Data describing the solution structure of the WW3* domain from human Nedd4-1

The third WW domain (WW3*) of human Nedd4-1 (Neuronal precursor cell expressed developmentally down-regulated gene 4-1) interacts with the poly-proline (PY) motifs of the human epithelial Na+ channel (hENaC) subunits at micromolar affinity. This data supplements the article (Panwalkar et al., 2015) [1]. We describe the NMR experiments used to solve the solution structure of the WW3* domain. We also present NOE network data for defining the rotameric state of side chains of peptide binding residues, and complement this data with χ1 dihedral angles derived from 3J couplings and molecular dynamics simulations data.


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The third WW domain (WW3*) of human Nedd4-1 (Neuronal precursor cell expressed developmentally down-regulated gene 4-1) interacts with the poly-proline (PY) motifs of the human epithelial Na þ channel (hENaC) subunits at micromolar affinity. This data supplements the article (Panwalkar et al., 2015) [1]. We describe the NMR experiments used to solve the solution structure of the WW3* domain. We also present NOE network data for defining the rotameric state of side chains of peptide binding residues, and complement this data with χ 1 dihedral angles derived from 3

Value of the data
The NOE network defines clearly the side chain orientations of particular ligand-binding residues; MD simulations provide atomistic descriptions of conformational fluctuations within the WW3 * domain that are not observed in the NMR-derived structure of the domain; This data set serves as a reference for future studies involving WW domains.

Data
We have collected 1592 NOE distance restraints from three-dimensional 15 N-edited and 13 C-edited NOESY spectra, which were processed using NMRPipe [2] and analyzed using CcpNMR Analysis [3]. The NOE dataset consists of 390 sequential, 416 intra-residue, 266 medium-range and 256 long-range NOE distance restraints. In addition, 60 dihedral angle restraints and five sidechain χ 1 angle restraints determined from combined 3 J αβ and 3 J Nβ couplings were used for structure calculation. The NOEs were picked manually and assigned in a semi-automated manner using the Aria 2.3.1 [4] software package. The structure calculation was carried out by a combination of Aria 2.3.1 and CNS version 1.21 [5] using the PARALLHDG force field. The protocol employed by Aria for calculation of the solution structure of the WW3 * domain is provided as supplementary material. The experiments performed to acquire chemical shift assignments, 3 J couplings and NOE distance restraints are summarized in Table 1. The 3 J couplings and the subsequently determined rotameric state for the WW3* domain are given in Table 2.
We provide, as examples, the NOE networks for two key peptide binding residues I440 and T447 (Figs. 1 and 2), side chain rotamers of which differ between NMR and the crystal structures [6]. MD simulations data of χ 1 rotameric states of six key peptide binding residues (R430, F438, I440, H442, T447 and W449) over 100 ns in the apo and hENaC peptide bound state of the WW3 * domain is provided (Fig. 3).    Fig. 1C). The δ1 methyl protons of I440 do not show NOEs to E428 and R430 but show NOEs to the amide proton and the α proton of H442 (black dashed lines in Fig. 1C). This NOE pattern defines the side chain conformation of I440.

NMR spectroscopy
Standard heteronuclear multidimensional NMR experiments [9] were performed on samples containing 1.5-1.8 mM WW3 * domain ( 13 C, 15 N-labeled) from human Nedd4-1 in 20 mM sodium phosphate buffer (pH 6.5), 50 mM NaCl, 0.1% (w/v) NaN 3 and 1 mM DSS in a 93%/7% (v/v) H 2 O/D 2 O Fig. 2. Strips from a 13 C-edited NOESY spectrum for the β proton (A) and the γ2 methyl protons (B) of the residue T337 of the WW3* domain are shown. The NOE network that gives rise to a gaucheþ rotamer is mapped onto the structure (C). This NOE pattern defines the side chain conformation of T447. mixture. NMR spectra were recorded at 25°C on NMR spectrometers equipped with cryogenically cooled z-gradient probes operating at 1 H frequencies of 600 and 900 MHz. 1 H, 15 N and 13 C chemical shift assignments of the WW3* domain were obtained using experiments in Table 1. An example of a backbone sequential walk using three-dimensional (3D) HNCA and CBCA(CO)NH spectra between residues F438 and H442 is presented in Fig. 4. Near complete backbone (193/200 or 96.5%) and side chain assignments (302/319 or 94.5%) were obtained. To derive NOE distance restraints for structure calculation, 15 N-edited and 13 C-edited NOESY spectra were recorded using mixing times between 150 and 180 ms. Backbone dihedral angles were obtained from TALOS þ [10] using a combination of backbone ( 1 H N , 1 H α , 13 C α , 13 C' and 15 N) and 13 C β chemical shifts. Sidechain χ 1 dihedral angles were obtained from a combination of 3 J αβ and 3 J Nβ couplings derived from 3D HNHB [11] and 3D HAHB (CACO)NH [12] experiments (Table 2).

MD simulations
MD simulations were performed using parameters described in [1].