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Post-translational modifications modulate ligand recognition by the third PDZ domain of the MAGUK protein PSD-95.

Murciano-Calles J, Corbi-Verge C, Candel AM, Luque I, Martinez JC - PLoS ONE (2014)

Bottom Line: The α3 helix is an extra structural element that is not present in other PDZ domains, which links PDZ3 with the following SH3 domain in the PSD-95 protein.This regulatory mechanism was confirmed experimentally via thermodynamic and NMR chemical shift perturbation analyses, discarding intra-domain long-range effects.Taken together, the results presented here reveal the molecular basis of the regulatory role of the α3 extra-element and the effects of post-translational modifications of PDZ3 on its binding affinity, both energetically and dynamically.

View Article: PubMed Central - PubMed

Affiliation: Department of Physical Chemistry and Institute of Biotechnology, Faculty of Sciences, University of Granada, Granada, Spain.

ABSTRACT
The relative promiscuity of hub proteins such as postsynaptic density protein-95 (PSD-95) can be achieved by alternative splicing, allosteric regulation, and post-translational modifications, the latter of which is the most efficient method of accelerating cellular responses to environmental changes in vivo. Here, a mutational approach was used to determine the impact of phosphorylation and succinimidation post-translational modifications on the binding affinity of the postsynaptic density protein-95/discs large/zonula occludens-1 (PDZ3) domain of PSD-95. Molecular dynamics simulations revealed that the binding affinity of this domain is influenced by an interplay between salt-bridges linking the α3 helix, the β2-β3 loop and the positively charged Lys residues in its high-affinity hexapeptide ligand KKETAV. The α3 helix is an extra structural element that is not present in other PDZ domains, which links PDZ3 with the following SH3 domain in the PSD-95 protein. This regulatory mechanism was confirmed experimentally via thermodynamic and NMR chemical shift perturbation analyses, discarding intra-domain long-range effects. Taken together, the results presented here reveal the molecular basis of the regulatory role of the α3 extra-element and the effects of post-translational modifications of PDZ3 on its binding affinity, both energetically and dynamically.

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The structure and sequence of the PDZ3 domain of PSD-95.The panel on the upper left shows a structural representation of the PDZ3 domain of PSD-95 in complex with the hexapeptide KKETAV (orange), modelled from the X-ray structure of the PDZ3-CRIPT complex (Protein Data Bank ID: 1BE9). The α1, α2, and α3 helices are shown in green, light blue and blue, respectively. The β2–β3 loop is shown in red and the β2 chain is shown in yellow. The dashed line indicates the binding pocket. The panel on the upper right is a detailed view of the interface of the α3 helix at the C-terminus of PDZ3 showing the spatial arrangement of the Phe, Tyr, Asp, and Glu residues. The lower panel shows the sequence of the PDZ3 domain and its secondary structures. Numbering of the protein residues is relative to their positions in the full-length PSD-95 protein. Numbering of the KKETAV peptide residues is from 0 (C-terminal Val residue) to −5 (N-terminal Lys residue).
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pone-0090030-g002: The structure and sequence of the PDZ3 domain of PSD-95.The panel on the upper left shows a structural representation of the PDZ3 domain of PSD-95 in complex with the hexapeptide KKETAV (orange), modelled from the X-ray structure of the PDZ3-CRIPT complex (Protein Data Bank ID: 1BE9). The α1, α2, and α3 helices are shown in green, light blue and blue, respectively. The β2–β3 loop is shown in red and the β2 chain is shown in yellow. The dashed line indicates the binding pocket. The panel on the upper right is a detailed view of the interface of the α3 helix at the C-terminus of PDZ3 showing the spatial arrangement of the Phe, Tyr, Asp, and Glu residues. The lower panel shows the sequence of the PDZ3 domain and its secondary structures. Numbering of the protein residues is relative to their positions in the full-length PSD-95 protein. Numbering of the KKETAV peptide residues is from 0 (C-terminal Val residue) to −5 (N-terminal Lys residue).

Mentions: An α-helical segment in PSD-95 (Figures 1 and 2; α3 helix), which connects the PDZ3 domain to the subsequent SH3 domain, packs the PDZ3 domain at a different face to that of the binding pocket, thereby enabling allosteric modulation of the protein, as demonstrated by a 21-fold drop in affinity for the C-terminal sequence of cysteine-rich PDZ-binding protein (CRIPT) after removal of the α3 helix [9]. A recent nuclear magnetic resonance (NMR) and small-angle X-ray scattering study of the tandem PDZ3-SH3 in PSD-95 demonstrated that the PDZ3 domain favours an orientation where its ligand binding site faces the SH3 domain, although this inter-domain interaction appears to be weak [10]. In this context, the α3 linking element can be positioned between the PDZ3 and SH3 domains or away from both of them when a C-terminal partner of PDZ3 displaces their weak interaction. Phospho-mimicking mutations at residues Tyr397 (located in the α3 helix), Ser415, and Ser418 of PSD-95 also weaken the interaction between the PDZ3 and SH3 domains [11]. PSD-95 can be phosphorylated at these sites in mouse brain, but very little is known about the consequences of these modifications in vivo. Considering that phosphorylation is the most common post-translational modification of proteins, the accumulation of phosphorylation sites in PSD-95 suggests that the α3-helix region is a key target for MAGUK regulation in vivo[11].


Post-translational modifications modulate ligand recognition by the third PDZ domain of the MAGUK protein PSD-95.

Murciano-Calles J, Corbi-Verge C, Candel AM, Luque I, Martinez JC - PLoS ONE (2014)

The structure and sequence of the PDZ3 domain of PSD-95.The panel on the upper left shows a structural representation of the PDZ3 domain of PSD-95 in complex with the hexapeptide KKETAV (orange), modelled from the X-ray structure of the PDZ3-CRIPT complex (Protein Data Bank ID: 1BE9). The α1, α2, and α3 helices are shown in green, light blue and blue, respectively. The β2–β3 loop is shown in red and the β2 chain is shown in yellow. The dashed line indicates the binding pocket. The panel on the upper right is a detailed view of the interface of the α3 helix at the C-terminus of PDZ3 showing the spatial arrangement of the Phe, Tyr, Asp, and Glu residues. The lower panel shows the sequence of the PDZ3 domain and its secondary structures. Numbering of the protein residues is relative to their positions in the full-length PSD-95 protein. Numbering of the KKETAV peptide residues is from 0 (C-terminal Val residue) to −5 (N-terminal Lys residue).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3935999&req=5

pone-0090030-g002: The structure and sequence of the PDZ3 domain of PSD-95.The panel on the upper left shows a structural representation of the PDZ3 domain of PSD-95 in complex with the hexapeptide KKETAV (orange), modelled from the X-ray structure of the PDZ3-CRIPT complex (Protein Data Bank ID: 1BE9). The α1, α2, and α3 helices are shown in green, light blue and blue, respectively. The β2–β3 loop is shown in red and the β2 chain is shown in yellow. The dashed line indicates the binding pocket. The panel on the upper right is a detailed view of the interface of the α3 helix at the C-terminus of PDZ3 showing the spatial arrangement of the Phe, Tyr, Asp, and Glu residues. The lower panel shows the sequence of the PDZ3 domain and its secondary structures. Numbering of the protein residues is relative to their positions in the full-length PSD-95 protein. Numbering of the KKETAV peptide residues is from 0 (C-terminal Val residue) to −5 (N-terminal Lys residue).
Mentions: An α-helical segment in PSD-95 (Figures 1 and 2; α3 helix), which connects the PDZ3 domain to the subsequent SH3 domain, packs the PDZ3 domain at a different face to that of the binding pocket, thereby enabling allosteric modulation of the protein, as demonstrated by a 21-fold drop in affinity for the C-terminal sequence of cysteine-rich PDZ-binding protein (CRIPT) after removal of the α3 helix [9]. A recent nuclear magnetic resonance (NMR) and small-angle X-ray scattering study of the tandem PDZ3-SH3 in PSD-95 demonstrated that the PDZ3 domain favours an orientation where its ligand binding site faces the SH3 domain, although this inter-domain interaction appears to be weak [10]. In this context, the α3 linking element can be positioned between the PDZ3 and SH3 domains or away from both of them when a C-terminal partner of PDZ3 displaces their weak interaction. Phospho-mimicking mutations at residues Tyr397 (located in the α3 helix), Ser415, and Ser418 of PSD-95 also weaken the interaction between the PDZ3 and SH3 domains [11]. PSD-95 can be phosphorylated at these sites in mouse brain, but very little is known about the consequences of these modifications in vivo. Considering that phosphorylation is the most common post-translational modification of proteins, the accumulation of phosphorylation sites in PSD-95 suggests that the α3-helix region is a key target for MAGUK regulation in vivo[11].

Bottom Line: The α3 helix is an extra structural element that is not present in other PDZ domains, which links PDZ3 with the following SH3 domain in the PSD-95 protein.This regulatory mechanism was confirmed experimentally via thermodynamic and NMR chemical shift perturbation analyses, discarding intra-domain long-range effects.Taken together, the results presented here reveal the molecular basis of the regulatory role of the α3 extra-element and the effects of post-translational modifications of PDZ3 on its binding affinity, both energetically and dynamically.

View Article: PubMed Central - PubMed

Affiliation: Department of Physical Chemistry and Institute of Biotechnology, Faculty of Sciences, University of Granada, Granada, Spain.

ABSTRACT
The relative promiscuity of hub proteins such as postsynaptic density protein-95 (PSD-95) can be achieved by alternative splicing, allosteric regulation, and post-translational modifications, the latter of which is the most efficient method of accelerating cellular responses to environmental changes in vivo. Here, a mutational approach was used to determine the impact of phosphorylation and succinimidation post-translational modifications on the binding affinity of the postsynaptic density protein-95/discs large/zonula occludens-1 (PDZ3) domain of PSD-95. Molecular dynamics simulations revealed that the binding affinity of this domain is influenced by an interplay between salt-bridges linking the α3 helix, the β2-β3 loop and the positively charged Lys residues in its high-affinity hexapeptide ligand KKETAV. The α3 helix is an extra structural element that is not present in other PDZ domains, which links PDZ3 with the following SH3 domain in the PSD-95 protein. This regulatory mechanism was confirmed experimentally via thermodynamic and NMR chemical shift perturbation analyses, discarding intra-domain long-range effects. Taken together, the results presented here reveal the molecular basis of the regulatory role of the α3 extra-element and the effects of post-translational modifications of PDZ3 on its binding affinity, both energetically and dynamically.

Show MeSH