Structures of KLK protease substrate transition states

Kallikrein-related peptidases (KLKs) are 15 (chymo)trypsin-like serine proteases that are secreted to a varying extent in nearly all tissues of humans. Some KLKs are required for fertilization, tooth development, and desquamation of the skin, while they are also critically involved in various diseases, e.g. prostate cancer or atopic dermatitis. Moreover, KLK3 (PSA) is a widely used clinical marker for prostate cancer. Up to now, information on substrate recognition and catalytic mechanisms of the KLKs on the structural level is scarce. Thus, studies of the enzyme substrate transition states are desired. To this end, substrate analog complexes in crystals will be investigated by X-ray diffraction measurements. Crystallization conditions for all KLKs from 1 to 8 and KLK10 are known. As first stage of the project, KLKs will be purified according to established or recently developed protocols, using E. coli, L. tarentolae, and HEK293 cells. Glycosylated proteins can be trimmed with deglycosylating enzymes, in order to facilitate crystallization. Crystals will be grown and soaked with substrate analogs, in order to form complexes with the respective KLK. These complexes represent either non-covalent enzyme substrate ("Michaelis") complexes, tetrahedral transition states, acyl intermediates, or enzyme product complexes. For stabilization of the usually short-lived transition states, inactive mutants can be used, e.g. KLK2-Ser195Ala with hexa- to octapeptides. Also non-cleavable surrogates of the scissile peptide bond serve this purpose, such as reduced amides (-CH 2 -NH-). Tetrahedral transition states could be mimicked by phosphinates (-PO(OPhe)-) and difluoromethylene ketones (-CO-CF2 -). Eventually, aldehyde or boronic acid peptide inhibitors can mimick acyl intermediates, while product complexes may be captured in shock frozen crystals as well. Too labile complexes can be stabilized by disulfide tethering of a KLK- Cys mutant and a Cys-containing compound prior to crystallization. The best locations of such disulfides around the active site have been determined by modelling using known crystal structures. Based on the previous investigations, the second project stage will comprise the complex formation of active KLKs with pro-KLKs and other natural substrates that are available as commercial products or recombinantly expressed proteins. Among them are semenogelins, enamel matrix and corneodesmosomal proteins. As special case of very slowly turned over substrates Kazal-type inhibitors (LEKTIs) in complex with KLKs shall be crystallized. Selected substrate fragments can be chemically ligated to suitable synthetic compounds, in order to yield stable KLK transition states complexes, which may be supported by disulfide tethering. Ideally, a series of all transition state analogs should be generated with a full length substrate. Analysis of such complexes will greatly expand our understanding of the substrate recognition and cleavage by KLKs and serine proteases in general. Eventually, it can be expected that the methods and compounds of the proposal will open new ways for basic research and bioorthogonal or even pharmaceutical applications.

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