Crystal structure analysis in Zn2+-bound state and biophysical characterization of CLas-ZnuA2

- Candidatus L. asiaticus is the causative agent of Huanglongbing disease of citrus.
- Structure of CLas-ZnuA2 has been determined in Zn2+-bound state.
- CLas-ZnuA2 binds to metal ions with unique square pyramidal coordination geometry.
- SPR and CD studies suggest that CLas-ZnuA2 has relatively lower binding affinity.

A periplasmic solute binding protein from second of the two gene clusters of Znu system in CLA (CLas-ZnuA2) belong to Cluster A1 family of solute binding proteins (SBPs). The crystal structures in metal-free, intermediate and metal-bound states, in the previous study, revealed the unusual mechanism of metal binding and release for CLas-ZnuA2. Although CLas-ZnuA2 showed maximum sequence identity to the Mn/Fe-specific SBPs, the mechanistic resemblance seems to be closer to Zn-specific SBPs of Cluster A-I family. The present study reports the binding affinity studies using SPR and CD and crystal structure of CLas-ZnuA2 in Zn2+-bound state. Despite a similar overall structure, there are noticeable differences at the metal binding site. The SPR and CD analysis confirmed our previous observation that CLas-ZnuA2 exhibits a low metal-binding affinity. The low metal-binding affinity of CLas-ZnuA2 could be attributed to the presence of a proline in linker helix resulting in relatively higher bending and rigidity of the same. This structural feature fixes the C-domain similar to metal-bound states of related SBPs. Further, the binding of both Mn2+ and Zn2+ occurs pentavalently with square pyramidal geometry not preferred by either. The site-specific positive Darwinian selection analysis showed that the proline in linker helix is under purifying selection and might have diverged long ago. Our structural and evolutionary analyses suggest that CLasZnua2 might have evolved, particularly for plant pathogens, to facilitate transport of both Mn2+ and Zn2+, with reversible binding to Zn2+, unlike other Mn-binding SBPs (PsaA).