Deciphering the mechanism of interaction between two key subunits from the Type IV Secretion System core complex: VirB9 and VirB7

• Ano de defesa: 2022
• Autor(a) principal: Macias, Angy Liseth Davalos
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: eng
• Instituição de defesa: Biblioteca Digitais de Teses e Dissertações da USP
• Palavras-chave em Português: Biofísica; Biologia estrutural; Cinética de proteínas; Mecanismos de reconhecimento molecular; Molecular biophysics; Molecular recognition mechanisms; Nuclear magnetic resonance spectroscopy; Protein kinetics; Ressonância magnética nuclear; Sistema de secreção tipo IV; Structural biology; Type IV secretion system
• Link de acesso: https://www.teses.usp.br/teses/disponiveis/46/46131/tde-02122022-124841/

Intrinsically disordered proteins (IDPs) are implicated in the regulation of many important processes within the cell, a factor that explains their abundance in the proteome. Often, IDPs undergo local or global conformational rearrangements coupled to binding. However, the mechanisms by which IDPs interact with their partners have been a topic of debate and remain poorly understood. Here, we characterized the dynamics of the VirB9 C-terminal domain (VirB9Ct), and its binding mechanism to the N-terminal tail of VirB7 (VirB7Nt). The interaction between the two domains is essential for the assembly of a supramolecular complex, the Type IV Secretion System from the phytopathogen Xanthomonas citri, which is responsible for the secretion of toxins that lead to bacterial killing. VirB7Nt is completely disordered in the unbound state, while VirB9Ct has characteristics of a molten globule. The unbound state of VirB9Ct was characterized by a combination of Circular Dichroism spectroscopy, Isothermal Titration Calorimetry, Differential Scanning Calorimetry, Chemical Exchange Saturation Transfer (CEST) NMR experiments, and ANS fluorescence assays. We found that, in the unbound state, VirB9Ct has similar secondary and tertiary structures as when bound to VirB7Nt. Furthermore, in the unbound state VirB9Ct samples the bound-conformation even in the absence of VirB7Nt, which points to conformational selection as recognition pathway. The interaction mechanism between VirB9Ct and VirB7Nt was elucidated by quantitative analysis of CEST and fluorescence stopped flow experiments. The results support the view that their interaction occurs through conformational selection at 25°C, but becomes more complex at higher temperatures due to the intrinsic dynamics of VirB9Ct, and, in this case a combined CS-IF mechanism predominates. Overall, these results highlight the need of combining different biophysical methods to elucidate protein-protein interaction mechanisms, and contribute to the understanding of molecular recognition pathways and their complexity.