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residue is part of a Pin1 recognition motif. Furthermore, the interaction was not disturbed by mutations in either the WW-domain or isomerase domain of Pin1 Pin1:W34A and Pin1:K63A, respectively (Figure 2D). Finally, these interactions were specific, as control pull-downs without peptide did not precipitate Pin1, nor did TFCD peptides precipitate green fluorescent protein (GFP), a protein not predicted to bind the TFCD (Figure 2D).
We next investigated the effect of phosphorylation on the interaction between Pin1 and TF using cysteine-linked peptides encoding the TFCD with varying states of phos- phorylation. These experiments suggest that Pin1 has the highest affinity for the TFCD peptide phosphorylated at Ser258 (Figure 2E). However, it should be noted that these co-immunoprecipitation experiments are not quantitative. Furthermore, the observation that Pin1 seems to bind unphosphorylated and Ser253-phosphorylated TFCD peptides may also be due to TF-phosphorylating kinases present in the whole-cell lysates used for these pull- downs. The pull-down reactions were specific, as neither a pull-down without peptide or a pull-down for GFP resulted in precipitation of the target protein (Figure 2E).
Taken together, these results confirm that Pin1 interacts with TF and strongly suggest that this interaction involves the predicted pSer258-Pro259 Pin1 recognition motif in the TFCD.
Structural modalities mediating Pin1–TFCD complex formation
To study the interaction between the TFCD and Pin1 in more detail, we performed isothermal titration calorime- try (ITC) with Ser253 or Ser258-phosphorylated TFCD- encoding peptides and purified Pin1 WW-domain (Online Supplementary Figure S1). Consistent with our pull-down experiments, these assays showed that the Pin1 WW- domain interacts specifically with the TFCD peptide phosphorylated at Ser258 with a fitted K of approximate-
assignment and NMR structure determination. Analysis of the Cα and Cβ values based upon the weighted chemical shift index highlighted the three β-strands, which are char- acteristic of WW-domains (Online Supplementary Figure S2A). A total of 203 unique intra- and intermolecular dis- tance constraints were derived from the analysis of the NOE spectroscopy (NOESY) experiments. We used these constraints together with the weighted chemical shift index (Online Supplementary Table S1 and Online Supplementary Figure S2A) to calculate the solution struc- ture of the Pin1 WW-domain and TFCD complex (see Methods for more details). The 20 lowest energy con- formers of the Pin1 WW-domain yielded a root-mean- square deviation (RMSD) of 1 Å (residues 6-39) (Figure 3B and Online Supplementary Figure S2B). The Pin1 WW- domain is a canonical WW-domain18 consisting of three twisted anti-parallel β-sheet strands with a conserved Trp- Trp motif located at the N-terminus of the first β-strand and C-terminus of the third β-strand, respective- ly.
Analysis of the Pin1 WW-domain–TFCD complex reveals that TFCD residues Asn257, pSer258 and Pro259 are involved in the binding interface of the Pin1 WW- domain, consistent with our pull-down assays. While the Pin1 WW-domain as a whole shows a well-defined, single conformation, the β1-β2 loop1 binding region of the WW- domain (residues 17 to 21) and the pSer258-Pro259 motif of TFCD show two distinct ensembles of conformers (Figure 3B and C), consistent with the fact that less NOEs were detected for the β1-β2 loop1. The features that prin- cipally drive the complex formation are the charge-charge interaction and the hydrophobic interaction between Trp34 (the second invariant Trp of the WW motif) with invariant Pro259. The ionic interactions between the phosphate of pSer258 with the positively charged guani- dinium groups of Arg14, Arg17, and Arg21 also appeared to stabilize the complex. However, as mentioned earlier, the resonances for Arg17, located at position 1 of loop1, disappeared during the NMR titration due to intermediate exchange in the NMR timescale (Figure 3A), suggesting high flexibility around loop1. Arg21 connects loop1 to the β2-strand into two conformations and involves multiple polar contacts with the double phosphorylated TFCD at pSer258 (Figure 3B and C). Similarly, Trp34-driven hydrophobic packing of Pro259 induces two ensembles of Trp34 side chain rotamers and Pro259 configurations, and both ensemble states still adopt a trans-configuration of the pSer258-Pro259 peptide bond (Online Supplementary Table S1). For comparison, the previously determined interactions of Pin1 WW-domain with Cell division cycle 25 (Cdc25) and the C-terminal domain of the RNA poly- merase II largest subunit (RNAP II-CTD) are also shown (Figure 3D).29,30
In order to further confirm the reliability of our struc- tures, step-wise energy minimization was performed on all Pin1 WW-domain-TFCD complexes. The RMSD of heavy atoms/all atoms before and after energy minimiza- tion was less than 0.45 Å, while the RMSD of the back- bone was less than 0.3 Å (Online Supplementary Figure S2B). Therefore, the interactions between the Pin1 WW- domain and the TFCD, such as the electrostatic interac- tions with pSer258 and the hydrophobic interactions of Pro259 with the WW-domain are consistent with the NMR structures calculated. The trans conformation of the pSer258-Pro259 peptide bond in the TFCD was stable dur-
d
ly 137 mM (Online Supplementary Figure S1B and D).
To characterize the molecular basis of the WW- domain/TFCD interactions, we titrated the 15N-labeled WW-domain with an increasing concentration of pSer253/pSer258 TFCD peptide. Analysis of the 2D 1H/15N heteronuclear single-quantum correlation (HSQC) spectra showed that in all titrations, binding kinetics were in the fast-to-intermediate exchange regime, with at least 11 residues showing a large chemical shift (δg > 0.1 ppm) (Figure 3A). Affected residues in the Pin1 WW-domain were located at the C-terminus of the β1-strand (S16), the β1-β2 loop (R17, S18, and G20), the β2-strand (R21, Y23, and F25), and the C-terminus of the β3-strand (W34 and E35) (Figure 3A; residues in bold). During our NMR titra- tions, the 1H/15N HSQC spectra showed line broadening for the Arg17 peak, resulting in its disappearance (Figure 3A). We attribute the broadening of the Arg17 line to its close proximity with the TFCD, with interactions occur- ing in the intermediate exchange regime in the NMR time scale (Figure 3B). A K of 133 ± 13 μM was determined
d
from the δg (ppm) for HN changes in Pin1 WW-domain
residue Ser18 (Figure 3A; box around moving chemical shift and inset graph), which is similar to our calorimetric measurements showing a fitted Kd value of ~137 mM (Online Supplementary Figure S1).
We next prepared 13C/15N- isotopically-labeled Pin1 WW-domain in complex with double phosphorylated pSer253/pSer258 TFCD peptide for sequential backbone
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