Swapped domain orders in ZO-1 PDZ3 fusion proteins – implications for binding of established and novel targets

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F61388963%3A_____%2F25%3A00639853" target="_blank" >RIV/61388963:_____/25:00639853 - isvavai.cz</a>

Nalezeny alternativní kódy

RIV/62690094:18470/25:50022749

Výsledek na webu

<a href="https://doi.org/10.1016/j.abb.2025.110634" target="_blank" >https://doi.org/10.1016/j.abb.2025.110634</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1016/j.abb.2025.110634" target="_blank" >10.1016/j.abb.2025.110634</a>

Jazyk výsledku

angličtina

Název v původním jazyce

Swapped domain orders in ZO-1 PDZ3 fusion proteins – implications for binding of established and novel targets

Popis výsledku v původním jazyce

PDZ domains play key roles in mediating protein-protein interactions by recognizing short PDZ-binding motifs, typically at the C-termini of target proteins. Zonula occludens 1 (ZO-1) is a scaffolding protein that links tight junction proteins to the actin cytoskeleton, and contains three PDZ domains. Here, we focus on its third PDZ (PDZ3_ZO-1) domain, which interacts with the C-terminus of junctional adhesion protein A as well as connexin 45. To investigate how the domain context of the PDZ3_ZO-1 domain affects its folding and function, we previously established two distinct fusions of PDZ3_ZO-1 and a Trp-cage mini-protein. These fusions with swapped domain order result in FD3A with Trp-cage fused C-terminally and FD4A with Trp-cage fused N-terminally. This study aims to explore the extent to which the distinct Trp-cage fusions affect the function of PDZ3_ZO-1 domain in peptide binding. We find that PDZ3_ZO-1 retains its function, interaction with the connexin 45 peptide, also as part of the fusion proteins. Furthermore, using a phage display approach, we identified a new PDZ3_ZO-1 binding peptide derived from the C-terminal region of methylcytosine dioxygenase TET3. Subsequent validation revealed a significantly higher affinity of PDZ3_ZO-1 for the TET3 peptide as compared to the connexin 45 peptide. Thermodynamic analyses revealed that the swapped domain order conferred distinct effects on the thermodynamic parameters. These results provide insights into the structural and functional adaptability of PDZ domains in engineered proteins, and offer useful principles for the rational design of functional fusion proteins.

Název v anglickém jazyce

Swapped domain orders in ZO-1 PDZ3 fusion proteins – implications for binding of established and novel targets

Popis výsledku anglicky

PDZ domains play key roles in mediating protein-protein interactions by recognizing short PDZ-binding motifs, typically at the C-termini of target proteins. Zonula occludens 1 (ZO-1) is a scaffolding protein that links tight junction proteins to the actin cytoskeleton, and contains three PDZ domains. Here, we focus on its third PDZ (PDZ3_ZO-1) domain, which interacts with the C-terminus of junctional adhesion protein A as well as connexin 45. To investigate how the domain context of the PDZ3_ZO-1 domain affects its folding and function, we previously established two distinct fusions of PDZ3_ZO-1 and a Trp-cage mini-protein. These fusions with swapped domain order result in FD3A with Trp-cage fused C-terminally and FD4A with Trp-cage fused N-terminally. This study aims to explore the extent to which the distinct Trp-cage fusions affect the function of PDZ3_ZO-1 domain in peptide binding. We find that PDZ3_ZO-1 retains its function, interaction with the connexin 45 peptide, also as part of the fusion proteins. Furthermore, using a phage display approach, we identified a new PDZ3_ZO-1 binding peptide derived from the C-terminal region of methylcytosine dioxygenase TET3. Subsequent validation revealed a significantly higher affinity of PDZ3_ZO-1 for the TET3 peptide as compared to the connexin 45 peptide. Thermodynamic analyses revealed that the swapped domain order conferred distinct effects on the thermodynamic parameters. These results provide insights into the structural and functional adaptability of PDZ domains in engineered proteins, and offer useful principles for the rational design of functional fusion proteins.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

—

OECD FORD obor

10608 - Biochemistry and molecular biology

Projekt

—

Návaznosti

I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Rok uplatnění

2025

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Název periodika

Archives of Biochemistry and Biophysics

ISSN

0003-9861

e-ISSN

1096-0384

Svazek periodika

774

Číslo periodika v rámci svazku

December

Stát vydavatele periodika

US - Spojené státy americké

Počet stran výsledku

10

Strana od-do

110634

Kód UT WoS článku

001593308600001

EID výsledku v databázi Scopus

2-s2.0-105017553705