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

Abstract

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.