Limits...
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.

Show MeSH
Interactions between C-PH and Ins(1,2,3,5,6)P5. A. and B. Two stereo views illustrating C-PH binding interactions with Ins(1,2,3,5,6)P5. Amino acid side chains and backbone atoms from C-PH making specific ligand contacts are indicated. The two water molecules mediating protein-ligand binding are shown as green balls.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2200656&req=5

Figure 3: Interactions between C-PH and Ins(1,2,3,5,6)P5. A. and B. Two stereo views illustrating C-PH binding interactions with Ins(1,2,3,5,6)P5. Amino acid side chains and backbone atoms from C-PH making specific ligand contacts are indicated. The two water molecules mediating protein-ligand binding are shown as green balls.

Mentions: The final model of IP5-bound C-PH was determined at exceptionally high resolution and consequently the interactions observed between ligand and protein (Figure 3) can be reported with a high degree of certainty. Table 2 lists the specific interactions and distances observed within the crystal structure between Ins(1,2,3,5,6)P5 and C-PH. Ins(1,2,3,5,6)P5 was bound to C-PH in the β1–β2 loop region (residues 253–264 and 277), making numerous interactions through all but one of its phosphate groups (Figure 3). The only phosphate group that does not interact with any residues of C-PH is that in the 1-position of myo-inositol. Although exposed to solvent, this phosphate remained ordered to the point of generating clear electron density. All amino acids that form stabilizing interactions with Ins(1,2,3,5,6)P5 are located in the β1–β2 loop region with the exception of Y277 which contributes a hydrogen bond and is situated on the β3 strand of C-PH. The 2-phosphate is the only phosphate that adopts an axial position. This configuration is strongly stabilized through interactions with the side chains of R264 and K253. Unlike the 2-phosphate, the 3-phosphate makes only a single interaction, in this case with Y277. As already mentioned, the 4-phosphate is not present in the structure. However the remaining 4-OH group, which represents the location where the additional phosphate group of IP6 would be, does make an interaction with the side chain of H256 at a distance of 3.5 Å. All attempts to model in a phosphate group at this position resulted in steric clashes with H256 and the main chain of residues R257 or G255, depending on the position of the phosphate chosen. In addition, the electron density for H256 is well-ordered indicating that this side chain is not mobile and could not adopt a conformation that would accommodate an additional phosphate group at the 4-position. The 5-phosphate of Ins(1,2,3,5,6)P5 interacts with main chain atoms of H256, R257 and N260 (water-mediated), as well as the side chain of R257. The final position on the inositol ring is occupied by the 6-phosphate and interacts with the side chain of R258 in addition to the main chain of N260 through a water molecule. C-PH residues shown here to interact with Ins(1,2,3,5,6)P5 are also known to be involved in binding PtdIns(3,4)P2 [18]. This demonstrates that Ins(1,2,3,5,6)P5 competes directly with PtdIns(3,4)P2 for binding to C-PH. While it is not known exactly how PtdIns(3,4)P2 interacts with residues in the binding loop, it seems reasonable that two of the phosphates from Ins(1,2,3,5,6)P5 will directly compete with the phosphates from PtdIns(3,4)P2 while the remaining phosphates will provide additional stabilizing interactions.


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)

Interactions between C-PH and Ins(1,2,3,5,6)P5. A. and B. Two stereo views illustrating C-PH binding interactions with Ins(1,2,3,5,6)P5. Amino acid side chains and backbone atoms from C-PH making specific ligand contacts are indicated. The two water molecules mediating protein-ligand binding are shown as green balls.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2200656&req=5

Figure 3: Interactions between C-PH and Ins(1,2,3,5,6)P5. A. and B. Two stereo views illustrating C-PH binding interactions with Ins(1,2,3,5,6)P5. Amino acid side chains and backbone atoms from C-PH making specific ligand contacts are indicated. The two water molecules mediating protein-ligand binding are shown as green balls.
Mentions: The final model of IP5-bound C-PH was determined at exceptionally high resolution and consequently the interactions observed between ligand and protein (Figure 3) can be reported with a high degree of certainty. Table 2 lists the specific interactions and distances observed within the crystal structure between Ins(1,2,3,5,6)P5 and C-PH. Ins(1,2,3,5,6)P5 was bound to C-PH in the β1–β2 loop region (residues 253–264 and 277), making numerous interactions through all but one of its phosphate groups (Figure 3). The only phosphate group that does not interact with any residues of C-PH is that in the 1-position of myo-inositol. Although exposed to solvent, this phosphate remained ordered to the point of generating clear electron density. All amino acids that form stabilizing interactions with Ins(1,2,3,5,6)P5 are located in the β1–β2 loop region with the exception of Y277 which contributes a hydrogen bond and is situated on the β3 strand of C-PH. The 2-phosphate is the only phosphate that adopts an axial position. This configuration is strongly stabilized through interactions with the side chains of R264 and K253. Unlike the 2-phosphate, the 3-phosphate makes only a single interaction, in this case with Y277. As already mentioned, the 4-phosphate is not present in the structure. However the remaining 4-OH group, which represents the location where the additional phosphate group of IP6 would be, does make an interaction with the side chain of H256 at a distance of 3.5 Å. All attempts to model in a phosphate group at this position resulted in steric clashes with H256 and the main chain of residues R257 or G255, depending on the position of the phosphate chosen. In addition, the electron density for H256 is well-ordered indicating that this side chain is not mobile and could not adopt a conformation that would accommodate an additional phosphate group at the 4-position. The 5-phosphate of Ins(1,2,3,5,6)P5 interacts with main chain atoms of H256, R257 and N260 (water-mediated), as well as the side chain of R257. The final position on the inositol ring is occupied by the 6-phosphate and interacts with the side chain of R258 in addition to the main chain of N260 through a water molecule. C-PH residues shown here to interact with Ins(1,2,3,5,6)P5 are also known to be involved in binding PtdIns(3,4)P2 [18]. This demonstrates that Ins(1,2,3,5,6)P5 competes directly with PtdIns(3,4)P2 for binding to C-PH. While it is not known exactly how PtdIns(3,4)P2 interacts with residues in the binding loop, it seems reasonable that two of the phosphates from Ins(1,2,3,5,6)P5 will directly compete with the phosphates from PtdIns(3,4)P2 while the remaining phosphates will provide additional stabilizing interactions.

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