# Structural & Functional Studies of TLR/IL-1R Signaling

> **NIH NIH R37** · BOSTON CHILDREN'S HOSPITAL · 2020 · $531,000

## Abstract

Toll-like receptors (TLRs) and receptors for pro-inflammatory cytokines IL-1 and IL-18 share a
common TIR domain in their intracellular region and belong to the TLR/IL1-R superfamily. TLRs
recognize pathogen-associated molecular patterns (PAMPs) to initiate protective immune
responses. The molecular pathways for these receptors are complex and their dysregulation is
associated with many human diseases both within and beyond the immune system. Signal
transduction of these receptors is initiated by the approximation of the receptor TIR domains
upon binding of PAMPs and cytokines. This leads to the recruitment of intracellular TIRcontaining
adaptors such as MyD88, TIRAP/Mal, TRIF and TRAM. MyD88 is critical for
signaling responses of IL-1, IL-18, and all TLRs except TLR3. In addition to its C-terminal TIR
domain, MyD88 contains an N-terminal death domain (DD). Through the DD, MyD88 interacts
with IRAKs, including IRAK1, IRAK2, IRAK4 and IRAK-M, which are characterized by an Nterminal
DD and a C-terminal Ser/Thr kinase or kinase-like domain. Eventually, the ensuing
pathway activates transcription factors NF-κB, AP-1, and IRFs to elicit anti-pathogen responses
and inflammation. Despite the biological importance of the TLR/IL-1R signaling system, limited
structural and mechanistic information is available. In this application for a MERIT extension, we
propose to continue our studies in this system using structural methods such as X-ray
crystallography and cryo-electron microscopy, as well as pharmacological and light microscopy
approaches.
In the extension period, we will focus on four related areas:
1. Elucidating the molecular mechanism of TIR-TIR interactions in wild-type and disease
mutant adaptor proteins. We will build from our preliminary data in this regard and
pursue high-resolution structure determination of these oligomeric complexes.
2. Interrogating TLR/IL-1R signal transduction using reconstituted systems on lipid bilayers
and total internal reflection fluorescence microscopy. These studies will better mimic
cellular signaling because they provide the membrane-based, two-dimensional platform
for signal complex assembly. In our preliminary data, we have established the feasibility
of reconstitution and imaging.
3. Elucidating TLR/IL-1R signal transduction and the functional interaction with other innate
immune pathways in CRISPR-modified macrophages in which an endogenous signaling
protein is replaced by the fluorescently tagged version. We have already generated a
number of these cell lines and shown that they can be activated. We will image these
cells under different activating conditions to obtain quantitative analysis on the temporal
and spatial control of these receptors.
4. Identifying small molecule inhibitors for IRAKs in the treatment of lymphomas. We have
previously crystallized IRAK4 and revealed an inactive conformation of the kinase during
the process of trans-autophosphorylation. In our preliminary data, we have crystallized...

## Key facts

- **NIH application ID:** 9893784
- **Project number:** 5R37AI050872-20
- **Recipient organization:** BOSTON CHILDREN'S HOSPITAL
- **Principal Investigator:** Hao Wu
- **Activity code:** R37 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $531,000
- **Award type:** 5
- **Project period:** 2002-01-01 → 2022-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9893784

## Citation

> US National Institutes of Health, RePORTER application 9893784, Structural & Functional Studies of TLR/IL-1R Signaling (5R37AI050872-20). Retrieved via AI Analytics 2026-06-14 from https://api.ai-analytics.org/grant/nih/9893784. Licensed CC0.

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