Development of an inhibitory TTC7B selective nanobody that blocks EFR3 recruitment of PI4KA

Sep 29, 2026

Uploaded by Marglet

Phosphatidylinositol 4 kinase IIIα (PI4KIIIα/PI4KA) is a key lipid kinase that specifies plasma membrane identity by recruiting PI4KA to the membrane. PI4KA recruitment depends on EFR3A/EFR3B interactions with TTC7 and FAM126 isoform combinations. The study maps TTC7–FAM126 pairing preferences, identifies a TTC7B-containing complex with altered EFR3A affinity, and shows that EFR3B phosphorylation reduces binding. A TTC7B-selective nanobody isolated by yeast display, validated by CryoEM and hydrogen deuterium exchange mass spectrometry, sterically blocks EFR3 binding, lowering membrane recruitment and PI4P production for tool-like therapeutic targeting.

LanguageEnglish

CategoryHealthcare

FormatPDF

Pages21

Reading Time53 min

AccessFree to download

Introduction

PI4KA complex recruitment and function

Disease relevance of PI4KA regulation

Results

EFR3–TTC7–FAM126 interaction mapping

Generation and characterization of a TTC7B-selective nanobody

Functional impact on membrane recruitment and PI4P production

Methods overview

Yeast display selection

CryoEM and hydrogen deuterium exchange mass spectrometry

RESEARCH ARTICLE Development of an inhibitory TTC7B selective nanobody that blocks EFR3 recruitment of PI4KA Received for publication, July 29, 2025, and in revised form, October 17, 2025 Published, Papers in Press, November 4, 2025 [https://doi.org/10.1016/j.jbc.2025.1](https://doi.org/10.1016/j.jbc.2025.1)10886 Sushant Suresh1, Alexandria L. Shaw 1,2, Damilola K. Akintola 1, Martine Lunke 1, Sophia Doerr3,4, Pooja Rohilla5, Tamas Balla5, Calvin K. Yip2, Scott D. Hansen3,6, Jennifer A. Cobb 1, and John E. Burke 1,2,7,* From the 1Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia, Canada; 2Department of Biochemistry and Molecular Biology, The University of British Columbia, Vancouver, British Columbia, Canada; 3Institute of Molecular Biology, University of Oregon, Eugene, Oregon, USA; 4Department of Biology, University of Oregon, Eugene, Oregon, USA; 5Section on Molecular Signal Transduction, Eunice Kennedy Shriver National Institute of Child Health and Human Development, NIH, Bethesda, Maryland, USA; 6Department of Chemistry and Biochemistry, University of Oregon, Eugene, Oregon, USA; 7University of Victoria Genome BC Proteomics Centre, Victoria, British Columbia, Canada Reviewed by members of the JBC Editorial Board. Edited by George M. Carman Phosphatidylinositol 4 kinase IIIα (PI4KIIIα/PI4KA) is an essential lipid kinase that plays a critical role in regulating plasma membrane (PM) identity. PI4KA is primarily recruited to the PM through the targeted recruitment by the proteins, EFR3A and EFR3B, which bind to the PI4KA accessory proteins, TTC7 (TTC7A/B) and FAM126 (FAM126A/B). Here, we characterized how both EFR3 isoforms interact with all possible TTC7–FAM126 combinations and developed ananobody that specifically blocked EFR3-mediated PI4KA recruitment in TTC7B-containing complexes. Most EFR3– TTC7–FAM126 combinations show similar binding affinities, with the exception of EFR3A–TTC7B–FAM126A, which binds with a 􀀙 10-fold higher affinity. Moreover, we showed that EFR3B phosphorylation markedly decreased binding to TTC7–FAM126. Using a yeast display approach, we isolated a TTC7B selective nanobody that blocked EFR3 binding. CryoEM and hydrogen deuterium exchange mass spectrometry showed an extended interface with both PI4KA and TTC7B that sterically blocks EFR3 binding. The nanobody caused decreased membrane recruitment both on lipid bilayers and in cells, with decreased PM production of phosphatidylinositol 4-phosphate. Collectively, these findings provide new insights into PI4KA regulation and provide a tool for manipulating PI4KA complexes, which may be valuable for therapeutic targeting. Phosphoinositides are critical mediators of many biological processes at the plasma membrane (PM), with the lipid phosphatidylinositol (PI) 4-phosphate (PI4P) being an essential PM lipid that is generated by the lipid kinase type III phosphatidylinositol 4 kinase (referred to by its gene name PI4KA) (1–3). PI4P is essential for defining the lipid identity of the PM (4), and it is the precursor for the downstream signaling lipids, PIP2 and PIP3, which play crucial roles in multiple signaling processes (5). The generation of PI4P at the * For correspondence: John E. Burke, [jeburke@uvic.ca](jeburke@uvic.ca). PM is essential for enrichment of phosphatidylserine (PS) as PI4P provides the energy necessary for transport of PS from the endoplasmic reticulum against its concentration gradient (6). The predominant form of PI4KA exists as a complex with its accessory proteins, TTC7 (two possible isoforms: TTC7A and TTC7B) and FAM126 (two possible isoforms: FAM126A and FAM126B) forming an 􀀙 750 kDa dimer of heterotrimers, referred to here as the PI4KA complex (7, 8). Both TTC7 and FAM126 contribute toward complex stability, with FAM126 not directly interacting with PI4KA but instead acting as a stabilizer of TTC7 (9). The PI4KA complex has to be recruited to the PM for its activity

FAQs

What role does PI4KA play at the plasma membrane?

PI4KA generates PI4P, which defines plasma membrane lipid identity and supports downstream signaling lipids and related membrane processes.

How does the EFR3–TTC7–FAM126 system control PI4KA recruitment?

PI4KA recruitment is mediated by palmitoylated EFR3A/EFR3B proteins through EFR3 interactions with TTC7 and FAM126 within PI4KA accessory protein complexes.

What does the TTC7B-selective inhibitory nanobody do?

The nanobody sterically blocks EFR3 binding to TTC7B-containing complexes, reducing PI4KA membrane recruitment and decreasing plasma membrane PI4P production.

Sep 29, 2026

Uploaded by Marglet