Structuraj investigations on the Ddi1 (DNA-damage inducible protein 1) of Leishmania major and on the rotavirus nonstructural protein NSP4 were carried out. Ddi1 belongs to the ubiquitin receptor family of proteins. One of its domains is similar to the retroviral aspartic proteinases. It has been shown that this domain is the target of HIV-protease inhibitors that were being used in the treatment of AIDS and it was observed that these drugs effectively controlled opportunistic diseases caused by many parasitic protozoa such as Leishmania and Plasmodium species. The retroviral protease-like domains present in Ddi1 proteins of these organisms were identified as the targets of these drugs. Structural studies on Ddi1 from L. major have been carried out, in an attempt to provide a platform for the design of anti-protozoal compounds. Rotavirus NSP4, the first viral enterotoxin to be identified, is a multifunctional glycoprotein that plays critical roles in viral pathogenesis and morphogenesis. As part of an ongoing project on the structural characterization of NSP4, we determined the structure of the diarrhea-inducing region of this protein from the rotavirus strain MF66. Chapter 1 presents an overview of Ddi1 and NSP4 of the rotavirus with an emphasis on their structural features. The methods employed during the course of the present work are described in Chapter 2. Structural studies on the retroviral protease-like domain of Ddi1 (Ddi1-RVP) of L. major is presented in Chapter 3. Apart from this domain, Ddi1 of L. major also has a ubiquitin-associated and ubiquitin-like domains whereas P. falciparum has only the ubiquitin-associated domain. Activity of the full length Ddi1 of L. major and the retroviral protease domain of P. falciparum using an HIV protease substrate was shown to be inhibited by an HIV protease inhibitor, saquinavir. Binding of saquinavir to the proteins was also confirmed by Biolayer Interferometry studies. The crystal structure of the retroviral protease domain of L. major Ddi1 has been determined. It forms a homodimeric structure similar to that of HIV protease and the reported structure of the same domain from Saccharomyces cerevisiae. The loops in Ddi1-RVP are similar to the 'flap' regions of the HIV protease which close-in upon substrate/inhibitor binding; they are visible in the electron density maps, unlike the case of the S. cerevisiae protein. Though the native form of the domain shows an open dimeric structure, normal mode analysis reveals that it can take up a closed conformation resulting from relative movements of the subunits. The present structure of Ddi1-RVP of L. major with the defined 'flap'-like loops will be helpful in the design of effective drugs against protozoal diseases, starting with HIV protease inhibitors as the lead compounds. Chapter 4 describes the structural investigations carried out on the diarrhea-inducing region of the nonstructural protein NSP4 of the rotavirus strain MF66 which forms an α-helical coiled-coil structure. Crystal structures of a synthetic peptide and of two recombinant proteins spanning this region showed parallel tetrameric organization of this domain with a bound Ca2+ ion at the core. Subsequently, we determined the structure of NSP4 from a different strain as a pentamer without the bound Ca2+ ion. This new structure provides more insights into understanding some of the functions of NSP4 such as the release of ions into the cytoplasm and binding to the double-layered particle (DLP). We also established conditions responsible for these structural transitions. The crystal structure of the coiled-coil domain of NSP4 presented in this chapter shows an entirely different structure which is an antiparallel tetramer. This explains our failure to determine the structure by the molecular replacement method using known oligomers. The structure was solved by the Sulphur-SAD method using diffraction data collected with Cr Ka radiation. The study reveals that the structural diversity of NSP4 is not limited. We could relate sequence variations and pH conditions to the differences in oligomeric assemblies. Surface properties of the domain suggest that the new form is likely to interact with different sets of proteins compared to those that interact with the parallel tetramers or pentamers. Further investigations are needed to establish this property.

Die ER-assoziierte Proteindegradation (ERAD) ist Teil des Qualitätskontrollsystems am ER, um der Akkumulation von fehlgefalteten Proteinen im ER entgegenzuwirken. Hierbei werden ERAD-Substrate mit Hilfe von E3-Ligasen wie z.B. HRD1 ubiquityliert und anschließend durch den p97-Ufd1-Npl4 Komplex aus der ER-Membran extrahiert. Im Zytosol werden diese extrahierten Proteine vom 26S Proteasom abgebaut. Für die Retrotranslokation von ERAD- Substraten werden zudem die Membranproteine Derlin-1 und VIMP benötigt, welche mit p97 assoziieren und einen Proteinkomplex bilden. HERP ist ein ER-lokalisiertes Protein, dessen Synthese durch den UPR (unfolded protein response) als Antwort auf die Akkumulation von fehlgefalteten Proteinen im ER induziert wird. Dies deutet auf eine Rolle von HERP im ERAD hin. Interessanterweise besitzt HERP eine sogenannte UBL-Domäne. Für andere Proteine mit UBL-Domäne konnte eine Interaktion dieser Domäne mit dem Proteasom nachgewiesen werden. Daher kann angenommen werden, dass HERP ebenfalls mit dem Proteasom interagiert und dies zur ER- Membran rekrutiert, wo es für den Abbau von ERAD-Substraten benötigt wird. Das Ziel der vorliegenden Arbeit war es, die Rolle von HERP innerhalb des UPR zu ermitteln. Die hier präsentierten Daten zeigen, dass HERP essentiell für den Abbau des ERAD-Modell- Substrates CD3-delta ist. Somit hat HERP tatsächlich eine Rolle im ERAD. Außerdem wird eine direkte Interaktion von HERP mit der E3-Ligase HRD1 nachgewiesen. Es wird zudem gezeigt, dass HERP und HRD1 einen Proteinkomplex mit p97, Derlin-1 und eventuell auch mit VIMP bilden. Dieser ERAD Komplex ist folglich sowohl für die Ubiquitylierung als auch die Retrotranslokation von ERAD-Substraten verantwortlich und garantiert somit die effiziente Prozessierung von Proteinen aus dem ER. Zudem wird gezeigt, dass die UBL-Domäne von HERP im Gegensatz zu anderen UBL- Domänen nicht mit dem Proteasom interagiert. Somit kann nicht mehr davon ausgegangen werden, dass Proteasombindung eine Gemeinsamkeit aller Proteine mit UBL-Domäne ist. Dagegen wird eine Interaktion der UBL-Domäne von HERP mit dem deubiquitylierenden Enzym USP7 nachgewiesen. Dies deutet darauf hin, dass auch Deubiquitylierung eine wichtige Rolle im ERAD-Prozess spielt. / ER-associated protein degradation (ERAD) is part of the ER quality control system dealing with the accumulation of misfolded proteins in the ER. This process requires polyubiquitylation of ERAD substrates involving E3 ligases, such as HRD1, and their subsequent extraction from the ER membrane by the p97-Ufd1-Npl4 complex. Retrotranslocation of substrates into the cytosol for degradation by the 26S proteasome also involves the membrane proteins Derlin-1 and VIMP, which are associated with p97 to form a protein complex. The ER-resident protein HERP was shown to be upregulated by the unfolded protein response pathway (UPR) upon the accumulation of misfolded proteins in the ER. It was therefore considered to function in ERAD. Interestingly, HERP contains a UBL domain. In other proteins this domain facilitates an interaction with the proteasome, suggesting that HERP might recruit the proteasome to the ER membrane for efficient ERAD. The aim of this study was to investigate the function of HERP within the UPR. The findings presented here demonstrate that HERP is essential for the degradation of a model ERAD substrate. Thus, HERP indeed has a role in ERAD. Moreover, the data show that HERP directly interacts with the E3 ligase HRD1 and the two proteins form a common protein complex with p97, Derlin-1 and possibly also with VIMP. This suggests that both ubiquitylation and retrotranslocation of ER proteins are performed by one protein complex, enabling an efficient processing of ERAD substrates. This study also demonstrates that the UBL domain of HERP does not share the proteasome binding property of other UBL domains, suggesting that proteasome binding cannot be considered a general feature of all UBL domains. Instead, the HERP UBL domain is able to interact with the deubiquitylating enzyme USP7. Therefore, deubiquitylation might also be an important aspect in the proteasome-dependent degradation of misfolded ER proteins.