title: Design and characterization of stabilized derivatives of human CD4D12 and CD4D1.
abstract: CD4 is present on the surface of T-lymphocytes and is the primary cellular receptor for HIV-1. CD4 consists of a cytoplasmic tail, one transmembrane region, and four extracellular domains, D1-D4. A construct consisting of the first two domains of CD4 (CD4D12) is folded and binds gp120 with similar affinity as soluble 4-domain CD4 (sCD4). However, the first domain alone (CD4D1) was previously shown to be largely unfolded and had 3-fold weaker affinity for gp120 when compared to sCD4 [Sharma, D.; et al. (2005) Biochemistry 44, 16192-16202]. We now report the design and characterization of three single-site mutants of CD4D12 (G6A, L51I, and V86L) and one multisite mutant of CD4D1 (G6A/L51I/L5K/F98T). G6A, L51I, and V86L are cavity-filling mutations while L5K and F98T are surface mutations which were introduced to minimize the aggregation of CD4D1 upon removal of the second domain. Two mutations, G6A and V86L in CD4D12 increased the stability and yield of the protein relative to the wild-type protein. The mutant CD4D1 (CD4D1a) with the 4 mutations was folded and more stable compared to the original CD4D1, but both bound gp120 with comparable affinity. In in vitro neutralization assays, both CD4D1a and G6A-CD4D12 were able to neutralize diverse HIV-1 viruses with similar IC(50)s as 4-domain CD4. These stabilized derivatives of human CD4 can be useful starting points for the design of other more complex viral entry inhibitors.
authors.affiliation: Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012, India.
authors.forename: Piyali
authors.initials: P
authors.lastname: Saha
authors.forename: Bipasha
authors.initials: B
authors.lastname: Barua
authors.forename: Sanchari
authors.initials: S
authors.lastname: Bhattacharyya
authors.forename: M M
authors.initials: MM
authors.lastname: Balamurali
authors.forename: William R
authors.initials: WR
authors.lastname: Schief
authors.forename: David
authors.initials: D
authors.lastname: Baker
authors.forename: Raghavan
authors.initials: R
authors.lastname: Varadarajan
mesh_terms: D000998:Antiviral Agents / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology; D015704:CD4 Antigens / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D015699:HIV Envelope Protein gp120 / Q000037:antagonists & inhibitors / Q000378:metabolism; D006801:Humans; D009154:Mutation; D011485:Protein Binding / Q000235:genetics; D017510:Protein Folding; D055550:Protein Stability; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D053586:Virus Internalization
chemical_list: D000998:Antiviral Agents; D015704:CD4 Antigens; D015699:HIV Envelope Protein gp120