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Multi-target Parallel Processing Approach for Gene-to-structure Determination of the Influenza Polymerase PB2 Subunit
Published on: June 28, 2013
Solution and crystallographic structures of the central region of the phosphoprotein from human metapneumovirus
Cedric Leyrat1, Max Renner, Karl Harlos
1Division of Structural Biology, University of Oxford, Oxford, United Kingdom.
Abstract:
Human metapneumovirus (HMPV) of the family Paramyxoviridae is a major cause of respiratory illness worldwide. Phosphoproteins (P) from Paramyxoviridae are essential co-factors of the viral RNA polymerase that form tetramers and possess long intrinsically disordered regions (IDRs). We located the central region of HMPV P (P(ced)) which is involved in tetramerization using disorder analysis and modeled its 3D structure ab initio using Rosetta fold-and-dock. We characterized the solution-structure of P(ced) using small angle X-ray scattering (SAXS) and carried out direct fitting to the scattering data to filter out incorrect models. Molecular dynamics simulations (MDS) and ensemble optimization were employed to select correct models and capture the dynamic character of P(ced). Our analysis revealed that oligomerization involves a compact central core located between residues 169-194 (P(core)), that is surrounded by flexible regions with α-helical propensity. We crystallized this fragment and solved its structure at 3.1 Å resolution by molecular replacement, using the folded core from our SAXS-validated ab initio model. The RMSD between modeled and experimental tetramers is as low as 0.9 Å, demonstrating the accuracy of the approach. A comparison of the structure of HMPV P to existing mononegavirales P(ced) structures suggests that P(ced) evolved under weak selective pressure. Finally, we discuss the advantages of using SAXS in combination with ab initio modeling and MDS to solve the structure of small, homo-oligomeric protein complexes.
Insights
Researchers elucidated the tetrameric structure of human metapneumovirus phosphoprotein (HMPV P) using advanced modeling and SAXS. This reveals a compact core crucial for viral RNA polymerase function in respiratory illness.
Area of Science:
- Structural Biology
- Virology
- Biochemistry
Background:
- Human metapneumovirus (HMPV) is a significant cause of global respiratory illness.
- Paramyxoviridae phosphoproteins (P) are vital RNA polymerase co-factors, forming tetramers with intrinsically disordered regions (IDRs).
- Understanding HMPV P structure is key to its role in viral replication.
Purpose of the Study:
- To determine the 3D structure of the tetramerization domain of HMPV P (P(ced)).
- To investigate the oligomerization mechanism and structural dynamics of HMPV P.
- To validate computational modeling approaches for protein structure determination.
Main Methods:
- Disorder analysis and ab initio modeling (Rosetta fold-and-dock) to predict P(ced) structure.
- Small-angle X-ray scattering (SAXS) for solution structure characterization and model validation.
- Molecular dynamics simulations (MDS) and ensemble optimization for dynamic analysis.
- X-ray crystallography to solve the high-resolution structure of the P(core) fragment.
Main Results:
- Identified a compact central core (P(core), residues 169-194) essential for HMPV P tetramerization.
- Validated ab initio models against SAXS data, achieving high accuracy (RMSD of 0.9 Å) with experimental crystal structure.
- Revealed that HMPV P(ced) structure evolved under weak selective pressure compared to other mononegavirales.
Conclusions:
- The study successfully determined the high-resolution structure of the HMPV P tetramerization domain.
- Combined SAXS, ab initio modeling, and MDS is a powerful strategy for studying dynamic oligomeric protein complexes.
- Structural insights into HMPV P may inform antiviral strategies against respiratory infections.
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