A monovalent mutant of cyanovirin-N provides insight into the role of multiple interactions with gp120 for antiviral activity.
Cyanovirin-N (CV-N) is a 101 amino acid cyanobacterial lectin with potent antiviral activity against HIV, mediated by high-affinity binding to branched N-linked oligomannosides on the viral surface envelope protein gp120. The protein contains two carbohydrate-binding domains, A and B, each of which binds short oligomannosides independently in vitro. The interaction to gp120 could involve either a single domain or both domains simultaneously; it is not clear which mode would elicit the antiviral activity. The model is complicated by the formation of a domain-swapped dimer form, in which part of each domain is exchanged between two monomers, which contains four functional carbohydrate-binding domains. To clarify whether multivalent interactions with gp120 are necessary for the antiviral activity, we engineered a novel mutant, P51G-m4-CVN, in which the binding site on domain A has been knocked out; in addition, a [P51G] mutation prevents the formation of domain-swapped dimers under physiological conditions. Here, we present the crystal structures at 1.8 A of the free and of the dimannose-bound forms of P51G-m4-CVN, revealing a monomeric structure in which only domain B is bound to dimannose. P51G-m4-CVN binds gp120 with an affinity almost 2 orders of magnitude lower than wt CV-N and is completely inactive against HIV. The tight binding to gp120 is recovered in the domain-swapped version of P51G-m4-CVN, prepared under extreme conditions. Our findings show that the presence of at least two oligomannoside-binding sites, either by the presence of intact domains A and B or by formation of domain-swapped dimers, is essential for activity.
Fromme
Raimund
R
Department of Chemistry and Biochemistry, Arizona State University, Tempe, Arizona 85287-1604, USA.
Katiliene
Zivile
Z
Giomarelli
Barbara
B
Bogani
Federica
F
Mc Mahon
James
J
Mori
Toshiyuki
T
Fromme
Petra
P
Ghirlanda
Giovanna
G
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics*; D019380:Anti-HIV Agents / Q000737:chemistry* / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D001665:Binding Sites / Q000235:genetics; D002241:Carbohydrates / Q000737:chemistry; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D018360:Crystallography, X-Ray; D015699:HIV Envelope Protein gp120 / Q000378:metabolism* / Q000502:physiology; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D013778:Tetrazolium Salts / Q000378:metabolism; D013816:Thermodynamics
D019380:Anti-HIV Agents; D001426:Bacterial Proteins; D002241:Carbohydrates; D002352:Carrier Proteins; D015699:HIV Envelope Protein gp120; D013778:Tetrazolium Salts; C059087:2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-((phenylamino)carbonyl)-2H-tetrazolium hydroxide; C107733:cyanovirin N