Human cystatin C (HCC) is a family 2 cystatin inhibitor of papain‐like (C1) and legumain‐related (C13) cysteine proteases. In pathophysiological processes, the nature of which is not understood, HCC is codeposited in the amyloid plaques of Alzheimer’s disease or Down’s syndrome. The amyloidogenic properties of HCC are greatly increased in a naturally occurring L68Q variant, resulting in fatal cerebral amyloid angiopathy in early adult life. In all crystal structures of cystatin C studied to date, the protein has been found to form 3D domain‐swapped dimers, created through a conformational change of a β‐hairpin loop, L1, from the papain‐binding epitope. We have created monomer‐stabilized human cystatin C, with an engineered disulfide bond (L47C)–(G69C) between the structural elements that become separated upon domain swapping. The mutant has drastically reduced dimerization and fibril formation properties, but its inhibition of papain is unaltered. The structure confirms the success of the protein engineering experiment to abolish 3D domain swapping and, in consequence, amyloid fibril formation. It illustrates for the first time the fold of monomeric cystatin C and allows verification of earlier predictions based on the domain‐swapped forms and on the structure of chicken cystatin. Importantly, the structure defines the so‐far unknown conformation of loop L1, which is essential for the inhibition of papain‐like cysteine proteases.

In this work, the influence of an internal electric field upon the crystallization of lysozyme and thaumatin is explored using a modified design of the gel-acupuncture setup. From a crystallographic point of view, the orientation of crystals that grow preferentially over different types of electrodes inside capillary tubes is also evaluated. Finally, the crystal quality and the three-dimensional structure of these proteins grown with and without the electric field influence are analyzed by means of X-ray diffraction methods.

Plant L-asparaginases and their bacterial homologs, such as EcAIII found in Escherichia coli, form a subgroup of the N-terminal nucleophile (Ntn)-hydrolase family. In common with all Ntn-hydrolases, they are expressed as inactive precursors that undergo activation in an autocatalytic manner. The maturation process involves intramolecular hydrolysis of a single peptide bond, leading to the formation of two subunits (␣ and ␤) folded as one structural domain, with the nucleophilic Thr residue located at the freed N terminus of subunit ␤. The mechanism of the autocleavage reaction remains obscure. We have determined the crystal structure of an active site mutant of EcAIII, with the catalytic Thr residue substituted by Ala (T179A). The modification has led to a correctly folded but unprocessed molecule, revealing the geometry and molecular environment of the scissile peptide bond. The autocatalytic reaction is analyzed from the point of view of the Thr 179 side chain rotation, identification of a potential general base residue, and the architecture of the oxyanion hole.Posttranslational modifications of proteins can be divided into reactions leading to covalent attachment, usually at a side chain, of a specific chemical group, such as phosphate or carbohydrate, and into reactions that lead to cleavage or cleavage/ rearrangement of the polypeptide backbone. The latter processes can be enzyme-catalyzed or occur without an additional biocatalyst. An important class of backbone rearrangement is connected with autocatalytic processes in maturating proteins. Notable examples in this category include intein splicing and simple backbone cleavage. Protein splicing requires cleavage of two peptide bonds that surround the so-called intein, followed by ligation of the flanking polypeptides, the N-and C-exteins. The process consists of four steps: acyl rearrangement, transesterification, cyclization of an asparagine residue, and a second acyl rearrangement (1). In maturation following the autocleavage pathway, only a single peptide cleavage occurs, and the mechanism includes only acylation and water-dependent deacylation. Although the mechanism of protein splicing has been extensively studied and seems to be well understood, the autoproteolysis reactions are rather obscure. The known examples of autoproteolytic proteins include a large group of N-terminal nucleophile (Ntn) 2 -hydrolases (2). In Ntn enzymes, a cleavage of a precursor molecule is required to generate a catalytic residue at the newly formed N terminus, which can be a threonine, serine, or cysteine. The family of Ntn-hydrolases includes such enzymes as aspartylglucosaminidases (3, 4), penicillin acylases (5-7), taspase1 (8), and plant-type L-asparaginases (9 -12). Sequence similarity within the Ntn-hydrolase family is very limited, but despite the variation of primary structure, the proteins share a common sandwich-like ␣␤␤␣ fold created by two ␤-sheets surrounded by two layers of ␣-helices (13). The autocatalyzed maturation of Ntn-hydrolases involves either the remo...

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