**3.3. Specificity of effector protein orientation in nucleosome complex formation (PRC1)**

Besides determining the binding mode, synergetic interactions can also provide the structural basis for specificity of effector protein activity. This was shown in the crystal structure, also from the Tan lab, of the polycomb repressive complex 1 (PRC1) that ubiquitinates H2A K119 in a highly specific manner [55]. On its surface, the nucleosome displays various lysine residues that can be ubiquitinated by the respective writer proteins. However, the downstream response wildly differs depending on the position of the ubiquitinated lysine. Thus, target specificity is of high importance for ubiquitin writer proteins. In case of PRC1, this is based on two distinct binding processes. For one, there is the interaction between acidic patch and the arginine anchor of the Ring1B/Bmi1 subunit. In addition, the E2 subunit UbcH5c engages the nucleosomal DNA. Combined, both contributions are responsible for exact positioning of the catalytic center of the ubiquitin carrying E2 to the target H2A K119 (**Figure 2B**).

Besides LANA, RCC1 and PRC1, other crystal structures of nucleosome complexes offered further insights into nucleosome recognition. In particular, the structure of the nucleosome complex of the SAGA DUB deubiquitination module showed a non-canonical acidic patch binding. Morgan *et al.* found that the SAGA nucleosome-binding DUB module possesses three equally crucial arginine residues distributed over an α-helix [56] (**Figure 2A**). This perhaps points towards yet other acidic patch interaction modes.

Recently, also cryo-EM-derived structures of nucleosome-protein complexes have been published. The first structure, solved in 2016, yielded the structure of the complex with 53BP1, a reader protein for post-translational histone modifications [40]. Subsequently, the structures of Snf2 and CENP-N were solved and published [71–73].

Since the first crystal structure two decades ago, the list of nucleosome complexes deposited in the RCSB PDB protein databank is continuously growing. Still, the 12 high-resolution structures solved to date only encompass a fraction of all nucleosome-protein interactions. This discrepancy highlights the need for alternative techniques in chromatin structural biology.

**4. Data-driven modeling**

in **Table 4**.

An attractive alternative to traditional structure determination methods is the modeling of structures of complexes based on some sort of experimental information on the interaction [79, 80]. In such data-driven modeling of a complex structure, the two interaction partners are docked together, guided by the experimental data, and respecting their biophysical properties. The exact binding interface and relative orientation of the binding partners are typically refined over several steps. Prerequisite for this approach is the availability of the 3D structures of the interacting partners. Several molecular docking programs allow the incorporation and use of experimental data and so increase the accuracy of resulting structures [81]. Hence, data from diverse biophysical techniques are translated into restraints guiding the docking process [82–84]. The type of information includes interaction interface, distances or shape of the complex and its subunits. Techniques that can provide these information are listed in **Table 3**. Interestingly, all three classes of information can be provided by NMR spectroscopy. It is possible to gather data on intermolecular distances and shape by paramagnetic relaxation enhancement (PRE) and the nuclear Overhauser effect (NOE) as well as information on binding interfaces and binding affinity through chemical shift perturbation (CSP). The use of these NMR methods in docking studies is reviewed in detail elsewhere [79]. An overview of publications that used data-driven docking to investigate nucleosome-protein complexes is listed

Recognition of Nucleosomes by Chromatin Factors: Lessons from Data-Driven Docking-Based…

http://dx.doi.org/10.5772/intechopen.81016

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**4.1. Bringing data-driven modeling to nucleosome complexes (LSD1-CoREST)**

**Interaction interface Distances Shape** Mutagenesis XL-MS Cryo-EM

**Table 3.** Biochemical and biophysical techniques for structural analysis of protein complexes.

A pioneer study for data-driven modeling of a nucleosome complex was successfully applied for the lysine-specific demethylase 1 and CoREST complex [86]. Both proteins cooperate in the demethylation of mono- and dimethylated H3K4. While it was possible to solve the crystal structure of LSD1-CoREST, their nucleosome-bound state remains elusive. Yang et al. gained insight into the molecular basis of LSD1-CoREST interaction by identifying point mutations that interfere with the LSD1-CoREST ability to demethylate a methylated peptide model of the histone H3 tail. Since it was previously shown that LSD1 recognizes a specific stretch of the H3 tail [94], it was possible to employ modeling to identify intermolecular interactions between the peptide and both the LSD1 active site and the LSD1-CoREST interface (**Figure 3B**). Lastly, NMR titration experiments of the CoREST SANT2 domain with DNA revealed a DNA-binding interface on SANT2. These pieces of interaction data were used to

H/D exchange Forster resonance energy transfer (FRET) Small angle X-ray or neutron scattering (SAXS/

SANS)

Electron paramagnetic resonance (EPR) Ion-mobility mass spectrometry (IM-MS)

**Figure 2.** A detailed depiction of the acidic patch region and the triad of H2A that complexes the canonical arginine anchor. (A) LANA was the first peptide model of a binding domain to be crystalized (green, pdb: 1zla). (B) The first crystalized protein domain was RCC1 (pink, pdb: 3mvd). (C) They all share the acidic patch as a common binding epitope for the arginine anchor residue, as is also the case for the PRC1 complex with its acidic patch binding RING domain (cyan, 4r8p). (D) Interestingly, acidic patch binding is not necessarily limited to one single arginine anchor residue. As for the nucleosome-bound structure of the deubiquitinase complex SAGA-DUB (yellow, pdb: 4zux), three arginine residues are essential part of the acidic patch binding, of which none occupies the position in the center of the acidic triad. Combination of nucleosomal DNA and acidic patch binding is shown in the structure of RCC1 and PRC1. (E) PRC1 (cyan, pdb: 4r8p) that besides the acidic patch also engages DNA with its UbcH5c subunit. (F) The same holds true for RCC1 (pink, pdb: 3mvd) that contacts to DNA with the unstructured tail region.
