Filament structure activates and regulates CRISPR-Cas “protein scissors”

CRISPR-Cas systems help protect bacteria from viruses. Several different types of CRISPR-Cas defense systems are found in bacteria, which differ in their composition and functions. Among them, the most studied proteins today are Cas9 and Cas12, also known as DNA or “genetic scissors”, which have revolutionized the field of genome editing, allowing scientists to edit genomes and accurately correct pathogenic mutations.

Researchers from the Institute of Biotechnology of the Vilnius University Life Sciences Center — Dalia Smalakytė, Audronė Rukšėnaitė, Dr. Giedrius Sasnauskas, Dr. Giedrė Tamulaitienė and Dr. Gintautas Tamulaitis — revealed the structure of the CRISPR-protein scissors Cases found in bacteria and provided mechanistic details of how they work. The results of their study were published in Molecular cell.

A team of researchers led by Dr. Tamulaitis is studying the bacterial defense system CRISPR-Cas10, which acts as a sensor. When a virus attacks the bacteria, it sends a “message” by synthesizing unique signal molecules called cyclic oligoadenylates.

These signaling molecules are recognized by different effectors, that is to say accessory proteins of the system which reinforce the bacterial defense against viruses. A recent computational analysis predicted that CRISPR-Cas10 effectors could have diverse enzymatic activities, allowing bacteria to defend against viruses in several ways.

“The discovery of cyclic oligoadenylates and the understanding of the mechanism of CRISPR-Cas10 have sparked great scientific interest and a breakthrough in signaling pathway research. Recently, a similar protective principle has been identified in other defense systems bacteria: CBASS, Pycsar and Thoeris “In this study, we investigated the tripartite effector CalpL-CalpT-CalpS which is activated by CRISPR-Cas10 signaling molecules and we explained how this complex system works and how it is regulated. “, explains Dr. Tamulaitis.

The CalpL-CalpT-CalpS effector consists of three key proteins: CalpL, which acts as a signal-recognizing protein scissor; CalpS, a protein that regulates gene expression; and CalpT, an inhibitor of the CalpS protein. The researchers used a combination of biochemical, biophysical, bacterial survival and cryogenic electron microscopy (cryo-EM) tests to study this system. They found that when CalpL binds to a molecule signaling viral infection, it forms a polymer filament of varying composition.

The filament structure allows the CalpT-CalpS heterodimer to attach, positioning the active CalpL scissor center near the CalpT inhibitor and allowing it to cleave it. Once CalpT is divided, CalpS is released from the heterodimer and can regulate gene expression to protect the bacteria against viral infection.

One of the authors, Dalia Smalakytė, points out that the activity of CRISPR-Cas protein scissors is tightly regulated over time. Protein scissors have an internal timer mechanism that is activated upon binding of signaling molecules and formation of filaments. This mechanism is unique compared to other similar signal-sensing effector proteins.

The newly discovered mechanism of the CRISPR-Cas10 system illustrates the complexity of the bacterial defense system. These studies pave the way for the practical application of CRISPR-Cas regulated protein scissors as a molecular indicator of infection.