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Characterization and molecular basis of the oligomeric structure of HIV-1 nef protein.

ABSTRACT: The Nef protein of human immunodeficiency virus type I (HIV-1) is an important determinant for the onset of AIDS disease. The self-association properties of HIV-1 Nef are analyzed by chemical cross-linking, dynamic light scattering, equilibrium analytical ultracentrifugation, and NMR spectroscopy. The experimental data show that the HIV-1 Nef core domain forms stable homo-dimers and trimers in solution, but not higher oligomers. These Nef homomers are not covalently linked by disulfide bridges, and the equilibrium between these forms is dependent on the Nef concentration. We further provide the molecular basis for the Nef core dimers and trimers obtained by analysis of crystallographic models. Oligomerization of biological polypeptides is a common tool used to trigger events in cellular signaling and endocytosis, both of which are targeted by Nef. The quaternary structure of Nef may be of physiological importance and may help to connect its cellular targets or to increase affinity of the viral molecule for its ligands. The herein described models for Nef dimers and trimers will allow further mutational studies to elucidate their role in vivo. These results provide novel insight into the structural and functional relationships of this important viral protein. Moreover, the oligomer interface may represent a novel target for the design of antiviral agents.

SUBMITTER: Arold S

PROVIDER: S-EPMC2144662 | biostudies-other | 2000 Jun

REPOSITORIES: biostudies-other

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Characterization and molecular basis of the oligomeric structure of HIV-1 nef protein.

Arold S S Hoh F F Domergue S S Birck C C Delsuc M A MA Jullien M M Dumas C C

Protein science : a publication of the Protein Society 20000601 6

The Nef protein of human immunodeficiency virus type I (HIV-1) is an important determinant for the onset of AIDS disease. The self-association properties of HIV-1 Nef are analyzed by chemical cross-linking, dynamic light scattering, equilibrium analytical ultracentrifugation, and NMR spectroscopy. The experimental data show that the HIV-1 Nef core domain forms stable homo-dimers and trimers in solution, but not higher oligomers. These Nef homomers are not covalently linked by disulfide bridges, ...[more]

PMID: 10892807

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Project description:The tendency of HIV-1 Nef to form aggregates in solution, particularly at pH values below 8, together with its large fraction of highly mobile residues seriously complicated determination of its three-dimensional structure, both for heteronuclear solution NMR (Grzesiek et al., 1996a, Nat Struct Biol 3:340-345) and for X-ray crystallography (Lee et al., 1996, Cell 85:931-942). Methods used to determine the Nef structure by NMR at pH 8 and 0.6 mM concentration are presented, together with a detailed description of Nef's secondary and tertiary structure. The described techniques have general applicability for the NMR structure determination of proteins that are aggregating and/or have limited stability at low pH values. Extensive chemical shift assignments are reported for backbone and side chain 1H, 13C, and 15N resonances of the HIV-1 Nef deletion mutants NEF delta 2-39, NEF delta 2-39, delta 159-173, and of NEF delta 2-39, delta 159-173 in complex with the SH3 domain of the Hck tyrosine protein kinase. Besides a type II polyproline helix, Nef's structure consists of three alpha-helices, a 3(10) helix, and a five-stranded anti-parallel beta-sheet. The analysis of 15N relaxation parameters of the backbone amide sites reveals that all the secondary structure elements are non-mobile on the picosecond to nanosecond and on the millisecond time scale. A large number of slowly exchanging amide protons provides evidence for the stability of the Nef core even on the time scale of hours. Significant internal motions on the ps to ns time scale are detected for residues 60 to 71 and for residues 149 to 180, which form solvent-exposed loops. The residues of the HIV-1 protease cleavage site (W57/L58) do not exhibit large amplitude motions on the sub-nanosecond time scale, and their side chains insert themselves into a hydrophobic crevice formed between the C-terminus of helix 1 and the N-terminus of helix 2. A refined structure has been determined based on additional constraints for side-chain and backbone dihedral angles derived from a large number of three-bond J-coupling and ROE data.

Project description:BackgroundProtein-protein association is essential for a variety of cellular processes and hence a large number of investigations are being carried out to understand the principles of protein-protein interactions. In this study, oligomeric protein structures are viewed from a network perspective to obtain new insights into protein association. Structure graphs of proteins have been constructed from a non-redundant set of protein oligomer crystal structures by considering amino acid residues as nodes and the edges are based on the strength of the non-covalent interactions between the residues. The analysis of such networks has been carried out in terms of amino acid clusters and hubs (highly connected residues) with special emphasis to protein interfaces.ResultsA variety of interactions such as hydrogen bond, salt bridges, aromatic and hydrophobic interactions, which occur at the interfaces are identified in a consolidated manner as amino acid clusters at the interface, from this study. Moreover, the characterization of the highly connected hub-forming residues at the interfaces and their comparison with the hubs from the non-interface regions and the non-hubs in the interface regions show that there is a predominance of charged interactions at the interfaces. Further, strong and weak interfaces are identified on the basis of the interaction strength between amino acid residues and the sizes of the interface clusters, which also show that many protein interfaces are stronger than their monomeric protein cores. The interface strengths evaluated based on the interface clusters and hubs also correlate well with experimentally determined dissociation constants for known complexes. Finally, the interface hubs identified using the present method correlate very well with experimentally determined hotspots in the interfaces of protein complexes obtained from the Alanine Scanning Energetics database (ASEdb). A few predictions of interface hot spots have also been made based on the results obtained from this analysis, which await experimental verification.ConclusionThe construction and analysis of oligomeric protein structure networks and their comparison with monomeric protein structure networks provide insights into protein association. Further, the interface hubs identified using the present method can be effective targets for interface de-stabilizing mutations. We believe this analysis will significantly enhance our knowledge of the principles behind protein association and also aid in protein design.

Dataset Information

Characterization and molecular basis of the oligomeric structure of HIV-1 nef protein.

Publications

Characterization and molecular basis of the oligomeric structure of HIV-1 nef protein.

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