Substrate Binding Drives Active-Site Closing of Human Blood Group†B Galactosyltransferase as Revealed by Hot-Spot Labeling and NMR Spectroscopy Experiments.

Crystallography has shown that human blood group A (GTA) and B (GTB) glycosyltransferases undergo transitions between "open", "semiclosed", and "closed" conformations upon substrate binding. However, the timescales of the corresponding conformational reorientations are unknown. Crystal structures show that the Trp and Met residues are located at "conformational hot spots" of the enzymes. Therefore, we utilized 15 N side-chain labeling of Trp residues and 13 C-methyl labeling of Met residues to study substrate-induced conformational transitions of GTB. Chemical-shift perturbations (CSPs) of Met and Trp residues in direct contact with substrate ligands reflect binding kinetics, whereas the CSPs of Met and Trp residues at remote sites reflect conformational changes of the enzyme upon substrate binding. Acceptor binding is fast on the chemical-shift timescale with rather small CSPs in the range of less than approximately 20 Hz. Donor binding matches the intermediate exchange regime to yield an estimate for exchange rate constants of approximately 200-300 Hz. Donor or acceptor binding to GTB saturated with acceptor or donor substrate, respectively, is slow (<10 Hz), as are coupled protein motions, reflecting mutual allosteric control of donor and acceptor binding. Remote CSPs suggest that substrate binding drives the enzyme into the closed state required for catalysis. These findings should contribute to better understanding of the mechanism of glycosyl transfer of GTA and GTB.

Protein NMR Studies of Substrate Binding to Human Blood Group†A and B Glycosyltransferases.

Donor and acceptor substrate binding to human blood group A and B glycosyltransferases (GTA, GTB) has been studied by a variety of protein NMR experiments. Prior crystallographic studies had shown these enzymes to adopt an open conformation in the absence of substrates. Binding either of the donor substrate UDP-Gal or of UDP induces a semiclosed conformation. In the presence of both donor and acceptor substrates, the enzymes shift towards a closed conformation with ordering of an internal loop and the C-terminal residues, which then completely cover the donor-binding pocket. Chemical-shift titrations of uniformly 2 H,15 N-labeled GTA or GTB with UDP affected about 20 % of all crosspeaks in 1 H,15 N TROSY-HSQC spectra, reflecting substantial plasticity of the enzymes. On the other hand, it is this conformational flexibility that impedes NH backbone assignments. Chemical-shift-perturbation experiments with δ1-[13 C]methyl-Ile-labeled samples revealed two Ile residues-Ile123 at the bottom of the UDP binding pocket, and Ile192 as part of the internal loop-that were significantly disturbed upon stepwise addition of UDP and H-disaccharide, also revealing long-range perturbations. Finally, methyl TROSY-based relaxation dispersion experiments do not reveal micro- to millisecond timescale motions. Although this study reveals substantial conformational plasticity of GTA and GTB, the matter of how binding of substrates shifts the enzymes into catalytically competent states remains enigmatic.

Studies on the Chitin Binding Property of Novel Cysteine-Rich Peptides from Alternanthera sessilis.

Hevein-like peptides make up a family of cysteine-rich peptides (CRPs) and play a role in plants in their defense against insects and fungal pathogens. In this study, we report the isolation and characterization of six hevein-like peptides, aSG1-G3 and aSR1-R3, collectively named altides from green and red varieties of Alternanthera sessilis, a perennial herb belonging to the Amaranthaceae family. Proteomic analysis of altides revealed they contain six cysteines (6C), seven glycines, four prolines, and a conserved chitin-binding domain (SXYGY/SXFGY). Thus far, only four 6C-hevein-like peptides have been isolated and characterized; hence, our study expands the existing library of these peptides. Nuclear magnetic resonance (NMR) study of altides showed its three disulfide bonds were arranged in a cystine knot motif. As a consequence of this disulfide arrangement, they are stable against thermal and enzymatic degradation. Gene cloning studies revealed altides contain a three-domain precursor with an endoplasmic reticulum signal peptide followed by a mature CRP domain and a short C-terminal tail. This indicates that the biosynthesis of altides is through the secretory pathway. (1)H NMR titration experiments showed that the 29-30-amino acid altides bind to chitin oligomers with dissociation constants in the micromolar range. Aromatic residues in the chitin-binding domain of altides were involved in the binding interaction. To the best of our knowledge, aSR1 is the smallest hevein-like peptide with a dissociation constant toward chitotriose comparable to those of hevein and other hevein-like peptides. Together, our study expands the existing library of 6C-hevein-like peptides and provides insights into their structure, biosynthesis, and interaction with chitin oligosaccharides.

Thermodynamic signature of substrates and substrate analogs binding to human blood group B galactosyltransferase from isothermal titration calorimetry experiments.

It has been observed earlier that human blood group B galactosyltransferase (GTB) hydrolyzes its donor substrate UDP-Galactose (UDP-Gal) in the absence of acceptor substrate, and that this reaction is promoted by the presence of an acceptor substrate analog, α-L-Fuc-(1,2)-ß-D-3-deoxy-Gal-O-octyl (3DD). This acceleration of enzymatic hydrolysis of UDP-Gal was traced back to an increased affinity of GTB toward the donor substrate in the presence of 3DD. Herein, we present new thermodynamic data from isothermal titration calorimetry (ITC) on the binding of donor and acceptor substrates and analogs to GTB. ITC data are supplemented by surface plasmon resonance and STD-NMR titration experiments. These new data validate mutual allosteric control of binding of donor and acceptor substrates to GTB. It is of note that ITC experiments reveal significant differences in enthalpic and entropic contributions to binding of the natural donor substrate UDP-Gal, when compared with its analog UDP-Glucose (UDP-Glc). This may reflect different degrees of ordering of an internal loop (amino acids 176-194) and the C-terminus (amino acids 346-354), which close the binding pocket on binding of UDP-Gal or UDP-Glc. As both ligands have rather similar dissociation constants KD and almost identical modes of binding this finding is unexpected. Another surprising finding is that an acceptor analog, α-L-Fuc-(1,2)-ß-D-3-amino-3-deoxy-Gal-O-octyl (3AD) as well as the constituent monosaccharide ß-D-3-amino-3-deoxy-Gal-O-octyl (3AM) effectively inhibit enzymatic hydrolysis of UDP-Gal. This is unexpected, too, because in analogy to the effects of 3DD one would have predicted acceleration of enzymatic hydrolysis of UDP-Gal. It is difficult to explain these observations based on structural data alone. Therefore, our results highlight that there is an urgent need of experimental studies into the dynamic properties of GTB.

Nuclear magnetic resonance spectral assignments of α-1,4-galactosyltransferase LgtC from Neisseria meningitidis: substrate binding and multiple conformational states.

Lipopolysaccharide α-1,4-galactosyltransferase C (LgtC) from Neisseria meningitidis is responsible for a key step in lipooligosaccharide biosynthesis involving the transfer of α-galactose from the sugar donor UDP-galactose to a terminal acceptor lactose. Crystal structures of the complexes of LgtC with Mn(2+) and the sugar donor analogue UDP-2-deoxy-2-fluorogalactose in the absence and presence of the sugar acceptor analogue 4'-deoxylactose provided key insights into the galactosyl-transfer mechanism. Combined with kinetic analyses, the enzymatic mechanism of LgtC appears to involve a "front-side attack" S(N)i-like mechanism with a short-lived oxocarbenium-phosphate ion pair intermediate. As a prerequisite for investigating the required roles of structural dynamics in this catalytic mechanism by nuclear magnetic resonance techniques, the transverse relaxation-optimized amide (15)N heteronuclear single-quantum correlation and methyl (13)C heteronuclear multiple-quantum correlation spectra of LgtC in its apo, substrate analogue, and product complexes were partially assigned. This was accomplished using a suite of complementary spectroscopic approaches, combined with selective isotopic labeling and mutagenesis of all the isoleucine residues in the protein. Only ~70% of the amide signals could be detected, whereas more than the expected number of methyl signals were observed, indicating that LgtC adopts multiple interconverting conformational states. Chemical shift perturbation mapping provided insights into substrate and product binding, including the demonstration that the sugar donor analogue (UDP-2FGal) associates with LgtC only in the presence of a metal ion (Mg(2+)). These spectral assignments provide the foundation for detailed studies of the conformational dynamics of LgtC.