Rational and computational design of stabilized variants of cyanovirin-N that retain affinity and specificity for glycan ligands.
Cyanovirin-N (CVN) is an 11 kDa pseudosymmetric cyanobacterial lectin that has been shown to inhibit infection by the human immunodeficiency virus by binding to high-mannose oligosaccharides on the surface of the viral envelope glycoprotein gp120. In this work, we describe rationally designed CVN variants that stabilize the protein fold while maintaining high affinity and selectivity for their glycan targets. Poisson-Boltzmann calculations and protein repacking algorithms were used to select stabilizing mutations in the protein core. By substituting the buried polar side chains of Ser11, Ser20, and Thr61 with aliphatic groups, we stabilized CVN by nearly 12 °C against thermal denaturation, and by 1 M GuaHCl against chemical denaturation, relative to a previously characterized stabilized mutant. Glycan microarray binding experiments confirmed that the specificity profile of carbohydrate binding is unperturbed by the mutations and is identical for all variants. In particular, the variants selectively bound glycans containing the Manα(1→2)Man linkage, which is the known minimal binding unit of CVN. We also report the slow denaturation kinetics of CVN and show that they can complicate thermodynamic analysis; in particular, the unfolding of CVN cannot be described as a fixed two-state transition. Accurate thermodynamic parameters are needed to describe the complicated free energy landscape of CVN, and we provide updated values for CVN unfolding.
Department of Applied Mathematics and Statistics, Stony Brook University, Stony Brook, New York 11794, United States.
Vadim
V
Patsalo
Daniel P
DP
Raleigh
David F
DF
Green
D019943:Amino Acid Substitution; D019380:Anti-HIV Agents / Q000737:chemistry / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002240:Carbohydrate Sequence; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002942:Circular Dichroism; D015195:Drug Design; D019791:Guanidine / Q000737:chemistry; D008024:Ligands; D046228:Microarray Analysis; D008969:Molecular Sequence Data; D009154:Mutation; D011134:Polysaccharides / Q000378:metabolism*; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary
D019380:Anti-HIV Agents; D001426:Bacterial Proteins; D002352:Carrier Proteins; D008024:Ligands; D011134:Polysaccharides; C107733:cyanovirin N; D019791:Guanidine