Scientists Find Small Changes in an Antibody Binding Site Can Completely Alter Protein Activity

By Eric Sauter

When it comes to recognizing antigens, the immune system is confronted with a paradox. First, to be effective, it must be able to recognize the vast universe of foreign antigens. However, that recognition has to be specific and selective enough to prevent the system from attacking the host, initiating a reaction that could lead to autoimmune diseases such as lupus, rheumatoid arthritis, diabetes, or multiple sclerosis.

On top of that, there are far fewer immune system cell lines than potential antigens, so the immune system starts out at a severe numerical disadvantage.

Now, scientists at The Scripps Research Institute have uncovered new evidence that the immune system can produce extremely subtle changes in antibody binding sites to expand its ability to recognize a wider variety of threats. These findings ultimately could lead to deeper insight into how the immune system is able to recognize and react to foreign threats.

The study was published August 4, 2008, in an advanced, online edition of the journal Proceedings of the National Academy of Science.

"We had two closely related antibodies, but with completely different specificities and functions," said Professor Ian A. Wilson, a Scripps Research investigator and member of The Skaggs Institute for Chemical Biology, whose laboratory conducted the study with Don Hilvert and his group at the Eldgenossische Technische Hochschule (ETH) in Zurich. "One, the Diels–Alderase antibody 1E9 was a catalytic antibody, while the other was the steroid binding antibody DB3. When we first looked at their sequences and structure, we realized they were highly related, derived from same antibody germ line in fact, so it was somewhat surprising they had such completely different activities. Our basic challenge in this study was to convert one protein activity into another."

To understand how these binding site changes alter and convert antibody specificity and function, Wilson and his colleagues first determined the crystal structures of the newly paired antibodies.

Then, through the mutation of a virtual handful of amino acid residues, Wilson and his colleagues were able to turn 1E9 into a high-affinity steroid receptor with a ligand recognition profile similar to DB3. Moreover, they found that the introduction of only two mutations in the 1E9 binding site increased the antibody's affinity for steroids as much as 14,000 times.

However, the study revealed that, despite close functional mimicry of DB3, the modified1E9 antibody developed a unique steroid binding mechanism that included extensive structural rearrangements in the combining site.

"We fully expected to find that the two antibodies would then bind in the same way and we expected the structure to confirm that," Wilson said. "However, what we found were fundamentally different modes of binding. That was a fairly big surprise."

The fusion of the 1E9 framework with the DB3 mutations not only changed the ligand specificity in 1E9, creating a binding site that accommodated steroids, but did so without sacrificing affinity for differently configured steroids.

"This shows how the immune system actually works, how each antibody can have multiple specificities which you can then fine tune," Wilson said. "As a result, our findings may show a generalized way to attain a particular specificity for immune receptors and extend their recognition range. Unfortunately, this method also carries with it the risk of increased receptor promiscuity, where antibodies bind to host antigens. This is basically what happens in autoimmune diseases—the system reacts to something foreign and then cross reacts with the host."

The first author of the study, Closely Related Antibody Receptors Exploit Fundamentally Different Strategies for Steroid Recognition, is Petra Verdino of The Scripps Research Institute. In addition to Wilson, Hilvert, and Verdino, authors include Caroline Aldag of the Eldgenossische Technische Hochschule, Zurich, Switzerland. For more information, see http://www.pnas.org/content/early/2008/08/07/0801783105.abstract .

The study was supported by Erwin-Schrodinger Fellowship of the Austrian Science Fund, the National Institutes of Health, and Eidgenossische Technische Hochschule, Zurich.

Send comments to: mikaono[at]scripps.edu

"This shows how the immune system actually works, how each antibody can have multiple specificities which you can then fine tune."

—Ian Wilson