Molecular characterization of a NAD+ hydrolase active bacterial TIR domain-containing protein 4540

Abstract Description Bacterial TIR proteins identified in pathogenic, nonpathogenic, commensal, and archaea have identified NADase activity, raising new and exciting questions about how bacterial TIR domains function in immunity. To answer questions about the role of bacterial TIR NADase activity and the regulation of human Toll and IL-1R signaling, we have sought to characterize the molecular mechanism of a bacterial TIR protein with NADase activity. Accordingly, we have determined the structure of the Acinetobacter baumannii Toll-Interleukin-1 receptor resistance domain protein (AbTIR), confirmed its enzymatic function in NAD+ hydrolysis, and mapped its interaction with NAD+ using HDX-MS. Upon binding NAD+, AbTir-TIR exhibits unique EX1 kinetics and large cooperative conformational changes. As part of this molecular characterization, we recently carried out a Course-Based Undergraduate Research Experience (CURE), which identified residues critical for bacterial TIR domain NADase activity. Students used bioinformatics to identify residues they hypothesized would play a role in NADase function. The results from these student-led studies support observations that NAD+ binding induces conformational changes within the bacteria TIR domain, as identified by hydrogen-deuterium exchange mass spectrometry studies and cryogenic electron microscopy. Collectively, these studies provide new insight and understanding into the molecular mechanisms of NADase-active bacterial TIR proteins. Funding Sources HHMI Inclusive Excellence Grant to Towson University Topic Categories Microbial, Parasitic, and Fungal Immunology (MPF)