Aberrant proteins represent an extreme hazard to cells. Therefore, molecular chaperones and proteases have to carry out protein quality control in each cellular compartment. In contrast to the ATP-dependent cytosolic proteases and chaperones, the molecular mechanisms of extracytosolic factors are largely unknown. To address this question, we studied the protease function of DegP, the central housekeeping protein in the bacterial envelope. Our data reveal that DegP processively degrades misfolded proteins into peptides of defined size by employing a molecular ruler comprised of the PDZ1 domain and the proteolytic site. Furthermore, peptide binding to the PDZ domain transforms the resting protease into its active state. This allosteric activation mechanism ensures the regulated and rapid elimination of misfolded proteins upon folding stress. In comparison to the cytosolic proteases, the regulatory features of DegP are established by entirely different mechanisms reflecting the convergent evolution of an extracytosolic housekeeping protease.allosteric regulation ͉ chaperones ͉ HtrA ͉ protein quality control ͉ molecular ruler

Flaviviruses comprise a number of important human pathogens including yellow fever, dengue, West Nile, Japanese encephalitis and tick-borne encephalitis viruses. They are small enveloped viruses that enter cells by receptor-mediated endocytosis and release their nucleocapsid into the cytoplasm by fusing their membrane with the endosomal membrane. The fusion event is triggered by the acidic pH in the endosome and is mediated by the major envelope protein E. Based on the atomic structures of the pre- and post-fusion conformations of E, a fusion model has been proposed that includes several steps leading from the metastable assembly of E at the virion surface to membrane merger and fusion pore formation trough conversion of E into a stable trimeric post-fusion conformation. Using recombinant subviral particles of tick-borne encephalitis virus as a model, we have defined individual steps of the molecular processes underlying the flavivirus fusion mechanisms. This includes the identification of a conserved histidine as being part of the pH sensor in the fusion protein that responds to the acidic pH and thus initiates the structural transitions driving fusion.

The fusion of enveloped viruses with cellular membranes is mediated by proteins that are anchored in the lipid bilayer of the virus and capable of triggered conformational changes necessary for driving fusion. The flavivirus envelope protein E is the only known viral fusion protein with a double membrane anchor, consisting of two antiparallel transmembrane helices (TM1 and TM2). TM1 functions as a stop-transfer sequence and TM2 as an internal signal sequence for the first nonstructural protein during polyprotein processing. The possible role of this peculiar C-terminal helical hairpin in membrane fusion has not been investigated so far. We addressed this question by studying TM mutants of tick-borne encephalitis virus (TBEV) recombinant subviral particles (RSPs), an established model system for flavivirus membrane fusion. The engineered mutations included the deletion of TM2, the replacement of both TM domains (TMDs) by those of the related Japanese encephalitis virus (JEV), and the use of chimeric TBEV-JEV membrane anchors. Using these mutant RSPs, we provide evidence that TM2 is not just a remnant of polyprotein processing but, together with TM1, plays an active role in fusion. None of the TM mutations, including the deletion of TM2, affected early steps of the fusion process, but TM interactions apparently contribute to the stability of the postfusion E trimer and the completion of the merger of the membranes. Our data provide evidence for both intratrimer and intertrimer interactions mediated by the TMDs of E and thus extend the existing models of flavivirus membrane fusion.Membrane fusion is a crucial step during the cell entry of enveloped viruses and is mediated by specific membrane-anchored viral surface proteins (fusion proteins) (11, 24). According to their molecular architecture, these have been assigned to three different structural classes (classes I, II, and III) (11,41). They all drive fusion by conformational changes that are triggered by interactions with the host cell (such as receptor binding or exposure to acidic pH) and presumably involve protein-protein interactions at the fusion site (41). Classes I and III, and the class II fusion proteins of alphaviruses, possess a single transmembrane (TM) domain that functions as a membrane anchor and is followed by a cytoplasmic tail of varying length (41). In contrast, the flavivirus class II viral fusion protein E is unique in possessing a hairpin-like doublemembrane-spanning carboxy terminus, derived from a special combination of stop-transfer and internal signal sequences, required for the intracellular sorting and processing of the flaviviral polyprotein (21) (Fig. 1A).Flaviviruses are members of the genus Flavivirus (family Flaviviridae) and comprise a number of important human pathogens, including the dengue viruses, Japanese encephalitis virus (JEV), yellow fever virus, West Nile virus, and tick-borne encephalitis virus (TBEV) (10, 40). They are small, enveloped, positive-strand RNA viruses that are assembled in the endoplasmic reticulum (ER) ...

The current model of flavivirus membrane fusion is based on atomic structures of truncated forms of the viral fusion protein E in its dimeric prefusion and trimeric postfusion conformations. These structures lack the two transmembrane domains (TMDs) of E as well as the so-called stem, believed to be involved in an intra-and intermolecular zippering reaction within the E trimer during the fusion process. In order to gain experimental evidence for the functional role of the stem in flavivirus membrane fusion, we performed a mutagenesis study with recombinant subviral particles (RSPs) of tick-borne encephalitis virus, which have fusion properties similar to those of whole infectious virions and are an established model for viral fusion. Mutations were introduced into the stem as well as that part of E predicted to interact with the stem during zippering, and the effect of these mutations was analyzed with respect to fusion peptide interactions with target cells, E protein trimerization, trimer stability, and membrane fusion in an in vitro liposome fusion assay. Our data provide evidence for a molecular interaction between a conserved phenylalanine at the N-terminal end of the stem and a pocket in domain II of E, which appears to be essential for the positioning of the stem in an orientation that allows zippering and the formation of a structure in which the TMDs can interact as required for efficient fusion.Flaviviruses form a genus within the family Flaviviridae and include important human pathogens such as the dengue viruses, Japanese encephalitis virus, West Nile virus, yellow fever virus, and tick-borne encephalitis virus (TBEV) (41). These small enveloped viruses enter cells by receptor-mediated endocytosis and fuse their membrane with that of the endosome. Fusion is triggered by the acidic pH of this compartment and is mediated by the viral envelope protein E, a class II viral fusion protein (37). During virus assembly, heterodimers of E and the precursor membrane protein prM together with the nucleocapsid bud into the lumen of the endoplasmic reticulum (ER), thus leading to the formation of nonfusogenic immature particles (10,24). In the course of their exocytosis, the prM protein is processed by the host protease furin in the transGolgi network, and only the membrane-anchored part of M remains associated with secreted mature virions (42, 43). The maturation process results in the formation of smooth-surfaced particles with a herringbone-like arrangement of metastable E homodimers on the virus surface (18, 28).X-ray crystallography of carboxy-terminally truncated soluble E (sE) proteins revealed an organization into three structurally distinct domains (domain I [DI], DII, and DIII) linked by short flexible regions (Fig. 1A) (15,25,27,30,31,46). DII contains the highly conserved fusion peptide (FP) loop at its tip. In the full-length protein, DIII is followed by the ϳ50-amino-acid-long "stem" that connects the ectodomain to the membrane anchor and consists of two amphipathic alpha helices (H1 and H2) flanking...