A glimpse into the structural properties of the intermediate and transition state in the folding of bromodomain 2 domain 2 by Φ value analysis

Bromodomains (BRDs) are small protein interaction modules of about 110 amino acids that selectively recognize acetylated lysine in histones and other proteins. These domains have been identified in a variety of multi‐domain proteins involved in transcriptional regulation or chromatin remodeling in eukaryotic cells. BRD inhibition is considered an attractive therapeutic approach in epigenetic disorders, particularly in oncology. Here, we present a Φ value analysis to investigate the folding pathway of the second domain of BRD2 (BRD2(2)). Using an extensive mutational analysis based on 25 site‐directed mutants, we provide structural information on both the intermediate and late transition state of BRD2(2). The data reveal that the C‐terminal region represents part of the initial folding nucleus, while the N‐terminal region of the domain consolidates its structure only later in the folding process. Furthermore, only a small number of native‐like interactions have been identified, suggesting the presence of a non‐compact, partially folded state with scarce native‐like characteristics. Taken together, these results indicate that, in BRD2(2), a hierarchical mechanism of protein folding can be described with non‐native interactions that play a significant role in folding.