| Summary: | 博士 === 國立清華大學 === 生命科學系 === 89 === Solution NMR structure of CTX A3 from Taiwan cobra (Naja atra) was determined at neutral pH by conventional 2D 1H NMR techniques. The molecular dynamics investigated using a model-free analysis of the 13C NMR spin-lattice relaxation experiments to help understanding of the relationship between structure and motional property. The order parameter (S2) of the polypeptide backbone and overall correlation time (tR) of the relatively flat CTX A3 are presented. As estimated by 13C NMR relaxation, overall tumbling motion of CTX A3 molecule can be concluded to be isotropic with a correlation time of about 3.8 - 4.5 ns. Recent studies of cobra P-type CTXs have shown that the loop II plays a crucial role in binding to biological membranes. A bound water molecule was then identified in the interior of loop II in CTX A3 by 1H NOESY/ROESY. In order to understand the role of bound water in the loop, the dynamics of the bound water was determined by a comprehensive analysis of 17O transverse triple-quantum filtered NMR and computer simulation. The single water molecule was found to be tightly hydrogen bonded to the NH of Met26, CO of Thr29 and CO of Val32. Surprisingly, despite the relatively long residence time (from 5 ns to 100 ms), the bound water molecule of CTX A3 is located within a dynamic (S2 ~ 0.7) and solvent accessible loop. Comparing to several P-type CTXs, the consensus sequence of MxAxPxVPV was derived. It is proposed that the exchange rate of the bound water may play a role in regulating the lipid binding mode of amphiphilic CTX molecules.
Glycosaminoglycans (GAGs) have also been suggested to be another potential target for CTX with high affinity and specificity via a cationic belt at the concave surface of the polypeptide. The interaction of GAGs, such as high molecular weight heparin, with CTXs can not only induce aggregation of CTX molecules but also enhance their penetration into membranes. Aiming to understand the interaction of CTXs and heparin, the binding of short chain heparin-derived disaccharide to CTX A3 was investigated by proton 1D NMR selective methods first. A novel heparin binding site on the convex side of the CTX, near the rigid disulfide-bond tightened core region of Cys38, was then identified due to the observation of intermolecular NOEs between protein and carbohydrate. The derived carbohydrate conformation indicated that the glycosidic linkage and the ring conformation change upon binding to CTX A3. Specifically, comparative studies of several heparin disaccharide homologues with CTX Tg and CTX A3 indicated that the electrostatic interaction between different sulfation pattern and Lysine residues played an important role on binding. These results also suggest a model on how the CTX-heparin interaction may regulate heparin-induced aggregation of the toxin via the second heparin binding site.
Although the bound form conformation of heparin disaccharide to cobra cardiotoxin has been determined, it remains a challenging issue to perform a similar study on heparin with longer chain length. Heparin-derived polysaccharide of biological origin is heterogeneous with different sulfation patterns and difficult to purify by using chromatographic method. The 1H NMR signals of long chain heparin are also difficult to resolve since they are severely overlapping to each other. Herein, we demonstrate that 1D selective excitation methods may have an advantage in studying a mixture of heparin-derived tetrasaccharide and their interaction with cobra cardiotoxin. A complete assignment of 1H NMR signal for three major tetrasaccharides, i.e., DUA2S-GlcNS6S-IdoA2S-GlcNS6S, DUA2S-GlcNS6S-GlcA-GlcNS6S and DUA2S-GlcNS6S-IdoA2S-GlcNS is demonstrated for the first time by using 1D selective excitation experiment. In addition, by comparing the relaxation time of the selectively excited anomeric proton in the absent and present of CTX A3, we concluded that the component with fully sulfation pattern owns the highest affinity with CTX A3.
By taking lysophosphatidylcholine (LPC) micelles as a membrane model, we have shown that the binding of heparin-derived hexasaccharide (Hep-6) to CTX at the b-strand region can induce conformational changes of CTX near its membrane binding loops and promote the binding activity of CTX towards to LPC. The Fourier-transform infrared spectra and NOE values of Hep-6/CTX and CTX/LPC complex in aqueous buffer also supplemented the aforementioned observation. So the detected conformational change may presumably be due to the result of structural coupling between the connecting loops and its b-strands. This is the first documentation of results showing how the association of hydrophilic carbohydrate molecules with amphiphilic proteins can promote hydrophobic protein-lipid interaction via the stabilization of its membrane bound form. A similar mechanism involving tripartite interactions of heparin, protein, and lipid molecules may be operative near the extracellular matrix of cell membranes.
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