Post-Translational Modifications Modulate Ligand Recognition by the Third PDZ Domain of the MAGUK Protein PSD-95

Murciano-Calles, Javier; Corbi‐Verge, Carles; Candel, Adela M.; Luque, Irene; Martínez, José C.

doi:10.1371/journal.pone.0090030

Cited by 20 publications

(61 citation statements)

References 40 publications

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“…Finally, mutation D332P was designed to emulate the succinimide ring formation at residue Asp332 previously described by us [20]. Another previous study [18] has shown that Asp332 is solvent exposed at the β2–β3 loop, not contacting with any residues of the ligand or of the domain; the cyclation of this position drives to a strong modification of the conformational dynamics of the loop, being this effect the responsible of a net drop in the binding affinity of short linear peptides. These effects can be considered essentially the same when a Pro or a succinimide are present, despite the ring occurs at different positions of the backbone.…”

Section: Resultsmentioning

confidence: 99%

“…This titration behaviour might be attributed to the protonation equilibriums of some Glu and/or Asp residues, the only ones whose pK a values are within the range of pH 3–4 [23]. This evidence strongly pointed to some of the above mentioned Glu residues, concretely to Glu334 and Glu401, since these are the main responsible for the packing of the extra α3-helix to the domain through salt-bridges with residues Lys355 and Arg399, respectively [18]. The question still unknown is to which extent these electrostatic interactions may also determine the folding/misfolding behaviour of PDZ3, since both salt-bridges would be influenced when Glu residues protonate below pH 3, being the packing of the extra-helix to the whole PDZ3 altered.…”

Section: Introductionmentioning

confidence: 89%

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The Impact of Extra-Domain Structures and Post-Translational Modifications in the Folding/Misfolding Behaviour of the Third PDZ Domain of MAGUK Neuronal Protein PSD-95

et al. 2014

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The modulation of binding affinities and specificities by post-translational modifications located out from the binding pocket of the third PDZ domain of PSD-95 (PDZ3) has been reported recently. It is achieved through an intra-domain electrostatic network involving some charged residues in the β2–β3 loop (were a succinimide modification occurs), the α3 helix (an extra-structural element that links the PDZ3 domain with the following SH3 domain in PSD-95, and contains the phosphorylation target Tyr397), and the ligand peptide. Here, we have investigated the main structural and thermodynamic aspects that these structural elements and their related post-translational modifications display in the folding/misfolding pathway of PDZ3 by means of site-directed mutagenesis combined with calorimetry and spectroscopy. We have found that, although all the assayed mutations generate proteins more prone to aggregation than the wild-type PDZ3, those directly affecting the α3 helix, like the E401R substitution or the truncation of the whole α3 helix, increase the population of the DSC-detected intermediate state and the misfolding kinetics, by organizing the supramacromolecular structures at the expense of the two β-sheets present in the PDZ3 fold. However, those mutations affecting the β2–β3 loop, included into the prone-to-aggregation region composed by a single β-sheet comprising β2 to β4 chains, stabilize the trimeric intermediate previously shown in the wild-type PDZ3 and slow-down aggregation, also making it partly reversible. These results strongly suggest that the α3 helix protects to some extent the PDZ3 domain core from misfolding. This might well constitute the first example where an extra-element, intended to link the PDZ3 domain to the following SH3 in PSD-95 and in other members of the MAGUK family, not only regulates the binding abilities of this domain but it also protects PDZ3 from misfolding and aggregation. The influence of the post-translational modifications in this regulatory mechanism is also discussed.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 89%

The Impact of Extra-Domain Structures and Post-Translational Modifications in the Folding/Misfolding Behaviour of the Third PDZ Domain of MAGUK Neuronal Protein PSD-95

et al. 2014

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“…In fact, a ∼100-fold stronger binding of the PDZ domain of Pals1 to the Crb tail is achieved upon intra-or inter-molecular interactions between the Src homology 3 (SH3)-and the guanylate kinase (GUK)-domain of Pals1 (Kantardzhieva et al, 2005;Li et al, 2014). Similarly, the affinity of the PDZ domain of the postsynaptic density protein PSD-95 to its ligand is reduced upon phosphorylation of a tyrosine residue in a linker region between the third PDZ domain and the subsequent SH3-domain, thereby weakening the intramolecular interaction between the PDZ and the SH3 domain (Murciano-Calles et al, 2014;Zhang et al, 2011).…”

Section: Discussionmentioning

confidence: 99%

AP-2-complex-mediated endocytosis of Drosophila Crumbs regulates polarity by antagonizing Stardust

Lin

Currinn

Pocha

et al. 2015

Journal of Cell Science

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Maintenance of epithelial polarity depends on the correct localization and levels of polarity determinants. The evolutionarily conserved transmembrane protein Crumbs is crucial for the size and identity of the apical membrane, yet little is known about the molecular mechanisms controlling the amount of Crumbs at the surface. Here, we show that Crumbs levels on the apical membrane depend on a well-balanced state of endocytosis and stabilization. The adaptor protein 2 (AP-2) complex binds to a motif in the cytoplasmic tail of Crumbs that overlaps with the binding site of Stardust, a protein known to stabilize Crumbs on the surface. Preventing endocytosis by mutation of AP-2 causes expansion of the Crumbspositive plasma membrane domain and polarity defects, which can be partially rescued by removing one copy of crumbs. Strikingly, knocking down both AP-2 and Stardust leads to the retention of Crumbs on the membrane. This study provides evidence for a molecular mechanism, based on stabilization and endocytosis, to adjust surface levels of Crumbs, which are essential for maintaining epithelial polarity.

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“…Thus, the heat release when such interactions are formed reflects a favourable enthalpic contribution at neutral pH. The binding entropy counterbalances enthalpy by approximately 15% to 20% and arises from losing conformational freedom because of the structural arrangement of the peptide ligand as a β‐strand in the binding pocket of the PDZ domain . Overall, the net Gibbs energy change is favourable, enthalpically driven, and entropically hindered.…”

Section: Resultsmentioning

confidence: 99%

“…Calorimetric titrations were performed at different temperatures on an isothermal titration calorimetry (ITC) 200 titration microcalorimeter (Microcal Inc) as described elsewhere . Briefly, titrations were made by a series of injections of 2 μL of PSD95‐PDZ2 or HRNTVV solutions into the calorimetric cell containing nNOS‐PDZ or the complex PSD95‐PDZ2/nNOS‐PDZ, respectively.…”

Section: Methodsmentioning

confidence: 99%

PDZ/PDZ interaction between PSD‐95 and nNOS neuronal proteins

Murciano-Calles

Coello

Cámara-Artigas

et al. 2019

J of Molecular Recognition

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N‐Methyl‐D‐aspartate (NMDA) receptors are key components in synaptic communication and are highly relevant in central nervous disorders, where they trigger excessive calcium entry into the neuronal cells causing harmful overproduction of nitric oxide by the neuronal nitric oxide synthase (nNOS) protein. Remarkably, NMDA receptor activation is aided by a second protein, postsynaptic density of 95 kDa (PSD95), forming the ternary protein complex NMDA/PSD95/nNOS. To minimize the potential side effects derived from blocking this ternary complex or either of its protein components, a promising approach points to the disruption of the PSD‐95/nNOS interaction which is mediated by a PDZ/PDZ domain complex. Since the rational development of molecules targeting such protein‐protein interaction relies on energetic and structural information herein, we include a thermodynamic and structural analysis of the PSD95‐PDZ2/nNOS‐PDZ. Two energetically relevant events are structurally linked to a “two‐faced” or two areas of recognition between both domains. First, the assembly of a four‐stranded antiparallel β‐sheet between the β hairpins of nNOS and of PSD95‐PDZ2, mainly enthalpic in nature, contributes 80% to the affinity. Second, binding is entropically reinforced by the hydrophobic interaction between side chains of the same nNOS β‐hairpin with the side chains of α2‐helix at the binding site of PSD95‐PDZ2, contributing the remaining 20% of the total affinity. These results suggest strategies for the future rational design of molecules able to disrupt this complex and constitute the first exhaustive thermodynamic analysis of a PDZ/PDZ interaction.

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Post-Translational Modifications Modulate Ligand Recognition by the Third PDZ Domain of the MAGUK Protein PSD-95

Cited by 20 publications

References 40 publications

The Impact of Extra-Domain Structures and Post-Translational Modifications in the Folding/Misfolding Behaviour of the Third PDZ Domain of MAGUK Neuronal Protein PSD-95

The Impact of Extra-Domain Structures and Post-Translational Modifications in the Folding/Misfolding Behaviour of the Third PDZ Domain of MAGUK Neuronal Protein PSD-95

AP-2-complex-mediated endocytosis of Drosophila Crumbs regulates polarity by antagonizing Stardust

PDZ/PDZ interaction between PSD‐95 and nNOS neuronal proteins

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