Characterization of the noncovalent complex of human immunodeficiency virus glycoprotein 120 with its cellular receptor CD4 by matrix-assisted laser desorption/ionization mass spectrometry.
The initial event in infection by the human immunodeficiency virus type 1 (HIV-1) is the interaction of the viral envelope glycoprotein (HIV-gp120) with its primary cellular receptor, the glycoprotein CD4. Molecular structure information about the HIV-gp120/CD4 complex can provide information relevant to an understanding of the basic processes occurring in HIV infection and to development of therapies that can inhibit AIDS. Previous studies by sugar gradient sedimentation of the interaction of HIV-gp120 with a cytoplasmic domain truncated soluble CD4 (sCD4) suggested that a one-to-one complex was formed. The stoichiometry, however, of the sCD4/HIV-gp120 complex remained to be confirmed by an independent method because (i) recent X-ray examination revealed dimerization of sCD4 and (ii) the low resolution and low accuracy of molecular weight determination by sugar gradient sedimentation can lead to artifactual data. Therefore, in this study matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was used to determine the molecular mass of the complex of fully glycosylated HIV-gp120 and sCD4. A mass of 145 kDa was measured, which is exactly the sum of the molecular masses of one HIV-gp120 and one sCD4 molecule. Complexes of higher order of stoichiometry were not detected. Identical results were obtained by chemically cross-linking the HIV-gp120/sCD4 complex with subsequent analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and MALDI-MS. This study confirms the earlier suggestions of the stoichiometry of the sCD4/HIV-gp120 complex in solution and also demonstrates the potential of MALDI-MS in investigations of specific noncovalent complexes of glycoproteins.
Borchers
C
C
Laboratory of Structural Biology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
Tomer
K B
KB
D015704:CD4 Antigens / Q000737:chemistry / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D015699:HIV Envelope Protein gp120 / Q000737:chemistry / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D015704:CD4 Antigens; D003432:Cross-Linking Reagents; D015699:HIV Envelope Protein gp120