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PreprintCited by 9Bacillus and Francisella bacterial researchEnzyme Structure and FunctionProtein Structure and Dynamics

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Structural and biophysical properties of FopA, a major outer membrane protein of Francisella tularensis

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Nagaratnam, Nirupa, Martin-Garcia, Jose M., Yang, Jay-How, Goode, Matthew R., Ketawala, Gihan, Craciunescu, Felicia M., Zook, James D., Grant, Thomas D., Fromme, Raimund, Hansen, Debra T., Fromme, Petra·2022·PLoS ONE
DOI 10.1101/2022.04.08.487631PMC PMC9342783

Preprint — published as 10.1371/journal.pone.0267370

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Abstract

Francisella tularensis is an extremely infectious pathogen and a category A bioterrorism agent. It causes the highly contagious zoonosis, Tularemia. Currently, FDA approved vaccines against tularemia are unavailable. F. tularensis outer membrane protein A (FopA) is a well-studied virulence determinant and protective antigen against tularemia. It is a major outer membrane protein (Omp) of F. tularensis . However, FopA-based therapeutic intervention is hindered due to lack of complete structural information for membrane localized mature FopA. In our study, we established recombinant expression, monodisperse purification, crystallization and X-ray diffraction (∼6.5 Å) of membrane localized mature FopA. Further, we performed bioinformatics and biophysical experiments to unveil its structural organization in the outer membrane. FopA consists of 393 amino acids and has less than 40% sequence identity to known bacterial Omps. Using comprehensive sequence alignments and structure predictions together with existing partial structural information, we propose a two-domain organization for FopA. Circular dichroism spectroscopy and heat modifiability assay confirmed FopA has a β-barrel domain consistent with alphafold2’s prediction of an eight stranded β-barrel at the N-terminus. Small angle X-ray scattering (SAXS) and native-polyacrylamide gel electrophoresis revealed FopA purified in detergent micelles is predominantly dimeric. Molecular density derived from SAXS at 31 Å shows putative dimeric N-terminal β-barrels surrounded by detergent corona and connected to C-terminal domains via flexible linker. Disorder analysis predicts N- and C-terminal domains are interspersed by a long intrinsically disordered region and alphafold2 predicts this region to be largely unstructured. Taken together, we propose a dimeric, two-domain organization of FopA in the outer membrane: the N-terminal β-barrel is membrane embedded, provides dimerization interface and tethers to membrane extrinsic C-terminal domain via long flexible linker. Structure determination of membrane localized mature FopA is essential to understand its role in pathogenesis and develop anti-tularemia therapeutics. Our results pave the way towards it.

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Metadata

Authors
Nagaratnam, N., Martin-Garcia, J. M., Yang, J.-H., Goode, M. R., Ketawala, G., Craciunescu, F. M., Zook, J. D., Grant, T. D., Fromme, R., Hansen, D. T., & Fromme, P.
Affiliations
  • Arizona State University (US)
  • University at Buffalo, State University of New York (US)
Corresponding author
Fromme, Petra
ORCID
Venue
PLoS ONE
Publisher
openRxiv
Published
2022-04-09
Volume
17
Issue
8
Pages
e0267370-e0267370
ISSN
1932-6203 (ISSN-L)
DOI
10.1101/2022.04.08.487631
PMC
PMC9342783
OpenAlex
W4289262218
Cited by
9
References
1
Citations by year
2026: 3 · 2025: 1 · 2024: 4 · 2023: 1
FWCI
0.49
Type
journal article
Language
en
Open access
Licence
cc-by
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • National Science Foundation (DE-AC02–06CH11357, 1542160, 1231306)
  • U.S. Department of Energy (DE‐ AC02‐06CH11357, 1542160, Contract No. DE-AC02-06CH11357., AC02‐06CH11357, 06CH11357, No. DE-AC02-06CH11357, P30 GM138395, NNCI-ECCS-1542160, DE-AC02-)
  • Arizona State University (NNCI‐ECCS‐1542160, ECCS-1542160)
  • National Institutes of Health (NNCI-ECCS-1542160, NIH-R01, DE-AC02–06CH11357, NIH U54, 1s10od018090-01, GM094599)
  • Directorate for Biological Sciences (BIOXFEL STC 1231306)
  • Office of Science (Contract No. DE-AC02-06CH11357, AC02‐06CH11357, DE-AC02, No. DE-AC02-06CH11357., 06CH11357, DE-AC02-06CH11357)
  • Biodesign Institute, Arizona State University (PG06509)
  • Eyring Materials Center, Arizona State University (NNCI-ECCS-1542160)
  • National Institute of General Medical Sciences (GM095583, GM138395, U54GM094599, 1S10OD018090, R01GM133998, DE-AC02-06CH11357, R01GM095583, GM094599, P30GM138395)
  • Division of Electrical, Communications and Cyber Systems (1542160, ECCS-1542160, NNCI-ECCS-1542160)
  • Argonne National Laboratory (DE- AC02-06-CH11357, DE-AC02, AC02–06CH11357, No. DE-AC02-06CH11357, 06CH11357, Contract No. DE-AC02-06CH11357., 1S10OD018090, P30 GM138395)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Bacillus and Francisella bacterial research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Structural Biology › Enzyme Structure and Function
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Structure and Dynamics
Keywords
Francisella tularensis, outer membrane proteins, tularemia, protein structure, C-terminal domain, intrinsically disordered regions, protein dimerization, detergent micelles, protective antigen, virulence factors
MeSH terms
Detergents, Francisella tularensis, Humans, Tularemia, X-Ray Diffraction, Scattering, Small Angle

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