Background: Oligopeptidases are serine proteases cleaving only short peptides. Results: The complex channel system found within a hexameric oligopeptidase presents a rigid, double-gated model for size-based substrate selection. Conclusion:The substrate selection mechanism applied by an oligopeptidase depends on its multimerization state. Significance: Degradation of cytotoxic and misfolded proteins is aided by oligopeptidases, which are thus possible targets of cancer therapy.

The structure of tetrameric mammalian acylaminoacyl peptidase – a key upstream regulator of the proteasome – was determined by cryo-EM (and elucidated by MD), showing a “shutters-and-channels” substrate selection apparatus created by oligomerization.

Enzymes of the prolyl oligopeptidase family (S9 family) recognize their substrates not only by the specificity motif to be cleaved but also by size - they hydrolyze oligopeptides smaller than 30 amino acids. They belong to the serine-protease family, but differ from classical serine-proteases in size (80 kDa), structure (two domains) and regulation system (size selection of substrates). This group of enzymes is an important target for drug design as they are linked to amnesia, schizophrenia, type 2 diabetes, trypanosomiasis, periodontitis and cell growth. By comparing the structure of various members of the family we show that the most important features contributing to selectivity and efficiency are: (i) whether the interactions weaving the two domains together play a role in stabilizing the catalytic triad and thus their absence may provide for its deactivation: these oligopeptidases can screen their substrates by opening up, and (ii) whether the interaction-prone β-edge of the hydrolase domain is accessible and thus can guide a multimerization process that creates shielded entrance or intricate inner channels for the size-based selection of substrates. These cornerstones can be used to estimate the multimeric state and selection strategy of yet undetermined structures.

The structure of porcine AAP (pAAP) in a covalently bound complex with meropenem was determined by cryo-EM to 2.1 Å resolution, showing the mammalian serine-protease inhibited by a carbapenem antibiotic....

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Serial femtosecond crystallography (SFX) using X-ray free electron laser (XFEL) significantly increases the realm of possibility of obtaining structural information from membrane proteins. So far access to the technology is limited to 2 XFEL facilities (LCLS, USA and SACLA, Japan) severely restricting application of this revolutionary technique. LeadXpro brings the industrial expertise of structure-based drug design on membrane protein and has premium access to SwissFEL when it starts operation towards the end of 2017. With capabilities for gene-to-structure, we employ a diversity of technique and industrial expertise including the crystallization of membrane protein (GPCRs, transporters and ion channels), traditional/serial crystallography and electron microscopy, coupled to easy access to synchrotron beamlines at the SLS and the collective scientific expertise at the Paul Scherrer institute. In collaboration with scientists at the SLS, we are also committed to further develop technology for application in serial crystallography. Our goal is to apply these methods for structure-based drug discovery to study difficult membrane protein target implicated in a variety of human diseases. Here we describe the drug discovery pipeline and technology we employed at LeadXPro, and show some examples of the range of activities in the company.

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