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Structural analysis of the carboxy terminal PH domain of pleckstrin bound to D-myo-inositol 1,2,3,5,6-pentakisphosphate.

Jackson SG, Zhang Y, Haslam RJ, Junop MS - BMC Struct. Biol. (2007)

Bottom Line: These domains are often found in signaling proteins and function predominately by targeting their host proteins to the cell membrane.Examination of the resulting electron density unexpectedly revealed the bound ligand to be D-myo-inositol 1,2,3,5,6-pentakisphosphate.The discovery of D-myo-inositol 1,2,3,5,6-pentakisphosphate in the crystal structure suggests that the inhibitory effects observed in the binding studies may be due to this ligand rather than IP6.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Canada. jacksosg@mcmaster.ca

ABSTRACT

Background: Pleckstrin homology (PH) domains are one of the most prevalent domains in the human proteome and represent the major phosphoinositide-binding module. These domains are often found in signaling proteins and function predominately by targeting their host proteins to the cell membrane. Inositol phosphates, which are structurally similar to phosphoinositides, are not only known to play a role as signaling molecules but are also capable of being bound by PH domains.

Results: In the work presented here it is shown that the addition of commercial myo-inositol hexakisphosphate (IP6) inhibited the binding of the carboxy terminal PH domain of pleckstrin (C-PH) to phosphatidylinositol 3,4-bisphosphate with an IC50 of 7.5 muM. In an attempt to characterize this binding structurally, C-PH was crystallized in the presence of IP6 and the structure was determined to 1.35 A. Examination of the resulting electron density unexpectedly revealed the bound ligand to be D-myo-inositol 1,2,3,5,6-pentakisphosphate.

Conclusion: The discovery of D-myo-inositol 1,2,3,5,6-pentakisphosphate in the crystal structure suggests that the inhibitory effects observed in the binding studies may be due to this ligand rather than IP6. Analysis of the protein-ligand interaction demonstrated that this myo-inositol pentakisphosphate isomer interacts specifically with protein residues known to be involved in phosphoinositide binding. In addition to this, a structural alignment of other PH domains bound to inositol phosphates containing either four or five phosphate groups revealed that the majority of phosphate groups occupy conserved locations in the binding pockets of PH domains. These findings, taken together with other recently reported studies suggest that myo-inositol pentakisphosphates could act to regulate PH domain-phosphoinositide interactions by directly competing for binding, thus playing an important role as signaling molecules.

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Conserved binding positions of inositol phosphates to PH domains. Superimposed inositol phosphates in complex with PH domains from five different proteins: DAPP1/PHISH-Ins(1,3,4,5)P4, red, 1FAO; Grp1-Ins(1,3,4,5)P4, yellow, 1FGY; ARNO-Ins(1,3,4,5)P4, blue, 1U27; Grp1-Ins(1,3,4,5,6)P5, green, 1FHW; C-PH-Ins(1,2,3,5,6)P5, gray, 2I5F. The four conserved phosphate binding positions are boxed.
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Figure 5: Conserved binding positions of inositol phosphates to PH domains. Superimposed inositol phosphates in complex with PH domains from five different proteins: DAPP1/PHISH-Ins(1,3,4,5)P4, red, 1FAO; Grp1-Ins(1,3,4,5)P4, yellow, 1FGY; ARNO-Ins(1,3,4,5)P4, blue, 1U27; Grp1-Ins(1,3,4,5,6)P5, green, 1FHW; C-PH-Ins(1,2,3,5,6)P5, gray, 2I5F. The four conserved phosphate binding positions are boxed.

Mentions: From this structural comparison it can be seen that in both cases the D-myo-inositol pentakisphosphate ligands interact with conserved side chains that would be involved in binding phosphoinositides. This suggests that the D-myo-inositol pentakisphosphates would directly compete with phosphoinositides for binding to the PH domains. In addition to this, it was found that four out of the five phosphates occupy overlapping positions within the binding cleft despite the differences in orientation and even stereochemistry. This overlap in phosphate positions is not simply coincidence, since a structural alignment of various PH domains solved in complex with either Ins(1,3,4,5)P4 or the two D-myo-inositol pentakisphosphates used in the comparison above reveals that the overlapping positions are conserved in these structures as well (Figure 5). The mode of interaction observed in our structure is therefore consistent with the possibility that myo-inositol polyphosphates could act to regulate PH domain-phosphoinositide associations by directly competing with phosphoinositides for binding to PH domains.


Structural analysis of the carboxy terminal PH domain of pleckstrin bound to D-myo-inositol 1,2,3,5,6-pentakisphosphate.

Jackson SG, Zhang Y, Haslam RJ, Junop MS - BMC Struct. Biol. (2007)

Conserved binding positions of inositol phosphates to PH domains. Superimposed inositol phosphates in complex with PH domains from five different proteins: DAPP1/PHISH-Ins(1,3,4,5)P4, red, 1FAO; Grp1-Ins(1,3,4,5)P4, yellow, 1FGY; ARNO-Ins(1,3,4,5)P4, blue, 1U27; Grp1-Ins(1,3,4,5,6)P5, green, 1FHW; C-PH-Ins(1,2,3,5,6)P5, gray, 2I5F. The four conserved phosphate binding positions are boxed.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2200656&req=5

Figure 5: Conserved binding positions of inositol phosphates to PH domains. Superimposed inositol phosphates in complex with PH domains from five different proteins: DAPP1/PHISH-Ins(1,3,4,5)P4, red, 1FAO; Grp1-Ins(1,3,4,5)P4, yellow, 1FGY; ARNO-Ins(1,3,4,5)P4, blue, 1U27; Grp1-Ins(1,3,4,5,6)P5, green, 1FHW; C-PH-Ins(1,2,3,5,6)P5, gray, 2I5F. The four conserved phosphate binding positions are boxed.
Mentions: From this structural comparison it can be seen that in both cases the D-myo-inositol pentakisphosphate ligands interact with conserved side chains that would be involved in binding phosphoinositides. This suggests that the D-myo-inositol pentakisphosphates would directly compete with phosphoinositides for binding to the PH domains. In addition to this, it was found that four out of the five phosphates occupy overlapping positions within the binding cleft despite the differences in orientation and even stereochemistry. This overlap in phosphate positions is not simply coincidence, since a structural alignment of various PH domains solved in complex with either Ins(1,3,4,5)P4 or the two D-myo-inositol pentakisphosphates used in the comparison above reveals that the overlapping positions are conserved in these structures as well (Figure 5). The mode of interaction observed in our structure is therefore consistent with the possibility that myo-inositol polyphosphates could act to regulate PH domain-phosphoinositide associations by directly competing with phosphoinositides for binding to PH domains.

Bottom Line: These domains are often found in signaling proteins and function predominately by targeting their host proteins to the cell membrane.Examination of the resulting electron density unexpectedly revealed the bound ligand to be D-myo-inositol 1,2,3,5,6-pentakisphosphate.The discovery of D-myo-inositol 1,2,3,5,6-pentakisphosphate in the crystal structure suggests that the inhibitory effects observed in the binding studies may be due to this ligand rather than IP6.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Canada. jacksosg@mcmaster.ca

ABSTRACT

Background: Pleckstrin homology (PH) domains are one of the most prevalent domains in the human proteome and represent the major phosphoinositide-binding module. These domains are often found in signaling proteins and function predominately by targeting their host proteins to the cell membrane. Inositol phosphates, which are structurally similar to phosphoinositides, are not only known to play a role as signaling molecules but are also capable of being bound by PH domains.

Results: In the work presented here it is shown that the addition of commercial myo-inositol hexakisphosphate (IP6) inhibited the binding of the carboxy terminal PH domain of pleckstrin (C-PH) to phosphatidylinositol 3,4-bisphosphate with an IC50 of 7.5 muM. In an attempt to characterize this binding structurally, C-PH was crystallized in the presence of IP6 and the structure was determined to 1.35 A. Examination of the resulting electron density unexpectedly revealed the bound ligand to be D-myo-inositol 1,2,3,5,6-pentakisphosphate.

Conclusion: The discovery of D-myo-inositol 1,2,3,5,6-pentakisphosphate in the crystal structure suggests that the inhibitory effects observed in the binding studies may be due to this ligand rather than IP6. Analysis of the protein-ligand interaction demonstrated that this myo-inositol pentakisphosphate isomer interacts specifically with protein residues known to be involved in phosphoinositide binding. In addition to this, a structural alignment of other PH domains bound to inositol phosphates containing either four or five phosphate groups revealed that the majority of phosphate groups occupy conserved locations in the binding pockets of PH domains. These findings, taken together with other recently reported studies suggest that myo-inositol pentakisphosphates could act to regulate PH domain-phosphoinositide interactions by directly competing for binding, thus playing an important role as signaling molecules.

Show MeSH