Dataset Information

Addressing the role of the ?-helical extension in the folding of the third PDZ domain from PSD-95.

ABSTRACT: PDZ domains are one of the most important protein-protein interaction domains in human. While presenting a conserved three dimensional structure, a substantial number of PDZ domains display structural extensions suggested to be involved in their folding and binding mechanisms. The C-terminal ?-helix extension (?3) of the third PDZ domain from PSD-95 (PDZ3) has been reported to have a role in function of the domain as well as in the stabilization of the native fold. Here we report an evaluation of the effect of the truncation of this additional helix on the folding and unfolding kinetics of PDZ3. Fluorescent variants of full length and truncated PDZ3 were produced and stopped-flow fluorescence measurements were made under different experimental conditions (pH, ionic strength and temperature) to investigate the folding kinetics of the respective variant. The results show that folding of PDZ3 is robust and that the mechanism is only marginally affected by the truncation, which contributes to a destabilization of the native state, but otherwise do not change the overall observed kinetics. Furthermore, the increase in the unfolding rate constants, but not the folding rate constant upon deletion of ?3 suggests that the ?-helical extension is largely unstructured in the folding transition state.

SUBMITTER: Gautier C

PROVIDER: S-EPMC5626748 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature

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Addressing the role of the α-helical extension in the folding of the third PDZ domain from PSD-95.

Gautier Candice C Visconti Lorenzo L Jemth Per P Gianni Stefano S

Scientific reports 20171003 1

PDZ domains are one of the most important protein-protein interaction domains in human. While presenting a conserved three dimensional structure, a substantial number of PDZ domains display structural extensions suggested to be involved in their folding and binding mechanisms. The C-terminal α-helix extension (α3) of the third PDZ domain from PSD-95 (PDZ3) has been reported to have a role in function of the domain as well as in the stabilization of the native fold. Here we report an evaluation o ...[more]

PMID: 28974728

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Project description: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 ?2-?3 loop (were a succinimide modification occurs), the ?3 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 ?3 helix, like the E401R substitution or the truncation of the whole ?3 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 ?-sheets present in the PDZ3 fold. However, those mutations affecting the ?2-?3 loop, included into the prone-to-aggregation region composed by a single ?-sheet comprising ?2 to ?4 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 ?3 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.

Dataset Information

Addressing the role of the ?-helical extension in the folding of the third PDZ domain from PSD-95.

Publications

Addressing the role of the α-helical extension in the folding of the third PDZ domain from PSD-95.

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