The crystal structure of chicken egg white cystatin has been solved by X‐ray diffraction methods using the multiple isomorphous replacement technique. Its structure has been refined to a crystallographic R value of 0.19 using X‐ray data between 6 and 2.0A. The molecule consists mainly of a straight five‐turn alpha‐helix, a five‐stranded antiparallel beta‐pleated sheet which is twisted and wrapped around the alpha‐helix and an appending segment of partially alpha‐helical geometry. The ‘highly conserved’ region from Gln53I to Gly57I implicated with binding to cysteine proteinases folds into a tight beta‐hairpin loop which on opposite sides is flanked by the amino‐terminal segment and by a second hairpin loop made up of the similarly conserved segment Pro103I ‐ Trp104I. These loops and the amino‐terminal Gly9I ‐ Ala10I form a wedge‐shaped ‘edge’ which is quite complementary to the ‘active site cleft’ of papain. Docking experiments suggest a unique model for the interaction of cystatin and papain: according to it both hairpin loops of cystatin make major binding interactions with the highly conserved residues Gly23, Gln19, Trp177 and Ala136 of papain in the neighbourhood of the reactive site Cys25; the amino‐terminal segment Gly9I ‐ Ala10I of bound cystatin is directed towards the substrate subsite S2, but in an inappropriate conformation and too far away to be attacked by the reactive site Cys25. As a consequence, the mechanism of the interaction between cysteine proteinases and their cystatin‐like inhibitors seems to be fundamentally different from the ‘standard mechanism’ defined for serine proteinases and most of their protein inhibitors.
From the lysosomal cysteine proteinase cathepsin B, isolated from human liver in its two‐chain form, monoclinic crystals were obtained which contain two molecules per asymmetric unit. The molecular structure was solved by a combination of Patterson search and heavy atom replacement methods (simultaneously with rat cathepsin B) and refined to a crystallographic R value of 0.164 using X‐ray data to 2.15 A resolution. The overall folding pattern of cathepsin B and the arrangement of the active site residues are similar to the related cysteine proteinases papain, actinidin and calotropin DI. 166 alpha‐carbon atoms out of 248 defined cathepsin B residues are topologically equivalent (with an r.m.s. deviation of 1.04 A) with alpha‐carbon atoms of papain. However, several large insertion loops are accommodated on the molecular surface and modify its properties. The disulphide connectivities recently determined for bovine cathepsin B by chemical means were shown to be correct. Some of the primed subsites are occluded by a novel insertion loop, which seems to favour binding of peptide substrates with two residues carboxy‐terminal to the scissile peptide bond; two histidine residues (His110 and His111) in this “occluding loop' provide positively charged anchors for the C‐terminal carboxylate group of such polypeptide substrates. These structural features explain the well‐known dipeptidyl carboxypeptidase activity of cathepsin B. The other subsites adjacent to the reactive site Cys29 are relatively similar to papain; Glu245 in the S2 subsite favours basic P2‐side chains. The above mentioned histidine residues, but also the buried Glu171 might represent the group with a pKa of approximately 5.5 near the active site, which governs endo‐ and exopeptidase activity. The “occluding loop' does not allow cystatin‐like protein inhibitors to bind to cathepsin B as they do to papain, consistent with the reduced affinity of these protein inhibitors for cathepsin B compared with the related plant enzymes.
The crystal structure of the porcine heart catalytic subunit of cAMP‐dependent protein kinase in a ternary complex with the MgATP analogue MnAMP‐PNP and a pseudosubstrate inhibitor peptide, PKI(5‐24), has been solved at 2.0 A resolution from monoclinic crystals of the catalytic subunit isoform CA. The refinement is presently at an R factor of 0.194 and the active site of the molecule is well defined. The glycine‐rich phosphate anchor of the nucleotide binding fold motif of the protein kinase is a beta ribbon acting as a flap with conformational flexibility over the triphosphate group. The glycines seem to be conserved to avoid steric clash with ATP. The known synergistic effects of substrate binding can be explained by hydrogen bonds present only in the ternary complex. Implications for the kinetic scheme of binding order are discussed. The structure is assumed to represent a phosphotransfer competent conformation. The invariant conserved residue Asp166 is proposed to be the catalytic base and Lys168 to stabilize the transition state. In some tyrosine kinases Lys168 is functionally replaced by an Arg displaced by two residues in the primary sequence, suggesting invariance in three‐dimensional space. The structure supports an in‐line transfer with a pentacoordinate transition state at the phosphorus with very few nuclear movements.
The crystal structure of the aldehyde oxido-reductase (Mop) from the sulfate reducing anaerobic Gram-negative bacterium Desulfovibrio gigas has been determined at 2.25 A resolution by multiple isomorphous replacement and refined. The protein, a homodimer of 907 amino acid residues subunits, is a member of the xanthine oxidase family. The protein contains a molybdopterin cofactor (Mo-co) and two different [2Fe-2S] centers. It is folded into four domains of which the first two bind the iron sulfur centers and the last two are involved in Mo-co binding. Mo-co is a molybdenum molybdopterin cytosine dinucleotide. Molybdopterin forms a tricyclic system with the pterin bicycle annealed to a pyran ring. The molybdopterin dinucleotide is deeply buried in the protein. The cis-dithiolene group of the pyran ring binds the molybdenum, which is coordinated by three more (oxygen) ligands.
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