Results from a CURE course project reveal novel insights into the structure of bacterial Toll/interleukin-1 receptor (TIR) domain proteins

Abstract

Abstract Toll-like receptors (TLRs) are critical for innate immune signaling, and various bacteria express Toll/interleukin-1 receptor (TIR) domains to evade this signaling. Bacterial TIRs exhibit NADase activity, although it is unclear what role NADase activity plays in evasion of innate responses. To better characterize the NADase activity of the TIR from Acenitobacter baumanii (AbTIR), we used site directed mutagenesis to identify amino acids predicted to be important for NADase activity. Studies were carried out in a Course-Based Undergraduate Research Experience (CURE) involving 16–20 undergraduate research students per year. Students used Chimera to analyze the AbTIR structure and hypothesize amino acids they predicted would play a role in enzymatic activity. They then performed site-directed mutagenesis to create plasmids for expression of mutant AbTIR. Mutant TIRs were expressed, purified and tested for NADase activity. So far, our mutagenesis results suggest a potential conformational change in the NAD binding site upon NAD binding. Identifying amino acids critical for enzymatic activity may inform drug development for diseases associated with dysregulated TIR activity. In addition, the application of the CURE class to carry out these studies has provided project-based research training experiences for approximately 80 undergraduate students, about half of whom are members of groups traditionally underrepresented in science. Several students, including some who had limited research experience before completing the course, are currently pursuing graduate studies and research-based careers, highlighting the value of CURE-based projects for facilitating the inclusion of students from diverse groups in research. TVS and TO were supported by the NIH Bridges to Baccalaureate Grant R25GM058264; CP was supported by the NIH Bridges to the Doctorate Grant R25GM119970, and the CURE course was supported by an HHMI Inclusive Excellence Grant