Solid-State Nuclear Magnetic Resonance Analysis Reveals a Possible Calcium Binding Site of Pradimicin A

Pradimicin A (PRM-A) is a unique natural product that recognizes d-mannopyranoside (Man) in the presence of Ca²⁺ ion. Although the Man binding geometry of PRM-A is largely understood, the molecular basis of Man recognition has yet to be established because of the lack of information regarding Ca²⁺ binding geometry. In this work, to examine the Ca²⁺ binding site of PRM-A, we performed a solid-state nuclear magnetic resonance experiment using ¹¹¹Cd²⁺ as a surrogate probe for Ca²⁺. Evaluation of ¹³C-¹¹¹Cd distances in the [PRM-A/¹¹¹Cd²⁺] complexes by rotational-echo double resonance (REDOR) and ¹¹¹Cd frequency selective REDOR (FSR) revealed that PRM-A binds ¹¹¹Cd²⁺ at the anthraquinone moiety, which contradicts the previous hypothesis of the alanine moiety being the Ca²⁺ and Cd²⁺ binding sites of PRM-A. The distances between Cd²⁺ and the carbon atoms at the binding site of PRM-A were found to be 3.5 ± 0.2 Å. Importantly, Man binding was shown not to alter the distances, indicating that [PRM-A/Ca²⁺] and [PRM-A/Ca²⁺/Man] complexes have similar Ca²⁺ binding geometries. This study provides an important clue to understanding the molecular basis of Man recognition of PRM-A.