*3.1. Protein–Peptide Binding*

The CABS-dock method has been extensively tested using the PeptiDB benchmark set of protein–peptide complexes [62,65,87]. One of the benchmark cases is the androgen receptor ligand binding domain (AR) in complex with a peptide with the FxxLF motif [88] (PDB code: 1T7R). To further analyze the interaction details of this complex, we performed blind global docking (using no knowledge about the binding site and peptide conformation) using CABS-dock [62]. As the input we used information on peptide sequence (incorporating the FxxLF motif: SSRFESLFAGEKESR), peptide secondary structure information assigned by the DSSP method [89] and the structure of the AR protein receptor. In this docking study, the peptide structure was simulated as fully flexible, while fluctuations of the protein receptor were limited to small backbone movements around the input structure (around 1 Å). The docking simulation started from random peptide conformations placed in random positions around the receptor structure. During simulation, the peptide remained unstructured until it was bound to the receptor binding site (Figure 5a). The docking simulations provided a set of high-quality models—the best model was characterized by a peptide-RMSD (root-mean-square deviation) value of 1.97 Å—and contact maps in strong agreement with the experimental data. As expected from the experimentally obtained structures and sequence analysis [88] the FxxLF interaction motif residues were most frequently involved in stabilizing hydrophobic interactions with the receptor. These high-frequency contacts are clearly visible in Figure 5a.

The study of the pKID/KIX system [63] involved performing a folding simulation of an intrinsically disordered protein (pKID) and its binding to a well-structured KIX receptor (Figure 5b). According to the experimental studies, the pKID structure is disordered in its unbound form with a slight propensity toward a helix (for detailed description on how one-dimensional secondary structure information is used in the CABS model see [78]). In the complex with the KIX protein, pKID adopts a characteristic conformation of two perpendicular helices that wrap around the receptor. However, most simulation results for the coupled folding and binding of this system published prior to the CABS-based study used models which biased pKID toward its native conformation (see the discussion in [63]). Using our method for studying this system enabled fully flexible treatment of the pKID protein. The obtained results [63] suggested the binding mechanism that involve two encounter complexes and were in well agreement with the available NMR experimental data. The predicted models presented high fractions of native contacts and allowed identification of residues essential for the binding and stabilization of the complex.

In the simulation of MDM2/p53 binding [64], the most challenging task was to adequately model the flexibility of the relatively long, unstructured regions of the protein receptor in addition to the fully flexible peptide [64,90] (Figure 5c). To provide a detailed insight into MDM2/p53 binding, we performed CABS-dock simulations and captured system behavior in agreement with the experimental data [64]. During the simulation, the flexible N- and C- terminal MDM2 fragments remained significantly disordered. The best resulting model was characterized by a peptide-RMSD value of 2.76 Å and 54% of the native contacts while the top ranked model by 3.74 Å and 60%, respectively. During simulations, we observed ensembles of models in which the peptide adopted different conformations loosely bound to the binding site and models in which the N-terminal highly flexible MDM2 fragment was interacting with the binding site. These findings are in agreement with the experimental data suggesting that p53-MDM2 binding is affected by significant rearrangements of the N-terminal MDM2 fragment (see discussion in [64]).

**Figure 5.** Case studies of modeling disordered or unfolded structures of proteins with CABS-based tools. In the figures, red or cyan marks structure fragments simulated as fully flexible (cyan was used to mark regions of interest discussed in the text), while beige marks regions whose motions were confined to small backbone movements (around 1 Å from the input structure). (**a**) Modeling of the dynamics of a flexible peptide representing the FxxLF motif in the proximity of the binding site of AR protein together with an averaged contact map showing frequency of residue–residue contacts during the docking simulation. (**b**) Modeling of coupled folding and binding of the disordered pKID to the KIX domain [63]; the map presents the frequency of contacts of near-native conformations obtained in the simulation. (**c**) Modeling of p53 peptide binding to the MDM2 receptor [64], which includes fully-flexible regions of the protein receptor (shown in cyan) interacting with a fully-flexible peptide (shown in red). (**d**) Modeling of barnase folding [52] in the de novo fashion (using no knowledge about the structure); the map is a residue–residue contact map showing relative contact frequencies in denaturing conditions; the protein fragments that form the folding nucleation site are colored in cyan in the presented folded structure of barnase.
