Abstract
HIV-1 entry into host cells is mediated by the sequential binding of the envelope glycoprotein gp120 to CD4 and a chemokine receptor. Antibodies binding to epitopes overlapping the CD4-binding site on gp120 are potent inhibitors of HIV entry, such as the llama heavy chain antibody fragment VHH D7, which has cross-clade neutralizing properties and competes with CD4 and mAb b12 for high affinity binding to gp120. We report the crystal structure of the D7 VHH at 1.5 Å resolution, which reveals the molecular details of the complementarity determining regions (CDR) and substantial flexibility of CDR3 that could facilitate an induced fit interaction with gp120. Structural comparison of CDRs from other CD4 binding site antibodies suggests diverse modes of interaction. Mutational analysis identified CDR3 as a key component of gp120 interaction as determined by surface plasmon resonance. A decrease in affinity is directly coupled to the neutralization efficiency since mutations that decrease gp120 interaction increase the IC50 required for HIV-1 IIIB neutralization. Thus the structural study identifies the long CDR3 of D7 as the key determinant of interaction and HIV-1 neutralization. Furthermore, our data confirm that the structural plasticity of gp120 can accommodate multiple modes of antibody binding within the CD4 binding site.
Highlights
The envelope glycoprotein (Env) from the human immunodeficiency virus type 1 (HIV-1) forms a heterotrimer composed of the receptor binding subunit gp120 and the membrane anchored fusion protein subunit gp41
MAbs 2F5, 4E10 and Z13 recognize epitopes within the membrane proximal region of gp41 [18,19,20,21], mAb 2G12 recognizes a carbohydrate motif [22,23], b12 interacts within the CD4 binding site [24,25], HJ16 overlaps with the CD4 binding site [26] and antibodies PG9 and PG16 are specific for the trimeric Env conformation [27]
CDR3 is composed of 18 residues (Lys95 – Tyr102) (Figure 2)
Summary
The envelope glycoprotein (Env) from the human immunodeficiency virus type 1 (HIV-1) forms a heterotrimer composed of the receptor binding subunit gp120 and the membrane anchored fusion protein subunit gp. A main problem in HIV-1 vaccine research is the generation of cross-subtype neutralizing antibodies, which is due to the fact that HIV-1 employs a number of strategies to evade the immune response This includes highly variable gp120 regions, a carbohydrate shield [10] and conformational masking of the receptor binding site [11]. The crystal structure of gp120 in complex with b12 revealed the molecular details including a substantial conserved gp120 surface overlapping between both the CD4- and b12-bound states [14] The similarities of both interactions is highlighted by the fact that b12 employs Tyr to fill the hydrophobic pocket in gp120 that is otherwise occupied by CD4 Phe43 [14]. Mutagenesis of selected CDR residues abrogate or enhance gp120 interaction in vitro and correlate with the neutralization activity of D7 against the B-clade HIV-1 IIIB providing a molecular model for D7-gp120 reactivity
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