# Structural and Mechanistic Elucidation of NAIP/NLRC4 Inflammasomes in Innate Immunity

> **NIH NIH R00** · OREGON HEALTH & SCIENCE UNIVERSITY · 2021 · $56,980

## Abstract

Summary of the Funded Grant:
The goal of the proposed work is to understand the molecular mechanism of NAIP/NLRC4
inflammasome regulation. The NAIP/NLRC4 inflammasomes are defenders against cytosolic infections
by bacteria such as Salmonella and Pseudomonas through recruitment and activation of pro-caspase-1,
leading to the production of interleukin-1β (pro-IL-1β) and IL-18, and subsequent pyroptotic cell death.
The NAIP/NLRC4 inflammasomes are comprised with three components, the bacterial ligands such as
flagellin, the NAIPs, and the NLRC4 protein. NAIPs recognize different bacterial ligands and determine
the specificity of NAIP/NLRC4 inflammasomes. Upon activation, ligand/NAIP complex activate NLRC4
through a huge conformational change on NLRC4 protein, which exposes the nucleation surface and
leads to the polymerization of NLRC4 molecule.
Even though the activation mechanism of NAIP/NLRC4 inflammasome is well studied, we still don't
know how the NAIP protein stays inhibited before ligand invasion. In the study proposed here, we will
illustrate the structural mechanism of NAIP inhibition through crystallography, cryo-EM studies and
cellular assays and screen for small molecules that inhibit the inflammatory pathway. We anticipate that
these studies will help us understand the molecular mechanism of auto-inflammatory diseases that
involve abnormal activation of NAIP/NLRC4 complex. The specific aims of this proposal are:
Aim 1. Elucidating the mechanism of ligand dependent activation of NAIP/NLRC4
inflammasomes.
In this aim, I will use a combination of biochemical characterization, structure determination and
mutational analysis in cells to address how bacterial ligands are recognized and how this recognition
activates NAIP proteins and initiates NLRC4 oligomerization. Unlike the previous proposal of NAIP
proteins as the sole receptor, I found that NLRC4 may also directly interact with ligands and act as a
co-receptor. As such, simultaneous interactions of ligands with both a NAIP protein and NLRC4 likely
facilitate cooperative ligand- induced conformational changes. I have reconstituted several
NAIP/NLRC4 inflammasomes and am on the way for cryo-EM 3D reconstruction of these important
complexes. Collectively, these studies will reveal the molecular basis for ligand specificity and ligand-
induced conformational transitions.
Aim 2. Establishing the inhibitory mechanism of NAIP proteins.
In this aim, I will investigate the auto-inhibited conformation of NAIP proteins using structure
determination and mutational analysis in cells. I anticipate that before ligand stimulation, NAIP proteins
may exist in a closed conformation with extensive intra-molecular interactions. Furthermore, the unique
N-terminal BIR domains of NAIP proteins may play a role in this process. As preliminary data, I have
overcome the difficulty in expressing and purifying these large, and aggregation-prone proteins (~160
kDa).
Aim 3. Identifying small molecule inhibitors of CARD...

## Key facts

- **NIH application ID:** 10138387
- **Project number:** 3R00AI137300-04S1
- **Recipient organization:** OREGON HEALTH & SCIENCE UNIVERSITY
- **Principal Investigator:** Liman Zhang
- **Activity code:** R00 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $56,980
- **Award type:** 3
- **Project period:** 2021-02-10 → 2022-12-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10138387

## Citation

> US National Institutes of Health, RePORTER application 10138387, Structural and Mechanistic Elucidation of NAIP/NLRC4 Inflammasomes in Innate Immunity (3R00AI137300-04S1). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10138387. Licensed CC0.

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