The structure of the harmonin/sans complex reveals an unexpected interaction mode of the two Usher syndrome proteins - PubMed (original) (raw)

The structure of the harmonin/sans complex reveals an unexpected interaction mode of the two Usher syndrome proteins

Jing Yan et al. Proc Natl Acad Sci U S A. 2010.

Abstract

The hereditary hearing-vision loss disease, Usher syndrome I (USH1), is caused by defects in several proteins that can interact with each other in vitro. Defects in USH1 proteins are thought to be responsible for the developmental and functional impairments of sensory cells in the retina and inner ear. Harmonin/USH1C and Sans/USH1G are two of the USH1 proteins that interact with each other. Harmonin also binds to other USH1 proteins such as cadherin 23 (CDH23) and protocadherin 15 (PCDH15). However, the molecular basis governing the harmonin and Sans interaction is largely unknown. Here, we report an unexpected assembly mode between harmonin and Sans. We demonstrate that the N-terminal domain and the first PDZ domain of harmonin are tethered by a small-domain C-terminal to PDZ1 to form a structural and functional supramodule responsible for binding to Sans. We discover that the SAM domain of Sans, specifically, binds to the PDZ domain of harmonin, revealing previously unknown interaction modes for both PDZ and SAM domains. We further show that the synergistic PDZ1/SAM and PDZ1/carboxyl PDZ binding-motif interactions, between harmonin and Sans, lock the two scaffold proteins into a highly stable complex. Mutations in harmonin and Sans found in USH1 patients are shown to destabilize the complex formation of the two proteins.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

The multi-dentate interaction between harmonin and Sans. (A) A schematic diagram showing the domain organizations of harmonin and Sans. The NPDZ1 and SAM-PBM boundaries used in this study are indicated. (B) GST-fusion protein-based pull-down assay showing that NPDZ1 binds to the SAM domain alone and the SAM-PBM of Sans. No interaction between the N-domain and the SAM domain was detected. (C) Analytical gel-filtration analysis showing that harmonin NPDZ1 and Sans SAM-PBM forms a 1∶1 stoichiometric complex. (D) ITC-based measurements of the binding affinities of NPDZ1 with Sans PBM, SAM, and SAM-PBM.

Fig. 2.

Fig. 2.

The overall structure of the NPDZ1/SAM-PBM complex. (A) Ribbon diagram of the NPDZ1/SAM-PBM complex structure. In this drawing, the N-domain is shown in Marine Blue, PDZ1 domain in Forest Green, and mini domain in Limon Green. SAM-PBM is drawn in Orange. The positions of the two point mutations, K157 in harmonin and K437 in Sans, are indicated with two Dashed Circles. (B) Surface representation showing the overall architecture of the NPDZ1/SAM-PBM complex with the same color scheme as in panel A.

Fig. 3.

Fig. 3.

Assembling of the NPDZ1 supramodule. (A) Cartoon diagram showing the NPDZ1 supramodule. In this drawing, the N-domain is shown in Blue, PDZ1 domain in Green, and the C-terminal mini-domain in Pinkish Red. (B) Surface representation showing the domain architecture of the NPDZ1 supramodule. (C and D) Stereo views of the molecular details of the N-domain/mini-domain and PDZ1/mini-domain interfaces, resp. The salt bridges and hydrogen bonds in the interfaces are shown as Dashed Lines.

Fig. 4.

Fig. 4.

Molecular details of the harmonin NPDZ1 and Sans SAM-PBM interaction. (A and B) The combined surface charge representation (NPDZ1) and the stick-ribbon model (SAM-PBM) showing the interaction interface between harmonin NPDZ1 and Sans SAM-PBM. The binding interface between the NPDZ1 and Sans PBM is better viewed in (A), whereas the binding interface between the NPDZ1 and Sans SAM is optimally depicted in (B). (C and D) Stereo views showing the detailed interactions between harmonin PDZ1 and Sans PBM (C), and between PDZ1 and Sans SAM (D). The hydrogen bonds and salt bridges involved in the interfaces are indicated as Dashed Lines. (E) A schematic cartoon diagram summarizing the three different binding modes of PDZ domains.

Fig. 5.

Fig. 5.

Syndromic mutations found in human patients destabilize the harmonin/Sans complex. (_A_-C) Detailed structural diagrams showing the three residues (D458 from Sans, R103, and V130 from harmonin) identified in USH patients. (A) The combined ribbon and the stick-dot-sphere representations showing the critical roles of Sans D458 and harmonin R103 residues in the harmonin PDZ1/Sans PBM interaction. (B) ITC-based measurements of the binding affinities of the NPDZ1/D458-SAM-PBM complex and the R103H-NPDZ1/SAM-PBM complex. (C) Enlarged view of the hydrophobic packing environment of harmonin V130, indicating that the substitution of this Val with Ile should not have major impact on the overall structure of the harmonin/Sans complex. (D_–_I) Both the PDZ1/PBM and PDZ1/SAM interactions are critical for the formation of the stable harmonin/Sans complex in HeLa cells. (D1_–_D3) When cotransfected in HeLa cells, harmonin a co-localizes with the Sans clusters in cytosol. (E1_–_E3) Deletion of SAM-PBM compromises the co-localization of harmonin a and Sans. (F1_–_F3) and (G1_–_G3) Mutations that weaken either the PDZ1/PBM or the PDZ1/SAM interactions attenuate the co-localization of harmonin and Sans. (H1_–_H3) and (I1_–_I3) Two mutants, Sans D458V (H1_–_H3) and harmonin R103H (I1_–_I3), identified in USH1 patients, also attenuate the colocalization of harmonin and Sans.

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