Zhen Li, Ashini Bolia, Jason D. Maxwell, Andrey A. Bobkov, Giovanna Ghirlanda, S. Banu Ozkan, Claudio J. Margulis

Mutations in the hinge region of cyanovirin-N (CVN) dictate its preferential oligomerization state. Constructs with the Pro51Gly mutation preferentially exists as monomers whereas wild type cyanovirin can form domain swapped dimers in certain conditions. Since 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 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. Based on 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 on a set of cyanovirin mutants confirms this hypothesis. Those possessing the Pro51Gly mutation are consistently inferior binders.