Conformation of the Phosphate d-Alanine Zwitterion in Bacterial Teichoic Acid from Nuclear Magnetic Resonance Spectroscopy

Abstract

The conformation of D-alanine (D-Ala) groups of bacterial teichoic acid is a central, yet untested, paradigm of microbiology. The D-Ala binds via the C-terminus, thereby allowing the amine to exist as a free cationic NH3+ group with the ability to form a contact-ion-pair with the nearby anionic phosphate group. This conformation hinders metal chelation by the phosphate because the zwitterion pair is charge neutral. To the contrary, the repulsion of cationic antimicrobial peptides (CAMPs) is attributed to the presence of the D-Ala cation; thus the ion-pair does not form in this model. Solid-state nuclear magnetic resonance (NMR) spectroscopy has been used to measure the distance between amine and phosphate groups within cell wall fragments of Bacillus subtilis. The bacteria were grown on media containing 15N D-Ala and β-chloroalanine racemase inhibitor. The rotational-echo double-resonance (REDOR) pulse sequence was used to measure the internuclear dipolar coupling and the results demonstrate: 1) the metal-free amine-to-phosphate distance is 4.4 Å and 2) the amine-to-phosphate distance increases to 5.4 Å in the presence of Mg2+ ions. As a result, the zwitterion exists in a nitrogen-oxygen ion-pair configuration providing teichoic acid with a positive charge to repel CAMPs. Additionally, the amine of D-Ala does not prevent magnesium chelation in contradiction to the prevailing view of teichoic acids in metal binding. Thus, the NMR-based description of teichoic acid structure resolves the contradictory models, advances the basic understanding of cell wall biochemistry, and provides possible insight into the creation of new antibiotic therapies.

Revised model of calcium and magnesium binding to the bacterial cell wall

“…Recent research with solid state NMR shows that Mg 2+ preferentially binds to the phosphate groups, pushing the D-alanine away from the phosphate (Garimella, Halye et al 2009). Solid state NMR experiments have also been performed to estimate the binding constant of wall teichoic acid using the 31 P chemical shift based on the magnesium concentration used in the experiments (Kern, Giffard et al 2010).…”

The pH behavior of a 2-aminoethyl dihydrogen phosphate zwitterion studied with NMR-titrations

“…Aminophosphate zwitterions are known to exist in a solid state [10], the phosphate group is known to interact with free cationic NH þ 3 groups, [11] and the protons of the phosphate group dissociate easily in water solutions [12], so it is possible for such zwitterions to exist in water solutions.…”

High-throughput, Highly Sensitive Analyses of Bacterial Morphogenesis Using Ultra Performance Liquid Chromatography

“…In bacteria, the cell wall is an important antibiotic target (4), with treatment often disrupting the integrity of the cell wall and eventually leading to cell lysis (5). The cell wall plays an important role in pathogenesis, in part due to the uncommon stereochemistry of the three D-isomers of amino acids that defend against most proteases that otherwise would degrade the cell wall (1,6). Furthermore, many surface proteins anchored to the cell wall are involved in pathogenic processes such as host-cell invasion and immune system interactions (7).…”