A Rigid Hinge Region Is Necessary for High-Affinity Binding of Dimannose to Cyanovirin and Associated Constructs.
Mutations in the hinge region of cyanovirin-N (CVN) dictate its preferential oligomerization state. Constructs with the Pro51Gly mutation preferentially exist as monomers, whereas wild-type cyanovirin can form domain-swapped dimers under certain conditions. Because the hinge region is an integral part of the high-affinity binding site of CVN, we investigated whether this mutation affects the shape, flexibility, and binding affinity of domain B for dimannose. Our studies indicate that the capability of monomeric wild-type CVN to resist mechanical perturbations is enhanced when compared to that of constructs in which the hinge region is more flexible. Our computational results also show that enhanced flexibility leads to blocking of the binding site by allowing different rotational isomeric states of Asn53. Moreover, at higher temperatures, this observed flexibility leads to an interaction between Asn53 and Asn42, further hindering access to the binding site. On the basis of these results, we predicted that binding affinity for dimannose would be more favorable for cyanovirin constructs containing a wild-type hinge region, whereas affinity would be impaired in the case of mutants containing Pro51Gly. Experimental characterization by isothermal titration calorimetry of a set of cyanovirin mutants confirms this hypothesis. Those possessing the Pro51Gly mutation are consistently inferior binders.
Department of Chemistry, University of Iowa , Iowa City, Iowa 52242, United States.
Zhen
Z
Li
Department of Chemistry and Biochemistry, Arizona State University , Tempe, Arizona 85287-1604, United States.
Ashini
A
Bolia
Department of Chemistry and Biochemistry, Arizona State University , Tempe, Arizona 85287-1604, United States.
Jason D
JD
Maxwell
Sanford Burnham Medical Research Institute , 10901 North Torrey Pines Road, La Jolla, California 92037, United States.
Andrey A
AA
Bobkov
Department of Chemistry and Biochemistry, Arizona State University , Tempe, Arizona 85287-1604, United States.
Giovanna
G
Ghirlanda
Center for Biological Physics, Department of Physics, Arizona State University , Tempe, Arizona 85287, United States.
S Banu
SB
Ozkan
Department of Chemistry, University of Iowa , Iowa City, Iowa 52242, United States.
Claudio J
CJ
Margulis
D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004187:Disaccharides / Q000378:metabolism*; D037241:Mannose-Binding Lectins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary
D001426:Bacterial Proteins; D002352:Carrier Proteins; D004187:Disaccharides; D037241:Mannose-Binding Lectins; C107733:cyanovirin N; C108662:1,3-mannosylmannose