**3. Crystal clear: lessons from crystallography and single particles**

A key role in the research of protein interactions are high-resolution three-dimensional structures of the complexes, typically obtained by crystallography and, increasingly, cryo-electron microscopy. These structures enable the identification of binding sites and intermolecular interactions, offering a guided approach to design binding-deficient mutants or competitive binders. The history of nucleosome structural biology peaked with the publication of the highresolution crystal structure of the nucleosome in 1997 [3]. Luger *et al.* achieved crystallization of the nucleosome together with a palindromic version of human α-satellite DNA [49]. This milestone study provided the foundation to also study the structures of nucleosomes together with chromatin factors in complexes. **Table 2** lists the structures of nucleosome-protein complexes solved to date by crystallography and cryo-electron microscopy [39, 50–58]. The most recent addition to this ever-growing list is the spectacular structures of the complex between the INO80 chromatin remodeler and the nucleosome [59, 60]. Below, we discuss a few cardinal studies to highlight the different nucleosomal binding modes of effector proteins.

**3.2. The first crystal structure of a nucleosome-bound protein domain (RCC1)**

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

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 per-

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

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.

formation and subsequent effects on chromatin structure.

haps points towards yet other acidic patch interaction modes.

of Snf2 and CENP-N were solved and published [71–73].

**(PRC1)**

The first structure of a protein bound to the nucleosome was the RCC1-nucleosome complex published by the Tan lab in 2010. RCC1 (regulator of chromosome condensation) is essential during mitosis by recruiting Ran GTPase, which plays a role in nucleus reorganization, to the nucleosome [65, 66]. A comparison with LANA highlighted the crucial and conserved interaction of arginine residues with the acidic patch triad [50]. Strikingly, RCC1 binds to the acidic patch using the canonical arginine anchor, here contained in a loop, and also binds the nucleosomal DNA through its N-terminal tail. Such synergetic interactions have been observed later in many other nucleosome-binding proteins [50, 55, 67–70]. This study was the first to show such complexity of nucleosomes as interaction platforms. It also highlights the importance of properly defining the boundaries of binding domains to capture all binding epitopes in order to reveal possible synergetic interactions and fully understand complex

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

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

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#### **3.1. The first crystal structure of a nucleosome complex (LANA)**

The first high-resolution structure of a nucleosome-protein complex was the crystal structure of a peptide model of Kaposi's sarcoma-associated herpesvirus LANA N-terminal region bound to the nucleosome [61]. The binding site identified in this study was the acidic patch. The atomistic resolution allowed to identify intermolecular side chain interactions including the arginine anchor bound to the acidic triad. Ever since, the LANA-nucleosome has become a golden standard for comparisons with other acidic patch interactions [50, 55]. Importantly, LANA is used to investigate the acidic patch binding ability of other proteins by competitive binding [62–64]. Interestingly, this exact epitope happened to be the binding interface also for the first full protein domain that was crystalized in its nucleosome-bound state.


**Table 2.** Structures of nucleosome-protein or nucleosome-peptide complexes deposited in the RCSB protein databank PDB.
