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Cited by 17Inflammasome and immune disordersImmune Response and Inflammationinterferon and immune responses

The DOI matches its Crossref record; 35 of the 35 references it cites were found in a registry or an index.

Structural basis for flagellin-induced NAIP5 activation

Open access
Paidimuddala, Bhaskar, Cao, Jianhao, Zhang, Liman·2023·Science Advances
DOI 10.1126/sciadv.adi8539PMC PMC10699770

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Abstract

The NAIP (NLR family apoptosis inhibitory protein)/NLRC4 (NLR family CARD containing protein 4) inflammasome senses Gram-negative bacterial ligand. In the ligand-bound state, the winged helix domain of NAIP forms a steric clash with NLRC4 to open it up. However, how ligand binding activates NAIP is less clear. Here, we investigated the dynamics of the ligand-binding region of inactive NAIP5 and solved the cryo-EM structure of NAIP5 in complex with its specific ligand, FliC from flagellin, at 2.9-Å resolution. The structure revealed a “trap and lock” mechanism in FliC recognition, whereby FliC-D0 C is first trapped by the hydrophobic pocket of NAIP5, then locked in the binding site by ID (insertion domain) and C-terminal tail of NAIP5. The FliC-D0 N domain further inserts into ID to stabilize the complex. According to this mechanism, FliC triggers the conformational change of NAIP5 by bringing multiple flexible domains together.

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Figures

Fig. 1. Dynamics of unliganded NAIP5, biochemical purification, and cryo-EM structure of the NAIP5/FliC complex. (A) Multi-body refinement of unliganded NAIP5 in Relion. From Relion multi-body refinement, we obtained 10 maps representing different conformations. Here, we only showed the 1st map (blue) and the 10th map (gray). The blue and gray region includes a part of HD2 and the entire LRR domains. (B) SEC profile of the NAIP5/FliC complex. (C) SDS-PAGE of fractions in (B). (D) Western blot of fractions in (B). (E) Representative cryo-EM micrograph and (F) 2D class averages showing side view and top/bottom views. (G) Cryo-EM map and structure of the NAIP5/FliC complex; individual domains are color-coded. (H) The missing densities of unliganded NAIP5 (black) are plotted in the newly resolved NAIP5/FliC structure. FliC is shown in salmon.
Fig. 1. Dynamics of unliganded NAIP5, biochemical purification, and cryo-EM structure of the NAIP5/FliC complex. (A) Multi-body refinement of unliganded NAIP5 in Relion. From Relion multi-body refinement, we obtained 10 maps representing different conformations. Here, we only showed the 1st map (blue) and the 10th map (gray). The blue and gray region includes a part of HD2 and the entire LRR domains. (B) SEC profile of the NAIP5/FliC complex. (C) SDS-PAGE of fractions in (B). (D) Western blot of fractions in (B). (E) Representative cryo-EM micrograph and (F) 2D class averages showing side view and top/bottom views. (G) Cryo-EM map and structure of the NAIP5/FliC complex; individual domains are color-coded. (H) The missing densities of unliganded NAIP5 (black) are plotted in the newly resolved NAIP5/FliC structure. FliC is shown in salmon.CC BY-NC · PubMed Central
Fig. 2. The FliC subunit in the NAIP5/FliC complex. (A) D0N and D0C helices of FliC in the cryo-EM map of NAIP5/FliC complex. The extra density that may correspond to part of remaining regions of FliC is labeled. (B) D0N and D0C in flagella filament. (C) Effect of different truncations of FliC on the complex formation. This experiment was repeated three independent times. (D) Illustration of the FliC truncations in (C).
Fig. 2. The FliC subunit in the NAIP5/FliC complex. (A) D0N and D0C helices of FliC in the cryo-EM map of NAIP5/FliC complex. The extra density that may correspond to part of remaining regions of FliC is labeled. (B) D0N and D0C in flagella filament. (C) Effect of different truncations of FliC on the complex formation. This experiment was repeated three independent times. (D) Illustration of the FliC truncations in (C).CC BY-NC · PubMed Central
Fig. 3. Interactions between FliC-D0C and NAIP5. (A) The C-terminal residues of D0C insert into the hydrophobic pocket of NAIP5 formed by NTD, BIR1, and HD1; NAIP5 domains are either color-coded (left) or shown with their surface electrostatic potentials (right). D0C is in gray. (B) The middle part of D0C (gray) is sandwiched by ID (red) and HD2 (orange). (C) The N-terminal part of D0C (gray) interacts with both ID (red) and LRR (cyan). (D) Effect of different D0C truncations in complex formation. This experiment was repeated three independent times.
Fig. 3. Interactions between FliC-D0C and NAIP5. (A) The C-terminal residues of D0C insert into the hydrophobic pocket of NAIP5 formed by NTD, BIR1, and HD1; NAIP5 domains are either color-coded (left) or shown with their surface electrostatic potentials (right). D0C is in gray. (B) The middle part of D0C (gray) is sandwiched by ID (red) and HD2 (orange). (C) The N-terminal part of D0C (gray) interacts with both ID (red) and LRR (cyan). (D) Effect of different D0C truncations in complex formation. This experiment was repeated three independent times.CC BY-NC · PubMed Central
Fig. 4. Interactions in the ID/CTT/D0C regions. (A) The CTT of NAIP5 inserts into the ID loop and forms extensive interactions with the LRR and ID of NAIP5. (B) Effects of mutations in the CTT in the complex formation. This experiment was repeated three independent times. (C) Residues mapped in the ID loop that are involved in ID/CTT and ID/D0C interactions. (D) D0N inserts into ID loop and interacts with both ID and HD2. (E) Effects of mutations in (C) and (D) in the complex formation. This experiment was repeated three independent times.
Fig. 4. Interactions in the ID/CTT/D0C regions. (A) The CTT of NAIP5 inserts into the ID loop and forms extensive interactions with the LRR and ID of NAIP5. (B) Effects of mutations in the CTT in the complex formation. This experiment was repeated three independent times. (C) Residues mapped in the ID loop that are involved in ID/CTT and ID/D0C interactions. (D) D0N inserts into ID loop and interacts with both ID and HD2. (E) Effects of mutations in (C) and (D) in the complex formation. This experiment was repeated three independent times.CC BY-NC · PubMed Central

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Metadata

Authors
Paidimuddala, B., Cao, J., & Zhang, L.
Affiliations
Oregon Health & Science University (US)
Corresponding author
Zhang, Liman
ORCID
Venue
Science Advances
Publisher
American Association for the Advancement of Science (AAAS)
Published
2023-12-08
Volume
9
Issue
49
Pages
eadi8539-eadi8539
ISSN
2375-2548 (ISSN-L)
DOI
10.1126/sciadv.adi8539
PMC
PMC10699770
OpenAlex
W4389371417
Cited by
17
References
35
Citations by year
2026: 9 · 2025: 5 · 2024: 3
FWCI
1.51
Type
journal article
Language
en
Open access
Licence
cc-by-nc
Locations
  • Science Advances (publishedVersion, cc-by-nc)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by-nc)
  • PubMed (publishedVersion)
  • OSTI OAI (U.S. Department of Energy Office of Scientific and Technical Information) (submittedVersion)
  • PubMed Central (submittedVersion, cc-by-nc-nd)
Funders
  • U.S. Department of Energy (grid.436923.9, 436923.9)
  • Oregon Health and Science University (grid.436923.9, 436923.9)
  • National Institutes of Health
  • Office of Science (grid.436923.9)
  • National Institute of General Medical Sciences (U24GM129547)
  • National Institute of Allergy and Infectious Diseases (R01AI165580)
  • Biological and Environmental Research (grid.436923.9)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Inflammasome and immune disorders
  • Life Sciences › Immunology and Microbiology › Immunology › Immune Response and Inflammation
  • Life Sciences › Immunology and Microbiology › Immunology › interferon and immune responses
Keywords
Naip5, NLRC4, inflammasome, flagellin, FliC, winged-helix domain, ligand binding, conformational change, cryo-EM structure, host-pathogen recognition, innate immunity

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CoChat. (2026). Citation check: Structural basis for flagellin-induced NAIP5 activation. https://app.cochat.ai/papers/10.1126/sciadv.adi8539
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Paidimuddala, B., Cao, J., & Zhang, L. (2023). Structural basis for flagellin-induced NAIP5 activation. Science Advances. https://doi.org/10.1126/sciadv.adi8539

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