Site-directed cross-linking with KLK proteases from prostate

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Many biological processes depend on proteins that bind other molecules, which are often proteins as well. Some of these complexes are very stable and can be analyzed in structural biology. However, there are also short-lived complexes, which are much more difficult to investigate. The current procedures for cross-linking molecular components of unstable complexes are rather unspecific. Thus, new approaches for site-directed linking are desirable. So-called click chemistry reactions result in the formation of covalent bonds at defined positions in proteins, among them the well-established 1,3-dipolar cycloaddition, also known as azide-alkyne click reaction, and the newly developed thiol-ene coupling for proteins. Both approaches are based on non-natural or non-canonical amino acids (nnAA), which can be incorporated into selected proteins by manipulating the genetic code and the protein synthesis in cell cultures. A suitable model system for protein-ligand interactions are the human kallikrein-related peptidases (KLKs) from prostate, in particular KLKs 2, 3, 4, 5, and 11. These serine proteases have important physiological functions in fertilization and altered roles under pathological conditions as prostate cancer.Knowledge of several KLK crystal structures is a good basis for placing nnAAs at selected positions of the proteases for linking them to other molecules. To this end, mainly inactive variants of these KLKs will be employed, which do not cleave the linked molecules after the click reactions. The two approaches of the click reactions will be applied in two steps: 1. Inactive KLK variants containing reactive nnAAs are linked with peptides containing an azide and an alkyne as nnAA reaction partner, or a cysteine and an olefinic side-chain, respectively. These peptides are derived from natural substrates, for which the prostatic KLKs are specific. 2. The nnAA-KLKs will be linked to natural substrates, with corresponding modifications by recombinant production. After purification of the stabilized complexes, they are biochemically characterized and crystallized. If this procedure yields suitable crystals for X-ray diffraction, most likely the structures of the protein complexes can be determined.Site-directed cross-linking of proteins with nnAAs could become a general method, which is applicable to protein complexes with other biomolecules, such as nucleic acids, lipids, and sugars. Especially in this project information can be gained on the three-dimensional structural elements of the KLKs, which determine their substrate specificity, which is highly valuable for elucidating biological and disease-related processes, e.g. in prostate cancer. Knowledge of this “tertiary specificity” of the KLK proteases will enable the preparation of novel drugs with unprecedented efficacy.