X-ray crystallographic studies of Thermobifida fusca Xeg74 catalytic domain, human UDP-GlcNAc pyrophosphorylase apo-AGX1, and quadruple mutants of staphylococcal nuclease and the structural analysis of clostridial collagenase collagen binding domain and S

Analysis of the X-ray crystal structure for Thermobifida fusca apo-Xeg74 xyloglucan specific endoglucanase as compared to the xyloglucan substrate bound structure of Clostridium thermocellum Xgh74A endoglucanase allowed for the observation of a narrower substrate binding cleft for apo-Xeg74 than observed in substrate-bound Xgh74A and the prediction of Xeg74 residues imperative for the enzyme's xyloglucan specificity as previously determined during the characterization of the protein. Xeg74 and Xgh74A are both endoglucanases classified as members of the CAZy GH74 family based on amino acid sequence similarity, overall fold similarity, and evolutionary relationship. Including Xeg74, structures have been determined for only three of the GH74 members; in addition, very few of the GH74 members have been fully characterized. A correlation was made between substrate specificities for characterized GH74 proteins and a sequence alignment of all GH74 protien sequences in the regions determined by Xeg74 structural analysis to be critical for xyloglucan specificity. Based on this correlation, predictions were made as to the structural binding sties and substrate specificities for other GH74 enzymes. A recommendation was also made that a subclassification method be developed for xyloglucan-specific endoglucanases in the GH74 family.;The stabilizing effect of calcium binding on the collagen binding domain of Clostridium histolyticum ColG collagenase, particularly in the N-terminal arm region, was revealed in the mass spectrometry analysis of limited proteolysis experiments for the protein in apo- and calcium-bound forms. Previously determined crystal structures of apo- and calcium bound collagen binding domains showed that a highly mobile N-terminal arm, observed as an alpha helix in apo-form, transitions to a stable beta sheet formation upon calcium binding. Based on mass spectrometry data for the digests, the highly mobile N-terminal arm was easily cleaved in apo-form and was determined to be an unstructured loop region of the protein with no secondary structural architecture. Determination of the architecture of this region is important due to its significance in the drug delivery mechanism of the protein.;No structure has been determined for the conserved clostridial collagenase catalytic domain. Biophysical characterization of the catalytic domains of Clostridium histolyticum ColH and ColG collagenases revealed that while calcium binding has no effect on the secondary or tertiary fold of the protein, calcium binding was significant for the crystallization of the protein and for prediction of its sub-domain structure, which was based on mass spectrometry and N-terminal sequencing analysis of limited proteolysis experiments for apo- and calcium-bound catalytic domains. The 32.4kDa C-terminal region of the protein was determined to be the minimum region of the protein necessary for catalysis since this sub-domain contains the components essential for activity. The 46.8kDa N-terminal domain of the protein is proposed to be responsible for unwinding the collagen substrate, regulating catalysis, and possibly binding to calcium. These sub domain regions are conserved in other clostridial collagenases. Future crystallization of the 32.4kDa domain will allow for structure determination of entire clostridial collagenase catalytic domains. (Abstract shortened by UMI.).;The apo-structure of human UDP-N-acetylglucosamine pyrophosphorylase AGX1 was refined at 1.9A resolution. Comparison of the apo-AGX1 structure to its previously determined product-bound structure revealed a large domain movement in the N-domain of the protein. For the product-bound structure, the N-domain is static and its position creates a closed active site; the large domain movement of the N-domain in the apo-structure alters the active site architecture by creating an open active site cleft. The N-domain movement was characterized as a combination of shear and hinge domain closure mechanisms, and from this characterized domain movement, the order of substrate binding and product release for the enzyme kinetic mechanism was predicted. The open active site of the apo-AGX1 structure also revealed that dissociation of the protein does not need to occur for substrate binding and product release, disproving the previously predicted kinetic mechanism for the protein.