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. 2022 Jun 30;479(12):1393-1405.
doi: 10.1042/BCJ20220147.

Deafness-related protein PDZD7 forms complex with the C-terminal tail of FCHSD2

Huang Wang #  1 , Dange Zhao #  1 , Haibo Du #  2 , Xiaoyan Zhai  2 , Shaoxuan Wu  1 , Lin Lin  1 , Zhigang Xu  2   3 , Qing Lu  1   4   5   6
Affiliations

Deafness-related protein PDZD7 forms complex with the C-terminal tail of FCHSD2

Huang Wang et al. Biochem J. .

Abstract

In cochlea, deafness-related protein PDZD7 is an indispensable component of the ankle link complex, which is critical for the maturation of inner-ear hair cell for sound perception. Ankle links, connecting the different rows of cochlear stereocilia, are essential for the staircase-like development of stereocilia. However, the molecular mechanism of how PDZD7 governs stereociliary development remains unknown. Here, we reported a novel PDZD7-binding partner, FCHSD2, identified by yeast two-hybrid screening. FCHSD2 was reported to be expressed in hair cell, where it co-operated with CDC42 and N-WASP to regulate the formation of cell protrusion. The association between FCHSD2 and PDZD7 was further confirmed in COS-7 cells. More importantly, we solved the complex structure of FCHSD2 tail with PDZD7 PDZ3 domain at 2.0 Å resolution. The crystal structure shows that PDZD7 PDZ3 adopts a typical PDZ domain topology, comprising five β strands and two α helixes. The PDZ-binding motif of FCHSD2 tail stretches through the αB/βB groove of PDZD7 PDZ3. Our study not only uncovers the interaction between FCHSD2 tail and PDZD7 PDZ3 at the atomic level, but also provides clues of connecting the ankle link complex with cytoskeleton dynamics for exploiting the molecular mechanism of stereociliary development.

Keywords: FCHSD2; PDZD7; ankle link; complex structure; stereocilia.

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Conflict of interest statement

The authors declare that there are no competing interests associated with the manuscript.

Figures

Figure 1.
Figure 1.. FCHSD2 is a PDZD7-binding partner.
(A,B) Western blots showing that (A) GFP-FCHSD2 is co-IPed with Myc-PDZD7, and (B) GFP-PDZD7 PDZ3 is co-IPed with Myc-FCHSD2. Expression vectors were transfected into HEK293T cells to express epitope-tagged proteins, and cell lysates were subjected to immunoprecipitation. IP indicates antibody used for immunoprecipitation, and WB indicates antibody used for detection. (C) Expression vectors were transfected into COS-7 cells and the subcellular localization of target proteins was determined using confocal microscopy. Scale bar, 20 μm.
Figure 2.
Figure 2.. FCHSD2 tail specifically binds to PDZD7 PDZ3.
(A) Schematic diagram showing the domain organizations of FCHSD2 and PDZD7. (B,C) Sequence alignments showing that FCHSD2 tail and PDZD7 PDZ3 are highly conserved among different species. The secondary structural elements of PDZD7 PDZ3 determined from this work are shown above its alignment. In these alignments, invariant residues are highlighted with red boxes, the conserved residues are colored in red. Specifically, residues indicated with black or red asterisk are responsible for the interaction between FCHSD2 tail and PDZD7 PDZ3. (D) ITC analysis showing that FCHSD2 tail binds to PDZD7 PDZ3 with a Kd value of ∼1.51 μM. △PBM abolishes FCHSD2 tail's binding to PDZD7 PDZ3. (E) SEC-MALS analysis showing that Trx-tagged FCHSD2 tail binds to Trx-tagged PDZD7 PDZ3 at the stoichiometry of 1 : 1. △PBM, the truncation of PBM; PR, proline rich; HHD, harmonin homology domain; PBM, PDZ-binding motif; Trx, thioredoxin.
Figure 3.
Figure 3.. The structure of the FCHSD2 tail/PDZD7 PDZ3 complex.
(A) Overall structure of the FCHSD2 tail/PDZD7 PDZ3 complex. FCHSD2 tail and PDZD7 PDZ3 are colored in orange and green, respectively. (B) Residues responsible for the formation of the hydrophobic core of PDZD7 PDZ3 are shown with stick model. PDZD7 PDZ3 is colored in green. Residues Ile870, Ile872, and Leu907 taken for mutation are colored in yellow. (C) SEC-MALS analysis showing that I870D and L907D compromise the homogeneity of PDZD7 PDZ3. (D) CD spectrum showing that I870D and L907D disrupt the normal PDZ folding of PDZD7 PDZ3.
Figure 4.
Figure 4.. Detailed interactions between FCHSD2 tail and PDZD7 PDZ3.
(A) Detailed interactions between FCHSD2 tail and PDZD7 PDZ3. Residues involved in protein interaction are shown with stick model. FCHSD2 tail and PDZD7 PDZ3 are colored in orange and green, respectively. Residues taken for mutations are highlighted in red boxes. Hydrogen bonds are shown as black dashed lines. (B) Summary of the binding affinities between WT or structural mutants of FCHSD2 tail and PDZD7 PDZ3. (C) ITC assays showing that mutant (c1) T738A or (c2) V740R abolishes FCHSD2 tail's binding to PDZD7 PDZ3. (D) ITC assays showing that mutant (d1) H920A or (d2) R928A abolishes PDZD7 PDZ3’ binding to FCHSD2 tail.
Figure 5.
Figure 5.. Mutations disrupt the cell colocalization of FCHSD2 and PDZD7.
(A) GFP-FCHSD2 and PDZD7-mCherry colocalize in the cytoplasm of COS-7 cells in a punctuate pattern. (B–E) T738A/V740R (B) or △PBM (C) disrupts GFP-FCHSD2's colocalization with PDZD7-mCherry; H920A/R928A (D) or △PDZ3 (E) disrupts PDZD7-mCherry's colocalization with GFP-FCHSD2. Scale bar, 20 μm. △PBM or △PDZ3, the truncation of PBM or PDZ3; PBM, PDZ-binding motif.
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