寡唾液酸內酯及Waglerin I蛋白片段8–14th之分子結構探討暨動態模擬研究

Abstract

Part 1. The conformation of the trisialic acid, α2,8-(NeuAc)3, lactone was analyzed by a combination of NMR spectroscopy, molecular modeling, and molecular dynamic (MD) calculations. The inter-residue NOEs provided 14 important distance restraints for the molecular simulation, and the final simulated structures showed a root mean square deviation of 0.78 A for all superimposed structures. Because of the steric hindrance from the spirobicyclic of d-lactone, the individual sialic acid pyranose rings are considered essential in the chair 5C2 conformation. In addition, the lactone I close to the non-reducing end adopted a half-chair A2HCB9 conformer, whereas the lactone II close to the reducing end adopted a skewed twist-boat C2SC9 conformer. In the NMR solution structure and in the 1.0-ns in-water MD calculation, the final simulated structures are in the exocyclic torsions (w7, w8) = (gauche-anti) surface of the energy adiabatic map, where the global minimum can be found. During the in-water MD simulation, a slight fluctuation of the structure was observed, reflecting the steady conformation of lactone and the middle residue of the trisaccharide. These data are consistent with a theoretical approach of polysialic acid (PSA) polylactone with torsions (w7, w8) = (65o, 175o) and (F, Y) = (75.8o, –112.4o). Thus, we conclude that the PSA polylactone is a right-hand helix with a rotation angle, m, of 240 o and a repeating unit, n, of 1.5 residues. The structural properties of the PSA lactone discussed within this context differ from the helical epitope of G2+ PSA and may serve in future PSA-related antigen designs. The approximate model of hydrolytic reactivity of tri-sialic acid, a2,8-(NeuAc)3, lactone is studied with 1.0-ns in-water molecular dynamics simulation and presented as the neutral hydrolysis of d-lactone with two water-layers. The initial state of this type of hydrolysis could be designated as a reactant complex model via water nucleophile with a proton transfer with another water molecule. In addition, increased probability of a water molecule localized at the hydrolytic center would result in better improved hydrolysis of d-lactone. The priority of stepwise de-lactonization of a2,8-(NeuAc)3 lactone relies on water attendance near the carbonyl carbon of lactones in the 3.5 A water-shell. From in-water molecular dynamics study, the motion of water molecules over the re-face of the carbonyl groups can be used for the quantitative description of the residence possibility, p, whose value is 0.12 for lactone I and 0.30 for lactone II. The geometric criteria used to determine the residence statistics are the distance of water-oxygen×××carbonyl carbon is less than 3.5 A; and the cone angle, q, of carbonyl O=C×××Owater lies in the range of 85~115°. With higher residence possibility, the hydrolytic reactivity of lactone II is faster. Both the radial g(r) and angular p(q) pair distribution functions of water oxygen and carbonyl groups of lactones ensure a better surrounding hydration encounter for lactone II. The main reason for the limited water activity around lactone I is deduced from steric hindrance shaped by the turn structure of a2,8-(NeuAc)3 lactone. Therefore, an expansive space over the re-face of lactone II is perceived. Part 2. A disulfide bridge linked heptapeptide PCHPPCH of the center loop (8th-14th) of Waglerin I behaving four conformers was analyzed by RP-HPLC, solution NMR technique and simulated annealing calculation. The conformation searching study about the prolyl cis/trans isomerization of the internal di-proline provides an interconverted mechanism, and concludes as followings: (1) the trans-cis conformer is classified as the global minimum; (2) the cis-cis conformer manners as an inter-convertible intermediate between trans-cis and cis-trans two major conformers; (3) another local minimum trans-medium form is equilibrated with trans-cis conformer. The conclusion described above is consistent with relative populations and thermodynamic data observed from RP-HPLC analysis approximately. Moreover, the RP-HPLC result and structural analysis confirmed the presence of the minor cis-trans conformer in the Waglerin I toxin. Therefore, this study can be regarded to complement the 1996 work performed by our group (Chuang, L.-C. et al. Biochim. Biophys. Acta 1996, 1292, 145.).