Structural and functional studies of the unique diadenylate cyclase enzyme and the unique YbbR-like protein in Staphylococcus aureus: proteins involved in c-di-AMP biosynthesis

Author(s)

Nathalya Cristina de Moraes Roso Mesquita

• Document type: Doctoral Thesis
• Press: São Carlos.
• Institution: Universidade de São Paulo (USP). Instituto de Física de São Carlos (IFSC/BT)
• Defense date: 2016-06-30

Examining board members

Marcos Vicente de Albuquerque Salles Navarro; Regina Lúcia Baldini; Lisandra Marques Gava Borges; Frederico José Gueiros Filho; José Luiz de Souza Lopes

• Advisor: Marcos Vicente de Albuquerque Salles Navarro

Abstract

Recently, a new bacterial signaling molecule, the dimeric cyclic AMP (c-di-AMP) has emerged as a central regulator of essential physiological processes, such as cell wall homeostasis, DNA integrity and bacterial virulence, among others. C-di-AMP is synthesized from two molecules of adenosine triphosphate (ATP) by proteins containing DisA_N domain, also called diadenilato cyclases (DACs). A survey in the Protein Families Database database (Pfam) found 2842 protein sequences containing the DAC domain, from 2386 different organisms. These proteins are divided into subfamilies and the three most abundant are: DacA (69,1%), a membrane protein associated with intracellular signaling resulting from an external environment change; DisA (24,1%), the first and most widely studied diadenilate cyclase, an intracellular protein found as active octamers in solution which indirectly controls cell division by DNA integrity verification; and DacB (5,5%), a cytoplasmic proteins, particularly expressed during bacterial spores formation. An interesting feature is that most organisms contain just a single and essential DAC-protein. Organisms containing two or more DAC-containing proteins, such as Clostridium and Bacillus spp., are exceptions. In Staphylococcus aureus (S. aureus), an opportunistic human pathogen responsible for some life-threating diseases, there is a single membrane attached diadenilate cyclase, hanging in the inner portion of the cell membrane (Sau_DacA). The activity of this protein is potentially regulated through direct interaction with YbbR, which contains an extracellular sensor domain. Sau_DacA conserves all key elements of bacterial di-adenylate cyclase, and for being the only di-adenylate cyclase from S. aureus, proves to be an excellent study target for new therapeutic purposes. However, to date, there is a lack of information about structure, c-di-AMP synthesis mechanism and regulation of nucleotide synthesis by Sau_DacA. Therefore, in this context the present work aims to contribute. Through a series of structural, calorimetric, spectroscopic and biochemical assays combined with site-directed mutations, we solved the structure of a soluble construct of Sau_DacA and identified a dimeric interface relevance for the conformational and thermal stability to the protein. This dimer is functionally active and highlights the importance of conserved motifs DGA (Aspartate-Glycine-Alanine) and RHR (Arginine-Histidine-Arginine) for the activity of Sau_DacA. The L5 loop, located between the active site and the dimer interface where is allocated the ATP binding motif (DGA), is stabilized in a favorable position for ATP binding, just in protein dimeric conformation. Our results combined with literary allowed us infer the synthesis of c-di-AMP occurs by face-to-face encounter of two distinct ATP binding site and its rate of synthesis could be regulated through direct protein interaction with. In this way, we contribute to a better understanding of Sau_DacA structure and function, assisting in its use as a target for new drugs development since it is known the biosynthesis of c-di-AMP is essential for most pathogens that synthesize. (AU)

• FAPESP's process: 12/01321-2 - Structural biochemistry of enzymes involved in biosynthesis of c-di-AMP in Staphylococcus aureus
• Grantee: Nathalya Cristina de Moraes Roso Mesquita
• Support Opportunities: Scholarships in Brazil - Doctorate