In the innate immune system, Toll-like receptors (TLRs) provide a front-line defense against invading bacteria, viruses, fungi and protozoa. Intriguingly, TLRs bind their """"""""non-self"""""""" cognate ligand without known maturation or selection. One set of TLR ligands, the Outer Membrane Proteins (OMPs), or porins, are transmembrane 2-barrel proteins. The method of universal recognition of a group of proteins that have large variability in their physical properties is difficult to envision. We hypothesize that TLRs initially scan outer membrane proteins based on electrostatic attraction. We further hypothesize that once TLRs are attracted to a membrane protein, they bind to main chain structural elements thus differentiating 2-strands from 1-helices. The goal of this 2-year proposal is to identify how electrostatics contribute to the recognition of OMPs by TLRs. Specifically, we plan to: 1. Identify the structure of the TLR2-PorB complex. We have already determined the structure of PorB by x-ray crystallography, such that both PorB and TLR2 now have available high-resolution structures. We have further co-purified the complex and taken initial electron microscopy imagers to show feasibility of determination of a co-structure. 2. Investigate the contributions of electrostatics to the affinity of the TLR2-PorB complex. We will use salt and chemical disruption to identify if charge-only effects contribute to the affinity of the TLR2-complex. Specifically, we will identify how methylation and acetylation of lysine side chains affects complex affinity. 3. Identify additional combinations of innate immunity receptors that bind OMPs in vitro. While TLR2 and PorB form one signaling complex, recognition of OMPs by other combinations of TLRs may result in different physiological responses. We have cloned 5 innate immunity receptors and 3 OMPs to identify which combinations of receptors and porins are capable of forming a complex.

Public Health Relevance

We are working to define the mechanisms of recognition between toll-like receptors and outer membrane proteins using a structural approach. We use a hybrid of electron microscopy, NMR, and crystallography to investigate this recognition complex, which spans two membranes in vivo.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Exploratory/Developmental Grants (R21)
Project #
1R21AI079558-01A1
Application #
7790153
Study Section
Biochemistry and Biophysics of Membranes Study Section (BBM)
Program Officer
Leitner, Wolfgang W
Project Start
2010-09-16
Project End
2012-08-31
Budget Start
2010-09-16
Budget End
2011-08-31
Support Year
1
Fiscal Year
2010
Total Cost
$229,991
Indirect Cost
Name
Vanderbilt University Medical Center
Department
Pharmacology
Type
Schools of Medicine
DUNS #
004413456
City
Nashville
State
TN
Country
United States
Zip Code
37212
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