Protein minimization of the gp120 binding region of human CD4.
CD4 is an important component of the immune system and is also the cellular receptor for HIV-1. CD4 consists of a cytoplasmic tail, one transmembrane region, and four extracellular domains, D1-D4. Constructs consisting of all four extracellular domains of human CD4 as well as the first two domains (CD4D12) have previously been expressed and characterized. All of the gp120-binding residues are located within the first N-terminal domain (D1) of CD4. To date, it has not been possible to obtain domain D1 alone in a soluble and active form. Most residues in CD4 that interact with gp120 lie within the region 21-64 of domain D1 of CD4. On the basis of these observations and analysis of the crystal structure of CD4D12, a mutational strategy was designed to express CD4D1 and region 21-64 of CD4 (CD4PEP1) in Escherichia coli. K(D) values for the binding of CD4 analogues described above to gp120 were measured using a Biacore-based solution-phase competition binding assay. Measured K(D) values were 15 nM, 40 nM, and 26 microM for CD4D12, CD4D1, and CD4PEP1, respectively. All of the proteins interact with gp120 and are able to expose the 17b-binding epitope of gp120. Structural content was determined using CD and proteolysis. Both CD4D1 and CD4PEP1 were partially structured and showed an enhanced structure in the presence of the osmolyte sarcosine. The aggregation behavior of all of the proteins was characterized. While CD4D1 and CD4PEP1 did not aggregate, CD4D12 formed amyloid fibrils at neutral pH within a week at 278 K. These CD4 derivatives should be useful tools in HIV vaccine design and entry inhibition studies.
Sharma
Deepak
D
Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012, India.
Balamurali
M M
MM
Chakraborty
Kausik
K
Kumaran
Sowmini
S
Jeganathan
Sadasivam
S
Rashid
Umar
U
Ingallinella
Paolo
P
Varadarajan
Raghavan
R
D015704:CD4 Antigens / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015699:HIV Envelope Protein gp120 / Q000378:metabolism*; D015497:HIV-1 / Q000378:metabolism; D006801:Humans; D011485:Protein Binding; D011487:Protein Conformation
D015704:CD4 Antigens; D015699:HIV Envelope Protein gp120