Structure insights, thermodynamic profiles, dsDNA melting activity, and liquid-liquid phase separation of the SARS-CoV-2 nucleocapsid N-terminal domain binding to DNA

• Published in: bioRxiv
• Main Authors: Caruso, Icaro Putinhon, Almeida, Vitor S, Amaral, Mariana J, Andrade, Guilherme C, Araujo, Gabriela R, Araujo, Talita S, Azevedo, Jessica M, Barbosa, Glauce M, Bartkevihi, Leonardo, Bezerra, Peter R, Katia Maria Dos Santos Cabral, Lourenço, Isabella O, Malizia-Motta, Clara L F, Marques, Aline L, Mebus-Antunes, Nathane C, Neves-Martins, Thais Cristtina, De Sá, Jessica M, Sanches, Karoline, Santana-Silva, Marcos Caique, Vasconcelos, Ariana A, Almeida, Marcius S, Amorim, Gisele C, Anobom, Cristiane D, Da Poian, Andrea T, Gomes-Neto, Francisco, Pinheiro, Anderson S, Almeida, Fabio C L
• Format: Paper
• Language: English
• Published: Cold Spring Harbor Cold Spring Harbor Laboratory Press 22.07.2021
• Subjects: Aptamers; Deoxyribonucleic acid; DNA; Hydrogen bonding; Infectivity; Melting; N protein; Nucleocapsids; Nucleotide sequence; Proteins; Regulatory sequences; Severe acute respiratory syndrome coronavirus 2; Thymidine; Transcription
• DOI: 10.1101/2021.07.21.453232

The SARS-CoV-2 nucleocapsid protein (N) is a multifunctional promiscuous nucleic acid-binding protein, which plays a major role in nucleocapsid assembly and discontinuous RNA transcription, facilitating the template switch of transcriptional regulatory sequences (TRS). Here, we dissect the structural features of the N protein N-terminal domain (N-NTD), either with or without the SR-rich motif (SR), upon binding to single and double-stranded TRS DNA, as well as their activities for dsTRS melting and TRS-induced liquid-liquid phase separation (LLPS). Our study gives insights on specificity for N-NTD/N-NTD-SR interaction with TRS, including an unfavorable energetic contribution to binding along with hydrogen bonds between the triple-thymidine (TTT) motif in the dsTRS and β-sheet II due to the defined position and orientation of the DNA duplex, a well-defined pattern (ΔH > 0 and ΔS > 0 for ssTRS, and ΔH < 0 and ΔS < 0 for dsTRS) for the thermodynamic profile of binding, and a preference for TRS in the formation of liquid condensates when compared to a non-specific sequence. Moreover, our results on DNA binding may serve as a starting point for the design of inhibitors, including aptamers, against N, a possible therapeutic target essential for the virus infectivity. Competing Interest Statement The authors have declared no competing interest.