy structures of murine SIgA and dIgA. Structures reveal two IgAs conjoined through four heavy-chain tailpieces and the JC that together form a β-sandwich-like fold. The two IgAs are bent and tilted with respect to each other, forming distinct concave and convex surfaces. In SIgA, SC is bound to one face, asymmetrically contacting both IgAs and JC. The bent and tilted arrangement of complex components limits the possible positions of both sets of antigen-binding fragments (Fabs) and preserves steric accessibility to receptor-binding sites, likely influencing antigen binding and effector functions.

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