Solid‐State NMR Spectroscopic Analysis of the Ca²⁺‐Dependent Mannose Binding of Pradimicin A

Abstract

Erleichterte Analyse: Das Ca2+‐abhängige Binden des nichtpeptidischen Kohlenhydratbinders Pradimicin A (PRM‐A) durch Mannose (Man) wurde im Festkörper an PRM‐A‐Aggregaten untersucht, was mit dem Dreikomponentengleichgewicht verknüpfte Probleme beseitigte. 113Cd‐NMR‐Spektroskopie und dipolgestützte zweidimensionale Rotationsresonanz ergaben die Mannosebindungsstelle von PRM‐A und die entscheidende Rolle des Ca2+‐Ions (siehe Bindungsmodell).

Mannose‐Binding Geometry of Pradimicin A

“…[19] Regarding the interaction with Ca 2 + ions, our previous study suggested the possibility that PRM-A binds Man in a Ca 2 + -mediated manner through its carboxylate group. [14] Unfortunately, lack of data regarding the geometry of Ca 2 + coordination prevented us from conducting further calculations in the presence of Ca 2 + ions to estimate the true mode of binding. However, it can be hypothesized that Man coordinates Ca 2 + ions through the 4-hydroxyl group, which is in the proximity of the carboxylate group of PRM-A in our binding model.…”

“…In addition, the H2 proton of Man lies over the A ring of PRM‐A at a distance of 3.03 Å, implying that the CH/π interaction between them could also contribute to stabilization of the complex as observed in many carbohydrate–receptor systems 19. Regarding the interaction with Ca 2+ ions, our previous study suggested the possibility that PRM‐A binds Man in a Ca 2+ ‐mediated manner through its carboxylate group 14. Unfortunately, lack of data regarding the geometry of Ca 2+ coordination prevented us from conducting further calculations in the presence of Ca 2+ ions to estimate the true mode of binding.…”

“…In our binding model, the C6 carbon atom of Man lies above the carboxylate group of PRM‐A (Figure 3 B). Since the Ca 2+ ion is assumed to bridge the carboxylate groups of two PRM‐A molecules to form the [PRM‐A 2 /Ca 2+ ] complex,11, 14 the 2‐deoxy‐Man residue of 2 bound in one binding site possibly covers a part of the other binding site, blocking its occupation by other molecules of 2 (Figure 5 A). On the other hand, the C1 carbon atom of Man is oriented opposite to the carboxylate group of PRM‐A, thereby enabling two molecules of 3 to be simultaneously accommodated without any steric hindrance (Figure 5 B).…”

“…To circumvent issues related to the complicated complex formation of PRMs with Man, while facilitating their interaction analysis, we have recently developed a conceptually novel analytical strategy in the solid state using PRM‐A, an original member of PRMs (Figure 1 A). 14, 15 The key to our analytical strategy is the use of the solid aggregate solely composed of the [PRM‐A 2 /Ca 2+ /Man 2 ] complex, which was prepared by the extensive washing of the aggregate containing the [PRM‐A 2 /Ca 2+ /Man 4 ] complex and non‐specifically absorbed Man. The simple 1:1 complexes of biosynthetically 13 C‐enriched PRM‐As and methyl α‐ D ‐[ 13 C 6 ]mannopyranoside ([ 13 C 6 ]Man‐OMe) enabled the identification of the primary Man binding site in PRM‐A by solid‐state NMR spectroscopy with avoidance of the problem associated with the complicated complex‐forming process.…”

Mannose-binding analysis and biological application of pradimicins

“…Our initial attempt to explore this issue was made by solid-state 113 Cd-NMR investigation using 113 Cd 2D with a spin of 1/2 as a surrogate probe for Ca 2D with a spin of 7/2. 34) Based on the previous report that PRMs bound Man in the presence of Cd 2D , 27) the [PRM-A 2 / 113 Cd 2D ] complex was prepared as aggregates, and subjected to solid-state 113 Cd-NMR analysis. Although the chemical shift of the 113 Cd signal in the 1D spectrum of the complex gave no information regarding the geometric arrangement of 113 Cd 2D coordination, subsequent rotational-echo double resonance (REDOR) and 111 Cd frequency-selective REDOR (FSR) experiments using the 111 Cd 2D probe gave an unexpected result.…”