A Glimpse into the Structural Properties of the Intermediate and Transition State in the Folding of Bromodomain 2 Domain 2 by Φ Value Analysis

• Main Authors: Leonore Novak, Maria Petrosino, Daniele Santorelli, Roberta Chiaraluce, Valerio Consalvi, Alessandra Pasquo, Carlo Travaglini-Allocatelli
• Format: Article
• Language: English
• Published: MDPI AG 2021-05-01
• Series: International Journal of Molecular Sciences
• Subjects: bromodomain; protein folding; Φ value analysis; protein stability; mutagenesis; folding kinetics
• Online Access: https://www.mdpi.com/1422-0067/22/11/5953
• ISSN: 1661-6596; 1422-0067

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.