Bio Design 2019; 7(4): 84-89
Published online December 30, 2019
© Korean Society for Structural Biology
Songwon Kim†, Na Jin Kim†, Subin Kim, Semi Hong, Jongmin Sung and Mi Sun Jin*
School of Life Sciences, GIST, 123 Cheomdan-gwagiro, Buk-gu, Gwangju 61005, Republic of Korea *Correspondence: email@example.com †These authors contributed equally to this work.
The fungal zinc metalloenzyme CafD from Aspergillus fumigatus is a minor β-carbonic anhydrase (β-CA) with low CO2 hydration activity. X-ray crystallography revealed that CafD forms a homodimer that possesses a zinc binding site in the dimer interface in addition to the catalytic zinc in the active site. However, the role of this ‘non-catalytic zinc’ has not been elucidated. In this study, we generated single (C39A or E54A) mutants targeting residues that form the non-catalytic zinc coordination sphere of CafD, and analyzed their structural and biochemical characteristics. Contrary to our expectations, these mutations strongly destabilized the three-dimensional fold of CafD, but the native homodimeric assembly was maintained even though the non-catalytic zinc binding site was disrupted. In the C39A mutant, the absence of the noncatalytic zinc permits flipping of a peptide bond, creating extra space for two water molecules that are vital in the formation of a water-mediated hydrogen bond network with the equivalent E54 and R56 residues of the two chains. By contrast, the E54A mutant contains an unexpected disulfide bond formed between the equivalent cysteine residues at position 39, which confers stability to the non-catalytic zinc-deficient CafD. Overall, our results demonstrate that a water-mediated hydrogen bond network and a disulfide bond may compensate for the loss of non-catalytic zinc at the dimer interface of CafD, and thereby contribute to structural integrity and/or thermal stability.