Structural characterization of the transmembrane and cytoplasmic domains of human CD4.
Cluster determinant 4 (CD4) is a type I transmembrane glycoprotein of 58 kDa. It consists of an extracellular domain of 370 amino acids, a short transmembrane region, and a cytoplasmic domain of 40 amino acids at the C-terminal end. We investigated the structure of the 62 C-terminal residues of CD4, comprising its transmembrane and cytoplasmic domains. The five cysteine residues of this region have been replaced with serine and histidine residues in the polypeptide CD4mut. Uniformly 15N and 13C labeled protein was recombinantly expressed in E. coli and purified. Functional binding activity of CD4mut to protein VpU of the human immunodeficiency virus type 1 (HIV-1) was verified. Close to complete NMR resonance assignment of the 1H, 13C, and 15N spins of CD4mut was accomplished. The secondary structure of CD4mut in membrane simulating dodecylphosphocholine (DPC) micelles was characterized based on secondary chemical shift analysis, NOE-based proton-proton distances, and circular dichroism spectroscopy. A stable transmembrane helix and a short amphipathic helix in the cytoplasmic region were identified. The fractional helicity of the cytoplasmic helix appears to be stabilized in the presence of DPC micelles, although the extension of this helix is reduced in comparison to previous studies on synthetic peptides in aqueous solution. The role of the amphipathic helix and its potentially variable length is discussed with respect to the biological functions of CD4.
Forschungszentrum Jülich, Institut für Neurowissenschaften und Biophysik (INB-2), 52425 Jülich, Germany.
Marc
M
Wittlich
Bernd W
BW
Koenig
Silke
S
Hoffmann
Dieter
D
Willbold
D000595:Amino Acid Sequence; D015704:CD4 Antigens / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D002247:Carbon Isotopes; D002942:Circular Dichroism; D054298:Human Immunodeficiency Virus Proteins / Q000378:metabolism; D006801:Humans; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009587:Nitrogen Isotopes; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D054334:Viral Regulatory and Accessory Proteins / Q000378:metabolism
D015704:CD4 Antigens; D002247:Carbon Isotopes; D054298:Human Immunodeficiency Virus Proteins; D009587:Nitrogen Isotopes; D054334:Viral Regulatory and Accessory Proteins