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Updated: Jun 17, 2026
From Constructs to Crystals – Towards Structure Determination of β-barrel Outer Membrane Proteins
Published on: July 4, 2016
Crystal structure of the Mp1p ligand binding domain 2 reveals its function as a fatty acid-binding protein
Shuang Liao1, Edward T K Tung, Wei Zheng
1From the Laboratory of Structural Biology, Tsinghua University, Beijing 100084, China.
Abstract:
Penicillium marneffei is a dimorphic, pathogenic fungus in Southeast Asia that mostly afflicts immunocompromised individuals. As the only dimorphic member of the genus, it goes through a phase transition from a mold to yeast form, which is believed to be a requisite for its pathogenicity. Mp1p, a cell wall antigenic mannoprotein existing widely in yeast, hyphae, and conidia of the fungus, plays a vital role in host immune response during infection. To understand the function of Mp1p, we have determined the x-ray crystal structure of its ligand binding domain 2 (LBD2) to 1.3 A. The structure reveals a dimer between the two molecules. The dimer interface forms a ligand binding cavity, in which electron density was observed for a palmitic acid molecule interacting with LBD2 indirectly through hydrogen bonding networks via two structural water molecules. Isothermal titration calorimetry experiments measured the ligand binding affinity (K(d)) of Mp1p at the micromolar level. Mutations of ligand-binding residues, namely S313A and S332A, resulted in a 9-fold suppression of ligand binding affinity. Analytical ultracentrifugation assays demonstrated that both LBD2 and Mp1p are mostly monomeric in vitro, no matter with or without ligand, and our dimeric crystal structure of LBD2 might be the result of crystal packing. Based on the conformation of the ligand-binding pocket in the dimer structure, a model for the closed, monomeric form of LBD2 is proposed. Further structural analysis indicated the biological importance of fatty acid binding of Mp1p for the survival and pathogenicity of the conditional pathogen.
Insights
Penicillium marneffei Mp1p mannoprotein binds fatty acids, crucial for its pathogenicity in immunocompromised individuals. Structural studies reveal ligand binding mechanisms essential for this dimorphic fungus.
Area of Science:
- Medical Mycology
- Structural Biology
- Biochemistry
Background:
- Penicillium marneffei is a dimorphic fungus endemic to Southeast Asia, causing infections primarily in immunocompromised individuals.
- The phase transition from mold to yeast is critical for its pathogenicity.
- Mp1p, a cell wall mannoprotein, is vital for the host immune response during P. marneffei infections.
Purpose of the Study:
- To elucidate the function of Mp1p by determining the x-ray crystal structure of its ligand binding domain 2 (LBD2).
- To investigate the interaction of Mp1p with potential ligands and its role in fungal pathogenicity.
Main Methods:
- X-ray crystallography to determine the structure of Mp1p LBD2 at 1.3 Å resolution.
- Isothermal titration calorimetry (ITC) to measure ligand binding affinity.
- Analytical ultracentrifugation to assess protein quaternary structure in solution.
- Site-directed mutagenesis to probe the role of specific residues in ligand binding.
Main Results:
- The crystal structure revealed a dimeric form of LBD2 with a palmitic acid molecule bound in a cavity formed at the dimer interface.
- ITC experiments showed Mp1p binds ligands with micromolar affinity.
- Mutations in key ligand-binding residues significantly reduced binding affinity.
- Analytical ultracentrifugation indicated that Mp1p and LBD2 are predominantly monomeric in solution, suggesting the observed dimer is likely a crystal packing artifact.
Conclusions:
- Fatty acid binding to Mp1p is biologically important for the survival and pathogenicity of Penicillium marneffei.
- A model for the closed, monomeric form of LBD2 was proposed based on structural insights.
- Understanding Mp1p's ligand-binding mechanism provides potential targets for antifungal therapies.
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