Abl Kinase
Created by Alison Underwood
Abl kinase (pdb id=3ms9) is a tyrosine kinase protein found in Mus musculus that exists in cells in a homodimeric state, consisting of two identical subunits, both with tyrosine kinase function (1). Tyrosine kinases function by transferring a phosphate group from ATP to a tyrosine residue on a substrate molecule, affecting the substrate’s activation state in signal transduction pathways (2). Abl kinases play a role in regulating the cytoskeleton and shape of a cell, proliferation and survival of the cell, cell adhesion, and cell migration (2, 3). There is evidence that the protein of interest (POI) functions in development and signal transduction in the central nervous system of mice, as well as in mouse heart growth and development (3, 4).
Corresponding to its function, each tyrosine kinase domain consists of two lobes and contains an ATP binding site between the lobes as well as a substrate binding site and an activation loop (5, 6). These three features contain a number of conserved residues. E-316 and M-318 are essential for forming hydrogen bonds with ATP in the ATP binding site (6). Other conserved residues of the ATP binding site are L-248, G-250, Y-253, A-269, K-271, E-286, M-290, V-299, T-315, N-322, L-370, A-380, and D-159. Conserved residues that comprise the substrate binding site are A-399, K-400, F-401, I-403, L-411, L-445, and Y-449. The activation loop consists of the twenty five residues from A-380 to K-404 (5). In addition to the tyrosine kinase domain, Abl kinase contains an N-terminal “cap”, an SH3 domain, and an SH2 domain, which have regulatory functions (7).
The secondary structure of Abl kinase is 39% helical with 17 helices in each kinase domain and is 15% beta sheet with 10 beta strands comprising three beta sheets. The remaining 46% of the structure consists of other types of secondary structures. The two kinase domain subunits are held together by 68 non-bonded contacts as well as hydrogen bonds between K-454, Y-456, Q-447, and D-444 of the first chain with K-454, Q-447, R-457, and Y-456 of the second chain, respectively. The protein of interest (POI) does not contain any prosthetic groups. The chloride ion is one of the POI’s ligands due to ionic interactions at Y-253, I-360, and T-319 (8).
The function of Abl kinase is strongly linked to the structure of the protein. In normally functioning cells, Abl kinase is tightly regulated and is held in an inactivated state by intramolecular interactions between a myristoyl residue on the N-terminus and the kinase domain, and between the SH2/SH3 domains and the kinase domain (PDB ID = 1opj for kinase domain bound to myristoyl group and 1opk for SH3, SH2 and kinase domain) (7, 9). The myristoyl group binds in a deep pocket formed by residues of several helices. With the myristoyl group bound, docking of the SH2 domain against the kinase domain requires a sharp bend in the C-terminal helix of the protein. The structural change results in the flipping of the side chains of Asp and Phe residues in a conserved D-381, F-382, G-383 motif in the activation loop to a DFG-out conformation, which contributes to the inactive state of the protein (PDB ID for DFG-in and DFG-out conformations = 3kf4, 3kfa). The inactive position of the Asp side chain prevents a magnesium ion from coordinating the ATP phosphate at the active site, inhibiting catalytic activity and holding the activation loop in a “ closed” configuration. In the inactive conformation, Y-393 is not phosphorylated. However, when Y-393 is phosphorylated, the protein becomes stabilized in its active conformation (6, 7). The active form of Abl kinase consists of a DFG-in conformation that allows magnesium to bind to the active site as well as an open activation loop and intramolecular interactions between K-271 and E-286 (10).
When Abl kinase is constitutively active due to disruption of its intramolecular interactions, it becomes an oncogenic protein and causes chronic myelogenous leukemia (CML). One such oncogenic protein, Bcr-Abl, is created by the translocation and fusion of the bcr and c-abl genes. The protein of interest (POI) is complexed with STI-571, also known as GleevecTM or imatinib, which is a drug used to treat CML that binds to the ATP binding site and inhibits Abl function in Bcr-Abl by preventing transfer of the phosphate group (9). The POI forms hydrogen bonds with imatinib at E-286, T-315, and M-318 (5, 6). Binding of Abl kinase to imatinib also displaces the DFG motif such that the activation loop is kept in its inactive form (11). Other drug-protein complexes are possible with the drug mimicking the myristoyl residue that is responsible for the autoinhibition of Abl kinase in healthy cells (7, 9). Ligands that bind at the myristate pocket and allow the bent conformation of the C-terminal helix like the myristoyl residue does can function as inhibitors. In contrast, ligands that bind in the myristate pocket and prevent the bent helix conformation due to steric constraints instead function as agonists of Abl kinase. The ligand MS9, which is bound to the POI in the myristate pocket, functions as an agonist rather than an antagonist of Abl kinase because of steric constraints that prevent the C-terminal helix from adopting the inactive bent conformation (9).
Proteins with similar primary structures often have similar functions due to conservation of amino acid residues within their active sites. A BLAST protein search was performed using the amino acid sequence of the POI to find other proteins with similar amino acid sequences. Abl kinase in Homo sapiens (PDB ID= 2E2B) had an E-value of 3e-172, suggesting significant primary structure similarity with the POI. Proteins with similar tertiary structures frequently have similar functions because tertiary structure determines function. The DALI server was used to find proteins with a three-dimensional structure similar to that of the POI. Protein 2E2B was also found to have significantly high tertiary structure similarity to the POI as shown by its RMSD value of 0.5 and Z score of 42.5 (12). Abl kinase in humans (2E2B) plays a role in the regulation of cell adhesion, the cytoskeleton, cell migration, and cell proliferation, functioning much like the POI (13). Although Abl kinase in humans is a different protein from Abl kinase in mice, the two proteins share the same function likely due to primary structure homology within active site regions and similar tertiary structures.
The importance of structure in determination of Abl kinase function can especially be seen in the T315I mutant form of Abl kinase (PDB ID = 2z60). The point mutation causes the protein to take on an active conformation with DFG-in, an open activation loop, and characteristic interactions between K-271 and E-286, making it resistant to drugs like imatinib. Despite the fact that Y-393 is not phosphorylated, which is usually necessary for activation, the mutant is constitutively active due to its structure (10). The DALI server was used to compare the mutant 3-D structure to that of the POI, and resulted in an RMSD of 1.4 and a Z-score of 36.5 indicating similar structure, but not as similar as those of other Abl kinase proteins in the inactive conformation (12). The T315I protein included an inhibitor, PPY-A, that prevented the mutant from functioning, returning its function to that of the POI (10). The function of Abl kinase relies on its structure so it is especially important to examine and mimic the inactive state in order to develop new therapeutic drugs (9).
Abl kinase has significant biological functions in both its natural and its mutant forms. In normally functioning cells, the POI regulates important cellular processes such as cell proliferation, shape, and migration and consequently plays a role in mammalian development. When mutated to a constitutively active form, Abl kinase causes carcinogenesis. Analysis of primary and tertiary structures can help determine protein function, and structure analysis is especially relevant when designing new drugs to cure cancers caused by Abl oncoproteins.