“The role of individual carbohydrate-binding sites in the function of the potent anti-HIV lectin Griffithsin”
Jie Xue, Yongguang Gao, Bart Hoorelbeke, Ioannis Kagiampakis, Bo Zhao, Borries Demeler, Jan Balzarini, and Patricia J. LiWang Molecular Pharmaceutics 4, 2613-2625 (2012).
Griffithsin (GRFT) is a lectin that has been shown to inhibit HIV infection by binding to high mannose glycan structures on the surface of gp120, and is among the most potent HIV entry inhibitors reported so far. However, important biochemical details on the antiviral mechanism of GRFT action remain unexplored. In order to understand the role of the three individual carbohydrate-binding sites (CBS) in GRFT, mutations were made at each site (D30A, D70A, and D112A), and the resulting mutants were investigated. NMR studies revealed that each GRFT variant was folded but showed significant peak movement on the carbohydrate-binding face of the protein. The wild-type and each point mutant protein appeared as tight dimers with a Kd below 4.2 µM. Mutation of any individual CBS on GRFT reduced binding of the protein to mannose, and ELISA assays revealed a partial loss of ability of each GRFT point mutant to bind gp120, with a near-complete loss of binding by the triple mutant D30A/D70A/D112A GRFT. A more quantitative surface plasmon resonance (SPR) examination showed a rather small loss of binding to gp120 for the individual GRFT point mutants (KD: 123 to 245 pM range versus 73 pM for wild-type GRFT), but dramatic loss of the triple mutant to bind gp120 derived from R5 and X4 strains (KD > 12 nM). In contrast to the 2- to 3-fold loss of binding to gp120, the single CBS point mutants of GRFT were significantly less able to inhibit viral infection, exhibiting a 26- to 1900-fold loss of potency, while the triple mutant was at least 875 fold less effective against HIV-1 infection. The disparity between HIV-1 gp120 binding ability and HIV inhibitory potency for these GRFT variants indicates that gp120 binding and virus neutralization do not necessarily correlate, and suggests a mechanism that is not based on simple gp120 binding.
“The griffithsin dimer is required for high potency inhibition of HIV-1: Evidence for manipulation of the structure of gp120 as part of the griffithsin dimer mechanism”
Jie Xue, Bart Hoorelbeke, Ioannis Kagiampakis, Borries Demeler, Jan Balzarini, and Patricia J. LiWang Antimicrobial Agents and Chemotherapy 57:8, 3976–3989 (2013).
Griffithsin (Grft) is a protein lectin derived from red algae that tightly binds the HIV envelope protein gp120 and potently inhibits virus infection. This inhibition is due to the binding by Grft of high-mannose saccharides on the surface of gp120. Grft has been shown to be a tight dimer, but the role of the dimer in function has not been fully explored. To investigate the role of the Grft dimer in anti-HIV function, an obligate dimer of Grft was designed by expressing the protein with a peptide linker between the two subunits. This “Grft-linker-Grft” is a folded protein dimer, apparently nearly identical in structural properties with the wild-type protein. A “one-armed” obligate dimer was also designed, with each of the three carbohydrate binding sites of one subunit mutated while the other subunit remained intact. While both constructed dimers retained the ability to bind gp120 and the viral surface, Grft-linker-Grft-OneArm was 84- to 1010-fold less able to inhibit HIV compared to wild-type Grft, while Grft-linker-Grft had near wild-type antiviral potency. Furthermore, while the wild-type protein demonstrated the ability to alter the structure of gp120 by exposing the CD4 binding site, Grft-linker-Grft-OneArm largely lost this ability. In experiments to investigate gp120 shedding, it was found that Grft has different effects on gp120 shedding for strains from subtype B and subtype C, and this might correlate with Grft function. Evidence is provided that the dimer form of Grft is critical to the function of this protein in HIV inhibition.