Gallery Structure of ReadCrystal——CBL-B

Cbl-b protein is a type of E3 ubiquitin ligase that negatively regulates immune function and shows great potential as a novel intracellular immune checkpoint drug target.

ReadCrystal Biotechnology has completed the expression and structural analysis of the Cbl-b target protein. Based on these experimental conditions and the available stock of proteins, ReadCrystal has shortened the turnaround time to 1-2 months, ensuring successful project completion.

Cbl-b, with the full name Casitas B lymphoma-b (Uniprot ID Q13191), is a RING-type E3 ubiquitin ligase and a member of the CBL family, encoded by the CBLB gene in humans. E3 ubiquitin ligases accept ubiquitin from specific E2 ubiquitin-conjugating enzymes and transfer it to substrates, thereby promoting the degradation of these substrates by the proteasome.

The full-length Cbl-b protein consists of 982 amino acids and is composed of various domains, including the highly conserved tyrosine kinase-binding (TKB) domain, the linker helix (LH) domain, the RING finger domain, and the C-terminal proline-rich region (PRR) and ubiquitin-associated (UBA) domain. The TKB domain contains three subdomains: a four-helix bundle (4H) sequence, a Ca2+-binding EF-hand sequence, and an SH2 sequence, which are connected to the RING zinc finger domain by a linker region (LHR). The TKB, LHR, and RING domains jointly perform the catalytic activity of the E3 ligase, with the TKB domain targeting phosphorylated tyrosine kinases (including growth factor receptors and cytokine receptors) as well as non-receptor tyrosine kinases.

Conformational changes between different domains of Cbl-b can result in the protein adopting either a closed, inactive conformation or an open, active conformation. The LH domain is clamped onto the TKB domain, restricting the movement of the RING domain, which can either be in a closed, autoinhibited state or an open state capable of binding to E2. When the conserved tyrosine (Tyr363, marked as Tyr) is phosphorylated (pTyr), the LH domain is released from the TKB domain, causing the RING domain to flip 180 degrees and move close to the substrate, transitioning Cbl-b from a self-inhibited closed state to an active open state. Inhibitor small molecules designed for the Cbl-b target generally bind between the TKB and LHR domains in the inactive conformation, stabilizing the protein in its inactive closed conformation, similar to the mechanism of allosteric inhibitors of SHP2.

Cbl-b can negatively regulate the signaling pathways of TCR (T-cell receptor), BCR (B-cell receptor), and FCER1 (high-affinity immunoglobulin epsilon receptor). Additionally, in the negative regulation processes such as endocytosis and lysosomal degradation of receptor tyrosine kinases (RTKs), c-Cbl and Cbl-b proteins play a key role as ubiquitin ligases and endocytic adaptor molecules. The expression of Cbl-b in T cells leads to ligand-induced downregulation of the T-cell receptor, thereby controlling the degree of T-cell activation during antigen presentation. Consequently, high expression of the ubiquitin ligase Cbl-b in cells can reduce the recognition and killing activity of immune cells against target cells. Cbl-b is highly expressed in certain tumor cells, such as melanoma tissues and gastric cancer tissues, where its elevated expression aids these tumor cells in immune evasion and is associated with the invasion and lymph node metastasis of gastric cancer. Therefore, inhibiting Cbl-b activity can enhance anti-tumor immune effects, similar to another immune target, PD-1.

Cancer immunotherapy (CI) relies on activating the patient's own immune cells to fight tumors and has revolutionized tumor treatment over the past decade. However, even in the field of lung cancer, where many of the most successful immune checkpoint blockade therapies have been approved, only a portion (25-30%) of patients respond to and benefit from these drugs. Thus, there is an urgent need for more CI drugs to help a broader range of cancer patients. As an important immunoregulatory target, Cbl-b is increasingly becoming a design target for anti-tumor drugs in the field of tumor immunotherapy, showing growing promise.

Therefore, elucidating the structure of Cbl-b in its various active states is of great significance for the development of related target drugs.

About ReadCrystal

ReadCrystal has successfully expressed, purified, and crystallized the 38-427 fragment of the Cbl-b protein, which includes the TKB, LHR, and RING domains. We have also accumulated extensive data on the co-crystal structures of this target and its inhibitors. In addition, We have experience in the expression, purification, and crystallization of proteins related to the Cbl-b pathway, both upstream and downstream, which allows us to shorten turnaround time to 1-2 months, ensuring successful project completion and meeting the needs of pharmaceutical development.