Gluten, a protein found in wheat, barley and rye, can cause severe immune reactions in some people. The immune system attacks the small intestine and, over time flattens the villi of the intestine. The damaged gut cannot absorb nutrients and people with the condition suffer from stomach pain, bloating and diarrhoea. Doctors call this condition celiac disease.
Currently, there is no cure for this problem. The only solution is a lifelong strict diet which avoids gluten containing food.
The problem is caused by mutations in an enzyme, transglutaminase 2, which modifies glutamine residues in gluten. When gluten is digested and is broken down into smaller fragments or peptides, the mutant form of transglutaminase 2 binds to gliadin, one of the peptides, and modifies it into a form that is recognizable to the immune system. This is what causes the immune reaction.
Scientists recognise that transglutaminase flips between open and closed conformations depending on the chemical environment to which it is exposed. Guanine tri-phosphate, GTP, keeps it closed whereas calcium makes it open.
What actually happens in the protein when transglutaminase 2 flips between open and closed? How do small changes in the transglutaminase 2 gene change its functions?
Ragothaman Yennamalli and team at the Jawaharlal Nehru University and the SASTRA University, decided to look more closely at this open versus closed transglutaminase 2 switch.
First, they collected all the known 3D shapes of human transglutaminase 2 that scientists have already worked out and stored in the Protein Data Bank, a public database. Some shapes were closed and bound to guanine di-phosphate or adenine tri-phosphate. Some were open.
The researchers simulated the molecular dynamics, a detailed, physics-based movie, of the protein, showing how every part of it jiggles, bends and moves over a simulated stretch of 200 nanoseconds. Though this an extremely short slice of real time, it is long enough at the atomic scale to see meaningful movements.
The researchers did this for four different versions of transglutaminase 2: open with calcium, open without calcium, closed with calcium, and closed without calcium.
They found that, when transglutaminase 2 is open, and has no calcium around, it moves the most. One region of the protein, a β2 domain, swings around by an angle of over 66 degrees, and shifts almost 38 Angstroms away from where it started, a major movement for a protein.
When calcium was added to the open shape, the same region barely moved, rotating only about 16 degrees. So, calcium, in a sense, holds the open shape steady, like a clamp that stops it from swinging too far. This keeps the site where gluten binds accessible and ready to work.
In the closed shape, with or without calcium, the β2 domain of transglutaminase 2 barely moved. This suggests that the closed shape is naturally compact and rigid, while the open shape is flexible and active. Calcium is what keeps that open shape usefully open rather than flopping around.
How does the open and closed configuration change the function of the protein?
To investigate the problem, the team used a coarse-grained model, where instead of tracking every single atom, each amino acid is treated as one connected bead on a spring-like network. This allows scientists to study slower, larger-scale motions of the whole protein without needing much computing time.
Using this method, the team found certain hotspot residues, small stretches of amino acids that bend and move around a lot. In the open shape, these hotspots sit very close to transglutaminase 2’s active site, the pocket where it binds and modifies gluten, about 8 to 9 Angstroms away.
In the closed shape, these same hotspots sit much farther from the active site. This closeness, in the open form, suggests that these flexible spots may help transglutaminase 2 bind to gluten. So calcium does not seem to be just switching transglutaminase 2 open. It may also be stabilising and protecting that open, working shape.
What happens when there is a mutation in transglutaminase 2?
Small, naturally occurring changes in a gene, where a single letter of DNA is swapped for another, may change the amino acid coded. Sometimes this does nothing. Sometimes it can quietly break the protein’s shape or function.
The team downloaded nearly a hundred such known mutations from SNPdbe, a public database. After removing duplicates, they were left with about sixty unique mutations to study.
Which of these mutations actually matter?
To estimate whether a change would destabilize transglutaminase 2 or interfere with its folding, the researchers ran each mutation through several different computer programs, some that look only at the protein sequence, and some that look at the actual 3D structure of the protein.
Using this approach, they narrowed the list down to ten mutations that were consistently flagged as damaging across multiple tools. Of these ten, four stood out as especially likely to cause major changes in the shape and behaviour of transglutaminase 2.
One of these, where cysteine at the 277th position is replaced by serine, sits exactly at the protein’s active site, the location where the open shape of transglutaminase 2 binds to RNA. A mutation that close to the active site could, at least in principle, disturb both the binding of transglutaminase 2 to gluten and its ability to switch shape and bind to RNA. Earlier RNA-binding studies showed that transglutaminase 2 binds to RNA only in its open form.
This computational study helps explain how transglutaminase 2 gets to that open form, why calcium is what keeps it there, and how a single mutation in the gene could unsettle that whole switch system.
Now that there are specific clues about the relationships between the structure and function of transglutaminase 2, the Yennamalli lab plans to identify small drug-like molecules that bind to specific sites to modify the calcium binding, the GTP binding and the RNA binding. This, they hope, will help develop treatments for celiac disease.
“Drugs for treating the condition are only one of the trajectories for our future efforts. About 14 million Indians are estimated to have celiac disease. But most cases are undiagnosed, since there is no simple diagnostic method. We hope to develop some diagnostics for the condition also,” says Ragothaman Yennamalli, Jawaharlal Nehru University.
Journal of Molecular Graphics and Modelling 148: 109498 (2026);
DOI: 10.1016/j.jmgm.2026.109498
Reported by Anuska Karmakar
PhD scholar, JNU
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