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Pin1 is an essential peptidyl-prolyl isomerase (PPIase) that catalyzes cis-trans prolyl isomerization in proteins containing pSer/Thr-Pro motifs. It has an N-terminal WW domain that targets these motifs and a C-terminal PPIase domain that catalyzes isomerization. Recently, Pin1 was shown to modify the conformation of phosphorylated histone H1 and stabilize the chromatin-H1 interaction by increasing its residence time. This Pin1-histone H1 interaction plays a key role in pathogen response, in infection, and in cell cycle control; therefore, anti-Pin1 therapeutics are an important focus for treating infections as well as cancer. Each of the H1 histones (H1.0-H1.5) contains several potential Pin1 recognition pSer/pThr-Pro motifs. To understand the Pin1-histone H1 interaction fully, we investigated how both the isolated WW domain and full-length Pin1 interact with three H1 histone substrate peptide sequences that were previously identified as important binding partners (H1.1, H1.4, and H1.5). NMR spectroscopy was used to measure the binding affinities and the interdomain dynamics upon binding to these sequences. We observed different K D values depending on the histone binding site, suggesting that energetics play a role in guiding the Pin1-histone interaction. While interdomain interactions vary between the peptides, we find no evidence for allosteric activation for the histone H1 substrates.

Using an evolved pyrrolysyl-tRNA synthetase-tRNA(Pyl) pair, a Se-alkylselenocysteine was genetically incorporated into histone H3 with a high protein expression yield. Quantitative oxidative elimination of Se-alkylselenocysteine followed by Michael addition reactions with various thiol nucleophiles generated biologically active mimics of H3 with posttranslational modifications including lysine methylation, lysine acetylation, and serine phosphorylation.

The three-dimensional structures of the two L-peptides, H-CGGIRGERA-OH, called L(A), and H-CGGIRGERG-OH, called L(G), corresponding or close to the IRGERA sequence present in the C-terminal region (residues 130-135) of histone H3, and their retro-inverso analogues HO-mAreGriGGC-NH2, called RI(mA), and HO-mGreGriGGC-NH2, called RI(mG), have been studied by two-dimensional 1H NMR and molecular dynamics calculations in association with a monoclonal antibody generated against L(A). At 25 degrees C, the affinity constants of the monoclonal antibody with respect to RI(mA) and RI(mG) were 75- and 270-fold higher than those measured with the homologous L(A) and L(G) peptides, respectively. Due to the spontaneous epimerization of the mA malonic residue, RI(mA) gave rise to two sets of resonances. With regard to the NH amide region, one set was similar to that for RI(mG) while the second was similar to those for the parent L-peptides L(A) and L(G). The antibody-bound conformations of the two couples of L- and retro-inverso peptides have been analyzed using molecular modeling calculations based on the transferred NOE interproton distances. Folded structures appeared in both cases with a type II' beta-turn in the parent GGIR sequence and a type I' beta-turn in the retro-inverso reGr sequence.