ABSTRACT
The majority of eukaryotic secretory and membrane proteins contain disulfide bonds, which are strongly conserved within protein families because of their crucial role in folding or function. The exact role of these disulfide bonds during folding is unclear. Using virus-driven evolution we generated a viral glycoprotein variant, which is functional despite the lack of an absolutely conserved disulfide bond that links two antiparallel beta-strands in a six-stranded beta-barrel. Molecular dynamics simulations revealed that improved hydrogen bonding and side chain packing led to stabilization of the beta-barrel fold, implying that beta-sheet preference codirects glycoprotein folding in vivo. Our results show that the interactions between two beta-strands that are important for the formation and/or integrity of the beta-barrel can be supported by either a disulfide bond or beta-sheet favoring residues.
Subject(s)
Conserved Sequence , Disulfides/chemistry , Evolution, Molecular , HIV Envelope Protein gp120/chemistry , HIV Envelope Protein gp120/metabolism , HIV-1/chemistry , Protein Folding , Amino Acid Sequence , Antibodies, Viral/chemistry , Computer Simulation , Glycoproteins/chemistry , Glycoproteins/metabolism , HIV-1/immunology , HIV-1/pathogenicity , HIV-1/physiology , HeLa Cells , Humans , Models, Molecular , Molecular Sequence Data , Mutant Proteins/chemistry , Mutant Proteins/metabolism , Protein Structure, Secondary , Virion/chemistry , Virus ReplicationABSTRACT
Protein folding in the endoplasmic reticulum goes hand in hand with disulfide bond formation, and disulfide bonds are considered key structural elements for a protein's folding and function. We used the HIV-1 Envelope glycoprotein to examine in detail the importance of its 10 completely conserved disulfide bonds. We systematically mutated the cysteines in its ectodomain, assayed the mutants for oxidative folding, transport, and incorporation into the virus, and tested fitness of mutant viruses. We found that the protein was remarkably tolerant toward manipulation of its disulfide-bonded structure. Five of 10 disulfide bonds were dispensable for folding. Two of these were even expendable for viral replication in cell culture, indicating that the relevance of these disulfide bonds becomes manifest only during natural infection. Our findings refine old paradigms on the importance of disulfide bonds for proteins.
Subject(s)
Disulfides , HIV Envelope Protein gp120/chemistry , HIV Envelope Protein gp160/chemistry , Alanine/chemistry , CD4-Positive T-Lymphocytes/virology , Cysteine/chemistry , Disulfides/chemistry , Endoplasmic Reticulum/metabolism , HeLa Cells , Humans , Mutagenesis, Site-Directed , Oxygen/chemistry , Protein Binding , Protein Folding , Protein Structure, Tertiary , Protein TransportABSTRACT
BACKGROUND: The HIV-1 envelope glycoprotein gp120, which mediates viral attachment to target cells, consists for approximately 50% of sugar, but the role of the individual sugar chains in various aspects of gp120 folding and function is poorly understood. Here we studied the role of the carbohydrate at position 386. We identified a virus variant that had lost the 386 glycan in an evolution study of a mutant virus lacking the disulfide bond at the base of the V4 domain. RESULTS: The 386 carbohydrate was not essential for folding of wt gp120. However, its removal improved folding of a gp120 variant lacking the 385-418 disulfide bond, suggesting that it plays an auxiliary role in protein folding in the presence of this disulfide bond. The 386 carbohydrate was not critical for gp120 binding to dendritic cells (DC) and DC-mediated HIV-1 transmission to T cells. In accordance with previous reports, we found that N386 was involved in binding of the mannose-dependent neutralizing antibody 2G12. Interestingly, in the presence of specific substitutions elsewhere in gp120, removal of N386 did not result in abrogation of 2G12 binding, implying that the contribution of N386 is context dependent. Neutralization by soluble CD4 and the neutralizing CD4 binding site (CD4BS) antibody b12 was significantly enhanced in the absence of the 386 sugar, indicating that this glycan protects the CD4BS against antibodies. CONCLUSION: The carbohydrate at position 386 is not essential for protein folding and function, but is involved in the protection of the CD4BS from antibodies. Removal of this sugar in the context of trimeric Env immunogens may therefore improve the elicitation of neutralizing CD4BS antibodies.
Subject(s)
Asparagine/chemistry , Carbohydrates/immunology , Dendritic Cells/metabolism , HIV Envelope Protein gp120/chemistry , HIV Envelope Protein gp120/immunology , HIV-1/immunology , Protein Folding , Asparagine/metabolism , Asparagine/physiology , Binding Sites , CD4 Antigens/immunology , Carbohydrates/chemistry , HIV Envelope Protein gp120/genetics , HIV-1/chemistry , HeLa Cells , Humans , Neutralization TestsABSTRACT
Most principles of protein folding emerged from refolding studies in vitro on small, soluble proteins, because large ones tend to misfold and aggregate. We developed a folding assay allowing the study of large proteins in detergent such that the extent of cellular assistance required for proper folding can be determined. We identified a critical step in the in vivo folding pathway of influenza virus hemagglutinin (HA). Only the formation of the first few disulfides in the top domain of HA required the intact endoplasmic reticulum. After that, HA proceeded to fold efficiently in a very dilute solution, despite its size and complexity. This study paves the way for detailed structural analyses during the folding of complex proteins.
