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From Constructs to Crystals – Towards Structure Determination of β-barrel Outer Membrane Proteins
Published on: July 4, 2016
Three-dimensional structure and biophysical characterization of Staphylococcus aureus cell surface antigen-manganese
Alexey Gribenko1, Lidia Mosyak, Sharmistha Ghosh
1Pfizer Vaccine Research, 401 North Middletown Road, Pearl River, NY 10965, USA. Alexey.Gribenko@pfizer.com
Abstract:
MntC is a metal-binding protein component of the Mn²⁺-specific mntABC transporter from the pathogen Staphylococcus aureus. The protein is expressed during the early stages of infection and was proven to be effective at reducing both S. aureus and Staphylococcus epidermidis infections in a murine animal model when used as a vaccine antigen. MntC is currently being tested in human clinical trials as a component of a multiantigen vaccine for the prevention of S. aureus infections. To better understand the biological function of MntC, we are providing structural and biophysical characterization of the protein in this work. The three-dimensional structure of the protein was solved by X-ray crystallography at 2.2Å resolution and suggests two potential metal binding modes, which may lead to reversible as well as irreversible metal binding. Precise Mn²⁺-binding affinity of the protein was determined from the isothermal titration calorimetry experiments using a competition approach. Differential scanning calorimetry experiments confirmed that divalent metals can indeed bind to MntC reversibly as well as irreversibly. Finally, Mn²⁺-induced structural and dynamics changes have been characterized using spectroscopic methods and deuterium-hydrogen exchange mass spectroscopy. Results of the experiments show that these changes are minimal and are largely restricted to the structural elements involved in metal coordination. Therefore, it is unlikely that antibody binding to this antigen will be affected by the occupancy of the metal-binding site by Mn²⁺.
Insights
MntC, a Staphylococcus aureus protein, binds manganese (Mn²⁺) and is a promising vaccine antigen. Its structure and metal-binding properties are characterized, showing minimal changes upon metal binding, unlikely to affect antibody recognition.
Area of Science:
- Microbiology
- Structural Biology
- Immunology
Background:
- MntC is a manganese-binding protein from Staphylococcus aureus, a pathogen causing significant infections.
- It is expressed during early infection stages and shows potential as a vaccine antigen against S. aureus and S. epidermidis.
- MntC is currently in clinical trials as part of a multi-antigen vaccine for S. aureus prevention.
Purpose of the Study:
- To elucidate the biological function of MntC through structural and biophysical characterization.
- To understand the metal-binding properties and their impact on protein structure and dynamics.
- To assess the potential influence of metal binding on antibody recognition for vaccine development.
Main Methods:
- X-ray crystallography for 3D structure determination at 2.2Å resolution.
- Isothermal titration calorimetry (ITC) for precise Mn²⁺ binding affinity determination.
- Differential scanning calorimetry (DSC) to confirm metal binding reversibility.
- Spectroscopic methods and deuterium-hydrogen exchange mass spectrometry (DXMS) for structural and dynamics analysis.
Main Results:
- The 3D structure reveals two potential metal binding modes, suggesting reversible and irreversible binding.
- ITC experiments precisely quantified Mn²⁺ binding affinity.
- DSC confirmed that MntC binds divalent metals both reversibly and irreversibly.
- Mn²⁺ binding induces minimal structural and dynamic changes, localized to metal coordination sites.
Conclusions:
- MntC exhibits complex metal-binding characteristics with potential for both reversible and irreversible manganese binding.
- Structural and dynamic changes upon Mn²⁺ binding are minimal and localized.
- These localized changes suggest that metal occupancy is unlikely to hinder antibody binding to MntC, supporting its use as a vaccine antigen.
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