Reverse Engineering Analysis of the High-Temperature Reversible Oligomerization and Amyloidogenicity of PSD95-PDZ3

• Published in: Molecules (Basel, Switzerland) Vol. 27; no. 9; p. 2813
• Main Authors: Onchaiya, Sawaros, Saotome, Tomonori, Mizutani, Kenji, Martinez, Jose C, Tame, Jeremy R H, Kidokoro, Shun-Ichi, Kuroda, Yutaka
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 28-04-2022; MDPI
• Subjects: Amyloidogenesis; amyloidogenicity; Buried structures; Calorimetry; Calorimetry, Differential Scanning; Circular Dichroism; Crystal structure; Dichroism; Differential scanning calorimetry; Disks Large Homolog 4 Protein; Fluorescence; Heat measurement; High temperature; high-temperature reversible oligomerization; Hydrophobicity; Kinases; Mutation; mutational analysis; oligomeric interface residues; Oligomerization; PDZ Domains; Point mutation; Postsynaptic density proteins; Protein Denaturation; Protein Structure, Secondary; Proteins; Reactive Oxygen Species; Reverse engineering; Synaptic density; Temperature; Thermal denaturation; Thermodynamics; high-temperature reversible oligomerization; thermal denaturation; amyloidogenicity; mutational analysis; oligomeric interface residues
• ISSN: 1420-3049; 1420-3049
• DOI: 10.3390/molecules27092813

PSD95-PDZ3, the third PDZ domain of the post-synaptic density-95 protein (MW 11 kDa), undergoes a peculiar three-state thermal denaturation (N ↔ In ↔ D) and is amyloidogenic. PSD95-PDZ3 in the intermediate state (I) is reversibly oligomerized (RO: Reversible oligomerization). We previously reported a point mutation (F340A) that inhibits both ROs and amyloidogenesis and constructed the PDZ3-F340A variant. Here, we “reverse engineered” PDZ3-F340A for inducing high-temperature RO and amyloidogenesis. We produced three variants (R309L, E310L, and N326L), where we individually mutated hydrophilic residues exposed at the surface of the monomeric PDZ3-F340A but buried in the tetrameric crystal structure to a hydrophobic leucine. Differential scanning calorimetry indicated that two of the designed variants (PDZ3-F340A/R309L and E310L) denatured according to the two-state model. On the other hand, PDZ3-F340A/N326L denatured according to a three-state model and produced high-temperature ROs. The secondary structures of PDZ3-F340A/N326L and PDZ3-wt in the RO state were unfolded according to circular dichroism and differential scanning calorimetry. Furthermore, PDZ3-F340A/N326L was amyloidogenic as assessed by Thioflavin T fluorescence. Altogether, these results demonstrate that a single amino acid mutation can trigger the formation of high-temperature RO and concurrent amyloidogenesis.