Structural Characterization of Whirlin Reveals an Unexpected and Dynamic Supramodule Conformation of Its PDZ Tandem

Abstract

Hearing relies on the transduction of sound-evoked vibrations into electric signals, occurring in the stereocilia bundle of hair cells. The bundle is organized in a staircase pattern formed by rows of packed stereocilia. This architecture is pivotal to transduction and involves a network of scaffolding proteins with hitherto uncharacterized features. Key interactions in this network are mediated by PDZ domains. Here, we describe the architecture of the first two PDZ domains of whirlin, a protein involved in these assemblies and associated with congenital deaf-blindness. C-terminal hairpin extensions of the PDZ domains mediate the transient supramodular assembly, which improves the binding capacity of the first domain. We determined a detailed structural model of the closed conformation of the PDZ tandem and characterized its equilibrium with an ensemble of open conformations. The structural and dynamic behavior of this PDZ tandem provides key insights into the regulatory mechanisms involved in the hearing machinery.

Probing the Architecture of a Multi-PDZ Domain Protein: Structure of PDZK1 in Solution

“…The SAXS profiles (scattering intensities as a function of momentum transfer s = 4p sinðqÞ=l where 2q is the scattering angle and l the X-ray wavelength) and atom-pair distance distributions (P(r) versus r) of the single domains are consistent with the expected globular shapes of these domains ( Figure S2E). The twodomain fragments associate to form compact but elongated structures, as already reported in earlier studies for two-domain PDZ constructs (Delhommel et al, 2017;Goult et al, 2007) ( Figure S2D). Expectedly, the construct D34 appears larger than D12 and D23, due to the long 49-residue linker between D3 and D4 (Table 1).…”

PDZ Domains as Drug Targets

“…Such extensions can be small secondary structural elements that add to the PDZ domain structural core, as exemplified by the third PDZ domain, PDZ3, of PSD‐95 that contains an additional C‐terminal α‐helix which influences ligand binding through an allosteric mechanism . In other cases, PDZ supramodules are formed by neighboring domains in either a homotypical or heterotypical fashion (Figure S2, Supporting Information). In the homotypical PDZ1‐2 tandem of PSD‐95, these two domains are restricted by a short linker, positioning the two domains in an antiparallel fashion.…”

“…In the homotypical PDZ1‐2 tandem of PSD‐95, these two domains are restricted by a short linker, positioning the two domains in an antiparallel fashion. In other proteins, such as Whirlin, a longer and more flexible linker allows for greater flexibility of the tandem PDZ domain . In general, the homotypical PDZ tandem domains can facilitate either binding of multiple copies of single proteins or multiple subunits of protein complexes (Figure S2, Supporting Information), such as the PSD‐95 GluN2B interaction.…”

“…[44,45] In addition to the carboxylate-dependent binding of PDZ domains, it has more recently been demonstrated that PDZ domains can also bind internal peptide motifs [42,[46][47][48][49] and phospholipids. [65] In other cases, PDZ supramodules are formed by neighboring domains in either a homotypical [36,[66][67][68][69][70] or heterotypical fashion ( Figure S2, Supporting Information). Here, the entire β-hairpin peptide (Figure 2f), comprising two β-strands connected by a sharp β-turn, is required for www.advancedsciencenews.com www.advtherap.com binding, thereby occupying more than twice the surface area of peptides binding to PDZ domains in the canonical carboxylatedependent fashion.…”

“…[50][51][52] The interaction between the neuronal nitric oxide synthetase (nNOS) β-hairpin structure and the syntrophin PDZ domain is an example of binding of internal peptide motifs. [67] In general, the homotypical PDZ tandem domains can facilitate either binding of multiple copies of single proteins or multiple subunits of protein complexes (Figure S2, Supporting Information), such as the PSD-95 GluN2B interaction. The carboxylate binding loop of the PDZ domain plays an essential role by coordinating to the backbone carbonyl of a Phe at P 0 .…”