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Prolidase (EC 3.4.13.9) is an enzyme that specifically hydrolyzes Xaa-Pro dipeptides into free amino acids. We previously studied kinetic behaviours and solved the crystal structure of wild-type (WT) Lactococcus lactis prolidase (Llprol), showing that this homodimeric enzyme has unique characteristics: allosteric behaviour and substrate inhibition. In this study, we focused on solving the crystal structures of three Llprol mutants (D36S, H38S, and R293S) which behave differently in v-S plots. The D36S and R293S Llprol mutants do not show allosteric behaviour, and the Llprol mutant H38S has allosteric behaviour comparable to the WT enzyme (Hill constant 1.52 and 1.58, respectively). The crystal structures of Llprol variants suggest that the active site of Llprol formed with amino acid residues from both monomers, i.e., located in an interfacial area of dimer. The comparison between the structure models of Llprol indicated that the two monomers in the dimers of Llprol variants have different relative positions among Llprol variants. They showed different interatomic distances between the amino acid residues bridging the two monomers and varied sizes of the solvent-accessible interface areas in each Llprol variant. These observations indicated that Llprol could adapt to different conformational states with distinctive substrate affinities. It is strongly speculated that the domain movements required for productive substrate binding are restrained in allosteric Llprol (WT and H38S). At low substrate concentrations, only one out of the two active sites at the dimer interface could accept substrate; as a result, the asymmetrical activated dimer leads to allosteric behaviour., Competing Interests: Declaration of competing interest Takuji Tanaka reports financial support was provided by Natural Sciences and Engineering Research Council of Canada. Shangyi Xu reports financial support was provided by Mitacs Canada. Takuji Tanaka reports equipment, drugs, or supplies was provided by Canadian Light Source Inc. Takuji Tanaka reports a relationship with University of Saskatchewan College of Agriculture and Bioresources that includes: employment. Shangyi Xu reports a relationship with University of Saskatchewan College of Agriculture and Bioresources that includes: funding grants. Pawel Grochulski reports a relationship with Canadian Light Source Inc. that includes: employment. Shangyi Xu reports a relationship with Canadian Light Source Inc. that includes: funding grants. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Lactococcus lactis prolidase (Xaa-Pro dipeptidase; EC.3.4.13.9) is unique from other prolidases by showing allosteric behaviour, substrate inhibition, and metal-dependent substrate specificity. We have previously shown several critical residues for these characteristics using site-directed mutagenesis and amino acid sequence-based models. In this present study, the three-dimensional structure of recombinant L. lactis prolidase was determined by X-ray crystallography at 2.25Å resolution in order to provide evidences of the proposed mechanism. Three molecules are located in the crystal asymmetric unit where molecule A forms a dimer with molecule B, while molecule C forms a dimer with molecule C' in the adjacent asymmetric unit. Of all the three molecules, molecule C is less defined and incomplete. While this fact compromises the overall quality of the refined model, the functional interpretation of the structure is not compromised since the biologically-functional homodimeric configuration of L. lactis prolidase is represented by well-defined molecules A and B. The refined model confirmed that there is a twelve-residue (residues 32-43) loop structure from one subunit over the active site of the other subunit, proving the existence of the putative loop structure in our previous study. This loop is three amino acids longer than the loops of prolidases of Pyrococcus furious (1pv9) and Pyrococcus horikoshii OT3 (1wy2). The crystal structure shows the loop structure can form two states ("open" and "closed" states) through interaction between the loop and active site proximity. It supports the proposed formation of allosteric site by the loop and Arg 293., (Copyright © 2017. Published by Elsevier B.V.)

Structures of metalloprotein active sites derived from X-ray crystallography frequently contain chemical anomalies such as unexpected atomic geometries or elongated bond-lengths. Such anomalies are expected from the known errors inherent in macromolecular crystallography (ca. 0.1-0.2Å) and from the lack of appropriate restraints for metal sites which are often without precedent in the small molecule structure literature. Here we review the potential of X-ray absorption spectroscopy to provide information and perspective which could aid in improving the accuracy of metalloprotein crystal structure solutions. We also review the potential problem areas in analysis of the extended X-ray absorption fine structure (EXAFS) and discuss the use of density functional theory as another possible source of geometrical restraints for crystal structure analysis of metalloprotein active sites., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Crystal structures of a genogroup II.4 human norovirus polymerase bound to an RNA primer-template duplex and the substrate analogue 2'-amino-2'-deoxycytidine-5'-triphosphate have been determined to 1.8 A resolution. The alteration of the substrate-binding site that is required to accommodate the 2'-amino group leads to a rearrangement of the polymerase active site and a disruption of the coordination shells of the active-site metal ions. The mode of binding seen for 2'-amino-2'-deoxycytidine-5'-triphosphate suggests a novel molecular mechanism of inhibition that may be exploited for the design of inhibitors targeting viral RNA polymerases.

The activated 65 kDa lepidopteran-specific CryIA(a) toxin from the commercially most important strain Bacillus thuringiensis var. kurstaki HD-1 has been investigated by X-ray diffraction and for its ability to form channels in planar lipid bilayers. Its three-dimensional structure has been determined by a multiple isomorphous replacement method and refined at 2.25 A resolution to an R-factor of 0.168 for data with I > 2 delta (I). The toxin is made of three distinct domains. The N-terminal domain is a bundle of eight alpha-helices with the central, relatively hydrophobic helix surrounded by amphipathic helices. The middle and C-terminal domains contain mostly beta-sheets. Comparison with the structure of CryIIIA, a coleopteran-specific toxin, shows that although the fold of these two proteins is similar, there are significant structural differences within domain II. This finding supports the conclusions from genetic studies that domain II is involved in recognition and binding to cell surface receptors. The distribution of electrostatic potential on the surface of the molecule is non-uniform and identifies one side of the alpha-helical domain as negatively charged. The predominance of arginine residues as basic residues ensures that the observed positive charge distribution is also maintained in the highly alkaline environment found in the lepidopteran midgut. Structurally important salt bridges that are conserved across Cry sequences were identified and their possible role in toxin action was postulated. In planar lipid bilayers, CryIA(a) forms cation-selective channels, whose conductance is significantly smaller than that reported for CryIIIA but similar to those of other Cry toxins.

A trypsin-activated CrylA(a) protein from Bacillus thuringiensis has been purified and crystallized. Crystals belong to orthorhombic space group P2(1)2(1)2(1), with cell dimensions a = 53.3, b = 111.3 and c = 154.7 A. The crystals diffract to at least 2.2 angstrum resolution and are suitable for X-ray structural analysis. They contain a single molecule in the asymmetric unit.

The title compound was synthesized as part of an effort to determine the identity of an abnormal steroid metabolite present in the urine of a patient exhibiting pronounced gynecomastia. The X-ray investigation of the synthesized compound showed that the 20-carbonyl of the 17 alpha oriented side chain lies under the D ring, and does not participate in hydrogen bonding in the crystal lattice. This conformation appears to be stable and sufficiently shielded that it is unlikely to make a major contribution to possible protein interactions.