C3 is central to many disease mechanisms, and the new findings from Aarhus University have already led to collaborations based on this new knowledge from the research team. A part of our immune system that is implicated in many diseases is called the complement system. This is a so-called cascade that, when activated, leads to inflammation and the marking of foreign organisms for elimination by immune cells. The central protein in this cascade is C3, which is one of the most abundant proteins in our blood—up to 1.5 g/L.

There are several ways in which C3 can be activated, but the least understood activation pathway involves a spontaneous reaction between C3 and a water molecule, resulting in a rearrangement of C3.

When the research project began, existing knowledge suggested that part of the C3 protein must pass through a very narrow channel when C3 reacts with water. This process is comparable to threading a string with a stopper through an opening much smaller than the stopper. In other activation pathways, an enzyme cuts off the stopper from C3, allowing the string to pass through easily, but this does not happen when C3 reacts with water.

In 2021, Ph.D. student Trine Gadeberg, under the supervision of Professor Gregers Rom Andersen at the Department of Molecular Biology and Genetics, used electron microscopy to investigate how this process occurs. Trine employed a laboratory trick using methylamine (MA), which induces the same rearrangement of C3 as water but at a much faster rate. The resulting product is called C3MA. Trine quickly succeeded in determining the three-dimensional structure of C3MA, confirming that the stopper had indeed been pulled through the narrow channel. However, this only intensified the interest in understanding how this step is possible.

The researchers proposed two models to explain the stopper-through-opening paradox: either the stopper deforms to fit through the opening, or the opening itself expands significantly. To investigate whether the stopper deformed, postdoctoral researcher Ana de Almeida tracked the same reaction using special UV radiation from the ASTRID2 synchrotron, located at the Department of Physics and Astronomy at Aarhus University. Combined with other data, Ana’s measurements showed that the shape of the stopper remained intact when C3 transformed into C3MA.

It has long been known that an intermediate state, called C3*, exists between C3 and C3MA. This state is sufficiently stable to be studied, even though C3* spontaneously converts to C3MA over a few days. With Trine’s prior success in electron microscopy in mind, Ph.D. student Martin Jørgensen set out to investigate C3*. Martin explains: “This turned out to be quite a challenge since C3* behaves like a ‘rubber’ molecule, adopting many different three-dimensional shapes.”

After months of analysis, Martin found that a small part of C3, which forms the narrow channel, is highly flexible in the C3* intermediate state, even though this part is normally fixed in both C3 and C3MA.

The researchers then searched their freezer for a molecule that binds to this flexible part of C3. Martin and Ana hypothesized that such a molecule would slow down the formation of C3MA, and indeed, they observed such a slowdown using multiple techniques.

With this new knowledge, the puzzle finally came together: the stopper can pass through an opening that had never been observed or predicted before—and which only exists in C3*.

The Research Paper

Cryo-EM Analysis of Complement C3 Reveals a Reversible Major Opening of the Macroglobulin Ring. Gadeberg, Trine Amalie Fogh, et al. Nature Structural & Molecular Biology, 2025, https://doi.org/10.1038/s41594-024-01467-4.

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Supplementary information

We strive to ensure that all our articles live up to the Danish universities' principles for good research communication. Against this background, the article is supplemented with the following information:

Study type:
Research article

External funding:
This work was supported by Lundbeck Foundation BRAINSTRUC center (R155-2015-2666, to G.R.A. and B.B.K.), the Novo Nordisk Foundation (NNF18OC0052105 and NNF20OC0065238, to G.R.A., NNF18OC0032724 BIO-MS to J.J.E. and NNF18OC0033926 to B.B.K.) and the Danish Council for Independent Research (10.46540/2032-00111B to J.J.E.).

Conflict of interest:
G.R.A., H.P. and R.K.J. are inventors on patents describing the hC3Nb2 nanobody and the EWEµH fusion protein. H.G.O. is co-founder of Commit Biologics, which has licensed these patents. The remaining authors declare no competing interests.

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For further information, please contact

Professor Gregers Rom Andersen
gra@mbg.au.dk
Department of Molecular Biology and Genetics
Aarhus University, Denmark