Synthetic antibodies against BRIL as universal fiducial marks for single−particle cryoEM structure determination of membrane proteins

Mukherjee, Somnath; Erramilli, Satchal K.; Ammirati, Mark; Alvarez, Frances Joan D.; Fennell, Kimberly F.; Purdy, Michael D.; Skrobek, Blazej M.; Radziwon, Katarzyna; Coukos, John S.; Kang, Ying; Dutka, Przemysław; Gao, Xiang; Qiu, Xiayang; Yeager, Mark; Xu, Hao; Han, Seungil; Kossiakoff, Anthony A.

doi:10.1038/s41467-020-15363-0

Cited by 74 publications

(54 citation statements)

References 65 publications

(76 reference statements)

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“…1b ). The purified fusion protein was incubated with Fab fragments of BRIL antibodies 18 , and the sample was analyzed by negative-stain electron microscopy (EM) (Supplementary Fig. 1c–e ).…”

Section: Resultsmentioning

confidence: 99%

Mechanism of membrane-curvature generation by ER-tubule shaping proteins

et al. 2021

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The endoplasmic reticulum (ER) network consists of tubules with high membrane curvature in cross-section, generated by the reticulons and REEPs. These proteins have two pairs of trans-membrane (TM) segments, followed by an amphipathic helix (APH), but how they induce curvature is poorly understood. Here, we show that REEPs form homodimers by interaction within the membrane. When overexpressed or reconstituted at high concentrations with phospholipids, REEPs cause extreme curvature through their TMs, generating lipoprotein particles instead of vesicles. The APH facilitates curvature generation, as its mutation prevents ER network formation of reconstituted proteoliposomes, and synthetic L- or D-amino acid peptides abolish ER network formation in Xenopus egg extracts. In Schizosaccharomyces japonicus, the APH is required for reticulon’s exclusive ER-tubule localization and restricted mobility. Thus, the TMs and APH cooperate to generate high membrane curvature. We propose that the formation of splayed REEP/reticulon dimers is responsible for ER tubule formation.

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Section: Resultsmentioning

confidence: 99%

Mechanism of membrane-curvature generation by ER-tubule shaping proteins

et al. 2021

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“…We employed an anti-BRIL Fab, BAG2 ( Mukherjee et al, 2020 ; Figure 1a ) with an anti-Fab nanobody (Nb) ( Ereño-Orbea et al, 2018 ) to complex with the BRIL fusion hFzd5 (hFzd5 ICL3 BRIL). The BRIL/Fab/Nb module was designed as a ‘fiducial’ marker for particle image alignment ( Mukherjee et al, 2020 ). We purified hFzd5 ICL3 BRIL from 293S GnTI - cells in a glycol-diosgenin/cholesterol hemisuccinate (GDN/CHS) micelle and incubated it with excess Fab and Nb before the size-exclusion chromatography step ( Figure 1—figure supplement 2a ).…”

Section: Resultsmentioning

confidence: 99%

“…The anti-BRIL Fab was expressed in E. coli and purified as described ( Mukherjee et al, 2020 ). The anti-Fab Nb ( Ereño-Orbea et al, 2018 ; Hermans et al, 2017 ) was cloned in pET26b+ vector with an N-terminal hexahistidine tag followed by a TEV protease site.…”

Section: Methodsmentioning

confidence: 99%

“…In order to optimize a rigid connection between hFzd5 and BRIL, we grafted two short linkers, each comprising five amino acid residues from A 2A adenosine receptor-TMs between TM5/6 of Fzd5 and BRIL (See Methods), as used for the first full-length Smo structure (Byrne et al, 2016). We employed an anti-BRIL Fab, BAG2 (Mukherjee et al, 2020) (Figure 1a) with an anti-Fab nanobody (Nb) (Ereño-Orbea et al, 2018) to complex with the BRIL fusion hFzd5 (hFzd5 ICL3 BRIL). The BRIL/Fab/Nb module was designed as a 'fiducial' marker for particle image alignment (Mukherjee et al, 2020).…”

Section: Structure Determination and Analysismentioning

confidence: 99%

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Structure of human Frizzled5 by fiducial-assisted cryo-EM supports a heterodimeric mechanism of canonical Wnt signaling

Tsutsumi

Mukherjee

Waghray

et al. 2020

eLife

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Frizzleds (Fzd) are the primary receptors for Wnt morphogens, which are essential regulators of stem cell biology, yet the structural basis of Wnt signaling through Fzd remains poorly understood. Here we report the structure of an unliganded human Fzd5 determined by single-particle cryo-EM at 3.7 Å resolution, with the aid of an antibody chaperone acting as a fiducial marker. We also analyzed the topology of low-resolution XWnt8/Fzd5 complex particles, which revealed extreme flexibility between the Wnt/Fzd-CRD and the Fzd-TM regions. Analysis of Wnt/β-catenin signaling in response to Wnt3a versus a 'surrogate agonist' that cross-links Fzd to LRP6, revealed identical structure-activity relationships. Thus, canonical Wnt/β-catenin signaling appears to be principally reliant on ligand-induced Fzd/LRP6 heterodimerization, versus the allosteric mechanisms seen in structurally analogous class A G protein-coupled receptors, and Smoothened. These findings deepen our mechanistic understanding of Wnt signal transduction, and have implications for harnessing Wnt agonism in regenerative medicine.

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“…In fact, antibody labeling methods for membrane proteins mediated by the fusion with a thermostabilized variant of apocytochrome b562 have been reported very recently (Miyagi et al, 2020, Mukherjee et al, 2020. Our method provides a new option for antibody labeling through exogenous epitopes.…”

Section: Discussionmentioning

confidence: 97%

Moving toward generalizable NZ-1 labeling for 3D structure determination with optimized epitope tag insertion

Tamura-Sakaguchi

Aruga

Hirose

et al. 2020

Preprint

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Antibody labeling has been extensively conducted for structure determination in both x-ray crystallography and EM analysis. However, establishing target-specific antibodies is a prerequisite for applying antibody-assisted structural analysis. To expand the applicability of this strategy, we have developed an alternative method to prepare an antibody-complex by inserting an exogenous epitope into the target. We here demonstrated that the Fab of monoclonal antibody NZ-1 could form a stable complex with the target containing an inserted PA14 tag as an epitope. By adopting a closed ring-like conformation inside the antigen-binding pocket, the inserted PA14 tag had less impact on the folding of the target. In fact, the PA14 tag could also be inserted into the sterically hindered loop for labeling. Using the improved labeling technique, we performed negative-stain EM on a bacterial site-2 protease, which enabled us to approximate the domain arrangement based on the docking mode of the NZ-1 Fab.

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Synthetic antibodies against BRIL as universal fiducial marks for single−particle cryoEM structure determination of membrane proteins

Cited by 74 publications

References 65 publications

Mechanism of membrane-curvature generation by ER-tubule shaping proteins

Mechanism of membrane-curvature generation by ER-tubule shaping proteins

Structure of human Frizzled5 by fiducial-assisted cryo-EM supports a heterodimeric mechanism of canonical Wnt signaling

Moving toward generalizable NZ-1 labeling for 3D structure determination with optimized epitope tag insertion

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