Multivalent interactions with gp120 are required for the anti-HIV activity of Cyanovirin.
Cyanovirin-N (CV-N) is a cyanobacterial lectin that binds to specific oligomannoses on the surface of gp120, resulting in nanomolar antiviral activity against HIV. In its monomeric form, CV-N contains two functional carbohydrate-binding domains, A and B. When refolded at high concentration, the protein can form a domain-swapped dimer. To clarify the role of multiple-binding sites in CV-N, we previously designed a monomeric mutant, P51G-m4-CVN, in which the binding site on domain A was rendered ineffective by four mutations (m4); in addition, a hinge region mutation (P51G) hinders the formation of a domain swapped dimer. The protein bound gp120 with diminished affinity and was completely inactive against HIV. Here, we present two mutants, DeltaQ50-m4-CVN and S52P-m4-CVN, which fold exclusively as domain-swapped dimers while containing the four mutations that abolish domain A. The dimers contain two intact B domains, thus restoring multivalency. DeltaQ50-m4-CVN and S52P-m4-CVN bind gp120 at low-nanomolar concentrations and recover in part the antiviral activity of wt CV-N. These results indicate that the number of carbohydrate binding domains, rather than their identity, is crucial to CV-N functionality.
Liu
Yinan
Y
Department of Chemistry and Biochemistry, Arizona State University, Tempe, AZ 85287-1604, USA.
Carroll
Jacob R
JR
Holt
Lindsey A
LA
McMahon
James
J
Giomarelli
Barbara
B
Ghirlanda
Giovanna
G
D019380:Anti-HIV Agents / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry*; D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry*; D002478:Cells, Cultured; D002942:Circular Dichroism; D019281:Dimerization; D015699:HIV Envelope Protein gp120 / Q000737:chemistry*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D013816:Thermodynamics
D019380:Anti-HIV Agents; D001426:Bacterial Proteins; D002352:Carrier Proteins; D015699:HIV Envelope Protein gp120; C107733:cyanovirin N