Characterization of the Structural Requirements for the NADase Activity of Bacterial Toll/IL-1R domains in a Course-based Undergraduate Research Experience

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.

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Publication Details

Journal
ImmunoHorizons
Published
2024-08-01
DOI
https://doi.org/10.4049/immunohorizons.2300062
Citations
2
Primary Topic
Antimicrobial Peptides and Activities
Type
article
Field-Weighted Citation Impact
0.69

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article

Characterization of the Structural Requirements for the NADase Activity of Bacterial Toll/IL-1R domains in a Course-based Undergraduate Research Experience

John E. Weldon, Katrina Schmidt, Greg A. Snyder, E. Pascoe et al.
2 citations
ImmunoHorizons
Antimicrobial Peptides and Activities
0.69
article

Characterization of the Structural Requirements for the NADase Activity of Bacterial Toll/IL-1R domains in a Course-based Undergraduate Research Experience

John E. Weldon, Katrina Schmidt, Greg A. Snyder, E. Pascoe, Terra Riddick, Alanna M. Stefano, Trinity Obiorah, Jasmine T Anderson, Sarah Hamilton, C. A. Palm, Michelle Snyder, Sumra Chaudhry, Raquel De Leon, J David, Alexis H. Scudder, P. B. Shoemaker, Phoebe Calkins, Tamara Vallejo-Schmidt, Victoria Akingbehin, Tahreem Riaz, Eber Guzman-Cruz, Britney C Erickson, Maimunah Conteh, Tope A Dada, Itohan R Eromosele, Nastaran Keshmiri, Mark A Mercanti, Michael A Ojo, Ariana M Poteat, Dalton Riedlbauer, Abra Rachida Koudjra, Lenora Paige Ingram, Daniel Fallah, Rachel Duff, Jaine Onwezi-Nwugwo, Lindsey Toneygay, Samina Sultana, Z. Alexander, Jaliyl K Jones, Alexa N Williams, Charlotte Bader, Sarah E Price, Louis T A Rolle, M. Sterling, Haley Collins, Sheeram Nallar
article en
2 citations

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.

ImmunoHorizonsVol. 8(8)
University of Maryland, Baltimore (US), Towson University (US)
National Science Foundation, University of Maryland, Baltimore County, Towson University, National Institutes of Health, Jess and Mildred Fisher College of Science and Mathematics, National Institute of General Medical Sciences
Openalex Percentile: Top 29%
Antimicrobial Peptides and Activities
0.69
DOIOpen Access PDFOpenAlex
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