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) wi th its primary cellular receptor, the glycoprotein CD4. Molecular structure information about the HIV-gp120/CD4 complex can provide information releva nt to an understanding of the basic processes occurring in HIV infection an d to development of therapies that can inhibit AIDS. Previous studies by su gar gradient sedimentation of the interaction of HIV-gp120 with a cytoplasm ic domain truncated soluble CD4 (sCD4) suggested that a one-to-one complex was formed. The stoichiometry, however, of the sCD4/HIV-gp120 complex remai ned to be confirmed by an independent method because (i) recent X-ray exami nation revealed dimerization of sCD4 and (ii) the low resolution and low ac curacy of molecular weight determination by sugar gradient sedimentation ca n lead to artifactual data. Therefore, in this study matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was used to determine th e 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 ma sses of one HIV-gp120 and one sCD4 molecule. Complexes of higher order of s toichiometry were not detected. Identical results were obtained by chemical ly cross-linking the HIV-gp120/sCD4 complex with subsequent analysis by sod ium dodecyl sulfate-polyacrylamide gel electrophoresis and MALDI-MS. This s tudy confirms the earlier suggestions of the stoichiometry of the sCD4/HIV- gp120 complex in solution and also demonstrates the potential of MALDI-MS i n investigations of specific noncovalent complexes of glycoproteins.