Abstract EN:

From the discovery of ubiquitin and its function as signal for proteasomal degradation over 20 years ago to this days, it became evident that ubiquitin is a universal signal in eukaryotic cells. Ubiquitin in its different forms is involved in many versatile cellular processes. Knowing that the ubiquitin signal is differently translated, depending on its occurrences as mono-ubiquitin or poly-ubiquitin, raises the question: how do cells distinguish between the different occurrences of ubiquitin and translate it into the proper response? Proteins interacting with ubiquitin contain so called ubiquitin binding domains (UBDs), whereas the affinities to ubiquitin vary from a few _M to mM. So far only three (K63, K48 and linear chains) out of the eight possible chain-linkages can be produced in sufficient amounts to characterize their interaction with UBDs. K48- and K63- linked ubiquitin chains regulate different cellular events and need to be recognized by different proteins. Thus, it is of prime importance to characterize the binding of different UBDs to these two kinds of ubiquitin chains, as it can give important clues related to the general mechanism of chain discrimination by ubiquitin adapter proteins. Some isolated UBDs exhibit a preference for one chain linkage type over the other, whereas others do not discriminate between mono-ubiquitin or K63- and K48-linked chains. Interestingly, many ubiquitin adapter proteins harbor more than one UBD. STAM2 is a ubiquitin adapter protein, that is involved in endosomal receptor sorting and supposed to preferentially bind mono-ubiquitin and K63- over K48-linked ubiquitin. STAM2 contains two UBDs (a VHS and UIM domain) that were shown to bind to ubiquitin . The current manuscript shows that STAM2’s SH3 domain binds ubiquitin as well. To understand the function of the sequential arrangement of three UBDs in one protein, first binding of the individual VHS and UIM domains to monoubiquitin as well as K48- and K63-linked di-ubiquitin was investigated. This work shows, that the VHS domain displays a different mode of binding for K63- and K48-linked diubiquitin. In spite of the fact, that the apparent Kd for both chains is the same, only one VHS domain can bind to K48-linked di-ubiquitin chains (with a preference for the distal domain), whereas K63-linked di-ubiquitin can accommodate two VHS domains at a time. Since no conclusion can be drawn with respect to the apparent Kds, the different binding modes might gain more impact in consideration of the ensemble of three UBDs. Results presented in this manuscript, based on a construct containing the VHS and UIM domain, show that binding to K63- but not K48-linked di-ubiquitin is cooperative