Emerging roles of the αC-β4 loop in protein kinase structure, function, evolution, and disease

Abstract

The faithful propagation of cellular signals in most organisms relies on the coordinated functions of a large family of protein kinases that share a conserved catalytic domain. The catalytic domain is a dynamic scaffold that undergoes large conformational changes upon activation. Most of these conformational changes, such as movement of the regulatory αC-helix from an "out" to "in" conformation, hinge on a conserved, but understudied, loop termed the αC-β4 loop, which mediates conserved interactions to tether flexible structural elements to the kinase core. We previously showed that the αC-β4 loop is a unique feature of eukaryotic protein kinases. Here, we review the emerging roles of this loop in kinase structure, function, regulation, and diseases. Through a kinome-wide analysis, we define the boundaries of the loop for the first time and show that sequence and structural variation in the loop correlate with conformational and regulatory variation. Many recurrent disease mutations map to the αC-β4 loop and contribute to drug resistance and abnormal kinase activation by relieving key auto-inhibitory interactions associated with αC-helix and inter-lobe movement. The αC-β4 loop is a hotspot for post-translational modifications, protein-protein interaction, and Hsp90 mediated folding. Our kinome-wide analysis provides insights for hypothesis-driven characterization of understudied kinases and the development of allosteric protein kinase inhibitors.

Keywords: Hsp-90; cancer mutation; conformational regulation; disease mutation; drug resistance; molecular brake; post-translational modifications; protein kinase.

FIGURE 1

Definition of the αC-β4 loop. (a) The αC-β4 loop (red) of protein kinase A (PDB ID: 1ATP). Structural regions near the αC-β4 loop are labeled for reference. At the top-right corner, a close-up shows the αC-β4 loop flanked by the RS3 and RS4 residues. (b) Sequence logo plots spanning from RS3 to RS4 are shown. This span of residues was used on all logo plots throughout the review. Sequence logo plots include amino acid sequences from Uniprot proteomes (top), amino acid sequences from PDB (middle), and secondary structure sequences from PDB (bottom). Secondary structure sequences were defined by DSSP where red = helix, blue = strand, black = coil. DSSP classifications: G = 310 helix, H = α-helix, I = π-helix, B = isolated β-bridge, E = extended β-strand, T = hydrogen bonded turn, S = non-hydrogen bonded bend, C = coil. (c) Histogram showing the distribution of αC-β4 loop lengths calculated from unique protein kinase structures in the PDB. The 15+ category includes lengths greater than or equal to 15. DSSP, Define Secondary Structure of Proteins; PDB, Protein Data Bank

FIGURE 2

Sequence conservation of the αC-β4 loop. (a) Sequence logo plots spanning from RS3 to RS4 are shown for different protein kinase groups. (b) The canonical HxN motif is shown in tyrosine kinase EphA3 (PDB ID: 3dzq) (left). The AGC-specific variant is shown in PKA (PDB ID: 1atp) (middle). The CK1- specific xxG motif is shown in CSNK1D (PDB ID: 4twc) (right). Residue numbers (not shown) are provided: 679–681 for the HxN motif in EphA3, 100–102 for xPF motif in PKA, and 62–64 for the xxG motif in CSNK1D. Side chains are not shown for the β8 strand. PDB, Protein Data Bank

FIGURE 3

Conserved interactions within the αC-β4 loop. Conservation was defined by the fraction of structures containing the interaction. All heatmaps use PKA numbering for residue positions. (a) Conserved contacts found in kinase structures are shown in an all-versus-all comparison of residues in the αC-β4 loop and β8 strand. Two examples of the two most highly conserved long-range contacts in the αC-β4 loop and β8 strand are shown in PKA (PDB ID: 1atp) and EphA3 (PDB ID: 3dzq). (b) Conserved water bridges found in kinase structures are shown in an all-versus-all comparison of residues in the αC-β4 loop. Two examples of a highly conserved water bridge is shown in EphA3 (PDB ID: 3dzq) and AuroraB (PDB ID: 2vrx). PDB, Protein Data Bank

FIGURE 4

Extended conformations of the αC-β4 loop in the protein kinome. (a) A phylogenetic tree shows αC-β4 loop lengths of human protein kinases.^, Branches containing longer αC-β4 loops are colored darker. The 18+ color category includes lengths greater than or equal to 18. (b) Structural examples of extended αC-β4 loops are shown in red. The β8 strand is shown for reference. Protein names are provided alongside its kinase group. Locations for αC-β4 loops are provided: 93–135 in scCKA1 (PDB ID: 4jr7), 147–160 in SRPK2 (PDB ID: 2×7g), 253–262 in HIPK2 (PDB ID: 6p5s), 212–222 in CLK3 (PDB ID: 6fyr), 92–112 in VRK1 (PDB ID: 6bru), 95–106 in Gilgamesh (PDB ID: 4 nt4), and 322–340 in Rhoptry kinase (PDB ID: 3byv). More information about these structures can be found in Table 1. PDB, Protein Data Bank

FIGURE 5

Missense mutations in the αC-β4 loop. For easy comparison, residue position on the x-axis is kept consistent throughout all graphs. (a) Bar graphs showing the number of missense mutations in the αC-β4 loop from 7 different protein kinase groups. The y-axis scale is consistent across all bar graphs to allow cross comparison. (b) The missense mutations are shown using a sequence logo plot. Similar to a sequence logo, each column shows the relative frequency of all substitutions occurring at that position. (c) A sequence logo for wildtype αC-β4 sequences from Uniprot is provided as reference

FIGURE 6

Post-translational modifications in the αC-β4 loop. For easy comparison, residue position on the x-axis is kept consistent throughout all graphs. (a) Bar graphs showing the number of PTMs found at each αC-β4 position separated by PTM. Please note that the y-axis scale is not consistent across bar graphs. (b) A sequence logo for wildtype αC-β4 sequences from Uniprot is provided as reference