Reverse Engineering Analysis of the High-Temperature Reversible Oligomerization and Amyloidogenicity of PSD95-PDZ3
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High-temperature reversible oligomerizationAmyloidogenicityOligomeric interface residuesThermal denaturationMutational analysis
Onchaiya, S... [et al.]. Reverse Engineering Analysis of the High-Temperature Reversible Oligomerization and Amyloidogenicity of PSD95-PDZ3. Molecules 2022, 27, 2813. [https://doi.org/10.3390/molecules27092813]
SponsorshipMEXT scholarship; Ministry of Education, Culture, Sports, Science and Technology, Japan (MEXT) Japan Society for the Promotion of Science 18H02385 21K05288 21K15049; TUAT's Institute of Global Innovation Research
PSD95-PDZ3, the third PDZ domain of the post-synaptic density-95 protein (MW 11 kDa), undergoes a peculiar three-state thermal denaturation (N <-> I-n <-> 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.