Intrinsic disorder and amino acid specificity modulate binding of the WW2 domain in kidney and brain protein (KIBRA) to synaptopodin

The second WW domain (WW2) of the kidney and brain scaffolding protein, KIBRA, has an isoleucine (Ile-81) rather than a second conserved tryptophan and is primarily unstructured. However, it adopts the canonical triple-stranded antiparallel β-sheet structure of WW domains when bound to a two-PPXY motif peptide of the synaptic protein Dendrin. Here, using a series of biophysical experiments, we demonstrate that the WW2 domain remains largely disordered when bound to a 69-residue two-PPXY motif polypeptide of the synaptic and podocyte protein synaptopodin (SYNPO). Isothermal titration calorimetry and CD experiments revealed that the interactions of the disordered WW2 domain with SYNPO are significantly weaker than SYNPO's interactions with the well-folded WW1 domain and that an I81W substitution in the WW2 domain neither enhances binding affinity nor induces substantial WW2 domain folding. In the tandem polypeptide, the two WW domains synergized, enhancing the overall binding affinity with the I81W variant tandem polypeptide 2-fold compared with the WT polypeptide. Solution NMR results showed that SYNPO binding induces small but definite chemical shift perturbations in the WW2 domain, confirming the disordered state of the WW2 domain in this complex. These analyses also disclosed that SYNPO binds the tandem WW domain polypeptide in an antiparallel manner, that is, the WW1 domain binds the second PPXY motif of SYNPO. We propose a binding model consisting of a bipartite interaction mode in which the largely disordered WW2 forms a “fuzzy” complex with SYNPO. This binding mode may be important for specific cellular functions.

KIBRA, also known as WWC1 (for WW and C2 domain containing), is a 125-kDa cytoplasmic scaffolding protein that is expressed mainly in the kidney and brain (1). It is a multifunc-tional protein implicated in the directional migration of podocytes (2)(3)(4), endosomal vesicle sorting (5), memory performance (6 -8), cell growth and tumor suppression (9 -11). Although these functional roles are well-documented, much less is known about how KIBRA engages partner proteins to facilitate these diverse functions.
The KIBRA polypeptide is organized into multiple protein-interacting modules. Of particular interest are two N-terminal WW domains, WW1 and WW2. The WW domain is a ϳ35 residue globular domain that folds into a twisted, three-stranded anti-parallel ␤ sheet and binds proline-rich motifs (12)(13)(14). Two conserved tryptophans (W), from which the WW name is derived, lie on opposite sides of the ␤ sheet. The first tryptophan is part of a hydrophobic core essential for domain stability, and the second tryptophan, located in a solvent-exposed region, is a hydrogen bond donor important in ligand binding (15).
Human KIBRA WW1 and WW2 domains are separated by a 14-residue segment and recognize proline-rich motifs of the type PPXY (where P is proline, X is any amino acid, and Y is tyrosine). The domains share 61% sequence identity with one major difference; the WW2 domain has an isoleucine (Ile-81) at the position normally occupied by the second conserved tryptophan in most WW domain proteins. WW domain proteins with a substitution of the second conserved tryptophan are not uncommon; and a couple of these "atypical" WW domain proteins that have tyrosine or phenylalanine substitutions show reduced or complete loss of binding to specific targets (16,17). Both human and mouse KIBRA WW2 domain display weak binding to PPXY-containing peptides (1,18). Furthermore, recent extensive work by Zhang and co-workers (18) show that the mouse KIBRA WW2 domain is unstructured but adopts the canonical WW domain structure when bound to two of the three PPXY sites of the synaptic protein Dendrin. The KIBRA WW domains bind the two Dendrin PPXY sites, which are separated by two residues, with an unusually high nanomolar affinity. Given that the dissociation constants of WW domain-PPXY interactions are generally in the low micromolar range (17,19), it is plausible that this high affinity binding induces folding of the WW2 domain.
Synaptopodin (SYNPO) is a 929-residue proline-rich actin cytoskeleton-associated protein, found in kidney podocytes and the dendrites of neurons (1,(20)(21)(22). In kidney podocytes, it is essential for the integrity of the podocyte actin cytoskeleton (23,24) and forms a WW domain-PPXY-mediated complex with KIBRA implicated in cell migration (4). In dendrites, it plays an important role in regulating spine dynamics and synaptic plasticity (25). SYNPO has two PPXY motifs, PPTY (residues 562-565) and PPSY (residues 581-584) separated by 15 amino acids and located in a region with striking sequence similarity to the region harboring the Dendrin PPXY motifs (4). Thus SYNPO is a good candidate to provide additional insight into the structural heterogeneity of the KIBRA WW2 domain.
In this work, we characterize the interaction between a 69-residue recombinant SYNPO polypeptide and variants of the human KIBRA WW domain polypeptide by a series of biophysical techniques. We show that recombinant human KIBRA WW2 domain is primarily disordered, binds SYNPO with relatively weak affinity and remains largely disordered in the complex. The two proteins interact in an antiparallel manner with the well-folded WW1 domain making substantial contacts with the SYNPO polypeptide, whereas the WW2 domain forms a "fuzzy" complex. This structural heterogeneity of the WW2 domain is discussed in the context of specific functions of the KIBRA WW domains.

SYNPO 545-613 is primarily unstructured
For this study, we used a 69-residue SYNPO polypeptide (SYNPO 545-613 ) that includes both PPXY motifs, located at residues 562-565 and 581-584, and ϳ28 flanking residues. The primary sequence of the SYNPO 545-613 polypeptide was analyzed with PONDR, a computational tool for predicting unstructured regions in proteins (26,27). The PONDR-generated disorder profile of SYNPO 545-613 ( Fig. 1) shows values greater than 0.5 indicative of a largely disordered polypeptide. To determine whether SYNPO 545-613 is disordered as predicted, we used far UV CD and NMR spectroscopy to investigate the solution structure of the polypeptide. A strong negative signal at 200 nm in the far UV CD spectrum (Fig. 1B) and limited dispersion in the amide proton region of the 1 H-15 N HSQC 2 spectrum (Fig. 1C) are consistent with a primarily disordered protein. To determine local structural propensities, backbone residue assignments were obtained for 57 of the 59 (Ser-596 and His-597 were not visible in the spectrum) nonproline residues. A plot of the deviations of the CA and CB chemical shift values (⌬C␣-⌬C␤) from standard random coil values (28) in Fig. 1D, shows no strong propensity for this segment of SYNPO to form ordered structure. These results agree with the significant disorder reported for other members of the SYNPO family (29).

Design of KIBRA constructs
Earlier studies assigned the KIBRA WW domain boundaries to residues 7-39 and 54 -86, and identified P37A and P84A as mutations in the WW1 and WW2 domains, respectively, which abrogate binding to PPXY motifs (1). Guided by these findings and sequence-based secondary structure prediction, we designed the KWW TD , KWW 1 , and KWW 2 constructs to include both, the first and the second WW domains, respectively, the P37A TD and P84A TD to probe binding to each WW domain in the context of the tandem WW domain construct, and the I81W mutants, I81W TD and KWW 2 -I81W, to restore the binding site tryptophan in the tandem and isolated WW domain constructs, respectively. A schematic of all the KIBRA constructs is shown in Fig. 2A.

The KIBRA WW domains adopt different secondary structures
PSIPRED (30), a structure prediction algorithm that gives estimates of secondary structure based on a protein's primary sequence, predicts ␤ strands for residues 22-26 and 32-35 in the WW1 domain, 70 -72 in the WW2 domain (designated by arrows in Fig. 2A), and disorder for the rest of the KWW TD sequence. The CD spectrum of KWW 1 shows a positive peak at 230 nm and a negative peak at 220 nm (black spectrum in Fig.  2B), which are signature WW domain peaks attributed to packing of the two tryptophans and ␤ strands, respectively. Thus, the CD spectrum of KWW 1 is consistent with a well-folded WW domain protein. In contrast, the spectra of KWW 2 and KWW 2 -I81W lack the positive peak and show strong negative signals at 200 nm indicative of predominantly unstructured proteins (orange and brown spectra in Fig. 2B).
CD spectra for the tandem WW domain constructs, KWW TD , I81W TD , P84A TD , and P37A TD are shown in Fig. 2C. KWW TD , I81W TD , and P84A TD all show positive signals at 230 nm, albeit more reduced than the corresponding signal in the spectrum of the isolated WW1 domain in Fig. 2A, and negative signals at 220 and 200 nm. The similarities in the CD spectra suggest that the P84A and I81W point mutations do not significantly alter the secondary structure of the tandem WW domain polypeptide. The spectrum of the KWW TD polypeptide is qualitatively identical to the sum of the spectra for each WW domain (data not shown), which strongly implies that the domains retain their folded and unfolded structures in the tandem construct. In contrast, the CD spectrum of P37A TD (black spectrum in Fig. 2C) does not show a positive peak at 230 nm but only the negative signals at 220 and 200 nm, which clearly demonstrates that this polypeptide is predominantly unfolded. To summarize, the CD data demonstrate that the WW1 domain is folded, the WW2 domain is unfolded, the I81W and P84A mutations in the WW2 domain do not result in substantial folding of the WW2 domain, and a P37A mutation in the WW1 domain is enough to disrupt the folded structure.

KIBRA-synaptopodin interaction
Trp indole NH signals rather than the four expected are observed in the indole region ( 1 H 10.2 ppm; 15 N 130 ppm) of the spectrum. Multiple peaks for the same NH are a strong indicator of multiple conformers in slow exchange. Peaks for the more folded KWW 1 domain, example Gly-11 at 8.8 ppm and Arg-40 at 7.4 ppm, are more dispersed, whereas most peaks in the linker segment and the KWW 2 domain are clustered in the middle of the spectrum.
The 1 H-15 N HSQC spectrum of P84A TD is shown in Fig. 3B. The spectrum is less heterogeneous than the KWW TD but is still missing the same peaks that were not visible in the spec-trum of the WT protein. Several peaks (labeled in red, Fig. 3B) are significantly more intense than equivalent peaks in the WT spectrum. Peaks for residues Asp-49, Cys-50, Ser-52, Glu-54, Glu-82, and Arg-89 are denoted by asterisks. These peaks display multiple conformations in the WT spectrum but coalesce into single peaks in the P84A TD spectrum. The more homogeneous P84A TD spectrum may be due to reduced cis-trans isomerization. The 1 H-15 N HSQC spectrum of I81W TD was comparable with the KWW TD spectrum (see Fig. 6). However, the 1 H-15 N HSQC spectrum of P37A TD was less dispersed in the amide proton region, indicative of unfolding or aggregation

KIBRA-synaptopodin interaction
of the polypeptide and consistent with the CD data (data not shown).

Backbone dynamics of the KIBRA tandem WW domain polypeptides
To gain insight into backbone dynamics of KWW TD , P84A TD , and I81W TD , we acquired steady-state heteronuclear NOE (HetNOE) data, which are sensitive to conformational dynamics in the picosecond to nanosecond time scale (31,32).

Binding studies monitored by ITC
Binding energetics for SYNPO 545-613 -WW domain complexes were determined from isothermal titration calorimetry (ITC) experiments. Representative binding isotherms are shown in Fig. S1 and thermodynamic parameters are summarized in Table 1. All the interactions are enthalpically driven. Binding affinities are reported as dissociation constants (K d ), and range between 1.7 and 27 M ( Table 1). Binding of SYNPO 545-613 to KWW TD is 2-fold weaker than I81W TD (K d values of 2.2 and 0.9 M, respectively) and ϳ1.7-fold higher than interactions with P84A TD (compare 2.2 to 3.7 M). The slight increase in the binding affinity of I81W TD relative to the WT KWW TD polypeptide suggests that the isoleucine (Ile-81) substitution in the WW2 domain reduces its affinity for SYNPO 545-613 . A stoichiometry of 1 is recorded for all interactions with the tandem WW domain polypeptides. As SYNPO 545-613 is not expected to bind mutant domains in P37A TD and P84A TD a stoichiometry of 1 is interpreted as binding of a single WW domain to a single PPXY motif (1:1). For KWW TD and I81W TD , simultaneous interactions with both WW domains (2:2) could also result in a stoichiometry of 1.
Thermodynamic parameters for the interaction of SYNPO 545-613 with the isolated KWW 1 , KWW 2 , and the WW2 domain mutant, KWW 2 -I81W are shown in Table 1. The K d for the KWW 1 domain-SYNPO 545-613 interaction is (5.2 M) ϳ1.5-2-fold weaker than the P84A TD mutant (binding to the WW2 domain is abolished) or the KWW TD construct. This suggests that residues in the WW1 domain alone are not sufficient for optimum stability of the complex. Isotherms for the SYNPO 545-613 -KWW 2 (Fig. S1F) and SYNPO 545-613 -KWW 2 -I81W (Fig. S1G) show low heat changes upon binding. The small magnitude of the heat changes make it difficult to fit the data and derive accurate thermodynamic parameters. Fixing the stoichiometry N, in the data analysis process to 1 (assuming binding to a single PPXY motif), rather than allowing it to be optimized as an additional fitting parameter, gave a minimum K d value of 60 M.

Solution NMR studies to map the KIBRA-SYNPO binding interface
To map the binding interface of the SYNPO-KIBRA complex, we monitored chemical shift changes in the 1 H-15 N HSQC spectrum of SYNPO 545-613 , KWW TD , or mutant tandem polypeptides without or with the unlabeled binding partner. A change is defined as a shift and/or a decrease in peak intensity. Data for 15 N-labeled SYNPO 545-613 with unlabeled KWW TD added at molar ratios of 1:0.5 and 1:1 (SYNPO:KWW TD ) are shown in Fig. 4. A plot of the normalized peak intensities of the 1:0.5 complex (Fig. 4A, gray plot) show a 43% decrease in peak intensities for residues 564 -570 and 578 -591. Increasing the molar ratio to 1:1 (orange plot in Fig. 4A and red spectrum in Fig.  4B) show complete loss of peaks corresponding to residues 558 -571 and 575-588. Missing resonances are attributed to exchange broadening of peaks at the binding interface, as well as to slower tumbling of the complex. N-terminal residues 545-556, inter-PPXY motif linker segment residues 572-574 and C-terminal residues 592-613 are not perturbed (labeled peaks in Fig. 4B). A 1:2 complex did not show additional changes in chemical shift and intensities (data not shown) indicating that binding is saturated in the 1:1 complex. These observations are consistent with binding of both WW domains to both PPXY motifs.

KIBRA-synaptopodin interaction
Normalized peak intensities for the 1:1 (gray area plot) and 1:2 (blue area plot) complexes are shown in Fig. 4C. The 1:1 complex shows complete disappearance of peaks corresponding to residues 579 -588, which include the second PPXY motif, and a few peaks corresponding to residues (564 -566) in the vicinity of the first PPXY motif. More peaks in the 560 -571 segment disappear when a higher concentration of unlabeled KWW 1 is added (blue plot). The pattern of peak disappearance clearly shows that KWW 1 first binds the second (PPSY) motif before it binds the first motif. In a reciprocal NMR titration experiment, we monitored chemical shift changes in the 1 H-15 N HSQC spectrum of KWW TD or I81W TD when bound to saturating concentrations of unlabeled SYNPO 545-613 . Spectra of unbound (black) and SYNPO 545-613 -bound KWW TD (red) or I81W TD (green) at molar ratios of 1:1 are shown in Fig. 5, A and B. Peaks that are significantly attenuated or shifted in the SYNPO 545-613 -bound spectra are labeled. We reasoned that attenuated peaks are directly or indirectly involved in binding, and attribute the broadened resonances to exchange between unbound and bound conformations as well as to slower tumbling of the complex. Increasing the concentration of added SYNPO 545-613 to a molar ratio of 1:2 did not result in additional changes in chemical shift or intensities (Fig. S2), a clear indication that binding is saturated in the 1:1 complex. Fewer WW2 domain peaks (labeled in magenta) are attenuated in the SYNPO 545-613bound KWW TD compared with the SYNPO 545-613 -bound I81W TD . This is consistent with the higher affinity of SYNPO 545-613 for the I81W mutant. Fig. 5C show plots of the chemical shift changes in the SYNPO-bound WW2 domain of KWW TD (red) and I81W TD (green). Small but distinct changes in chemical shift are observed for most residues except Thr-79 in the SYNPO-bound I81W TD spectrum, which is significantly shifted. To summarize, SYNPO 545-613 binding is accompanied by changes in chemical shift for select residues in both WW domains. This is clear indication that SYNPO binds both WW domains. The lack of chemical shift dispersion in the SYNPObound spectra of KWW TD and I81W TD is indication that the WW2 domain remains globally disordered in the SYNPObound complex.

Binding-induced folding of the KIBRA WW2 domain
Zhang and co-workers (18) reported that the mouse KIBRA WW2 domain adopts the canonical WW domain structure when bound to a two-PPXY motif-containing peptide of the neural and renal protein, Dendrin. The two PPXY sites in Dendrin are separated by only two residues, whereas the SYNPO 545-613 PPXY sites are separated by a 15-residue linker. Thus we asked the question whether a SYNPO polypeptide with a two-residue linker would induce folding of the WW2 domain. To address this question, we engineered the SYNPO variant SYNPO-2A. The two PPXY sites in this variant are separated by two non-native alanine residues (Fig. 6). The ITCmeasured dissociation constant of the SYNPO-2A-KWW TD interaction was 0.1 M (Fig. 6B), which represents a 20fold increase in binding affinity compared with the WT SYNPO 545-613 (K d of 2.2 M in Table 1). Next, we monitored  15 N-labeled SYNPO 545-613 at a 1:1 molar ratio. Peaks visible in the KIBRA-bound spectrum are labeled. C, plots of the relative intensity versus residue numbers of KWW 1bound SYNPO 545-613 at molar ratios of 1:1 (gray) and 1:2 (blue). Binding is accompanied by complete loss of peaks in the vicinity of the PPXY motifs. Peak intensities are relative to the intensity of the same peak in unbound SYNPO 545-613 , which is taken as one.

KIBRA-synaptopodin interaction
binding between 15 N-labeled KWW TD and SYNPO-2A by NMR spectroscopy. The 1 H-15 N HSQC spectrum of SYNPO-2A-bound KWW TD is shown in Fig. 6C. The spectrum is welldispersed, a clear indication that both WW domains are wellfolded in the complex.

Discussion
Despite decades of well-documented studies on the functional roles of the KIBRA WW domains, how the domains, particularly the WW2 domain, bind partner PPXY proteins is unclear. A recent structural study of a mouse KIBRA tandem WW domain polypeptide bound to a peptide of the synaptic protein Dendrin, shows that the isolated WW2 domain is unfolded but adopts a well-folded structure in the complex (18). In contrast, the biophysical studies reported here clearly demonstrate that the WW2 domain remains largely disordered when bound to the actin-binding synaptic and podocyte protein Synaptopodin. The different folds of the WW2 domain in these independent studies clearly demonstrate that different PPXY partners induce varying degrees of folding of the WW2 domain. The extent of folding may be important for the diverse functions of KIBRA.

The isolated KIBRA WW2 domain binds PPXY peptides with weak affinity
In this study and previously reported studies (1,18), the isolated WW2 domain displays weak binding to PPXY-containing peptides. Two unusual features of the KIBRA WW2 domain explain why it has a weak affinity for PPXY containing peptides: intrinsic disorder and the Ile-81 substitution of the conserved tryptophan. Disorder coupled to weak binding is inferred from observations that when the relatively well-folded WW1 domain is unfolded by introducing the P37A mutation, its affinity for the SYNPO polypeptide is also considerably weakened (Table  1). Pro-37 is a conserved WW domain residue and part of a hydrophobic core formed by the side chains of Trp-12 and Tyr-24. The hydrophobic core stabilizes the anti-parallel ␤ sheet of the KIBRA WW1 domain (18), therefore it is not surprising that disrupting this core unfolds the WW1 domain. A mutation of the conserved proline in the WW domains of Yes-associated protein (YAP) and Formin-binding protein, FBP28, also unfold the domains (33,34). Unexpectedly, alanine substitution in the equivalent position in the WW2 domain, Pro-84, leads to a substantial reduction in internal motions of the WW2 domain. This observation is intriguing because a stable hydrophobic core comprising Trp-59, Phe-71, and Pro-84 (equivalent to Trp-12, Tyr-24, and Pro-37 in the WW1 domain) is not expected to form in the primarily disordered WW2 domain. It is possible that Pro-84 is involved in cis-trans isomerization, which is abolished by the alanine mutation.
The second underlying cause of the weak affinity for PPXY peptides is the Ile-81 substitution in the WW2 domain. We find that as reported for the WW domains of Smad ubiquitination regulatory factor 2 and WW domain-containing oxidoreductase (16,17), the loss of the binding site tryptophan reduces the affinity of the tandem WW domain polypeptide for SYNPO 545-613 . Isoleucine or leucine is the preferred amino acid in this position for KIBRA isoforms from Drosophila to humans, which suggests that this substitution is optimized for the functions of the KIBRA WW domains.

Similarities and differences between the KIBRA-SYNPO and KIBRA-Dendrin complexes
The solution studies reported here provide valuable molecular level insights into how the KIBRA WW domains, particularly, the WW2 domain, bind SYNPO. The pattern of peak disappearance in the NMR titration experiments (Fig. 4) strongly suggests that the second PPXY motif binds the N-terminal WW1 domain in the same anti-parallel orientation previously reported for the Dendrin polypeptide (18). The PPXY polypeptides may bind in a similar anti-parallel manner, but clearly there are distinct differences in how they influence the structure of the WW2 domain. The 1 H-15 N HSQC spectrum of the Dendrin-bound mouse KIBRA tandem WW domains shows significant peak dispersion consistent with global folding of the WW2 domain (18). In contrast, the limited dispersion in the 1 H-15 N HSQC spectrum of the 1:1 (Fig. 5) and 1:2 ( Fig. S2) KIBRA-SYNPO complexes strongly argues against global folding of the WW2 domain. We do not rule out the possibility that in this dynamic complex, a population of the WW2 domain becomes folded upon binding to SYNPO 545-613 . However, this population is minimal compared with the population that remains disordered in the complex. Thus, whereas binding of Dendrin to the tandem WW domain of mouse KIBRA induces substantial folding of the WW2 domain, the data presented here clearly show that binding of SYNPO 545-613 to the tandem WW domains of human KIBRA does not induce substantial folding of the WW2 domain.
A second major difference between the two binding partners is that whereas the dissociation constant of the SYNPO 545-613 -KIBRA complex is in the low micromolar range, a typical range for most WW domain-PPXY interactions (17,35,36), the interaction with Dendrin occurs with an unusually high nanomolar dissociation constant. The two PPXY sites in the Dendrin peptide are separated by only two amino acids; and increasing the number of amino acids between the two PPXY sites substantially reduced the binding affinity for the KIBRA WW domains (18). On the contrary, the two PPXY sites in the SYNPO 545-613 polypeptide are separated by 15 residues. We demonstrate that a mutant SYNPO 545-613 polypeptide, SYNPO-2A, with a two-residue linker binds the KWW TD polypeptide with a 20-fold higher affinity compared with the WT SYNPO 545-613 polypeptide. Furthermore, initial NMR studies show that binding of SYNPO-2A to the KIBRA tandem WW domains induces substantial chemical shift changes in the 1 H-15 N HSQC spectrum (Fig. 6), similar to the KIBRA-Dendrin interaction (18). Clearly, whereas the WW2 domain remains largely disordered when bound to WT SYNPO, reducing the SYNPO PPXY linker to two residues induces substantial folding of the domain. The bound WW2 domain is best described as a fuzzy complex, a term used to describe IDP complexes that defy the classic stable and wellfolded binding interface (37)(38)(39) and retain conformational heterogeneity in the bound state. "Fuzziness" is reduced when the SYNPO PPXY sites are separated by two residues (panel 4). We speculate that partners in which the PPXY sites are separated by two residues will induce substantial folding of the WW2 domain, whereas partners with a longer linker will not. The structural variability of the WW2 domain may be critical for the diverse cellular functions of KIBRA.

Cloning of constructs
All constructs were prepared using the PCR-based Gibson assembly cloning protocol (New England Biolabs). Briefly, a 69-residue Synaptopodin (Uniprot ID Q8N3V7) construct (hereafter referred to as SYNPO 545-613 ), and KIBRA (Uniprot ID Q8IX03) WW domain constructs KWW TD (residues 1-91), KWW 1 (residues 1-48), and KWW 2 (residues 45-91) were generated by PCR. We also generated a SYNPO 545-613 variant, SYNPO-2A, with a two-alanine residue linker between the two PPXY sites. PCR products were cloned into a modified pET24 TM (Novagen) expression vector engineered with a tobacco etch virus protease recognition sequence to allow cleavage of the N-terminal poly-histidine tag. A Q5 TM PCRbased mutagenesis protocol (New England Biolabs) was used to generate single site mutations of the tandem WW domain construct, P37A TD and P84A TD , which abrogate binding of PPXY motifs to the WW1 and WW2 domains, respectively (1), and I81W (I81W TD ) and the isolated WW2 domain (KWW 2 -I81W). Primers for the PCR and mutagenesis reactions were purchased from Eurofins Genomics.

Recombinant protein production
Recombinant plasmids were transformed into Escherichia coli BL21 (DE3) cells. Cells were cultured at 37°C in Lysogeny broth, or 15 N-enriched MJ9 media supplemented with [ 12 C]-or [ 13 C]glucose to mid-log phase (optical density of 0.6 -0.7). The temperature was reduced to 20°C, and 0.1 mM isopropyl 1-thio-D-galactopyranoside was added to the culture to induce protein expression. Cultured cells were harvested after 12 h, lysed by sonication, and centrifuged to remove cell debris. The soluble His 6 -tagged proteins were first purified on a nickel-nitrilotriacetic acid affinity (Qiagen) column, followed by treatment with tobacco etch virus protease to remove the His 6 tag, and finally purified on a Superdex-75 (GE Life Sciences) size exclusion chromatography column. Purity of recombinant proteins were assessed by SDS-PAGE. Protein concentrations were determined from the absorbance at 280 nm and computed molar extinction coefficients (http://web.expasy. org/protparam).

CD spectroscopy
Far UV CD data were recorded at 25°C, on a JASCO 720 spectropolarimeter using a bandwidth of 1.0 nm and a path length of 1 mm. The experiments were collected on two independent samples prepared from two different protein preparations. Protein samples at concentrations of 10 -20 M were in buffer composed of 10 mM sodium phosphate, pH 6.8 or 8.0. Three scans were recorded on each sample. Reported data are the average of the experimental repeats.   (KWW TD, KWW 1 , KWW 2 ,  P37A TD , P84A TD , KWW 2 -I81W and I81W TD ) in buffer composed of 50 mM sodium phosphate, 50 mM NaCl, 5 mM ␤-mercaptoethanol, 1 mM sodium azide, pH 8, was titrated into 12-24 M SYNPO 545-613 in the same buffer. ITC data were analyzed with the Origin 7.0 software provided with the instrument. Data were fitted to a one-site model. Reported data are the average of two or three independent experiments performed under similar conditions using proteins from three different preparations. Three replicate titrations were carried out for each experiment. Standard deviations are computed from the average of the two experiments.
Titration data were collected on 15 N-labeled SYNPO 545-613 or 15 N-labeled KIBRA constructs bound to the unlabeled partner. Unlabeled proteins were added at molar ratios of 1:0.5, 1:1, and 1:2 (labeled:unlabeled protein). The peak corresponding to the last residue (91 in isotopically labeled KWW TD /I81W TD and 613 in isotopically labeled SYNPO 545-613 ) was used as an internal reference to correct for small changes in peak intensities across spectra. The relative intensity of each peak (measured as peak heights) was calculated as the ratio of the intensity of the peak in the spectrum of the bound protein to the intensity of the same peak in the spectrum of the unbound protein.
Steady-state 1 H-15 N heteronuclear NOEs data were collected as two two-dimensional interleaved spectra with and without proton saturation and a recycle delay of 8 s (43). NOE values were determined from the ratio of the peak intensities in the presence and absence of proton saturation. Errors associated with the data ( noe ) were estimated from the root mean square variation of the noise using Equation 1 (32). noe /NOE ϭ ͕͑ sat /I sat ͒ 2 ϩ ͑ unsat /I unsast ͒ 2 ͖ 1/ 2 (Eq. 1) Where sat and unsat are the standard deviation values of peak intensities with and without saturation, respectively. HetNOE experiments for KWW TD were collected on two independent samples prepared from two different protein preparations.

Secondary structure propensity from NMR chemical shifts
Random coil values for NMR-based chemical shift propensities were compiled from the web-based algorithm of Poulsen et al. (44 -46). Local structural propensities were calculated from the deviations of the experimental C␣ and C␤ chemical shifts from expected random coil values (28). A negative difference between the C␣ and the C␤ secondary chemical shifts (⌬C␣-⌬C␤) is suggestive of ␤ strand propensity; and a positive difference is attributed to helical propensity.

Chemical shift changes
Chemical shift for assigned residues in the C terminus (residue 45-91) of KWW TD and I81W TD were computed from the 1:1 titration point. The combined 1 H and 15 N chemical shift changes were calculated as follows, ⌬ ppm ϭ ͱ͑⌬␦HN͒ 2 ϩ ͑⌬␦N ϫ 0.17͒ 2 (Eq. 2) where HN and N are the 1 H and 15 N chemical shifts.

BMRB accession code
The chemical shifts for KIBRA KWW TD have been deposited in the Biological Magnetic Resonance Data Bank under accession code 27930.