An in cellulo-derived structure of PAK4 in complex with its inhibitor Inka1

Abstract

PAK4 is a metazoan-specific kinase acting downstream of Cdc42. Here we describe the structure of human PAK4 in complex with Inka1, a potent endogenous kinase inhibitor. Using single mammalian cells containing crystals 50 μm in length, we have determined the in cellulo crystal structure at 2.95 Å resolution, which reveals the details of how the PAK4 catalytic domain binds cellular ATP and the Inka1 inhibitor. The crystal lattice consists only of PAK4-PAK4 contacts, which form a hexagonal array with channels of 80 Å in diameter that run the length of the crystal. The crystal accommodates a variety of other proteins when fused to the kinase inhibitor. Inka1-GFP was used to monitor the process crystal formation in living cells. Similar derivatives of Inka1 will allow us to study the effects of PAK4 inhibition in cells and model organisms, to allow better validation of therapeutic agents targeting PAK4.

Figure 1. Inka1 is a potent kinase inhibitor.

(a) PAK4 architecture and alignment of the AID and the Inka1 iBox and iBox-C from frogs and human. Red asterisks indicate activation mutations in PAK4* (RR48/49AE). Red bars indicate pseudosubstrate sequences. (b) Co-immunoprecipitation of full-length HA-Inka1 by FLAG-tagged PAK4 constructs. (c) Kinase assays using 6His–PAK1 (activated) or PAK4cat, with GST-iBox as indicated. Activity was assessed by the phosphorylation of GST-Raf13 quantified by densitometry (lower right). The quality of the purified proteins is indicated (lower left). (d) The inhibition profile of GST-iBox and selected peptides of the iBox and iBox-C (n=3, error bars indicate s.e.m.). The IC₅₀ values were determined from the intercepts of the graphs.

Figure 2. Intracellular PAK4cat:Inka1 crystals.

(a) Inka1 and PAK4 show nuclear and cytoplasmic localization, respectively. (b) Co-expression leads to cytoplasmic enrichment of Inka1 (left panels). Inka1 and PAK4cat co-expression results in intracellular crystals (right panels), which immunostain for both proteins (middle panels). (c) Inka1 regions capable of generating co-crystals. A single chain fusion of iBox–PAK4cat efficiently generated intracellular crystals. (d) In-cellulo crystals of trypsinized cells. (e) A single cell mounted on a cryo-loop on a synchrotron beamline. The crystal (yellow), the cell membrane (red) and the nucleus (green) are highlighted.

Figure 3. The in-cellulo X-ray structure of the catalytic domain of PAK4 in complex with Inka1.

(a) The X-ray structure of the iBox–PAK4cat complex derived from diffraction of the in-vivo crystals. The typical kinase fold is observed with the iBox (red) binding the PAK4cat close to the phospho-Ser474 (orange), ATP and magnesium ions (mustard). (b) Overlay of in vitro and in vivo PAK4cat:Inka1 complex structure. Comparison between the α-carbon traces of Pak4cat:Inka crystallized in vivo (grey and red) and Pak4cat co-crystallized with a synthetic peptide iBox24 (see Fig. 1d). The PAK4cat with iBox24 yielded a structure at 2 Å, which was overlaid (backbone of the chains in yellow and cyan). The ATP and two Mg²⁺, found in the in vivo structure, are represented in stick and sphere format. On the right is the comparison of the electron density maps of the Inka1 core sequence in the two structures. Stereo images of portions of the 2Fo–Fc electron density maps contoured at 1.5σ and centred at P(0) in Inka is provided in Supplementary Fig. 6. (c) Conservation of the bond angles comparing the substrate serine with proline mimetic in Inka1. The local main-chain and side-chain orientation of the substrate serine (S0) and corresponding prolines in the substrate mimetics are as indicated. Values corresponding to these four residues mapped onto the standard Ramachandran plot indicate their similar orientation.

Figure 4. Inka1 inhibition of PAK4 activity through substrate mimicry.

(a) Left-to-right: PAK4:AID (red); the in-cellulo structure of PAK4:iBox (dark red); PAK4:substrate (purple). The inhibitor prolines (P0) are similarly positioned to the serine (S0) of the substrate. (b) To assess the inhibitors as ‘super-substrates' we tested 13aa synthetic peptides with Pro (0)Ser substitutions in an array. The contribution of each side chain to substrate binding was assessed via alanine substitutions. The Ser (0)Ala completely abolished phosphorylation in each case, confirming other serines were not phosphorylated. (c) iBox-PAK4 in-cellulo structure highlighting the cluster of hydrophobic contacts between the Inka1 side chains and the surface of the PAK4 (yellow). The hydrogen bonds are marked in orange.

Figure 5. Crystal packing of the PAK4cat:Inka crystals and the nature of the protein–protein interface.

(a) The in-cellulo construct and crystal packing of PAK4cat, which form the channel in the presence of Inka1 (red). The schematic of the construct is similarly coloured. (b) The N-lobes, which form the strands that run along the length of the channel. (c) The threefold axis involves hydrophobic interactions of the C-lobe, primarily involving proline residues as indicated. (d) The twofold interface involves primarily hydrophobic side-chain interactions between the B-subunit (blue) N-lobe α-helices including the F364 in the α-helix-C, which interacts with the β-strand sequences. The α-helix-C, a conserved feature of protein kinases, co-ordinates PAK4 kinase activity. PAK4cat (alternately yellow and cyan) and iBox (red). Numbers indicate fold axes. This schematic was generated using PyMOL Molecular Graphics System.

Figure 6. Incorporation of GFP into PAK4 crystals and their in-vivo dynamics.

(a) Schematic of the fluorescent Inka1 constructs generated and (b) the resultant in cellulo crystals when transfected with PAK4cat. (c) Structured illumination microscopy of a cell containing two crystals (SIM, left) and a single crystal observed by two-channel confocal (right) images of GFP–Inka1:PAK4cat crystals. The cross-sections (line) show the crystal enveloped by membrane (also see Supplementary Movie 4). (d) Effect of addition of PF3758309 (5 μM, arrow) on a growing GFP–Inka1:Flag-PAK4cat crystal. GFP incorporation appears to occur at both ends based on the obvious depletion of GFP signal in the growing crystal after PF3758309 is added. The recovery of signal at 1.5 h after drug addition may be due to drug depletion. Right: the measured growth rates of GFP–Inka1 crystals before and after drug addition (n=17, error bars indicate 1 s.d.).

Figure 7. Representative structures of complexes between known classes of endogenous inhibitors and their target protein kinases.

The orientation of the kinase domain (blue or green) in each case is positioned using the conserved secondary helices of the C-lobe. The organization of the inhibitor in each case is shown in red. In the case of p27 KIP, the cyclin A subunit (shown in yellow) provides an important helix to stabilize the CDK2 in an active state. It is noteworthy that the PKI and Inka1 extended region take up similar positions between the N- and C-lobes, although the helical region of each contacts are very different regions of the C-lobe.