Case study of hydrogen bonding in a hydrophobic cavity.

Protein internal hydrogen bonds and hydrophobicity determine protein folding and structure stabilization, and the introduction of a hydrogen bond has been believed to represent a better interaction for consolidating protein structure. We observed an alternative example for chicken IL-1ß. The native IL-1ß contains a hydrogen bond between the Y157 side-chain OηH and I133 backbone CO, whereby the substitution from Tyr to Phe abolishes the connection and the mutant without the hydrogen bond is more stable. An attempt to explain the energetic view of the presence of the hydrogen bond fails when only considering the nearly identical X-ray structures. Here, we resolve the mechanism by monitoring the protein backbone dynamics and interior hydrogen bond network. IL-1ß contains a hydrophobic cavity in the protein interior, and Y157 is one of the surrounding residues. The Y157 OηH group introduces an unfavorable energy in the hydrophobic cavity, therefore sequestering itself by forming a hydrogen bond with the proximate residue I133. The hydrogen bonding confines Y157 orientation but exerts a force to disrupt the hydrogen bond network surrounding the cavity. The effect propagates over the entire protein and reduces the stability, as reflected in the protein backbone dynamics observed by an NMR hydrogen-deuterium (H/D) exchange experiment. We describe the particular case in which a hydrogen bond does not necessarily confer enhanced protein stability while the disruption of hydrophobicity must be integrally considered.

NMR characterization of the electrostatic interaction of the basic residues in HDGF and FGF2 during heparin binding.

Electrostatic interaction is a major driving force in the binding of proteins to highly acidic glycosaminoglycan, such as heparin. Although NMR backbone chemical shifts have generally been used to identify the heparin-binding site on a protein, however, there is no correlation between the binding free energies and the perturbed backbone chemical shifts for individual residues. The binding event occurs at the end of a side chain of basic residue, and does not require causing significant alterations in the backbone environment at a distance of multiple bonds. We used the H2CN NMR pulse sequence to detect heparin binding through the side-chain resonances Hε-Cε-Nζ of Lys and Hδ-Cδ-Nε of Arg in the two proteins of hepatoma-derived growth factor (HDGF) and basic fibroblast growth factor (FGF2). H2CN titration experiments revealed chemical shift perturbations in the side chains, which were correlated with the free energy changes in various mutants. The residues K19 in HDGF and K125 in FGF2 demonstrated the most significant perturbations, consistent with our previous observation that the two residues are crucial for binding. The result suggests that H2CN NMR provides a precise evaluation for the electrostatic interactions. The discrepancy observed between backbone and side chain chemical shifts is correlated to the solvent accessibility of residues that the K19 and K125 backbones are highly buried with the restricted backbone conformation and are not strongly affected by the events at the end of the side chains.

Role of heparan sulfates and glycosphingolipids in the pore formation of basic polypeptides of cobra cardiotoxin.

Cobra venom contains cardiotoxins (CTXs) that induce tissue necrosis and systolic heart arrest in bitten victims. CTX-induced membrane pore formation is one of the major mechanisms responsible for the venom's designated cytotoxicity. This chapter examines how glycoconjugates such as heparan sulfates (HS) and glycosphingolipids, located respectively in the extracellular matrix and lipid bilayers of the cell membranes, facilitate CTX pore formation. Evidences for HS-facilitated cell surface retention and glycosphingolipid-facilitated membrane bilayer insertion of CTX are reviewed. We suggest that similar physical steps could play a role in the mediation of other pore forming toxins (PFT). The membrane pores formed by PFT are expected to have limited lifetime on biological cell surface as a result of membrane dynamics during endocytosis and/or rearrangement of lipid rafts.

Role of glycosphingolipid conformational change in membrane pore forming activity of cobra cardiotoxin.

The major cardiotoxin from Taiwan cobra (CTX A3) is a pore forming beta-sheet polypeptide that requires sulfatide (sulfogalactosylceramide, SGC) on the plasma membrane of cardiomyocytes for CTX-induced membrane leakage and cell internalization. Herein, we demonstrate by fluorescence spectroscopic studies that sulfatides induce CTX A3 oligomerization in sulfatide containing phosphatidylcholine (PC) vesicles to form transient pores with pore size and lifetime in the range of about 30 A and 10(-2) s, respectively. These values are consistent with the CTX A3-induced conductance and mean lifetime determined previously by using patch-clamp electrophysiological experiments on the plasma membrane of H9C2 cells. We also derived the peripheral binding structural model of CTX A3-sulfatide complex in sulfatide containing PC micelles by NMR and molecular docking method and compared with other CTX A3-sulfatide complex structure determined previously by X-ray in membrane-like environment. The NMR results indicate that sulfatide head group conformation changes from a bent shovel (-sc/ap) to an extended (sc/ap) conformation upon initial binding of CTX A3. An additional global reorientation of sulfatide molecule is also needed for CTX A3 dimer formation as inferred by the difference between the X-ray and NMR complex structure. Since the overall folding of CTX A3 molecules remained the same, sulfatide in phospholipid bilayer is proposed to play an active role by involving its local and global conformational changes to promote both the oligomerization and reorientation of CTX A3 molecule for its transient pore formation and cell internalization.

Structures of heparin-derived tetrasaccharide bound to cobra cardiotoxins: heparin binding at a single protein site with diverse side chain interactions.

Cobra cardiotoxins (CTXs) are three-fingered polypeptides with positively charged domains that have been shown to bind to anionic ligands of snake venom citrate, glycosaminoglycans, sulfoglycosphingolipid, and nucleotide triphosphate with various biochemical effects including toxin dimerization, cell surface retention, membrane pore formation, cell internalization and blocking of enzymatic activities of kinase and ATPase. The reported anionic binding sites, however, are found to be different among different CTX homologues for potentially different CTX activities. Herein, by NMR studies of the binding of inorganic phosphate, dATP (stable form of ATP), and heparin-derived tetrasaccharide to Naja atra CTX A1, a novel CTX molecule exhibiting in vivo necrotic activity on skeletal muscle, we demonstrate that diverse ligands binding to CTXs could also occur at a single protein site with flexible side chain interactions. The flexibility of such an interaction is also illustrated by the available heparin-CTX A3 complex structures with different heparin chain lengths binding at the same site. Our results provide a likely structural explanation on how the interaction between heparan sufate and proteins depends more on the overall charge cluster organization rather than on their fine structures. We also suggest that the ligand binding site of CTX homologues can be fine-tuned by nonconserved residues near the binding pocket because of their flexible side chain interaction and dimerization ability, even for the rigid CTX molecules tightened by four disulfide bonds.

Structural difference between group I and group II cobra cardiotoxins: X-ray, NMR, and CD analysis of the effect of cis-proline conformation on three-fingered toxins.

Natural homologues of cobra cardiotoxins (CTXs) were classified into two structural subclasses of group I and II based on the amino acid sequence and circular dichroism analysis, but the exact differences in their three-dimensional structures and biological significance remain elusive. We show by circular dichroism, NMR spectroscopic, and X-ray crystallographic analyses of a newly purified group I CTX A6 from eastern Taiwan cobra (Naja atra) venoms that its loop I conformation adopts a type VIa turn with a cis peptide bond located between two proline residues of PPxY. A similar "banana-twisted" conformation can be observed in other group I CTXs and also in cyclolinopeptide A and its analogues. By binding to the membrane environment, group I CTX undergoes a conformational change to adopt a more extended hydrophobic domain with beta-sheet twisting closer to the one adopted by group II CTX. This result resolves a discrepancy in the CTX structural difference reported previously between solution as well as crystal state and shows that, in addition to the hydrophobicity, the exact loop I conformation also plays an important role in CTX-membrane interaction. Potential protein targets of group I CTXs after cell internalization are also discussed on the basis of the determined loop I conformation.

Structural basis of citrate-dependent and heparan sulfate-mediated cell surface retention of cobra cardiotoxin A3.

Anionic citrate is a major component of venom, but the role of venom citrate in toxicity other than its inhibitory effect on the cation-dependent action of venom toxins is poorly understood. By immobilizing Chinese hamster ovary cells in microcapillary tubes and heparin on sensor chips, we demonstrated that heparan sulfate-mediated cell retention of the major cardiotoxin (CTX) from the Taiwan cobra, CTX A3, near membrane surfaces is citrate-dependent. X-ray determination of a CTX A3-heparin hexasaccharide complex structure at 2.4 A resolution revealed a molecular mechanism for toxin retention in which heparin-induced conformational changes of CTX A3 lead to citrate-mediated dimerization. A citrate ion bound to Lys-23 and Lys-31 near the tip of loop II stabilizes hydrophobic contact of the CTX A3 homodimer at the functionally important loop I and II regions. Additionally, the heparin hexasaccharide interacts with five CTX A3 molecules in the crystal structure, providing another mechanism whereby the toxin establishes a complex network of interactions that result in a strong interaction with cell surfaces presenting heparan sulfate. Our results suggest a novel role for venom citrate in biological activity and reveal a structural model that explains cell retention of cobra CTX A3 through heparan sulfate-CTX interactions.