An Integrative Approach to Understand the Effect of Sodium Thiocyanate on Human Carbonic Anhydrase 2

Abstract

Human Carbonic Anhydrase 2 (hCAII) is a model metalloenzyme whose Zn(II)diamagnetic center can be replaced by other transition metal ions, such as paramagnetic ones. Furthermore, hCAII has been used to understand the origin of the paramagnetic effect1. This study aims to understand the effect of the addition of an excess of sodium thiocyanate, an inhibitor and a chaotropic agent, on Cobalt(II) hCAII. Co(II) is a paramagnetic ion with S= 3/2. A previous study2 determined that Co(II) Bovine CA might bind more than one thiocyanate ion in the active center, in presence of an excess of ligand. We investigated the extra binding using 1H-15N HSQC NMR experiments as a primary source of information, complemented by X-ray crystallography and circular dichroism (CD). We titrated hCAII with thiocyanate and monitored by 1H-15N HSQC spectra. With this, we determined which residues interact with thiocyanate and how increasing the ligand concentration increases the number of interactions. Furthermore,1H-15N HSQC NMR relaxation experiments revealed that 1000-fold of thiocyanate concentration decreases the T1 relaxation parameter. The excess of thiocyanate can denature proteins, due to its chaotropic nature. However, CD experiments between 250 nm to 320 nm, revealed that the hydrophobic core of the enzyme remains unchanged, thus hCAII maintains its structure. Co(II)-hCAII and Zn(II)-hCAII were crystallized using the vapor diffusion technique and soaked with the ligand. The structures of both forms (to maximum resolutions of 1.7 Å and 1.75 Å, respectively) were solved by molecular replacement using a known structure of hCAII as search model (PDB code 3KS3). The structures revealed both forms only bind one thiocyanate molecule in the active center. This integrative approach proved to be crucial to understand how the protein 3D structure is affected by the presence of an excess of thiocyanate. Under these conditions, hCAII increases its hydrodynamic radius whilst maintaining its structure, thus explaining the changes of Co(II)-Bovine CA EPR spectra previously described. 1. Cerofolini, L. et al. J. Phys. Chem. Lett. 10, 3610–3614 (2019) 2. Bencini, A.et al. J. Inorg. Biochem. 14, 81–93 (1981)