Characterization of the Structural Requirements for the NADase Activity of Bacterial Toll/IL-1R domains in a Course-based Undergraduate Research Experience
Year: 2024
Type: article
Abstract: TLRs initiate innate immune signaling pathways via Toll/IL-1R (TIR) domains on their cytoplasmic tails. Various bacterial species also express TIR domain-containing proteins that contribute to bacterial evasion of the innate immune system. Bacterial TIR domains, along with the mammalian sterile α and TIR motif-containing protein 1 and TIRs from plants, also have been found to exhibit NADase activity. Initial X-ray crystallographic studies of the bacterial TIR from Acinetobacter baumannii provided insight into bacterial TIR structure but were unsuccessful in cocrystallization with the NAD+ ligand, leading to further questions about the TIR NAD binding site. In this study, we designed a Course-Based Undergraduate Research Experience (CURE) involving 16-20 students per year to identify amino acids crucial for NADase activity of A. baumannii TIR domain protein and the TIR from Escherichia coli (TIR domain-containing protein C). Students used structural data to identify amino acids that they hypothesized would play a role in TIR NADase activity, and created plasmids to express mutated TIRs through site-directed mutagenesis. Mutant TIRs were expressed, purified, and tested for NADase activity. The results from these studies provide evidence for a conformational change upon NAD binding, as was predicted by recent cryogenic electron microscopy and hydrogen-deuterium exchange mass spectrometry studies. Along with corroborating recent characterization of TIR NADases that could contribute to drug development for diseases associated with dysregulated TIR activity, this work also highlights the value of CURE-based projects for inclusion of a diverse group of students in authentic research experiences.
Source: ImmunoHorizons
Authors: Tamara Vallejo-Schmidt, Cheyenne Palm, Trinity Obiorah, Abra Rachida Koudjra, Katrina Schmidt, Alexis H Scudder, Eber Guzman-Cruz, Lenora Paige Ingram, Britney C Erickson, Victoria Akingbehin, Terra Riddick, Sarah Hamilton, Tahreem Riaz, Zachary Alexander, Jasmine T Anderson, Charlotte Bader, Phoebe H Calkins, Sumra S Chaudhry, Haley Collins, Maimunah Conteh, Tope A Dada, Jaira David, Daniel Fallah, Raquel De Leon, Rachel Duff, Itohan R Eromosele, Jaliyl K Jones, Nastaran Keshmiri, Mark A Mercanti, Jaine Onwezi-Nwugwo, Michael A Ojo, Emily R Pascoe, Ariana M Poteat, Sarah E Price, Dalton Riedlbauer, Louis T A Rolle, Payton Shoemaker, Alanna Stefano, Michaela K Sterling, Samina Sultana, Lindsey Toneygay, Alexa N Williams, Sheeram Nallar, John E Weldon, Greg A Snyder, Michelle L D Snyder
Institutions: Towson University, University of Maryland, Baltimore
Language: English
Subfield: Immunology
Field: Immunology and Microbiology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $1,802
Awards: Facilitating seamless transitions from community college to Towson University, University System of Maryland, Louis Stokes Alliance for Minority Participation, 2010-2015 (Senior-Level Alliance Project), Bridges to the Doctorate: A Partnership Between Towson University and University of Maryland School of Medicine, Bridges to the Doctorate at Towson University
OpenAlex ID: W4401768394