Structural Aspects of LIMK Regulation and Pharmacology

Figure 1. The LIMK conformational space. (A) LIMK1/2 and the phosphatase SSH1 control the dynamics of the actin cytoskeleton. ADF—actin depolymerizing factor. (B) Both LIMK1 and LIMK2 contain N-terminal protein interaction modules and a C-terminal kinase domain. (C) With ATP-γ-S bound, LIMK1 adopts the canonical active-kinase conformation. The catalytic and regulatory spines are fully formed (indicated in green). Please note the G-rich loop enclosing the co-substrate, the αC-in conformation, the attached activation loop, and the unusual orientation of the αG helix (all indicated in pink). PDB ID 5L6W. (D) Inhibitor binding can induce a rotated G-rich loop conformation. PDB IDs 5L6W and 5NXD. (E) The αC helix is capable of adopting both, the αC-in and the αC-out conformation. PDB IDs 5L6W and 5NXD. (F) The DFG motif in the base of the activation loop switches between the DFG-in and the DFG-out conformation. PDP IDs 5L6W and 5NXD. (G) Conformational plasticity is also observed in the activation loop, which can either be attached to the C lobe or flexible. Please note that the flexible loop is not fully resolved in the crystal structure. PDB IDs 5HVJ and 5NXC.

Figure 2. Comparison of kinase-substrate recognition modes. (A) Profile views of the AKT2 and LIMK kinase domains to showcase their overall appearance. (B) Snapshot of AKT2 with substrate and co-substrate bound in the moment of catalysis. The phosphoacceptor loop is tightly attached to the kinase, positioning the phosphoacceptor residue (serine S9) next to the ATP γ phosphate. PDB ID 1O6K [20]. (C) ATP interacts with the LIMK1 hinge, its adenine rings are sandwiched by hydrophobic residues, and its phosphates are oriented by polar residues. (D) LIMK1 with substrate and co-substrate bound, again in the moment of catalysis. The substrate protein attaches to the kinase solely with its anchor helix—the residues flanking the phosphoacceptor residue (serine S3) do not interact with the kinase. The arrow indicates the rocking movement that allows the phosphoacceptor to poke into the active site. The two CFL1 orientations are taken from independent crystal structures. Parts of CFL1 orientation 2 are omitted for clarity. PDB IDs 5HVJ and 5L6W [9,10].

Figure 3. Inhibitors bound to LIMKs. (A) The type-1 binder LIJTF500127 spans from the hinge to a pocket between the β5 strand and the DFG motif. PDB ID 7ATS. (B) The type-3 binder TH300 mainly exhibits hydrophobic interactions with kinase elements surrounding the back pocket. PDB ID 5NXD. (C) PF477736 occupies the front pocket only but interacts with charged residues from the β3 strand and the DFG motif. PDB ID 5NXC. (D) The pan-kinase inhibitor staurosporine binds due to its disc shape and its hinge interactions. PDB ID 3S95. The G-rich loops are omitted for clarity reasons.