Three-dimensional structure of the barley beta-D-glucan glucohydrolase in complex with a transition state mimic.

Glucophenylimidazole (PheGlcIm), a tetrahydroimidazopyridine-type inhibitor and 4H3 conformer mimic of a glucoside, binds very tightly to a barley beta-d-glucan glucohydrolase, with a Ki constant of 2 x 10(-9) m and a DeltaG of 51 kJ mol(-1). PheGlcIm binds to the barley beta-d-glucan glucohydrolase approximately 2 x 10(5) times tighter than laminarin, which is the best non-synthetic ground-state substrate found so far for this enzyme, 10(6) times tighter than 4-nitrophenyl beta-d-glucopyranoside, and 2 x 10(7) tighter than glucose. The three-dimensional structure of the beta-d-glucan glucohydrolase with bound PheGlcIm indicates that the complex resembles a hypothetical transition state during the hydrolytic cycle, that the enzyme derives substrate binding energy from the "aglycone" portion of the ligand, and that it also reveals an anti-protonation trajectory for hydrolysis. Continuous electron densities at the 1.6 sigma level form between the three active site residues Asp95, His207, and Asp285, and the C6OH, C7OH, C8OH, and C9OH groups of PheGlcIm. These electron densities correspond to the most favorable interactions in the three-dimensional structure of the beta-d-glucan glucohydrolase-PheGlcIm complex and indicate atomic distances equal to or less than 2.55 A. The crystallographic data were corroborated with ab initio molecular orbital calculations. The data indicate that the 4E conformation of the glucose part of PheGlcIm is critical for tight binding and provide the first evidence for probable substrate distortion during catalysis by this enzyme.

Single-chain Fv multimers of the anti-neuraminidase antibody NC10: the residue at position 15 in the V(L) domain of the scFv-0 (V(L)-V(H)) molecule is primarily responsible for formation of a tetramer-trimer equilibrium.

Single-chain variable fragment of the murine monoclonal antibody NC10 specific to influenza virus N9 neuraminidase, joined directly in the V(L) to V(H) orientation (scFv-0), forms an equilibrium mixture of tetramer and trimer with the tetramer as the preferred multimeric species. In contrast, the V(H)-V(L) isomer was previously shown to exist exclusively as a trimer. Computer-generated trimeric and tetrameric scFv models, based on the refined crystal structure for NC10 Fv domain, were constructed and used to evaluate factors influencing the transition between V(L)-V(H) trimer and tetramer. These model structures indicated that steric restrictions between loops spanning amino acid residues L55-L59 and L13-L17 from the two adjacent V(L) domains within the V(L)-V(H) trimer were responsible for four scFv-0 molecules assembling to form a tetramer. In particular, leucine at position L15 and glutamate at position L57 appeared to interfere significantly with each other. To minimize this steric interference, the site-directed mutagenesis technique was used to construct several NC10 scFv-0 clones with mutations at these positions. Size-exclusion chromatographic analyses revealed that several of these mutations resulted in the production of NC10 scFv-0 proteins with significantly altered tetramer-trimer equilibrium ratios. In particular, introduction of a polar residue, such as asparagine or threonine, at position L15 generated a highly stable NC10 scFv-0 trimer.

Structural basis for broad substrate specificity in higher plant beta-D-glucan glucohydrolases.

Family 3 beta-D-glucan glucohydrolases are distributed widely in higher plants. The enzymes catalyze the hydrolytic removal of beta-D-glucosyl residues from nonreducing termini of a range of beta-D-glucans and beta-D-oligoglucosides. Their broad specificity can be explained by x-ray crystallographic data obtained from a barley beta-D-glucan glucohydrolase in complex with nonhydrolyzable S-glycoside substrate analogs and by molecular modeling of enzyme/substrate complexes. The glucosyl residue that occupies binding subsite -1 is locked tightly into a fixed position through extensive hydrogen bonding with six amino acid residues near the bottom of an active site pocket. In contrast, the glucosyl residue at subsite +1 is located between two Trp residues at the entrance of the pocket, where it is constrained less tightly. The relative flexibility of binding at subsite +1, coupled with the projection of the remainder of bound substrate away from the enzyme's surface, means that the overall active site can accommodate a range of substrates with variable spatial dispositions of adjacent beta-D-glucosyl residues. The broad specificity for glycosidic linkage type enables the enzyme to perform diverse functions during plant development.