Structural and biochemical basis of NAD+ nucleosidase activity of TIR domain-containing proteins

Abstract

NAD+ (nicotinamide adenine dinucleotide) was first identified as a cofactor in many redox reactions. Subsequently, it has been found to synchronize many cellular functions through the regulation of NAD+ consuming enzymes like CD38, sirtuins, and PARPs (poly ADP-ribose polymerases). The latest addition of the NAD+ consuming enzyme family is the TIR (Toll/interleukin-1 receptor) domain-containing proteins, which are conserved from prokaryotes to eukaryotes. Intriguingly, the NAD+ nucleosidase activity of the TIR domain-containing proteins is linked to cell death in the three forms of life. In animals, SARM1 (sterile alpha and TIR motif containing 1) is the only TIR domain-containing protein with NAD+ nucleosidase activity, and the enzymatic activity of SARM1 is linked to neuronal cell death. In plants, some of the plant TIR domain- containing proteins consume NAD+, which causes localized cell death in response to infections. The NAD+ nucleosidase activity of some of the bacterial TIR domain-containing proteins is also linked to the bacterial cell death in response to phage infection. However, there have been no structural and biochemical studies on the NAD+ nucleosidase activity of the TIR domains. To understand the molecular basis of the TIR domains' enzymatic activity, I explored TIR domains from two forms of life (i.e., animals and bacteria), which are diverse in terms of sequence, three-dimensional structure, and domain architecture.Chapter 2 of my thesis describes the identification of the critical residues in the catalytic pocket based on the crystal structure of the human TIR domain of SARM1 (SARM1TIR), followed by functional assays using two different methods. The functional tests described in this chapter also proved that the crystallographic interfaces observed in the SARM1TIR are essential for the activation and regulation of the enzymatic activity. The crystal structures of the two interface mutants of human SARM1TIR showed how these interfaces regulate the enzymatic activity of SARM1TIR. Biochemical assays and negative stain electron microscopy with human SARM1 and its orthologs shed light on the role of oligomerization in the NAD+ nucleosidase activity of SARM1.In the rest of the chapters, I describe the structural and biochemical basis for the NAD+ nucleosidase activity of the bacterial TIR domain-containing proteins. Initial studies suggested that the bacterial TIR domain- containing proteins act as virulence factors by mimicking host TIR domain interactions; however, recent studies indicate that bacterial TIR domain-containing proteins also take part in the bacterial antiviral response. The structural and biochemical studies described in Chapters 3-4 revealed that the active site of the bacterial TIR domain-containing proteins is different from SARM1 or plant TIR domains, and the reaction products also vary from species to species.In a nutshell, my work presented in this thesis provided an in-depth structural and biochemical characterization of the enzymatic activity of the TIR domain-containing proteins from animals and bacteria.