Mutational evidence for an internal fusion peptide in flavivirus envelope protein E

The envelope protein E of the flavivirus tick-borne encephalitis (TBE) virus promotes cell entry by inducing fusion of the viral membrane with an intracellular membrane after uptake by endocytosis. This protein differs from other well-studied viral and cellular fusion proteins because of its distinct molecular architecture and apparent lack of involvement of coiled coils in the low-pH-induced structural transitions that lead to fusion. A highly conserved loop (the cd loop), which resides at the distal tip of each subunit and is mostly buried in the subunit interface of the native E homodimer at neutral pH, has been hypothesized to function as an internal fusion peptide at low pH, but this has not yet been shown experimentally. It was predicted by examination of the X-ray crystal structure of the TBE virus E protein (F. A. Rey et al., Nature 375:291-298, 1995) that mutations at a specific residue within this loop (Leu 107) would not cause the native structure to be disrupted. We therefore introduced amino acid substitutions at this position and, using recombinant subviral particles, investigated the effects of these changes on fusion and related properties. Replacement of Leu with hydrophilic amino acids strongly impaired (Thr) or abolished (Asp) fusion activity, whereas a Phe mutant still retained a significant degree of fusion activity. Liposome coflotation experiments showed that the fusion-negative Asp mutant did not form a stable interaction with membranes at low pH, although it was still capable of undergoing the structural rearrangements required for fusion. These data support the hypothesis that the cd loop may be directly involved in interactions with target membranes during fusion.     

Expression of olfactory receptors in the cribriform mesenchyme during prenatal development

Olfactory receptors (ORs) are expressed in sensory neurons of the nasal epithelium, where they are supposed to be involved in the recognition of suitable odorous compounds and in the guidance of outgrowing axons towards the appropriate glomeruli in the olfactory bulb. During development, some olfactory receptor subtypes have also been found in non-sensory tissues, including the cribriform mesenchyme between the prospective olfactory epithelium and the developing telencephalon, but it is elusive if this is a typical phenomenon for ORs. Monitoring the onset and time course of expression for several receptor subtypes revealed that 'extraepithelial' expression of ORs occurs very early and transiently, in particular between embryonic stages E10.25 and E14.0. In later stages, a progressive loss of receptor expressing cells was observed. Molecular phenotyping demonstrated that the receptor expressing cells in the cribriform mesenchyme co-express key elements, including Galpha(olf), ACIII and OMP, characteristic for olfactory neurons in the nasal epithelium. Studies on transgenic OMP/GFP-mice showed that 'extraepithelial' OMP/GFP-positive cells are located in close vicinity to axon bundles projecting from the nasal epithelium to the presumptive olfactory bulb. Moreover, these cells are primarily located where axons fasciculate and change direction towards the anterior part of the forebrain.     

Solution-phase synthesis of Aib-containing cyclic hexapeptides

The 18-membered Aib-containing cyclohexapeptides, cyclo(-Gly-Aib-Aib-Gly-Aib-Phe-) (22), cyclo(-Gly-Aib-Phe(2Me)-Gly-Aib-Aib-) (24a), cyclo(-Gly-Phe(2Me)-Aib-Gly-Aib-Aib-) (24b), and cyclo(-Gly-Phe(2Me)-Aib-Gly-Aib-Phe-) (25), have efficiently been synthesized by solution-phase techniques. The linear precursors 1a-1d were prepared by combining the 'azirine/oxazolone method' for incorporation of alpha,alpha-disubstituted alpha-amino acids (Aib, Phe(2Me)) into the peptide chains by classical peptide coupling methods for segment condensations. Deprotection of the amino and carboxy termini of 1a-1d, followed by cyclization with DEPC as the coupling reagent, gave the above-mentioned cyclic hexapeptides 22, 24a, 24b, and 25 in good yields (26-57%). The solid-state conformations of the linear hexapeptides 1d, 16 and 27, and of the cyclohexapeptides 22 and 25 have been established by X-ray crystallography.     

Evolution of class B floral homeotic proteins: obligate heterodimerization originated from homodimerization

The class B floral homeotic genes from the higher eudicot model systems Arabidopsis and Antirrhinum are involved in specifying the identity of petals and stamens during flower development. These genes exist in two different types termed DEF- and GLO-like genes. The proteins encoded by the class B genes are stable and functional in the cell only as heterodimeric complexes of a DEF- and a GLO-like protein. In line with this, heterodimerization is obligate for DNA binding in vitro. The genes whose products have to heterodimerize to be stable and functional are each other's closest relatives within their genomes. This suggests that the respective genes originated by gene duplication, and that heterodimerization is of relative recent origin and evolved from homodimerization. To test this hypothesis we have investigated the dimerization behavior of putative B proteins from phylogenetic informative taxa, employing electrophoretic mobility shift assays and the yeast two-hybrid system. We find that an ancestral B protein from the gymnosperm Gnetum gnemon binds DNA in a sequence-specific manner as a homodimer. Of the two types of B proteins from the monocot Lilium regale, the GLO-like protein is still able to homodimerize, whereas the DEF-like protein binds to DNA only as a heterodimeric complex with the GLO-like protein. These data suggest that heterodimerization evolved in two steps after a gene duplication that gave rise to DEF- and GLO-like genes. Heterodimerization may have originated after the gymnosperm-angiosperm split about 300 MYA but before the monocot-eudicot split 140-200 MYA. Heterodimerization may have become obligate for both types of flowering plant B proteins in the eudicot lineage after the monocot-eudicot split.     

The murine alpha(1)-proteinase inhibitor gene family: polymorphism,chromosomal location,and structure

alpha(1)-Proteinase inhibitor (alpha(1)-PI) is a member of the serpin superfamily of serine proteinase inhibitors, which function in maintaining homeostasis through regulation of numerous proteolytic processes. In laboratory mice (Mus musculus domesticus), alpha(1)-PI occurs in multiple isoforms encoded by a family of three to five genes that are polymorphic among inbred strains and that are located at the Serpina1 locus on chromosome 12. In the present study, we have characterized the alpha(1)-PI gene family of inbred mice in more detail. We show that mice express seven isoforms, all of which are encoded by genes that map to the Serpina1 locus. In addition, polymorphism at the locus is defined by three haplotypes (Serpina1(b), Serpina1(c), and Serpina1(l)) that differ with regard to both the number and identity of alpha(1)-PI genes. Finally, we present the complete sequence of an 84-kb region of Serpina1 containing a tandem repeat of two alpha(1)-PI genes.     

Capsid protein C of tick-borne encephalitis virus tolerates large internal deletions and is a favorable target for attenuation of virulence

Deletions ranging in size from 4 to 21 amino acid residues were introduced into the capsid protein of the flavivirus tick-borne encephalitis (TBE) virus. These deletions incrementally affected a hydrophobic domain which is present at the center of all flavivirus capsid protein sequences and part of which may form an amphipathic alpha-helix. In the context of the full-length TBE genome, the deletions did not measurably affect protein expression and up to a deletion length of 16 amino acid residues, corresponding to almost 17% of mature protein C, viable virus was recovered. This virus was strongly attenuated but highly immunogenic in adult mice, revealing capsid protein C as a new and attractive target for the directed attenuation of flaviviruses. Apparently, the larger deletions interfered with the correct assembly of infectious virus particles, and this disturbance of virion assembly is likely to be the molecular basis of attenuation. However, all of the mutants carrying large deletions produced substantial amounts of subviral particles, which as judged from density gradient analyses were identical to recombinant subviral particles as obtained by the expression of the surface proteins prM and E alone. The structural and functional flexibility of protein C revealed in this study and its predicted largely alpha-helical conformation are reminiscent of capsid proteins of other enveloped viruses, such as alphaviruses (N-terminal domain of the capsid protein), retroviruses, and hepadnaviruses and suggest that all of these may belong to a common structural class, which is fundamentally distinct from the classical beta-barrel structures of many icosahedral viral capsids. The possibility of attenuating flaviviruses by disturbing virus assembly and favoring the production of noninfectious but highly immunogenic subviral particles opens up a promising new avenue for the development of live flavivirus vaccines.     

Effects of multiple deletions of murein hydrolases on viability,septum cleavage,and sensitivity to large toxic molecules in Escherichia coli

The multiplicity of murein hydrolases found in most bacteria presents an obstacle to demonstrating the necessity of these potentially autolytic enzymes. Therefore, Escherichia coli mutants with deletions in multiple murein hydrolases, including lytic transglycosylases, amidases, and DD-endopeptidases, were constructed. Even a mutant from which seven different hydrolases were deleted was viable and grew at a normal rate. However, penicillin-induced lysis was retarded. Most of the mutants were affected in septum cleavage, which resulted in the formation of chains of cells. All three enzymes were shown to be capable of splitting the septum. Failure to cleave the septum resulted in an increase in outer membrane permeability, and thus the murein hydrolase mutants did not grow on MacConkey agar plates. In addition, the hydrolase mutants not only could be lysed by lysozyme in the absence of EDTA but also were sensitive to high-molecular-weight antibiotics, such as vancomycin and bacitracin, which are normally ineffective against E. coli.     

Nested MWC model describes hydrolysis of GroEL without assuming negative cooperativity in binding

Folding assistance and ATPase activity of GroEL are based on the existence of different conformations. In order to characterise these conformations, published data on steady state ATPase activity in the absence of GroES were reanalysed simultaneously in terms of the Nested MWC model. This model is a hierarchical extension of the symmetry-model of Monod et al. [J. Mol. Biol. 12 (1965) 88]. An unique set of GroEL specific parameters was obtained. This set was supported by comparison of predictions arising from this set of values with experimental data for hydrolysis of ATP in the presence of ADP and ATPgammaS, binding of ATPgammaS and ADP to GroEL in the absence of ATP, and binding of ATP as monitored by fluorescence labelling. Thus, for the first time, multiple data sets for the interaction of nucleotides with GroEL are described quantitatively by an allosteric model. A noteworthy feature of our model is that no negative cooperativity in ATP binding occurs in accordance to experimental observations. Furthermore, the model also includes the existence of a conformation with very high ATPase activity. Such a conformation might be of importance at a certain stage in the folding cycle.