Thermodynamic Analysis of Point Mutations Inhibiting High-Temperature Reversible Oligomerization of PDZ3

Abstract

Differential scanning calorimetry (DSC) indicated that PDZ3 undergoes a peculiar thermal denaturation exhibiting two endothermic peaks due to the formation of reversible oligomers at high temperature (N↔I 6 ↔D). This contrasts sharply with the standard 2-state denaturation model observed for small, globular proteins. We performed an alanine scanning analysis by individually mutating three hydrophobic residues at the crystallographic oligomeric interface (Phe340, Leu342, Ile389) and one away from the interface (Leu349, as a control). DSC analysis indicated that PDZ3-F340A and PDZ3-L342A exhibited a single endothermic peak. Furthermore, PDZ3-L342A underwent a perfect 2-state denaturation, as evidenced by the single endothermic peak, and confirmed by detailed DSC analysis, including global fitting of data measured at different protein concentrations. Reversible oligomerization (RO) at high temperatures by small globular proteins is a rare event. While we designed the mutations based on our previous study showing that a point mutation Val380 to a nonhydrophobic amino acid inhibited RO in DEN4 ED3, the results are nevertheless surprising since high-temperature RO involves proteins in a denatured state, as assessed by circular dichroism. Future studies will determine how and why mutations designed using crystal structures determined at ambient temperatures influence the formation of RO at high temperatures, and whether high-temperature ROs are related to the propensity of proteins to aggregate or precipitate at lower temperatures, which would provide a novel and unique way of controlling protein solubility and aggregation. Significance (120 words)Despite being a small globular protein, which normaly undergo a two-state unfolding, the thermal denaturation of PSD95-PDZ3, monitored by DSC, exhibited two

The order of PDZ3 and TrpCage in fusion chimeras determines their properties—a biophysical characterization

“…The absence of oligomers for the FD3A chimera suggests that FD3A preserves its one‐domain compact form and uses a different denaturation mechanism. It has been described that the PDZ3 oligomer intermediates are formed by interactions of PDZ β‐sheets 62 . Therefore, in the case of FD3A, a stable domain–domain interface between PDZ3 and TrpCage could be possibly preserved, and this could block the association of β‐sheets necessary to form the oligomeric intermediates.…”

“…The PDZ2 domain from the ZO-2 protein showed a T m around 40-50 C with an obvious protein concentration dependency. 61 In one of the previous studies, the T m of PDZ3 was estimated at a surprisingly high temperature of 72.58 C, 62 and the explanation of such high thermostability was provided as follows: the PDZ3 partially thermally unfolded intermediates form aggregates under native conditions, and they were detected by native-state hydrogen exchange experiments. It implies that two processes could take place during the thermal unfolding of the PDZ3 domain.…”

“…It has been described that the PDZ3 oligomer intermediates are formed by interactions of PDZ β-sheets. 62 Therefore, in the case of FD3A, a stable domain-domain interface between PDZ3 and TrpCage could be possibly preserved, and this could block the association of β-sheets necessary to form the oligomeric intermediates. Therefore, our data support the conclusion that PDZ3 and TrpCage interdomain communication within the FD3A and FD4A chimeras has a different character.…”

Biophysical studies of amorphous protein aggregation and in vivo immunogenicity

“…We called this state an RO state (Reversible Oligomer state; Nakamura et al 2012 ). The RO state has now been observed in native proteins and protein domains (Saotome et al 2016 , 2020 ; Onchaiya et al 2022 ), suggesting that the RO state is not just an artifact resulting from the addition of an artificial SCP tag.…”

Blocking PSD95‐PDZ3's amyloidogenesis through point mutations that inhibit high‐temperature reversible oligomerization (RO)

“…The BSA of three hydrophobic residues (Phe340, Leu342, and Ile389) was markedly large, and that of Val328 was moderate. Based on our previously observed correlation between the large BSA and the appearance of the high-temperature RO [17][18], we individually mutated these four residues to alanine (Group 1). Additionally, three residues that were not buried upon oligomerization (BSA = 0) in the asymmetric unit (Val315, Leu349, and Val365) were selected for comparison (Group 2).…”