Limits...
Structural and functional analyses of minimal phosphopeptides targeting the polo-box domain of polo-like kinase 1.

Yun SM, Moulaei T, Lim D, Bang JK, Park JE, Shenoy SR, Liu F, Kang YH, Liao C, Soung NK, Lee S, Yoon DY, Lim Y, Lee DH, Otaka A, Appella E, McMahon JB, Nicklaus MC, Burke TR, Yaffe MB, Wlodawer A, Lee KS - Nat. Struct. Mol. Biol. (2009)

Bottom Line: Comparative binding studies and analyses of crystal structures of the PLK1 PBD in complex with the minimal phosphopeptides revealed that the C-terminal SpT dipeptide functions as a high-affinity anchor, whereas the N-terminal residues are crucial for providing specificity and affinity to the interaction.Inhibition of the PLK1 PBD by phosphothreonine mimetic peptides was sufficient to induce mitotic arrest and apoptotic cell death.The mode of interaction between the minimal peptide and PBD may provide a template for designing therapeutic agents that target PLK1.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Metabolism, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA.

ABSTRACT
Polo-like kinase-1 (Plk1) has a pivotal role in cell proliferation and is considered a potential target for anticancer therapy. The noncatalytic polo-box domain (PBD) of Plk1 forms a phosphoepitope binding module for protein-protein interaction. Here, we report the identification of minimal phosphopeptides that specifically interact with the PBD of human PLK1, but not those of the closely related PLK2 and PLK3. Comparative binding studies and analyses of crystal structures of the PLK1 PBD in complex with the minimal phosphopeptides revealed that the C-terminal SpT dipeptide functions as a high-affinity anchor, whereas the N-terminal residues are crucial for providing specificity and affinity to the interaction. Inhibition of the PLK1 PBD by phosphothreonine mimetic peptides was sufficient to induce mitotic arrest and apoptotic cell death. The mode of interaction between the minimal peptide and PBD may provide a template for designing therapeutic agents that target PLK1.

Show MeSH
The nature of PBD binding and specificity. (a) Superposition of the phosphopeptide-binding pockets of PBDPL, PBDPP, PBDS+G, and PBDS. PBD is drawn in grey. PLHSpT is in green and its associated glycerol molecule is in yellow. PPHSpT is drawn in cyan. The glycerol molecule (two half-occupancy conformations at the Ser-1 position) of PBDS+G is drawn in magenta. The two sulfate anions of PBDS+G and PBDS are drawn with the sulfur atoms in black and oxygen atoms in red. The differences in the exact positions of sulfate and phosphate groups could be due to the fact that the sulfate is a free anion, whereas the phosphate is covalently linked to the phosphopeptide. PDB ID for PBDPL, 3HIK; PDB ID for PBDPP, 3C5L; PDB ID for PBDS+G and PBDS, 3HIH. (b) The PBD residues involved in binding of PLHSpT are labeled and shown in cyan. All water molecules that form an interface between the phosphopeptide and PBD are drawn in red mesh. (c) Superposition of PLHSpT (green), PPHSpT (cyan), MQSpTPL (magenta), and PMQSpTPL (grey). (d,e) The mixture of HeLa lysates expressing the kinase-inactive Flag-Plk1(K82M), Flag-Plk2(K108M), or Flag-Plk3(K52R) was subjected to pull-down assays as in Fig. 2a with the indicated 5-mer wild-type (PLHSpT) and mutants cross-linked to the beads. The respective non-phospho-T78 peptide (PLHST) was used as a control. The numbers at the top of the blot indicate the relative efficiency of Plk2 precipitation, whereas the numbers at the bottom denote the relative efficiency of Plk1 precipitation. (f) Illustration depicting the nature of the interactions between the SpT-containing peptides and the Plk1 PBD. Alignment of minimal p-T78 peptides (PLHST and LHSTA) and synthetic optimal peptides (PMQSTPL and MQSTPL) showed that, in addition to the critical SpT motif, the N-terminal Pro-4 and Met-3 residues are important to stabilize the interactions by docking into a hydrophobic core surrounded by the Trp414, Phe535 and Arg516 residues in Plk1 PBD. The His-2 residue is important for Plk1 specificity since substitution of Gln for His enhances Plk2 binding. The Ala+1 or Pro+1 residue is central for guiding a priming kinase to phosphorylate the Thr residue.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2721907&req=5

Figure 3: The nature of PBD binding and specificity. (a) Superposition of the phosphopeptide-binding pockets of PBDPL, PBDPP, PBDS+G, and PBDS. PBD is drawn in grey. PLHSpT is in green and its associated glycerol molecule is in yellow. PPHSpT is drawn in cyan. The glycerol molecule (two half-occupancy conformations at the Ser-1 position) of PBDS+G is drawn in magenta. The two sulfate anions of PBDS+G and PBDS are drawn with the sulfur atoms in black and oxygen atoms in red. The differences in the exact positions of sulfate and phosphate groups could be due to the fact that the sulfate is a free anion, whereas the phosphate is covalently linked to the phosphopeptide. PDB ID for PBDPL, 3HIK; PDB ID for PBDPP, 3C5L; PDB ID for PBDS+G and PBDS, 3HIH. (b) The PBD residues involved in binding of PLHSpT are labeled and shown in cyan. All water molecules that form an interface between the phosphopeptide and PBD are drawn in red mesh. (c) Superposition of PLHSpT (green), PPHSpT (cyan), MQSpTPL (magenta), and PMQSpTPL (grey). (d,e) The mixture of HeLa lysates expressing the kinase-inactive Flag-Plk1(K82M), Flag-Plk2(K108M), or Flag-Plk3(K52R) was subjected to pull-down assays as in Fig. 2a with the indicated 5-mer wild-type (PLHSpT) and mutants cross-linked to the beads. The respective non-phospho-T78 peptide (PLHST) was used as a control. The numbers at the top of the blot indicate the relative efficiency of Plk2 precipitation, whereas the numbers at the bottom denote the relative efficiency of Plk1 precipitation. (f) Illustration depicting the nature of the interactions between the SpT-containing peptides and the Plk1 PBD. Alignment of minimal p-T78 peptides (PLHST and LHSTA) and synthetic optimal peptides (PMQSTPL and MQSTPL) showed that, in addition to the critical SpT motif, the N-terminal Pro-4 and Met-3 residues are important to stabilize the interactions by docking into a hydrophobic core surrounded by the Trp414, Phe535 and Arg516 residues in Plk1 PBD. The His-2 residue is important for Plk1 specificity since substitution of Gln for His enhances Plk2 binding. The Ala+1 or Pro+1 residue is central for guiding a priming kinase to phosphorylate the Thr residue.

Mentions: To investigate the binding nature of the minimal peptides to the PBD, the crystal structures of the Plk1 PBD in complex with the phosphopeptides PLHSpT (hereon referred to as PBDPL) and PPHSpT (PBDPP; to examine the importance of the N-terminal residue for the interaction) were solved at 1.7 Å and at 2.3 Å resolution, respectively (Fig. 3a,b and Table 3). Additionally, we attempted to crystallize a complex by mixing the PBD (without phosphopeptide) and the kinase domain, each expressed and purified separately. However, the kinase domain precipitated and only the PBD was found in a diffraction quality crystal. This novel crystal form contained two PBD molecules per asymmetric unit, referred to as PBDS+G (with sulfate and glycerol) and PBDS (with sulfate only) for chains A and B, respectively (Fig. 3a). We found several strong peaks of positive difference density in the /Fo/−/Fc/ maps for PBDPL, PBDS+G and PBDS, which could not be interpreted as water molecules. These peaks were modeled as sulfate, glycerol, and ethylene glycol molecules. PBDPL contained a glycerol molecule in the phosphopeptide-binding cleft (Fig. 3a,b), occupying a cavity formed by the phosphopeptide, two water molecules, and PBD. The three hydroxyl groups of this glycerol molecule were involved in hydrogen bonding with the backbone carbonyls of the phosphopeptide and PBD, the phosphate group of p-Thr, and one of the water molecules. PBDS+G and PBDS contained a sulfate anion in the same pocket (Fig. 3a), in the region normally occupied by the phosphate of p-Thr. The choice of modeling the density in this pocket as sulfate instead of phosphate stemmed from the presence of 0.3 M lithium sulfate in the crystallization media. PBDS+G contained a glycerol molecule in the phosphopeptide-binding cleft (Fig. 3a). This glycerol molecule was located at the − 1 position, normally occupied by the Ser residue when a phosphopeptide is in the binding cleft (Fig. 3a). The L2 loop in PBDS was much less ordered than in the PBDS+G structure. Analysis of contacts with symmetry-related molecules showed that this difference in the degree of order observed in the L2 region is likely caused by crystal packing.


Structural and functional analyses of minimal phosphopeptides targeting the polo-box domain of polo-like kinase 1.

Yun SM, Moulaei T, Lim D, Bang JK, Park JE, Shenoy SR, Liu F, Kang YH, Liao C, Soung NK, Lee S, Yoon DY, Lim Y, Lee DH, Otaka A, Appella E, McMahon JB, Nicklaus MC, Burke TR, Yaffe MB, Wlodawer A, Lee KS - Nat. Struct. Mol. Biol. (2009)

The nature of PBD binding and specificity. (a) Superposition of the phosphopeptide-binding pockets of PBDPL, PBDPP, PBDS+G, and PBDS. PBD is drawn in grey. PLHSpT is in green and its associated glycerol molecule is in yellow. PPHSpT is drawn in cyan. The glycerol molecule (two half-occupancy conformations at the Ser-1 position) of PBDS+G is drawn in magenta. The two sulfate anions of PBDS+G and PBDS are drawn with the sulfur atoms in black and oxygen atoms in red. The differences in the exact positions of sulfate and phosphate groups could be due to the fact that the sulfate is a free anion, whereas the phosphate is covalently linked to the phosphopeptide. PDB ID for PBDPL, 3HIK; PDB ID for PBDPP, 3C5L; PDB ID for PBDS+G and PBDS, 3HIH. (b) The PBD residues involved in binding of PLHSpT are labeled and shown in cyan. All water molecules that form an interface between the phosphopeptide and PBD are drawn in red mesh. (c) Superposition of PLHSpT (green), PPHSpT (cyan), MQSpTPL (magenta), and PMQSpTPL (grey). (d,e) The mixture of HeLa lysates expressing the kinase-inactive Flag-Plk1(K82M), Flag-Plk2(K108M), or Flag-Plk3(K52R) was subjected to pull-down assays as in Fig. 2a with the indicated 5-mer wild-type (PLHSpT) and mutants cross-linked to the beads. The respective non-phospho-T78 peptide (PLHST) was used as a control. The numbers at the top of the blot indicate the relative efficiency of Plk2 precipitation, whereas the numbers at the bottom denote the relative efficiency of Plk1 precipitation. (f) Illustration depicting the nature of the interactions between the SpT-containing peptides and the Plk1 PBD. Alignment of minimal p-T78 peptides (PLHST and LHSTA) and synthetic optimal peptides (PMQSTPL and MQSTPL) showed that, in addition to the critical SpT motif, the N-terminal Pro-4 and Met-3 residues are important to stabilize the interactions by docking into a hydrophobic core surrounded by the Trp414, Phe535 and Arg516 residues in Plk1 PBD. The His-2 residue is important for Plk1 specificity since substitution of Gln for His enhances Plk2 binding. The Ala+1 or Pro+1 residue is central for guiding a priming kinase to phosphorylate the Thr residue.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2721907&req=5

Figure 3: The nature of PBD binding and specificity. (a) Superposition of the phosphopeptide-binding pockets of PBDPL, PBDPP, PBDS+G, and PBDS. PBD is drawn in grey. PLHSpT is in green and its associated glycerol molecule is in yellow. PPHSpT is drawn in cyan. The glycerol molecule (two half-occupancy conformations at the Ser-1 position) of PBDS+G is drawn in magenta. The two sulfate anions of PBDS+G and PBDS are drawn with the sulfur atoms in black and oxygen atoms in red. The differences in the exact positions of sulfate and phosphate groups could be due to the fact that the sulfate is a free anion, whereas the phosphate is covalently linked to the phosphopeptide. PDB ID for PBDPL, 3HIK; PDB ID for PBDPP, 3C5L; PDB ID for PBDS+G and PBDS, 3HIH. (b) The PBD residues involved in binding of PLHSpT are labeled and shown in cyan. All water molecules that form an interface between the phosphopeptide and PBD are drawn in red mesh. (c) Superposition of PLHSpT (green), PPHSpT (cyan), MQSpTPL (magenta), and PMQSpTPL (grey). (d,e) The mixture of HeLa lysates expressing the kinase-inactive Flag-Plk1(K82M), Flag-Plk2(K108M), or Flag-Plk3(K52R) was subjected to pull-down assays as in Fig. 2a with the indicated 5-mer wild-type (PLHSpT) and mutants cross-linked to the beads. The respective non-phospho-T78 peptide (PLHST) was used as a control. The numbers at the top of the blot indicate the relative efficiency of Plk2 precipitation, whereas the numbers at the bottom denote the relative efficiency of Plk1 precipitation. (f) Illustration depicting the nature of the interactions between the SpT-containing peptides and the Plk1 PBD. Alignment of minimal p-T78 peptides (PLHST and LHSTA) and synthetic optimal peptides (PMQSTPL and MQSTPL) showed that, in addition to the critical SpT motif, the N-terminal Pro-4 and Met-3 residues are important to stabilize the interactions by docking into a hydrophobic core surrounded by the Trp414, Phe535 and Arg516 residues in Plk1 PBD. The His-2 residue is important for Plk1 specificity since substitution of Gln for His enhances Plk2 binding. The Ala+1 or Pro+1 residue is central for guiding a priming kinase to phosphorylate the Thr residue.
Mentions: To investigate the binding nature of the minimal peptides to the PBD, the crystal structures of the Plk1 PBD in complex with the phosphopeptides PLHSpT (hereon referred to as PBDPL) and PPHSpT (PBDPP; to examine the importance of the N-terminal residue for the interaction) were solved at 1.7 Å and at 2.3 Å resolution, respectively (Fig. 3a,b and Table 3). Additionally, we attempted to crystallize a complex by mixing the PBD (without phosphopeptide) and the kinase domain, each expressed and purified separately. However, the kinase domain precipitated and only the PBD was found in a diffraction quality crystal. This novel crystal form contained two PBD molecules per asymmetric unit, referred to as PBDS+G (with sulfate and glycerol) and PBDS (with sulfate only) for chains A and B, respectively (Fig. 3a). We found several strong peaks of positive difference density in the /Fo/−/Fc/ maps for PBDPL, PBDS+G and PBDS, which could not be interpreted as water molecules. These peaks were modeled as sulfate, glycerol, and ethylene glycol molecules. PBDPL contained a glycerol molecule in the phosphopeptide-binding cleft (Fig. 3a,b), occupying a cavity formed by the phosphopeptide, two water molecules, and PBD. The three hydroxyl groups of this glycerol molecule were involved in hydrogen bonding with the backbone carbonyls of the phosphopeptide and PBD, the phosphate group of p-Thr, and one of the water molecules. PBDS+G and PBDS contained a sulfate anion in the same pocket (Fig. 3a), in the region normally occupied by the phosphate of p-Thr. The choice of modeling the density in this pocket as sulfate instead of phosphate stemmed from the presence of 0.3 M lithium sulfate in the crystallization media. PBDS+G contained a glycerol molecule in the phosphopeptide-binding cleft (Fig. 3a). This glycerol molecule was located at the − 1 position, normally occupied by the Ser residue when a phosphopeptide is in the binding cleft (Fig. 3a). The L2 loop in PBDS was much less ordered than in the PBDS+G structure. Analysis of contacts with symmetry-related molecules showed that this difference in the degree of order observed in the L2 region is likely caused by crystal packing.

Bottom Line: Comparative binding studies and analyses of crystal structures of the PLK1 PBD in complex with the minimal phosphopeptides revealed that the C-terminal SpT dipeptide functions as a high-affinity anchor, whereas the N-terminal residues are crucial for providing specificity and affinity to the interaction.Inhibition of the PLK1 PBD by phosphothreonine mimetic peptides was sufficient to induce mitotic arrest and apoptotic cell death.The mode of interaction between the minimal peptide and PBD may provide a template for designing therapeutic agents that target PLK1.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Metabolism, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA.

ABSTRACT
Polo-like kinase-1 (Plk1) has a pivotal role in cell proliferation and is considered a potential target for anticancer therapy. The noncatalytic polo-box domain (PBD) of Plk1 forms a phosphoepitope binding module for protein-protein interaction. Here, we report the identification of minimal phosphopeptides that specifically interact with the PBD of human PLK1, but not those of the closely related PLK2 and PLK3. Comparative binding studies and analyses of crystal structures of the PLK1 PBD in complex with the minimal phosphopeptides revealed that the C-terminal SpT dipeptide functions as a high-affinity anchor, whereas the N-terminal residues are crucial for providing specificity and affinity to the interaction. Inhibition of the PLK1 PBD by phosphothreonine mimetic peptides was sufficient to induce mitotic arrest and apoptotic cell death. The mode of interaction between the minimal peptide and PBD may provide a template for designing therapeutic agents that target PLK1.

Show MeSH