Multiple conformational states of the HPK1 kinase domain in complex with sunitinib reveal the structural changes accompanying HPK1 trans-regulation

Abstract

Hematopoietic progenitor kinase 1 (HPK1 or MAP4K1) is a Ser/Thr kinase that operates via the c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) signaling pathways to dampen the T-cell response and antitumor immunity. Accordingly, selective HPK1 inhibition is considered a means to enhance antitumor immunity. Sunitinib, a multi-receptor tyrosine kinase (RTK) inhibitor approved for the management of gastrointestinal stromal tumors (GISTs), renal cell carcinoma (RCC), and pancreatic cancer, has been reported to inhibit HPK1 in vitro In this report, we describe the crystal structures of the native HPK1 kinase domain in both nonphosphorylated and doubly phosphorylated states, in addition to a double phosphomimetic mutant (T165E,S171E), each complexed with sunitinib at 2.17-3.00-Å resolutions. The native nonphosphorylated cocrystal structure revealed an inactive dimer in which the activation loop of each monomer partially occupies the ATP- and substrate-binding sites of the partner monomer. In contrast, the structure of the protein with a doubly phosphorylated activation loop exhibited an active kinase conformation with a greatly reduced monomer-monomer interface. Conversely, the phosphomimetic mutant cocrystal structure disclosed an alternative arrangement in which the activation loops are in an extended domain-swapped configuration. These structural results indicate that HPK1 is a highly dynamic kinase that undergoes trans-regulation via dimer formation and extensive intramolecular and intermolecular remodeling of the activation segment.

Keywords: cancer; conformational change; conformational plasticity; crystal structure; dimerization; domain swapping; drug discovery; hematopoietic progenitor kinase 1 (HPK1); inhibitor; serine/threonine protein kinase; trans-regulation.

Figure 1.

Domain architecture and structure of HPK1. A, schematic of primary sequence and domains of full-length HPK1 (top) and kinase domains used for crystallization (bottom). B, subunit structure of the HPK1^+0P–sunitinib KD. N-lobe, cyan ribbon; C-lobe, green ribbon; AS, yellow; and P + 1 motif, orange. C, selected interactions at the HPK1^+0P dimer interface. Chain B is colored slate. Dashed lines with numbers show distance (Å).

Figure 2.

Activation loops of the HPK1^+0P and HPK1^+2P dimers. A, the activation loops of the +0P dimer in ribbon representation with chain A in green and chain B in blue. Arrows show areas of β-strand. The DFG motif and phosphorylation sites are drawn as magenta and yellow sticks, respectively. Dashed lines indicate hydrogen bonds. B, the activation loops of the +2P dimer. The coloring scheme is as described in A. Dashed lines show interactions between protein and phosphate groups.

Figure 3.

The configuration of HPK1–sunitinib dimers. A, HPK1^+0P KD in a tightly closed head-to-head dimer. B, HPK1^+2P KD in an open head-to-head dimer. C, HPK1^2PM KD in a head-to-head domain-swapped dimer. Chains A and B are colored green and cyan, respectively. Interface residues for chain A are shown as red lines, and inhibitor is drawn as yellow sticks.

Figure 4.

HPK1–sunitinib and comparable domain-swapped dimers from the PDB. A, the HPK1^2PM KD in a domain-swapped dimer. Chain A is shown as a green cartoon with transparent surface, and chain B is shown as a cyan cartoon. Inhibitor and WYMAPE residues are drawn as sticks. The active-site surface is colored yellow in subunit A. B, zoomed-in view of HPK1^2PM domain swap. Selected chain A side chains (green) surrounding YWMAPE residues from the P + 1 and APE motifs of chain B (cyan) are shown. C, equivalent view of HPK1^+0P chain A (dark green) to illustrate the duplication of interactions in domain-swapped and nonswapped dimers. D, crystal structures of selected kinase domains from the PDB that assemble as domain-swapped dimers.