Differential Binding Affinities and Allosteric Conformational Changes

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Differential binding affinities and allosteric conformational changes underlie interactions of Yorkie and a multivalent PPxY partner Afua Nyarko Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b00973 • Publication Date (Web): 19 Dec 2017 Downloaded from http://pubs.acs.org on December 20, 2017

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Biochemistry

Differential binding affinities and allosteric conformational changes underlie interactions of Yorkie and a multivalent PPxY partner

Afua Nyarko Department of Biochemistry and Biophysics, Oregon State University, Corvallis, OR 97331

KEYWORDS: WW domain, PPxY motif, intrinsically disordered protein, multivalent.

ACS Paragon Plus Environment

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ABSTRACT

Tondu domain growth inhibitor (Tgi) is among a growing number of multivalent PPxY proteins that regulate cell growth via interactions with the tandem WW domains of the transcription coactivator protein, Yorkie (Yki). These proteins are attractive candidates for targeted drug design, but the substantial amount of disorder predicted from their primary sequences make structural studies that are foundational to drug design challenging. We have successfully overexpressed full length recombinant Tgi and Yki, experimentally confirmed that intrinsic structural disorder is common to both proteins, and assessed binding of the Yki WW domains to the three Tgi PPxY motifs using NMR and isothermal titration calorimetry. We find that the tandem WW domains positively cooperate to engage all three PPxY sites with a broad range of affinities. The first PPxY motif that is quite distant from the other two serves as the “binding initiation” site, and is essential for high affinity interactions. Importantly, by monitoring binding to the full length/larger protein domains we obtain more physiologically relevant affinity information, and identify “long-range” residues that could be targeted to fine-tune binding. This expansion of protein functionality through modulation of residues outside the recognition sequences offers potential alternative targets for drug design.


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Biochemistry

INTRODUCTION Yorkie (Yki) is a non-DNA binding transcriptional co-activator in Drosophila, and the highly conserved orthologue of the mammalian oncoprotein, Yes-associated protein (YAP)1. Yki promotes cell proliferation by partnering with DNA-binding transcription factors such as Scalloped (Sd), a member of the transcriptional enhancer activator family of transcriptional factors, Homothorax, a TALE-homeodomain protein, and the homeotic protein Teashirt, to regulate the synthesis of genes critical to cell growth and/or anti-apoptosis2-5. Regulating the interaction between Yki and its DNA-binding transcription factors is therefore an important first step to prevent tissue overgrowth. The hippo signaling pathway is an evolutionary conserved regulator of organ size and tissue homeostasis and a key negative regulator of Yki-promoted tissue growth. When activated, hippo signaling integrates multiple proteins to phosphorylate and retain Yki in the cytoplasm for subsequent degradation1, 6. Dysregulation of hippo signaling is linked to tissue overgrowth, and other forms of tumors (reviewed in 7). In addition to hippo signaling, a number of tumor suppressor proteins physically interact with Yki to negatively regulate its activity8-10. Since most of these tumor suppressor proteins are conserved between Drosophila and humans, insight into their regulatory mechanisms will aid in the design of drugs to fight cancer and other tumors. The D. melanogaster yki gene encodes a 395-residue WW domain protein that is predicted by sequence-based secondary structure algorithms to be primarily unstructured (Figure 1). The WW domain is a protein-protein interaction module that folds into a three-stranded antiparallel beta sheet, and recognizes proline-rich sequences11-13. Yki has two WW domains which recognize a PPxY (Pro-Pro-Xaa-Tyr) sequence14. Several Yki regulators have multiple copies of this motif15-19 (Figure 1), but it has not yet been shown how the multiple sites influence


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binding and regulation. Structural studies on large intact domains that could address these questions have been limited due to the high degree of sequence disorder predicted for segments that include the recognition motifs (Figure 1), and the difficulty of studying disordered proteins. B

A 1

395

1

2467

1

1833

1

1427

1105

1 1

1

382 337

Figure 1: Yki binding partners have multiple PPxY motifs in intrinsically disordered regions. (A) Yki partner proteins Ncoa6, a receptor coactivator protein; Myopic, the Drosophila homolog of His-domain protein tyrosine phosphatase; Expanded, a FERM domain protein; Warts, a Dbf2-related kinase; Tondu domain-containing growth inhibitor (Tgi), a transcription repressor; and WW domain binding protein 2 (Wbp2), a transcription activator have multiple binding motifs (orange bars) in primarily unstructured regions (gray bars). Structure analysis of Yki is shown at the top for comparison. Predicted secondary structure (helices and sheets) is shown above the diagram for each protein. (B) Constructs used in this work were as follows: full length Yki, the tandem WW domains (WWTD), individual WW domains (WW1 or WW2), full length Tgi with three PPxY sites (orange ovals) designated P1, P2, and P3, Tgi variants Y91A, Y276A, and Y286A, each with two PPxY motifs, an N-terminal construct, Tgi1-97 with one PPxY motif, and a C-terminal construct Tgi258-382, with two PPxY motifs.

Tondu domain-containing growth inhibitor (Tgi), is a transcription co-repressor which was first identified in a gain-of-function screen for Drosophila genes whose overexpression resulted in decreased eye and wing sizes20. It was subsequently shown to compete with Yki for binding to Sd, while simultaneously forming a complex with Yki to retain it in the nucleus20 21. Tgi is a 382residue protein with three PPxY motifs, with the first (P1), separated from the second (P2) and


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Biochemistry

third (P3) motifs by a long stretch of residues with high predicted disorder (Figure 1). There are conflicting reports on how Yki associates with Tgi in cultured cells. One study reports that binding is mediated by P1 only20, while another study shows binding to all three PPxY sites21, raising the question of how all three PPxY motifs interact simultaneously with the two Yki WW domains. To better understand how Yki associates with the three PPxY sites of Tgi, here we provide the first characterization of full length Yki and Tgi, and study the interactions between them using a series of truncated and site-directed mutants. We use isothermal titration calorimetry (ITC) to identify interacting domains, and solution NMR to identify Tgi residues that mediate direct binding with Yki, as well as non-binding residues that could influence complex formation.

MATERIALS AND METHODS Cloning of constructs –The cDNA of Drosophila Yki (Uniprot ID: Q45VV3) was used as the template in PCR reactions to generate full length Yki, the tandem WW domains (WWTD, residues 232 – 352), the first WW domain, (WW1, residues 232 – 306), and the second WW domain (WW2, residues 303 – 352) constructs. PCR products were cloned into Nde1/Xho1 sites of a pET15bTM (Novagen) expression vector using the Gibson assemblyTM cloning protocol (New England Biolabs, MA). Full length Tgi, and two smaller constructs spanning N-terminal residues 1 – 97 (Tgi1-97) and C-terminal residues 258 – 382 (Tgi258-382) were generated from the cDNA of Tgi isoform B (Uniprot ID: Q8IQJ9A). The cDNA was obtained from the Drosophila Genomics Resource Center, (https://dgrc.bio.indiana.edu). Mutagenesis reactions – The PCR-based Q5-mutagenesis protocol (New England Biolabs, MA) was used to generate three Tgi variants Y91A, Y276A, and Y286A. This single tyrosine – alanine substitution “knocks out” a single PPxY site, and abolishes binding of the WW domain to


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the mutant site. Primers for the mutagenesis reaction were purchased from Eurofins Genomics (https://www.eurofinsgenomics.com). The sequences of cloned, and mutagenic constructs were verified by DNA sequencing after which they were transformed into E.coli BL21-DE3 cell lines for protein expression. Recombinant protein production – BL21-DE3 cells were grown at 37 oC in Luria broth, or 15

N- enriched MJ9 media supplemented with 12C or 13C glucose. The cultures were grown to an

optical density (OD600) of 0.6-0.7, at which point protein overexpression was induced with 0.1 mM IPTG (all Yki constructs) or 1 mM IPTG (all Tgi constructs). Cells were cultured for an additional 12 hours at 20 oC (Yki constructs) or 3 hours at 37 oC (Tgi constructs), after which they were harvested, lyzed by sonication, and centrifuged to remove cell debris. Recombinant His 6-tagged proteins were purified by Ni-NTA-affinity (Qiagen) chromatography, followed by size exclusion chromatography (SEC) on a Superdex75 or Superdex200 (GE life sciences) column. Protein concentrations were determined from the Beer-Lambert relationship using absorbance at 280 nm, and molar extinction coefficients obtained from http://web.expasy.org/protparam/. Peptide synthesis – Synthetic peptides that contain the three PPxY recognition motifs, QRASPPPPYREPLP (P1p), TPHHTPPRYNTPPP (P2p), and TPPPPPPAYGIAGT (P3p) were purchased from Genscript (Piscataway, NJ, USA). The concentration of each peptide was determined using the absorbance at 280 nm and an extinction coefficient of 1490 M-1 cm-1. Circular dichroism, fluorescence spectroscopy, and analytical SEC – Samples for far UV circular dichroism (CD) and fluorescence spectroscopy experiments were dialyzed against buffer composed of 10 mM sodium phosphate, 2 mM TCEP, pH 7.5, with or without 10 mM NaCl. Fluorescence data were collected on a Fluorolog 3 spectrofluorometer (Horiba Scientific Inc) using 3 µM protein samples in a 0.5 cm quartz cuvette. Emission spectra were recorded with a scan speed


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Biochemistry

of 1 nm/s, and excitation and emission slit widths of 2 nm. The excitation wavelength was set to 295 nm to selectively excite tryptophan residues. Reported data were corrected for signals from the experimental buffer. CD data were recorded on a JASCO 720 spectropolarimeter, using a pathlength of 1 mm, a bandwidth of 1.0 nm and 10 µM protein concentrations. Recorded data are the average of three scans. Analytical SEC experiments were performed at room temperature, on a Superdex200 10/300 GL (GE life sciences) pre-equilibrated with 50 mM sodium phosphate, 0.4 M NaCl, 5 mM DTT, pH 7.5. A sample volume of 100 µl with protein concentrations of 100 µM, was injected at a flow rate of 0.8 mL/min, and monitored at 280 nm. Isothermal titration calorimetry. A Microcal VP-ITC instrument (Malvern instruments Inc, MA) set to 25 oC, was used to record ITC data. 27 - 10 μl injections of 150 – 400 µM of Yki variants (WWTD, WW1 or WW2) in buffer composed of 50 mM sodium phosphate, 50 mM NaCl, 2 mM TCEP, 0.5 mM sodium azide, pH 7.5, was titrated into 15 – 30 µM of Tgi or its variants (Y91A, Y276A, Y286A, Tgi1-97, and Tgi258-382) in the same buffer. ITC data were also recorded for the titration of Yki variants with Tgi peptides P1p, P2p and P3p. The peptides were dissolved in the reaction buffer to a final concentration of 0.6 – 1 mM. ITC data were analyzed by singlesite fits of the thermograms using the Origin 7.0 software provided with the instrument. The free energy of binding (ΔG) was calculated from the relation: ΔG = −RTlnK, where K is the association constant. Unless specifically noted reported data are the average of three independent experiments, with error estimation from experimental repeats. NMR data collection and analysis. NMR experiments were performed on a Bruker Avance III, 800 MHz spectrometer (Bruker BioSpin) equipped with a triple resonance cryogenic probe. Protein concentrations were 60 – 250 μM, and data were recorded at 10 oC. TROSY-based 22 triple resonance experiments HNCACB, CBCA(CO)NH and HBHA(CO)NH , and a 2D 1H-15N HSQC


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experiment were used to assign backbone resonances. All experiments featured the BEST (band selective excitation short transient) sequences of Favier and Brutscher23, and non-uniform sampling (NUS) to minimize data collection times. Reconstruction of NUS data used the iterative shrinkage thresholding approach implemented in NMRpipe 24. All NMR spectra were processed in NMRpipe and visualized with the graphical program Sparky (Goddard T.D, and Kneller D.G, 2005, Sparky 3, University of California, San Francisco). Chemical shift differences (Δ13Cα–Δ13Cβ) were calculated by subtracting random coil chemical shifts compiled from https://spin.niddk.nih.gov/bax/nmrserver/Poulsen_rc_CS from the experimentally determined CA and CB chemical shifts. Experiments to monitor binding used 150 µM 15N-labeled Tgi1-97, or Tgi258-382, with 450 µM unlabeled WWTD, or 60 µM 15N-labeled Tgi with 15 µM or 180 µM unlabeled WWTD.

RESULTS Average structure and interactions of full length Yki and Tgi – To determine whether full length Yki and Tgi are significantly disordered as predicted from their sequences, the structures of the recombinant proteins were characterized by tryptophan fluorescence spectroscopy, and far UV CD (Figure 2). In fluorescence emission spectra (Figure 2A), the wavelength of maximum emission (λmax) of 352 nm for Tgi demonstrates the absence of tertiary packing in the vicinity of the three Tgi tryptophans (W34, W202, and W273), while a λmax of 346 nm for Yki is indicative of partial burial of one or more of the five Yki tryptophans (W247, W269, W316, W338, and W391). Since four of the five Yki tryptophans (W247, W269, W316, and W338) are within the WW domains these results suggest a relatively ordered structure for the Yki WW domains. The CD spectra of Yki, and Tgi (Figure 2B) show the characteristic strong negative signal at 203 nm


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indicative of predominant random coil-like structures. Taken together, the fluorescence and far

Intensity, A.U

1.6

[ϴ], deg x103 cm2 dmol-1

UV CD data are consistent with the predicted intrinsic disorder of Yki and Tgi. A

1.2 0.8 0.4 0 310

330

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390

0

B -4

-8

-12 190

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[ϴ], deg x103 cm2 dmol-1

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Wavelength, nm

Wavelength, nm

Intensity at 280 nm

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Biochemistry

0

D

-7

-14

-21 195

205

Time, min

215

225

235

245

Wavelength, nm

Figure 2: Solution studies of full length Yki and Tgi (A) Tryptophan fluorescence emission spectra, (B) Far UV CD spectra, and (C) Analytical SEC are shown for Yki (green), and Tgi (orange). The analytical SEC experiment also shows migration of an equimolar mixture of the two proteins (C, black plot). (D) The CD spectrum of a mixture of Yki and Tgi (black), and the sum of individual spectra of the two proteins (red). All experiments were recorded at 25 o

C, in sodium phosphate buffer, pH 7.5 with 10 mM (fluorescence) or 0.4 M (analytical SEC) NaCl.

Analytical size exclusion chromatography experiments confirm complex formation between Yki and Tgi (Figure 2C) by the presence of a single peak for a stoichiometric mixture of Yki and Tgi, which migrates faster than free Yki or Tgi. We also determined whether one or both proteins undergo significant secondary structure rearrangements upon complex formation by comparing the Far UV CD spectrum of a stoichiometric mixture of the two proteins to the sum of


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individual spectra of Yki and Tgi (Figure 2D). The similarity in the two spectra is indication that no notable change in secondary structure accompanies binding. Yki and Tgi interacting domains identified by ITC —Yki forms non-native aggregates at the relatively high concentrations required for ITC experiments, therefore for these experiments we used the better behaved tandem WW domain construct of Yki (WWTD), which corresponds to residues 232 – 352. The experiments used hexa-histidine tagged proteins to minimize nonspecific cleavage of these predominantly unstructured proteins. The hexa-histidine tag did not affect binding since recombinant proteins that had previously been treated with thrombin to remove all residues from the expression vector gave similar binding affinities as the tagged proteins (data not shown). WWTD binds Tgi with a stoichiometry of 1, and an apparent Kd of 6.5 µM (Figure 3A, table 1). Since there are two WW domains in Yki, and three PPxY sites in Tgi, a stoichiometry of 1 may be due to binding of both WW domains to two PPxY sites. To investigate which PPxY sites preferentially associate with the tandem WW domains, we monitored binding to the Tgi variants Y91A, Y276A, and Y286A. These variants were generated to “knock-out” binding to one motif at a time leaving only two sites available for binding. WWTD binds all three Tgi variants with a stoichiometry of 1, but with significantly different apparent Kd values: 15 µM for Y91A (Figure 3B), 5.8 µM for Y276A (Figure 3C), and 1.4 µM for Y286A (Figure 3D). The binding isotherm of the WWTD-Y91A interaction does not reach sufficient saturation and the midpoint of the binding curve is not well-defined. This is characteristic of weak interactions and typically result in relatively higher uncertainties in the computed thermodynamic parameters. From these results, we conclude that all the three PPxY sites can bind the tandem WW domains, but the results do not answer which interactions dominate in the WT Tgi – WWTD complex.


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As a stoichiometry of 1, may also be due to the binding of a single WW domain to a single PPxY motif, the relative affinities of each WW domain for WT Tgi were also measured. As with the WWTD – Y91A interaction, the binding isotherms for the WW1 –Tgi (Figure 3E), and WW2-Tgi (Figure 3F) interactions do not reach sufficient saturation at the concentrations used for the experiments, and the midpoints of the binding isotherms are not well defined. However, the weak binding affinity inferred from failure to reach saturation strongly implies that both WW domains are required for a relatively stable complex with Tgi. Time, mins

A µcal/sec

0

B 30

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-1

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D 0

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kcal/mol of injectant

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µcal/sec

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-2


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Biochemistry

0

1

2

-24

0.0

0.5

1.0

0

1

2

Molar ratio Figure 3: ITC binding isotherms of the Yki-Tgi interaction. Representative plots are shown for the interaction of WWTD with (A) Tgi (B) Y91A (C) Y276A, and (D) Y286A. Plots are also shown for the interaction between Tgi and (E) WW1 (F) WW2; between WWTD and (G) Tgi1-97 and (H) Tgi258-382. ITC data were recorded at 25 oC in 50 mM sodium phosphate buffer with 50 mM NaCl, 2 mM TCEP, and 0.5 mM NaN 3, pH 7.5.


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Table 1: Thermodynamics parameters for the Yki-Tgi interactions Syringe

Cell

PPxY Sites

N

Kd (μM)

∆H (kcal/mol)

T∆S (kcal/mol)

∆G (kcal/mol)

WWTD

Tgi

P1+P2+P3

1.0

6.5 ± 0.4

-45.8 ± 0.2

-38.7 ± 0.1

-7.08 ± 0.13

WWTD

Y91A

P2+P3

1.0

15 ± 0.5

-29.5 ± 0.3

-22.9 ± 0.3

-6.58 ± 0.02

WWTD

Y276A

P1+P3

1.0

5.8 ± 0.1

-10.8 ± 0.2

-3.69 ± 0.2

-7.14 ± 0.01

WWTD

Y286A

P1+P2

1.0

1.4 ± 0.1

-17.9 ± 0.6

-9.89 ± 0.6

-7.97 ± 0.04

WW1

Tgi

P1+P2+P3

1.0

20.6 ± 0.8

-16.4 ± 0.2

-10.0 ± 0.2

-6.39 ± 0.02

WW2

Tgi

P1+P2+P3

1.0

22.1± 2.8

-3.63 ± 0.5

2.73 ± 0.6

-6.36 ± 0.07

WWTD

Tgi1-97

P1

0.5

4.3 ± 0.01

-37.1 ± 0.1

-29.7 ± 0.1

-7.37 ± 0.01

WWTD

Tgi258-382

P2+P3

1.0

6.0 ± 0.1

-27.9 ± 0.1

-20.8 ± 0.1

-7.12 ± 0.01

The changes in enthalpy (ΔH°), entropy (− TΔS°) and free energy of binding (ΔG°) are shown for interactions between Yki WW domains and Tgi or its mutants. Values were determined at 25 °C from the average of three independent experiments with error estimation from experimental repeats.

ITC measurements were also used to determine the relative affinities of the WWTD construct for Tgi1-97 and Tgi258-382 which have one and two PPxY motifs, respectively. WWTD binds Tgi1-97 with a stoichiometry of 0.5, and a Kd of 4.3 μM (Figure 3G), while interactions with Tgi258-382 (Figure 3H) occur with a Kd of 6 μM, and a binding stoichiometry of 1. A stoichiometry of 0.5 (or 1:2) is interpreted as one WWTD polypeptide (with its two binding sites) binding two Tgi1-97 polypeptides. The stoichiometry of 1 for the Tgi258-382 construct is interpreted as a two-WW domain: two-PPxY site interaction. It is worth noting that the WWTD – Tgi258-382, interaction occurs with a tighter Kd than the WWTD – Y91A variant interaction, even though both constructs have the same (P2 + P3) binding sites. We return to this point later. In all these ITC experiments, binding is enthalpically driven suggesting energetically favorable intermolecular hydrogen bonds and van der Waals contacts (summarized in Table 1). Interactions with synthetic peptides monitored by ITC – To gain insight into the binding preference of specific PPxY sites, binding of the single PPxY-motif peptides P1p, P2p, and P3p to Yki variants were monitored by ITC. WWTD binds the P1p peptide with a stoichiometry of 2, and


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a Kd of 9.9 µM (Figure 4A, and Table 2). The other interactions are relatively weak, and are characterized by binding isotherms that do not reach sufficient saturation at the concentrations used for the experiments. As previously explained, this leads to high uncertainties in the computed thermodynamic parameters. The P2p – WWTD interaction (Figure 4D) and P2 – WW1/WW2 interactions (data not shown) were too weak to fit to a binding model, and are not included in the thermodynamic parameters in Table 2. Time, mins

A µcal/sec

30

60

0

90 120

30

60

90 120

0.0 -0.4 -0.8 -1.2 -1.6

-0.3

-3

-2

-6

-4

-2

-9

-6

-4

-12

-8

-6

0.0

0

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90 120

0

1

2

3

30

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1

2

0.0 -0.2 -0.4

-0.6

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5

D

E

0

0

30

60

90 120

1

2

60

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90 120

0.0

0.0

-0.3

-0.2

-0.04

-0.6

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-0.08

-0.9

-0.6

-0.12 -0.8

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F 30

µcal/sec


C

B

0


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Biochemistry

-1 -1

-1.2

-2

-2 -1.6

-3 0

1

2

-3 -4 0

1

2

3

4

5

0

3

4

Molar ratio Figure 4: ITC binding isotherms of the Yki -Tgi peptides interactions. Representative plots are shown for the interaction of Tgi peptide P1p with (A) WWTD (B) WW1 (C) WW2, between Tgi peptide P2p and (D) WWTD, and between Tgi peptide P3p and (E) WWTD (F) WW1. ITC data were recorded at 25 oC in 50 mM sodium phosphate buffer with 50 mM NaCl, 2 mM TCEP, and 0.5 mM NaN 3, pH 7.5.


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Table 2: Thermodynamics parameters for the Yki-Tgi peptides interactions Syringe

Cell

N

Kd (μM)

∆H kcal/mol)

T∆S (kcal/mol)

∆G (kcal/mol)

P1p

WWTD

2.0

9.9 ± 0.2

-16.2 ± 1.1

9.58 ± 1.4

-6.63 ± 0.2

WW1

1.0

16.1 ± 0.1

-14.4 ± 0.5

7.89 ± 0.4

-6.51± 0.04

WW2

1.0

14.9 ± 0.1

-14.9 ± 0.4

8.34 ± 0.3

-6.58± 0.03

WWTD

2.0

16.3 ± 0.3

-4.15 ± 0.4

-2.38 ± 0.5

-6.53 ± 0.02

WW1

1.0

22.9 ± 0.2

-3.18 ± 0.3

-3.13 ± 0.1

-6.31 ± 0.02

WW2

1.0

22.9 ± 0.3

-5.16 ± 0.2

-1.17 ± 0.3

-6.33 ± 0.01

P3p

Thermodynamic values were determined at 25 °C from the average of two independent experiments with error estimation from experimental repeats

NMR characterization of Tgi binding domains – 1H-15N HSQC spectra of Tgi, Tgi1-97 and Tgi258-382 are shown in Figure 5. The spectrum of Tgi (Figure 5A) displays a limited dispersion in the proton dimension, and significant resonance broadening that confirms its intrinsic disorder inferred from far UV CD and fluorescence spectra. Because of the extensive peak overlap in Tgi, we used the smaller Tgi1-97 and Tgi258-382 to aid the unambiguous assignments of backbone resonances. Any residual structure of the native protein appears not to be significantly altered in the smaller constructs since crosspeaks from the 1H-15N HSQC spectra of Tgi1-97 (Figure 5B) and Tgi258-382 (Figure 5C) overlap well with peaks from Tgi. Standard triple resonance NMR experiments on

13

C and

15

N uniformly labeled proteins

were used to obtain backbone assignments for Tgi1-97, and Tgi258-382. 1H-15N HSQC spectra with resonance assignments for 85 of the 87 non-proline residues of Tgi1-97 (peaks for R37, and R38 were not detected), and all 103 non-proline residues of Tgi258-382 are shown in Figure 5D and E, respectively. To determine local structural propensities, we used the deviations of the experimental Cα and Cβ chemical shifts from expected random coil values

25

, compiled from the web-based

algorithm of Poulsen et al 26-28. Substantial positive deviations for four or more sequential residues


14

Page 15 of 30

is indicative of α-helical propensity while negative deviations for three or more sequential residues represent extended structural elements. A plot of the deviations versus the residue numbers is shown in Figure 5F. Using a cut-off of 0.3 (the average deviation) we identify helical propensities for Tgi1-97 residues 4 – 14, 55 – 60, and 75 – 78, and Tgi258-382 residues 364 – 368, with regions between residues 20 and 35 having some indication of weak helical tendency. The remaining residues populate extended structural elements or have no apparent ordered structural preference.

B

C

(ppm)

A

15N

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

1

H (ppm)


15

Biochemistry

D

G262

G44

G51

G290

E

G379 G370

*

G378 G287

* G79

(ppm)

S20 T3 S53

15N

S75

T30 T31

S86 T77 S42

T297

T291

V293 S336 T315 T340 S344 T349 S309 S360 S317 T278 T357 T313 T355 S337 T303 S347 T292 T307 T338 S348 R263 Y286 T301H364 T299 V260 I288 H270 T372 F365 S352 H380 Q377 L369 V333 N305 N277 E371 K376 R275 L330 I351 * D267 N350 Y276 Q310 W373 K381 I321 E327 I353 V324 Q339 A259 R264 I322 A368 E358 A332 A366 I311 A295 A331 A359 A258 L298 V294 K325 L266 R323 A326 F354 E382 A285

A320 K356 T32

G97

S9 S70

N52 V7 N17/18 R66 D65 Y45 E56 V28 V76 Q83 M1 V15 K82 E2 Q40 R38 E54 E60 R39 E21 R23 E49 A12 L8 M68 E37 R10 R84 K27 M14 A80 K33 A11 Q16 L95 E93 D73 Q46 R92 A50 A24 A85 A4 R41 L29 M74 E64R57 L5 D69 D65/67 W34Nε

L376 A261

A335 H271

S26

E58/R5 Y91 9 W34

I265

M318

T13 T25

A289

A334

W373Nε

R63

L81R55 R35 M22 I72

A343

1

H (ppm)

F Predicted Observed

2 ∆Cα-∆Cβ

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 30

1 0 -1

-2

Figure 5: Resonance assignment and Secondary structure of Tgi constructs. The spectrum of (A) Tgi1-382 (black) superimposed with the spectrum of (B) Tgi1-97 (blue), and (C) Tgi 258-382 (red). 1H-15N HSQC spectra showing backbone assignments of non-proline residues in (D) Tgi1-97, and (E) Tgi258-382. (F) A plot of the chemical shift differences (∆13Cα - ∆13Cβ) as a function of the residue number. ΔCα − ΔCβ values > ± 0.3 ppm were considered significant. Sequence-based predicted, and chemical shift-determined helical propensities are shown as bars above the plots. Experiments were recorded at 10 oC on an 800 MHz Bruker Avance III spectrometer.


16

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Biochemistry

Yki binding interface on Tgi mapped by NMR spectroscopy – 1H and 15N chemical shifts of backbone amide groups are very sensitive to the local chemical environment and can be perturbed by binding of a partner and/or allosteric conformational changes 29. Perturbed residues may broaden beyond the limit of detection, and thus may not be observed in the spectrum of the bound protein. To identify residues that experience changes in their chemical environments upon binding, unlabeled WWTD were added to 15N labeled Tgi1-97, or Tgi258-382. 1H-15N HSQC spectra of unbound and WWTD - bound Tgi1-97 and Tgi258-382 are shown in Figure 6. Peaks that completely disappear from the WWTD-bound Tgi1-97 spectrum (Figure 6A, red spectrum) correspond to residues T25 – A43, and Q83 – L95 (Figure 6D). Undetected peaks in the WWTD – Tgi258-382 spectrum (Figure 6B, red spectrum) correspond to residues G262 – H271, and R275 – V293 (Figure 6D). Most residues not observed in the WWTD-bound spectrum are in the neighborhood of the recognition motifs (Q83 – L95, G262 – H271, and R275 – V293), but interestingly, some are distant from the recognition motifs (T25 – A43). We attribute the disappearance of the former to direct binding, and assign these regions as the binding segments of Yki on Tgi. Given the specificity of the WW domain - PPxY motif interaction direct binding to residues T25 – A43 is not anticipated, and we attribute the disappearance of these peaks to allosteric conformational changes. To determine the order of binding to the three PPxY sites, we assigned peaks in the 1H-15N HSQC spectrum of full length Tgi corresponding to residues S86, Y91, Y276, and Y286 that are close to or part of the recognition motifs, as well as S42 and the indole HN peak of W34 which are distant from the recognition motifs, but disappear from the spectrum of the WWTD-bound Tgi197

or Tgi258-382. We then monitored changes in peak intensities in titration experiments that used

two different concentrations of unlabeled WWTD. Figure 6C shows portions of the 1H-15N HSQC


17

Biochemistry

spectrum of unbound Tgi (black), and WWTD-bound Tgi at molar ratios (Tgi:WWTD) of 1:0.25 (red) and 1:3 (purple). Peaks corresponding to residues Y91 and S86 that are part of or close to the P1 recognition sequence completely disappear from the spectrum of the WWTD-bound protein at the lower concentration of unlabeled WWTD. The indole HN peak of W34 and S42 which are distant from the PPxY recognition sequences also disappear from the spectrum at this low concentration of added WWTD (highlighted in magenta, Figure 6D). The Y276 and Y286 residues of the P2 and P3 recognition motifs are attenuated (highlighted in cyan, Figure 6D), and completely disappear from the spectrum at a higher concentration of added unlabeled WWTD. From these results, we conclude that WWTD binds P1 before P2, and P3 A

C

B

S86

W202Nε

118.0 W34Nε 130. 0

119.0

W373Nε

N (ppm)

S42

8.35

15

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 30

8.25

10.1

Y276

Y286

121.0 120.0

122.0 Y91 8.3

8.2

8.1

8.0

1

H (ppm)

D 1 N

Tgi258-382

Tgi1-97 24

44

82

P1

96

261

P2

P3

294

382 C

……ATSKVLTTTKWRRERRQRSAG…KQRASPPPPYREPLP…AGRRILDTPHHTPPRYNTPPPPPPAYGIAGTTVV…… ……ATSKVLTTTKWRRERRQRSAG…KQRASPPPPYREPLP…AGRRILDTPHHTPPRYNTPPPPPPAYGIAGTTVV……

Tgi1-382


18

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Biochemistry

Figure 6: NMR studies of Yki-bound Tgi. 1H-15N HSQC spectra of unbound (black) and WWTD-bound (red) spectra of (A) Tgi1-97 and (B) Tgi258-382. (C) Portions of 1H-15N HSQC spectra of unbound Tgi (black) and WW TD-bound Tgi at molar ratios (Tgi:WWTD) of 1:0.25 (red) or 1:3 (purple). Labeled peaks correspond to residues that disappear or become less intense in the WWTD-bound spectrum. (D) A summary of results of titration experiments in frames A, B, and C mapped onto the residues which lose peak intensity or completely disappear in the WWTD-bound spectrum. The experimentally determined binding segments highlighted in yellow correspond to Tgi residues Q83 – L95, G262 – H271, and R275 – V293. The three PPxY sequences within these segments are underlined. Residues that completely disappear but are not at the recognition sites are highlighted in green. Also highlighted in pink and turquoise respectively are residues in frame C (red spectrum) that completely disappear or become less intense .

DISCUSSION In an ongoing effort to better understand how multiple site interactions influence binding and regulation of Yki, here we characterize interactions of Yki to full length Tgi at the molecular level. We find that binding is modulated by intrinsic protein disorder, positive cooperativity of the WW domains, and the ability of the Yki WW domains to engage all three Tgi binding sites with different affinities. Importantly we identify a segment on Tgi that undergoes allosteric conformational changes as a result of binding to the Yki WW domains, and speculate that residues in this segment could fine–tune binding and serve as targets in drug design. Tgi and Yki are predominantly unstructured proteins that remain disordered upon binding – We present the first biophysical characterization of full length Tgi, and Yki (Figure 2), that provide experimental evidence that both proteins are primarily disordered as predicted from their sequences. Though globally unstructured, local segments show propensity to adopt ordered structure - namely helical propensities at the N, and C termini of Tgi (Figure 5F), and a more ordered structure for the tryptophan-rich WW domains of Yki (Figure 2A). The assumption that the Yki WW domains adopt a more ordered structure is reasonable, given that YAP WW domains


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Page 20 of 30

which share more than 60 % sequence identity with Yki adopt a three-stranded anti-parallel beta sheet fold 12, 30. Yki and Tgi retain their predominantly unstructured conformations upon binding, as is evidenced by the absence of global structural ordering in the CD spectrum (Figure 2D). This phenomenon, termed dynamic “fuzzy” complex is not uncommon, and is a characteristic of some intrinsically disordered proteins (IDPs) such as Sic1, Cdc4, and p27Kip1 which function in cellcycle regulation31-33. Synergy, differential affinity, and allostery in the multisite-dependent interaction between Yki and Tgi – The combined ITC and NMR studies reveal novel molecular insights underlying complex formation. First, ITC experiments to identify binding domains show that each WW domain is independently capable of binding Tgi with relatively similar affinities, but binding is enhanced ~3-fold when both domains are present, and interactions occur simultaneously (Figure 3, Table 1). This clear demonstration of cooperativity between the tandem WW domains of Yki is in contrast with YAP WW domains which show no evidence of cooperativity, display noticeable differences in binding affinities for the same ligand 34 and when in tandem, binding is diminished by at least two-fold relative to that observed for the isolated domains 35. One major difference between the YAP WW domain studies and the results reported here is that all the studies with YAP used single PPxY motif peptides, even though some of the target partners have multiple copies of the PPxY motif. Comparable studies with single PPxY motif Tgi peptides show that binding to the tandem WW domains is diminished relative to that observed for multisite constructs, suggesting a role for multiple PPxY motifs in enhancing binding to the tandem WW domains. Secondly, quantitative analysis of binding affinities demonstrates different affinities of the WW domains for all three possible bivalent PPxY sites, with a relatively higher affinity for sites


20

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Biochemistry

that include the P1 motif. A careful analysis of PPxY sequences shows that the P1 sequence, PPPY, is more common among Yki regulators listed in Figure 1, as well as in several well-established PPxY partners of YAP34. Proline is unique among the other amino acids in that the Cδ of its side chain is cyclized to its backbone amide nitrogen which restricts its φ conformational torsion angle. Because of this conformational restriction, prolines confer rigidity in peptides 36, and a consensus sequence with three consecutive prolines may enhance binding by preorganizing the type II polyPro (PPII) helical conformation that PPxY motifs adopt when bound to their cognate WW domains12, 37. A rigid PPxY conformation would lower the entropic penalty for binding, and make binding more energetically favorable. In addition to energetically favorable interactions at P1, the charged pair of residues comprising, Q83 – R84, R93 – E94 (Figure 6D) flanking the core P1 motif suggest that electrostatic interactions may play a role in interactions at P1. Together, with the early disappearance of peaks at the P1 recognition motif in NMR titration experiments (Figure 6), which demonstrates that binding is initiated at this site, we conclude that P1 plays a critical role in the Yki-Tgi complex formation. Thirdly, interactions at P1 are coupled to some allosteric conformational changes in residues T25 – A43 that that are 40 residues upstream of the P1 motif (Figure 6D). Standard sequence-based secondary structure algorithms predict helical propensities for residues 32 – 38, that are part of the T25 – A43 segment. Though the low positive deviations in the NMR-detected structure do not support the formation of a strong helix (Figure 5C), they are consistent with the formation of a nascent helix either in the unbound or WWTD-bound protein. Peak disappearance in this segment of the protein which we attribute to conformational changes may be necessary to bring P1 closer to P2 and/or P3 for bivalent interactions with the tandem WW domains.


21

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Page 22 of 30

Limitations of peptide-based studies of modular protein-protein interactions –The ability of the tandem WW domains to associate with all three PPxY sites, is evidenced by ITC experiments (Figures 3, and 4), and the disappearance of peaks at all three recognition motifs in NMR experiments (Figure 6). However, there are noticeable differences in the affinity of the tandem WW domains for specific sites. An example is the 2-fold increase in binding affinity of the tandem WW domains for Tgi1-97 (4.3 µM) compared to the isolated P1p peptide (9.9 µM). These results suggest that as part of a larger domain, the recognition motif may experience conformational constraints that favor binding to the tandem WW domains. Furthermore, the higher affinity of the tandem WW domains for bivalent (P1+P2 or P1+P3) sites even though binding to isolated P2 and P3 sites is relatively weak supports observations from a number of multivalent site studies 38, 39 that show that having multiple motifs on the same polypeptide chain converts low affinity interactions to high affinity interactions. This enhanced binding to multivalent sites appears to strengthen the cooperativity of the Yki WW domains. For the full length protein which has all three PPxY sites available for binding, the puzzling observation that the Kd is 5-fold weaker than interactions at P1+P2 alone (Figure 3, and Table 1) suggest that interactions at sites P1+P3, and P2+P3 contribute negatively to the overall affinity. Furthermore, the marked difference in dissociation constants that accompany binding of the tandem WW domains to the full length Y91A variant, versus Tgi258-382 (compare Kd of 6 µM to 15 μM in Table 1) even though both constructs contain the same recognition motifs (P2 + P3), demonstrates an inhibitory effect of residues that are not directly involved in binding. Taken together, these results demonstrate the importance of examining multiple target motif interactions within their larger biological context and not as autonomous units.


22

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Biochemistry

In summary, molecular-level characterization of interactions between the two Yki WW domains and full length Tgi demonstrate how synergy between the WW domains, differential affinities for PPxY sites, allosteric conformational changes, and the inhibitory effect of regions outside the recognition sequences modulate complex formation. These findings offer novel insights into how interactions between tandem WW domains and multiple PPxY motifs are coordinated, highlight the importance of studying small binding motifs within the context of their full-length proteins, and identify potential alternative targets for drug design.


23

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Page 24 of 30

Corresponding Author Afua Nyarko, Department of Biochemistry and Biophysics, Oregon State University, Corvallis, OR 97331. Tel: (541) 737-4486; Fax: (541) 737-0481; E-mail: [email protected]

Funding Sources This work is supported by start-up funds from Oregon State University, and a New Investigator Award from the Medical Research Foundation of Oregon. ACKNOWLEDGMENT The author wishes to thank Prof. Kenneth Irvine (Rutgers University) for Yki cDNA clones, Profs Andy Karplus and Elisar Barbar for valuable discussions. Tgi clones were obtained from the Drosophila Genomics Resource Center, which is supported by NIH grant 2P40OD010949-10A1. NMR experiments were collected at the Oregon State University NMR Facility funded in part by the National Institutes of Health, HEI Grant 1S10OD018518, and by the M. J. Murdock Charitable Trust grant # 2014162.

ABBREVIATIONS TCEP, tris-2-carboxyethyl phosphine; DTT, Dithiothreitol; NMR, nuclear magnetic resonance spectroscopy; HSQC, heteronuclear single quantum coherence; WT, wild type.


24

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Biochemistry

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Differential Binding Affinities and Allosteric Conformational Changes

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