Computer-designed protein targets immune receptor linked to inflammation at a new undruggable site

LA JOLLA, CA-To sense their environment and respond accordingly, cells enlist membrane proteins as communication hubs, receiving molecular messages from outside and triggering responses inside. One of these proteins is Toll-like receptor 4 (TLR4), an immune receptor that plays an essential role in protecting against infections. But overactivity of TLR4 has been linked to inflammatory disorders like sepsis, arthritis and inflammatory bowel disease, making it an attractive target for therapies. Despite its appeal, TLR4 is difficult to precisely manipulate, and no FDA-approved drugs specifically block it.

Now, in a new PNAS study published on September 22, 2026, scientists at Scripps Research engineered a small synthetic protein that can bind to TLR4 within cell membranes and block subsequent inflammatory responses. The findings advance understanding of what TLR4's membrane-embedded region does: rather than a passive anchor, it's an active determinant of cross-membrane signaling-mechanistic insight that could guide design of a new class of anti-inflammatory treatments. More broadly, the study outlines new computational tools that other researchers can use to target proteins within membranes.

People assumed that the regions of TLR4 exposed outside and inside the cell were the main signaling drivers, but we showed that the membrane-spanning region is also critical for this function, says the study's first author Colleen Maillie, a research project analyst at Scripps Research.

The cell membrane is composed of two compact layers of oily molecules. This gives the membrane very different biochemical properties from the water-based environments found outside and within the cell, where the majority of proteins reside. Many of the rules governing how proteins fold and function within these oily membranes are still unclear, which has made it challenging for researchers to design drugs that act there.

But Maillie and a team of scientists in the labs of co-senior authors Assistant Professor Marco Mravic and Professor Andrew Ward of Scripps Research were determined to develop a new method to better access and manipulate membrane proteins. For this study, they selected TLR4.

TLR4 is a key sensor of bacteria that activates and mobilizes immune cells to fight infection, says Ward. It may be activated by adjuvants in vaccines to improve immune responses or inhibited to suppress inflammation, making it both a sensor and a dial to tune innate immunity.

Generally, when the exposed portion of TLR4 detects a bacterial molecule outside the cell, it triggers changes in the receptor that prompt it to form dimers, or pairs of two TLR4 proteins. These dimers then sometimes-but not always-switch on an inflammatory response in the cell. TLR4 can also detect some non-bacterial molecules, including ones associated with tissue damage.

Maillie and the rest of the team wondered if the transmembrane (or membrane-embedded) region of TLR4, which was thought to passively attach it to the membrane, might actually be playing an active role in determining these inflammatory responses.

To better understand the transmembrane region's role, the team introduced a fragment of TLR4-that included the membrane-spanning region and a small neighboring section of the protein-into human cells grown in the lab. They found that these fragments readily associate with TLR4 in the membrane, using a screening approach recently developed by the Mravic lab that emits light when tagged proteins are close together. The result shows that the approach, which was specifically developed to measure which synthetic proteins target membrane proteins, is useful in practice. Additionally, the team found that the presence of the fragments reduced overall NF- B signaling responses, one of the main inflammatory pathways triggered by TLR4.

Now that they knew that targeting the transmembrane region might be a feasible way to dial down TLR4-initiated inflammation, they aimed to design a synthetic protein that could bind there more strongly and alter its activity to a greater extent. The first step was using computer programs to generate 3D blueprints of protein structures.

Scientists have been using computers to help design proteins for decades, says Mravic. Models for protein interactions and structures living in water have become increasingly accurate. However, for membrane proteins, they are not. There are unique atomic details underlying molecular biophysics in lipid bilayers that current equations and AI models don't accurately capture.

Since the computer programs couldn't reliably identify how the proteins would interact with their targets in the membrane, the team used their predictions as starting points, then applied custom design criteria developed in the Mravic lab to optimize apolar packing, or how tightly the chemical structures of the designed proteins fit together with TLR4. They generated many structure options, then narrowed them down to the nine with the best predicted biophysical and chemical features to test in living cells.

Employing the same light-up approach used previously, the team tested the nine synthetic proteins and found that eight showed signs of associating with TLR4. Three stood out as the strongest candidates, and Design-6 showed the strongest evidence of interaction, along with other appealing qualities; for example, it didn't aggregate as much as the naturally occurring transmembrane fragments. Design-6 was also able to substantially reduce NF- B inflammatory signaling.

We had this theory, which we encoded into software, that maximizing apolar packing would make more stable protein interactions within the greasy membrane, says Mravic. The software now lets us design new practical molecules with potential clinical relevance that can insert and act within the membrane.

The aut

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