{"id":6095,"date":"2025-12-05T11:25:40","date_gmt":"2025-12-05T11:25:40","guid":{"rendered":"https:\/\/www.sygnaturediscovery.com\/?post_type=case-study&p=6095"},"modified":"2026-02-05T16:48:04","modified_gmt":"2026-02-05T16:48:04","slug":"generating-a-tl1a-crystal-structure","status":"publish","type":"case-study","link":"https:\/\/www.sygnaturediscovery.com\/fr\/case-study\/generating-a-tl1a-crystal-structure\/","title":{"rendered":"Generating a TL1A Crystal Structure"},"content":{"rendered":"\n

At Sygnature Discovery, we thrive on solving complex protein challenges\u2014and our recent success in solving the crystal structure of TL1A, a member of the TNF ligand superfamily, is a perfect example of how our expertise can accelerate drug discovery.<\/h2>\n\n\n\n

Background<\/h3>\n\n\n\n

TNF-like 1A (TL1A), also known as TNFSF15, is a cytokine that belongs to the tumour necrosis factor family of proteins, sharing homology with TNF\u03b1 . It has a broad range of functions, being involved in maintaining cellular homeostasis, inflammatory responses and determining cellular fate (1). Its dysregulation has been implicated in multiple auto-immune diseases, atherosclerosis, rheumatoid arthritis and cancer, making TL1A an attractive therapeutic target (1-2).<\/p>\n\n\n\n

The project began with a thorough review of the literature and structural databases to understand the behaviour of TL1A and any known challenges. In vivo, TL1A is expressed as a 251 amino acid transmembrane anchored protein that forms non-covalent homotrimers through its TNF homology domain and contains one intramolecular disulphide bond between cysteine residues, C95 and C135 (2-3). The N-terminal transmembrane domain (residues 1-71) is responsible for anchoring TL1A to the plasma membrane, whilst the C-terminal portion (residues 72-251) forms an extracellular soluble domain. The C-terminal portion can be cleaved off from the transmembrane domain by metalloproteinases such as TNF\u03b1 converting enzyme (TACE), generating a soluble species of TL1A, known as sTL1A (residues 72-251) (3-4). sTL1A is the species that is commonly used in recombinant protein production, with wild-type sTL1A expressed previously (2-3). However , Zhan et al reported disulphide bond heterogeneity between C95 and C135, when wild-type sTL1A was expressed in E. coli<\/em>. As such, they engineered a C95S\/C135S mutant to prevent disulphide mispairing (5).<\/p>\n\n\n\n

Guided by these literature precedents and prior experience with TNF\u03b1 production, we designed two constructs: wild-type TL1A and a C95S\/C135S mutant<\/p>\n\n\n\n

Purification and Quality Control<\/h4>\n\n\n\n

Both constructs expressed well, forming trimeric TL1A as expected. However, the wild-type protein showed increased aggregation and formed incorrect intermolecular disulphide bonds between TL1A protomers, as revealed by our non-reducing SDS-PAGE analysis (Figure 1). Whereas the SDS-PAGE and SEC-MALS analysis of the C95S\/C135S mutant confirmed a homogeneous protein preparation, that retained the correct trimer formation (Figure 2). As such, the C95S\/C135S construct was ideal to take forward for crystallography studies.<\/p>\n\n\n\n

\"SDS<\/figure>\n\n\n\n

Figure 1:<\/strong> Reducing and non-reducing SDS-PAGE analysis of wild-type TL1A (WT) and the C95S\/C135S double cysteine mutant [Cys].<\/em><\/p>\n\n\n\n

WT TL1A and the C95S\/C135S mutant were analysed under reducing (left panel) and non-reducing (right panel) conditions. Each panel includes molecular weight markers (Marker in kDa) and lanes containing 2 \u00b5g, 5 \u00b5g, and 10 \u00b5g of protein. Under reducing conditions, both WT and C95S\/C135S migrate predominantly as a single band consistent with their expected monomeric molecular weights (~20.5 kDa). This reflects the disruption of non-covalent trimeric interactions due to the presence of reductant and SDS in the gel. However, under non-reducing conditions, purification artefacts are observed for WT, indicating the formation of incorrect intermolecular disulfide-linked species. Whilst a single band was present for the C95S\/C135S mutant under both conditions, indicating its suitability for crystallography trials.<\/p>\n\n\n\n

\"SEC<\/figure>\n\n\n\n

Figure 2:<\/strong> SEC MALS analysis of wildtype TL1A (WT) and the C95S\/C135S double cysteine mutant [Cys].<\/em><\/p>\n\n\n\n

Size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) was performed to assess the oligomeric state and homogeneity of TL1A WT (red trace) and the C95S\/C135S mutant (green trace). Light scattering intensity at 90\u00b0 is plotted against elution time (minutes). Both samples elute at approximately 25 minutes, consistent with their expected trimeric molecular weight (~62 kDa). The C95S\/C135S mutant displays a sharper and more symmetrical peak compared to WT, indicating improved sample homogeneity. Moreover, the lack of an aggregation peak at approximately 21.5 minutes for C95S\/C135S indicates improved suitability for structural studies over the WT TL1A.<\/p>\n\n\n\n

Structural Biology<\/h4>\n\n\n\n

Using a combination of broad and literature-informed crystallisation screens, we obtained high-quality crystals of the TL1A mutant. The final structure was solved at 2.2 \u00c5 resolution using data collected at Diamond Light Source and belonged to the space group P 1 21 1 (Figure 3). Our structure revealed two trimers per asymmetric unit, offering a unique advantage for future asymmetric binder co-crystallisation. As such, this preliminary work provides a strong foundation for rapidly enabling the solution of TL1A-ligand structures by soaking or co-crystallisation.<\/p>\n\n\n\n

Sygnature TL1A [C95S\/C135S] Structure:<\/h3>\n\n\n\n
\"Sygnature<\/figure>\n\n\n\n

Data Collection & Refinement Statistics:<\/h4>\n\n\n\n
\"Sygnature<\/figure>\n\n\n\n

Figure 3:<\/strong> Crystal structure and data collection\/refinement statistics for TL1A [C95S\/C135S]<\/em><\/p>\n\n\n\n

The top panel shows the crystal structure of the Sygnature TL1A [C95S\/C135S] mutant, with each monomer coloured in blue, cyan, and orange. The bottom panel summarises the key data collection and refinement statistics.<\/p>\n\n\n\n

Conclusions<\/h3>\n\n\n\n

This project showcases our ability to:<\/p>\n\n\n\n