The saying in the design world is that form follows function. But in biology, and protein biology in particular, it would be more correct to say that form begets function. Shape and structure are the foundation for most protein-based interactions in cells, and are why basic functions like receptor binding, antibody neutralization and gene transcription work.
Two enzymes in the immune system’s B cells, called RAG1 and RAG2, are a perfect example. Together, they form a complex that splices antibody-producing genes together in unique combinations through a process called V(D)J recombination. They do a similar job in T cells to build antigen-binding T-cell receptors (TCRs). In either case, the enzymes are essential to a robust immune response.
In a recent Cell paper, a team led by Hao Wu, PhD, of the Program in Cellular and Molecular Medicine (PCMM) at Boston Children’s Hospital and Maofu Liao, PhD, at Harvard Medical School used electronic microscopy to reveal how RAG1 and 2 interact at a structural level, both with each other and with DNA. The structural biology images they’ve created show plainly what mutations in the genes for these proteins do to cause disease.
Come together, right now
While researchers have known about RAG1 and 2 for decades, only in the last year have the proteins’ individual structures been solved. But no one has ever before imaged the two together.
“People have been working on the RAG structures forever. Even two or three years ago there was barely anything,” says Wu, a structural biologist who recently received a National Institutes of Health Pioneer Award for efforts to exploit innate immune signaling proteins as drug targets. “It’s been a challenge to get the proteins to behave well so that you can actually do structure studies. It’s been a big RAG year.”
Her team’s images of the intact proteins’ structures reveal a lot about how the pair bind in complex to DNA, and how contact with DNA triggers a shape change that activates the complex’s DNA-cutting enzymatic activity, allowing different kinds of antibodies to be created.
“The images reveal the structural features in RAG1/2 that underlie the fidelity of V(D)J recombination,” Wu says, referring to the process by which the RAG complex shuffles antibody genes in B-cells and TCR genes in T-cells code for TCRs in such a way that the resulting antibodies or TCRs will function properly.
When RAG genes go wrong
When Wu and her colleagues looked at the impact of disease-causing mutations in the protein’s respective genes, they found that specific mutations (of which hundreds have been found for each protein) change the form of the protein complex and how it fits over its DNA target site.
“Most of the mutations disturb the structure, but many also affect how the proteins engage DNA,” Wu explains. “That not only can change the complex’s enzymatic activity, decreasing its efficiency, but also its specificity profile.”
The end result, Wu notes, is that patients with these RAG mutations have a much less diverse repertoire of B-cells and T-cells, leaving the body unable to react to many of the pathogens it might face. Some severe mutations nearly eliminate the complex’s DNA-cutting activity.
“By doing that you get a very severe combined immunodeficiency,” Wu says. “Some versions of these mutants change the immune repertoire to the point where you end up with immune compromise and autoimmunity all at once.”
Wu says that the study is more about understanding the structural basis of RAG-associated diseases, and less about finding potentially druggable targets. “You don’t really want to inhibit RAG1 or 2. It’s not a straightforward way of treating RAG deficiencies,” she explains. “The best way would still be bone marrow transplant or gene therapy that completely replace a patient’s defective gene.”
She mentions that other members of the PCMM and Boston Children’s faculty, including PCMM director Frederick Alt, PhD, and Luigi Notarangelo, MD, from Boston Children’s Immunology Program, are interested in RAG’s involvement in B-cell lymphomas and autoimmunity. Her team’s structural studies, she thinks, could form a baseline for understanding how RAG make cuts at places in the B-cell genome outside the cluster of antibody-producing genes. “We want to find out how RAG might work off-target and see if we can elucidate any disease-causing mechanisms.”