Structural analyses of inositol phosphate second messengers bound to signaling effector proteins

Abstract

The higher-order inositol phosphate second messengers inositol tetrakisphosphate (IP4), inositol pentakisphosphate (IP5) and inositol hexakisphosphate (IP6) are important signaling molecules that regulate DNA-damage repair, cohesin dynamics, RNA-editing, retroviral assembly, nuclear transport, phosphorylation, acetylation, crotonylation, and ubiquitination. This functional diversity has made understanding how inositol polyphosphates regulate cellular processes challenging to dissect. However, some inositol phosphates have been unexpectedly found in X-ray crystal structures, occasionally revealing structural and mechanistic details of effector protein regulation before functional consequences have been described. This review highlights a sampling of crystal structures describing the interaction between inositol phosphates and protein effectors. This list includes the RNA editing enzyme "adenosine deaminase that acts on RNA 2" (ADAR2), the Pds5B regulator of cohesin dynamics, the class 1 histone deacetylases (HDACs) HDAC1 and HDAC3, and the PH domain of Bruton's tyrosine kinase (Btk). One of the most important enzymes responsible for higher-order inositol phosphate synthesis is inositol polyphosphate multikinase (IPMK), which plays dual roles in both inositol and phosphoinositide signaling. Structures of phosphoinositide lipid binding proteins have also revealed new aspects of protein effector regulation, as mediated by the nuclear receptors Steroidogenic Factor-1 (SF-1, NR5A2) and Liver Receptor Homolog-1 (LRH-1, NR5A2). Together, these studies underscore the structural diversity in binding interactions between effector proteins and inositol phosphate small signaling molecules, and further support that detailed structural studies can lead to new biological discovery.

Keywords: ADAR2; Btk; HDAC1; HDAC2; HDAC3; Higher-order inositol phosphates; IP4; IP5; IP6; IP6 regulated gene expression; Inositol polyphosphate multikinase IPMK; Inositol regulated transcription; Inositol-binding proteins; NR5A1 nuclear receptor; Pds5b.

Figure 1:. Pds5B crystal structure (PDB:5HDT) reveals IP6 binding site.

A. The overall 2.7Å crystal structure of human Pds5B (residues 21–1120), showing the IP6 acquired natively from the insect cell expression system. B. Close-up of IP6 binding site, showing interactions between IP6 phosphates are Pds5B basic residues surrounding the IP6-binding pocket. Water molecules depicted as red spheres, all images generated in Pymol (Schrödinger).

Figure 2:. ADAR2 crystal structure (PDB: 1ZY7) reveal IP6 binding site.

A.The overall 1.7Å crystal structure of the catalytic domain of human ADAR2 (residues 299–701), showing the IP6 acquired natively from the yeast (Saccharomyces cerevisiae) expression system. B. Close-up of IP6 binding sites, showing interactions between IP6 phosphates basic residues of ADAR2 core surrounding the IP6-binding pocket. Water molecules depicted as red spheres, all images generated in Pymol (Schrödinger).

Figure 3:. Class 1 HDAC crystal structures (PDB:5ICN HDAC1 and PDB:4A69 HDAC3) reveal IP4 and IP6 binding site.

A.The overall 2.1Å crystal structure of full-length human HDAC3 in complex with the deacetylase activation domain of NCOR2 (SMRT) corepressor depicted in light pink, showing the inositol tetrakisphosphate (1,4,5,6)P4 (IP4) acquired natively from the HEK293 cell expression system. IP4 is an enzyme product of the inositol polyphosphate multikinase (IPMK). B. Close-up of the inositol tetrakisphosphate (1,4,5,6)P4 (IP4) binding site, showing interactions between IP4 phosphates and basic residues of the HDAC3 and NCOR2 proteins which form the IP4-binding pocket. C. The overall 3.0Å crystal structure of full-length human HDAC1 in complex with the ELM2-SANT domain from the MTA1 corepressor depicted in light pink, showing the IP6 acquired natively from the HEK293 cell expression system. D. Close-up of the IP6 binding site, showing interactions between IP6 phosphates and basic residues of the HDAC1 and MTA proteins which form the IP6-binding pocket. Water molecules depicted as red spheres, all images generated in Pymol (Schrödinger).

Figure 4:. Bruton’s tyrosine kinase (Btk) crystal structure (PDB:4Y94) reveal a novel IP6 binding site.

A. The overall 2.3Å crystal structure of bovine Btk PH-TH domain (residues 1–172) showing the IP6, which had been ectopically added prior to crystallization. Note Btk is a dimer, and the IP6 resides between in the dimers in a peripheral binding site. Monomer Btk(1) shown in pink, monomer Btk(2) shown in gray. The other two IP6 molecules are bound to the canonical PIP3 / IP4 binding site. B. Close-up of the IP6 in the peripheral binding site between monomers Btk(1) and Btk(2), showing interactions between IP6 phosphates and basic residues from the Btk(1) and Btk(2) peripheral IP6 binding site. C. Same as in B, rotated 180°. Water molecules depicted as red spheres, all images generated in Pymol (Schrödinger).

Figure 5:. Steroidogenic Factor-1 (SF-1) ligand binding domain (LBD) crystal structures bound to phosphoinositide lipids reveal PIP2 (PDB:4QK4) and PIP3 (PDB:4QJR) binding sites.

A.Superposition of the overall crystal structures of human SF-1 (residues 218–461) complexed with a peptide representing the transcriptional coactivator PGC1α (cyan) and either the lipid PI(4,5)P2 (PIP2) at 2.8Å or the lipid PI(3,4,5)P3 (PIP3) at 2.4Å, showing binding sites for the phosphoinositide lipids. B. Close-up of the PIP2 binding site, showing position of the axial 2-position going into the plane of the page. C. Close-up of the PIP3 binding site, showing position of the axial 2-position going out of the plane of the page. Water molecules depicted as red spheres, all images generated in Pymol (Schrödinger).

Figure 6:. Liver Receptor Homolog-1 (LRH-1) ligand binding domain (LBD) crystal structure bound to phosphoinositide lipid reveals PIP3 (PDB:4RWV) binding site.

A. The overall 1.9Å crystal structure of human LRH-1 (residues 297–539) complexed with a peptide representing the transcriptional coactivator DAX-1 (cyan) and the phosphoinositide lipid di-palmitoyl (C16:0) PI(3,4,5)P3 (PIP3) ectopically added prior to crystallization, showing binding sites for the PIP3 lipid. B. Close-up of the PIP3 binding site, showing position of the axial 2-position going out of the plane of the page. Water molecules depicted as red spheres, all images generated in Pymol (Schrödinger).