Force computations in automated docking

Automated docking refers to the problem of computing the optimal complementary fit of two molecules---a macromolecular receptor such as DNA or protein and a small molecule of interest (ligand). AutoDock is a docking software that computes the receptor-ligand binding energy to rank docked ligands. In this work, AutoDock\u27s grid-based method for energy evaluation was exploited to complement the binding energies with computed forces on docked ligand atoms. These forces and energies helped to provide insights into enzyme-substrate interaction mechanisms of three different enzymes--- Hypocrea jecorina Cel7A, a cellobiohydrolase, Fusarium oxysporum Cel7B, an endoglucanase, and Saccharomyces cerevisiae alpha-1,2-mannosidase. Cel7A and Cel7B are cellulose-degrading enzymes that, based on structural homology, belong to glycoside hydrolase Family 7. Cel7A binds crystalline cellulose and processively breaks cellobiose units from chain ends, while Cel7B targets amorphous cellulose and makes internal breaks in cellulose chain with limited processivity. The processive force on the substrate docked to the Cel7A catalytic domain (CD) is greater than twice that on the substrate docked to the Cel7B CD, explaining the difference in their processive behavior. Cel7A has a two-domain structure with a CD and a cellulose binding domain (CBD) joined by a highly glycosylated linker. Based on the interaction energies and forces on cellooligosaccharides docked to the CD and CBD, we propose a molecular machine model where the CBD wedges itself under a free chain end on the crystalline cellulose surface and feeds it to the CD active-site tunnel. alpha-1,2-Mannosidase from the endoplasmic reticulum, a Family 47 glycosyl hydrolase, is a key enzyme in the N-glycon synthesis pathway. AutoDock was used to dock alpha-D-mannopyranosyl-(1,2)-alpha-D-mannopyranose with its glycon in chair (1C4, 4C1), half-chair (3 H2, 3H4, 4H3), skew-boat (O S2, 3S1, 5S1), boat (2,5 B, 3,OB, B 1,4, B2,5), and envelope (3 E, 4E, E3, E4) conformations. Both docked energies and forces on docked ligand atoms were calculated to determine how the ligand distorts to the transition state. From these, we can conclude that the most likely binding pathways are 1C4 → 3H2 → OS 2 → 3,OB → 3 S1 → 3E and OS2 → 3,O B → 3S1 → 3E with 1C4 and OS2 as starting conformations, respectively

The fate of β-d-mannopyranose after its formation by endoplasmic reticulum α-(1→2)-mannosidase I catalysis

The automated docking program AutoDock was used to dock all 38 characteristic β-d-mannopyranose ring conformers into the active site of the yeast endoplasmic reticulum α-(1→2)-mannosidase I, a Family 47 glycoside hydrolase that converts Man9GlcNAc2 to Man8GlcNAc2. The subject of this work is to establish the conformational pathway that allows the cleaved glycon product to leave the enzyme active site and eventually reach the ground-state conformation. Twelve of the 38 conformers optimally dock in the active site where the inhibitors 1-deoxymannonojirimycin and kifunensine are found in enzyme crystal structures. A further 23 optimally dock in a second site on the side of the active-site well, while three dock outside the active-site cavity. It appears, through analysis of the internal energies of different ring conformations, of intermolecular energies between the ligands and enzyme, and of forces exerted on the ligands by the enzyme, that β-d-mannopyranose follows the path 3E→1C4→1H2→B2,5 before being expelled by the enzyme. The highly conserved second site that strongly binds β-d-mannopyranose-4C1 may exist to prevent competitive inhibition by the product, and is worthy of further investigation

Understanding protein structure-function relationships in Family 47 α-1,2-mannosidases through computational docking of ligands

Family 47 [Alpha]-1,2-mannosidases are crucial enzymes involved in N-glycan maturation in the endoplasmic reticulum (ER) and the Golgi apparatus of eukaryotic cells. They have also been implicated in playing a role in the quality control of newly synthesized proteins in the ER, by indirectly supplying the signal necessary for targeting misfolded proteins for degradation. High-resolution crystal structures of the human and yeast ER [Alpha]-1,2-mannosidases have been recently determined by others. In the crystal structure of the yeast enzyme, the N-glycan from one molecule extends into the active-site cavity of the adjacent symmetry-related molecule, forming what is believed to be the enzyme-substrate complex. However, due to the absence in the crystal structure of the terminal mannosyl residue that is cleaved by the enzyme, it has not been possible to unambiguously identify the catalytic proton donor and the nucleophile involved in the glycoside bond hydrolysis. The human [Alpha]-1,2-mannosidase crystal structure was determined by others in complex with inhibitors 1-deoxy-mannojirimycin and kifunensine, both of which bind in the active site in the unusual 1C4 conformation. In this work, [Alpha]-galactose, [Alpha]-glucose, and [Alpha]-mannose were docked in the active site in the energetically stable 4C1 conformation as well as in the 1C4 conformation to compare the energetics of interaction. From these docked structures, a model for substrate selectivity and conformer selectivity based on the dimensions of the active site was proposed. Specifically, the proposal was that the opening of the active-site neck is too narrow for the flatter and more extended 4C1 conformation to enter the-1 site of the catalytic domain, while the more compact 1C4 conformation can enter this site. [Alpha]-D-Galactopyranosyl-(l[Right pointing arrow]2)-[Alpha]-D-mannopyranose, [Alpha]-D-glucopyranosyl-(1Right pointing arrow]2)-[Alpha]-D-mannopyranose, and [Alpha]-D-mannopyranosyl-(1[Right pointing arrow]2)-[Alpha]-D-mannopyranose were also docked into the active site with the nonreducing-end residue in the 1C4 and E4 (representing the transition state) conformations. The results of these docking runs clearly show how this enzyme achieves transition-state stabilization. Based on the docked structure of [Alpha]-D-mannopyranosyl-E4-(1[Right pointing arrow]2)-[Alpha]-D-mannopyranose, the catalytic acid and base are Glu132 and Glu435, respectively

Targeted gene inactivation in Clostridium phytofermentans shows that cellulose degradation requires the family 9 hydrolase Cphy3367

Microbial cellulose degradation is a central part of the global carbon cycle and has great potential for the development of inexpensive, carbon-neutral biofuels from non-food crops. Clostridium phytofermentans has a repertoire of 108 putative glycoside hydrolases to break down cellulose and hemicellulose into sugars, which this organism then ferments primarily to ethanol. An understanding of cellulose degradation at the molecular level requires learning the different roles of these hydrolases. In this study, we show that interspecific conjugation with Escherichia coli can be used to transfer a plasmid into C. phytofermentans that has a resistance marker, an origin of replication that can be selectively lost, and a designed group II intron for efficient, targeted chromosomal insertions without selection. We applied these methods to disrupt the cphy3367 gene, which encodes the sole family 9 glycoside hydrolase (GH9) in the C. phytofermentans genome. The GH9-deficient strain grew normally on some carbon sources such as glucose, but had lost the ability to degrade cellulose. Although C. phytofermentans upregulates the expression of numerous enzymes to break down cellulose, this process thus relies upon a single, key hydrolase, Cphy3367