title: Kinetic and structural analysis of mutant CD4 receptors that are defective in HIV gp120 binding.
abstract: The T-cell antigen coreceptor CD4 also serves as the receptor for the envelope glycoprotein gp120 of HIV. Extensive mutational analysis of CD4 has implicated residues from a portion of the extracellular amino-terminal domain (D1) in gp120 binding. However, none of these proteins has been fully characterized biophysically, and thus the precise effects on molecular structure and binding interactions are unknown. In the present study, we produced soluble versions of three mutant CD4 molecules (F43V, G47S, and A55F) and characterized their structural properties, thermostability, and ability to bind gp120. Crystallographic and thermodynamic analysis showed minimal structural alterations in the F43V and G47S mutant proteins, which have solvent-exposed mutant side chains. In contrast, some degree of disorder appears to exist in the folded state of A55F, as a result of mutating a buried side chain. Real time kinetic measurements of the interaction of the mutant proteins with gp120 showed affinity decreases of 5-fold for G47S, 50-fold for A55F, and 200-fold for F43V. Although both rate constants for the binding reaction were affected by these mutations, the loss in affinity was mainly due to a decrease in on rates, with less drastic changes occurring in the off rates. These observations suggest the involvement of conformational adaptation in the CD4-gp120 interaction. Together, the structural and kinetic data confirm that F43V is a critical residue in gp120 recognition site, which may also include main chain interactions at residue Gly-47.
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032, USA.
authors.forename: H
authors.initials: H
authors.lastname: Wu
authors.forename: D G
authors.initials: DG
authors.lastname: Myszka
authors.forename: S W
authors.initials: SW
authors.lastname: Tendian
authors.forename: C G
authors.initials: CG
authors.lastname: Brouillette
authors.forename: R W
authors.initials: RW
authors.lastname: Sweet
authors.forename: I M
authors.initials: IM
authors.lastname: Chaiken
authors.forename: W A
authors.initials: WA
authors.lastname: Hendrickson
mesh_terms: D001665:Binding Sites; D015704:CD4 Antigens / Q000737:chemistry* / Q000378:metabolism*; D018360:Crystallography, X-Ray; D015699:HIV Envelope Protein gp120 / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013816:Thermodynamics; D014508:Urea
chemical_list: D015704:CD4 Antigens; D015699:HIV Envelope Protein gp120; D011994:Recombinant Proteins; D014508:Urea