Stephan Krapp

Structural basis for the activation of human procaspase-7

Caspases form a family of proteinases required for the initiation and execution phases of apoptosis. Distinct proapoptotic stimuli lead to activation of the initiator caspases-8 and -9, which in turn activate the common executioner caspases-3 and -7 by proteolytic cleavage. Whereas crystal structures of several active caspases have been reported, no three-dimensional structure of an uncleaved caspase zymogen is available so far. We have determined the 2.9-Å crystal structure of recombinant human C285A procaspase-7 and have elucidated the activation mechanism of caspases. The overall fold of the homodimeric procaspase-7 resembles that of the active tetrameric caspase-7. Each monomer is organized in two structured subdomains connected by partially flexible linkers, which asymmetrically occupy and block the central cavity, a typical feature of active caspases. This blockage is incompatible with a functional substrate binding site/active site. After proteolytic cleavage within the flexible linkers, the newly formed chain termini leave the cavity and fold outward to form stable structures. These conformational changes are associated with the formation of an intact active-site cleft. Therefore, this mechanism represents a formerly unknown type of proteinase zymogen activation.

Open and closed structures of the UDP-glucose pyrophosphorylase from Leishmania major.

Uridine diphosphate-glucose pyrophosphorylase (UGPase) represents a ubiquitous enzyme, which catalyzes the formation of UDP-glucose, a key metabolite of the carbohydrate pathways of all organisms. In the protozoan parasite Leishmania major, which causes a broad spectrum of diseases and is transmitted to humans by sand fly vectors, UGPase represents a virulence factor because of its requirement for the synthesis of cell surface glycoconjugates. Here we present the crystal structures of the L. major UGPase in its uncomplexed apo form (open conformation) and in complex with UDP-glucose (closed conformation). The UGPase consists of three distinct domains. The N-terminal domain exhibits species-specific differences in length, which might permit distinct regulation mechanisms. The central catalytic domain resembles a Rossmann-fold and contains key residues that are conserved in many nucleotidyltransferases. The C-terminal domain forms a left-handed parallel β-helix (LβH), which represents a rarely observed structural element. The presented structures together with mutagenesis analyses provide a basis for a detailed analysis of the catalytic mechanism and for the design of species-specific UGPase inhibitors.

Key Factors for Successful Generation of Protein–Fragment Structures: Requirement on Protein, Crystals, and Technology

In the past two decades, fragment-based approaches have evolved as a predominant strategy in lead discovery. The availability of structural information on the interaction geometries of binding fragments is key to successful structure-guided fragment-to-lead evolution. In this chapter, we illustrate methodological advances for protein-fragment crystal structure generation in order to offer general lessons on the importance of fragment properties and the most appropriate crystallographic setup to evaluate them. We analyze elaborate protocols, methods, and clues applied to challenging complex formation projects. The results should assist medicinal chemists to select the most promising targets and strategies for fragment-based crystallography as well as provide a tutorial to structural biologists who attempt to determine protein-fragment structures.

IR laser-induced protein crystal transformation

A novel method and the associated instrumentation for improving crystalline order (higher resolution of X-ray diffraction and reduced mosaicity) of protein crystals by precisely controlled heating is demonstrated. Crystal transformation is optically controlled by a video system.

High-resolution crystal structures of factor XIa coagulation factor in complex with nonbasic high-affinity synthetic inhibitors.

Factor XI (FXI) is a key enzyme in the coagulation pathway and an attractive target for the development of anticoagulant drugs. A small number of high-resolution crystal structures of FXIa in complex with small synthetic inhibitors have been published to date. All of these ligands have a basic P1 group and bind exclusively in the nonprime side of the active site of FXIa. Here, two structures of FXIa in complex with nonbasic inhibitors that occupy both the prime and nonprime sides of the active site are presented. These new structures could be valuable in the design and optimization of new FXIa synthethic inhibitors.

Identification and optimisation of 3,3-dimethyl-azetidin-2-ones as potent and selective inhibitors of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1)

3,3-Di-methyl-azetidin-2-ones were identified as potent and selective 11β-HSD1 inhibitors against the human and mouse forms of the enzyme. Structure guided optimisation of LLE was conducted, utilising a key polar interaction and identifying stereochemical preference for the 4S isomer. Metabolic stability was improved to afford oral exposure, providing tool compounds suitable for pre-clinical evaluation.