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Assessment of Immunologically Relevant Dynamic Tertiary Structural Features of the HIV-1 V3 Loop Crown R2 Sequence by ab initio Folding
Published on: September 16, 2010
Refined solution structure and backbone dynamics of HIV-1 Nef
1Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0520, USA. grzesiek@speck.niddk.nih.gov
Summary
This study presents novel NMR methods to determine the structure of HIV-1 Nef, a protein that aggregates easily. These techniques enable structural analysis of challenging, unstable proteins.
Area of Science:
- Structural Biology
- Biochemistry
- Virology
Background:
- HIV-1 Nef protein aggregation at low pH and high mobility of residues complicated structural determination.
- Previous attempts using NMR and X-ray crystallography faced significant challenges.
Purpose of the Study:
- To present methods for determining the three-dimensional structure of HIV-1 Nef using NMR at pH 8.
- To describe the secondary and tertiary structure of HIV-1 Nef.
- To offer generalizable techniques for NMR structure determination of aggregating and unstable proteins.
Main Methods:
- Heteronuclear solution NMR spectroscopy was employed at pH 8 and 0.6 mM concentration.
- Extensive chemical shift assignments were performed for various HIV-1 Nef mutants and complexes.
- 15N relaxation parameters and slowly exchanging amide protons were analyzed to assess protein dynamics and stability.
Main Results:
- The three-dimensional structure of HIV-1 Nef was determined, revealing alpha-helices, a 3(10) helix, and a beta-sheet.
- All secondary structure elements were found to be rigid on picosecond to nanosecond and millisecond timescales.
- Solvent-exposed loops (residues 60-71 and 149-180) exhibited significant internal motions.
- The HIV-1 protease cleavage site (W57/L58) showed no large-amplitude motions, with side chains occupying a hydrophobic crevice.
Conclusions:
- The developed NMR methods are effective for determining the structure of aggregating and unstable proteins like HIV-1 Nef.
- The study provides a detailed structural and dynamic characterization of HIV-1 Nef.
- The findings contribute to understanding HIV-1 protein structure-function relationships and potential therapeutic targets.
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