Inositol phosphate kinases: Expanding the biological significance of the universal core of the protein kinase fold

Abstract

The protein kinase family is characterized by substantial conservation of architectural elements that are required for both ATP binding and phosphotransferase activity. Many of these structural features have also been identified in homologous enzymes that phosphorylate a variety of alternative, non-protein substrates. A comparative structural analysis of these different kinase sub-classes is a portal to a greater understanding of reaction mechanisms, enzyme regulation, inhibitor-development strategies, and superfamily-level evolutionary relationships. To serve such advances, we review structural elements of the protein kinase fold that are conserved in the subfamily of inositol phosphate kinases (InsPKs) that share a PxxxDxKxG catalytic signature: inositol 1,4,5-trisphosphate kinase (IP3K), inositol hexakisphosphate kinase (IP6K), and inositol polyphosphate multikinase (IPMK). We describe conservation of the fundamental two-lobe kinase architecture: an N-lobe constructed upon an anti-parallel β-strand scaffold, which is coupled to a largely helical C-lobe by a single, adenine-binding hinge. This equivalency also includes a G-loop that embraces the β/γ-phosphates of ATP, a transition-state stabilizing residue (Lys/His), and a Mg-positioning aspartate residue within a catalytic triad. Furthermore, we expand this list of conserved structural features to include some not previously identified in InsPKs: a 'gatekeeper' residue in the N-lobe, and an 'αF'-like helix in the C-lobe that anchors two structurally-stabilizing, hydrophobic spines, formed from non-consecutive residues that span the two lobes. We describe how this wide-ranging structural homology can be exploited to develop lead inhibitors of IP6K and IPMK, by using strategies similar to those that have generated ATP-competing inhibitors of protein-kinases. We provide several examples to illustrate how such an approach could benefit human health.

Fig. 1.. The kinase activities of IPMK, IP3K, and IP6K in the pathway of inositol phosphate synthesis in mammals.

Each individual inositol phosphate is abbreviated as follows: ‘Ins’ refers to inositol; the subscripts denote the total number of phosphates (‘P’), and the numbers in parentheses describe the positions of the phosphate groups around the inositol ring. Note that ‘5-InsP₇’ denotes an ‘inositol pyrophosphate’ that has a diphosphate group at the 5-position. Arrows depict metabolic steps, catalyzed by the following enzymes, with E.C. numbers in parentheses: 1, IPMK, inositol polyphosphate multikinase (2.7.1.151; also known as IPK2); IP3K, inositol trisphosphate 3-kinase (2.7.1.127); INPP5, inositol polyphosphate 5-phosphatase, (3.1.3.56); ITPK1, inositol trisphosphate 6-kinase/ inositol tetrakisphosphate 1-kinase (2.7.1.134); IP5K, inositol pentakisphosphate 2-kinase (2.7.1.158; also known as IPK1); IP6K, inositol hexakisphosphate kinase (2.7.4.21).

Fig. 2.. A cartoon describing conserved secondary and tertiary structures among PI3Kγ, PKA, IPMK and IP3KA

Ribbon plots of the following enzymes are shown (PDB access codes in parentheses): A, Murine protein kinase A (catalytic subunit)/ADP complex, E.C. 2.7.11.11 (1L3R); B, Sus scrofa PI3Kγ (catalytic subunit)/ATP complex (E.C. 2.7.11.1) (1E8X). C, Human IP3KA/AMP-PNP complex (1W2C) D, Human IPMK/ADP/IP3 complex (5W2H). The α-helices and β-strands that are colored are conserved in at least two of the structures shown; in cases where individual structural elements have two labels separated by a forward slash, the first is the label given in the original PDB entry, and the second label corresponds to the corresponding element in the homologous protein (color coding of labels matches that of the originating lobe). Non-conserved structural elements are depicted in light gray. Broken lines are used to depict the expected G-loops in IP3K and IPMK (their actual structures are not known). Bound nucleotides are shown as stick models. Note that the structural element in PI3Kγ that we describe as a ‘G-loop’ (panel B) was originally named ‘P-loop’ (Walker et al., 1999). For an explanation, see the text.

Fig. 3.. Electrostatic surface plots of substrate binding sites for PKA and IPMK

Shown are electrostatic surface plot with blue and red coloration to denote positive and negative electrostatic potentials, respectively, at physiological pH, for A, murine PKA in complex with a 20 residue peptide inhibitor (PDB, 1L3R; (Madhusudan et al., 2002)), B, human IPMK (PDB, 5W2H) in complex with Ins(1,4,5)P₃; (Wang and Shears, 2017)). Substrates are colored green and red.

Fig. 4.. Cooperation between the lobes: R-Spines, gatekeepers, catalytic triads and other motifs.

A, B and C highlight selected, conserved elements of murine PKA (PDB, 1L3R), human IPMK (PDB, 5W2H) and Sus scrofa PI3Kγ (PDB, 1E8X), respectively, shown as a composite of ribbon plots, with selected key residues as space-filling representations and stick-model depictions. For PKA and PI3Kγ, the identities of residues comprising the gatekeeper, R-spine and the anchoring α-helix are provided in reviews of this topic (Taylor and Kornev, 2011; Vadas et al., 2011); the corresponding features in IPMK were identified from its alignment with PKA (using Pymol) in which bound nucleotides were superimposed. Panels D, E, F are corresponding stick-model close-ups of the catalytic pockets. The dashed gray line in panel F denotes that the actual structure of the G-loop is disordered in the crystal structure. The residues that we denote as comprising a ‘catalytic triad’ (see text) are as follows: PKA, Lys72, Glu91, Asp184; PI3Kγ, Lys833, Asp836, Asp964; IPMK, Lys75, Glu86, Asp385 (also see Table 1). Thin, broken black lines depict interactions between residues in the catalytic triad, and also those involving the nucleotide’s ribose ring. Magenta balls depict the metal binding sites occupied by magnesium (or, in the case of PI3Kγ, lutetium (Walker et al., 1999)). All other color schemes match those in Fig. 2. Note that the structural element in PI3Kγ that we describe as a ‘G-loop’ (panel E) was originally named ‘P-loop’ (Walker et al., 1999). For an explanation, see the text.

Fig. 5.. C-Spines in PKA, PI3K and IPMK

A, B and C describe the relative spatial positions of the individual residues (stick models) that comprise the C-spines of murine PKA (PDB, 1L3R), human IPMK (PDB, 5W2H) and Sus scrofa PI3Kγ (PDB, 1E8X), respectively. Individual residues were identified as described in the legend to Fig. 4. Color schemes match those in Fig. 2. Bound nucleotides are shown as green stick models (color schemes as in Fig. 2). D, shows the C-spine of IPMK as a space-filling model (yellow surface) anchored to the α7-helix.

Fig. 6.. Conserved aspects of nucleotide binding for PKA, PI3K and InsP kinases.

Shown is a graphical representation of highly conserved elements of protein kinases, PI3Ks and PDKG-InsPKs. Broken lines depict charged and polar contacts. For an explanation of catalytic triad, see Fig. 4 D,E,F and the text. ‘Catalytic K/H’ refers to the transition state stabilizing residue in the DxK/H motif (Table 1). See text and Table 1 for other details.