A point mutation within the ATP-binding site inactivates both catalytic functions of the ATP-dependent protease La (Lon) from Escherichia coli

AbstractA point mutant in the ATP-binding motif (GPPGVGK362T) of the ATP-dependent protease La from Escherichia coli was investigated in which the lysine at position 362 was replaced by an alanine. The catalytic efficiency of the K362A mutant is at least two orders of magnitude lower than that of wild-type protease La due to a decreased Vmax and an increased KM for ATP. Simultaneously, the peptidase activity of La K362A is almost completely eliminated. Since selective inactivation of the peptidase activity of La does not affect its intrinsic ATPase activity, coupling of proteolysis with ATP hydrolysis is only uni-directional in this energy-dependent protease

Oligosaccharyltransferase: the central enzyme of N-linked protein glycosylation

Abtract: N-linked glycosylation is one of the most abundant modifications of proteins in eukaryotic organisms. In the central reaction of the pathway, oligosaccharyltransferase (OST), a multimeric complex located at the membrane of the endoplasmic reticulum, transfers a preassembled oligosaccharide to selected asparagine residues within the consensus sequence asparagine-X-serine/threonine. Due to the high substrate specificity of OST, alterations in the biosynthesis of the oligosaccharide substrate result in the hypoglycosylation of many different proteins and a multitude of symptoms observed in the family of congenital disorders of glycosylation (CDG) type I. This review covers our knowledge of human OST and describes enzyme composition. The Stt3 subunit of OST harbors the catalytic center of the enzyme, but the function of the other, highly conserved, subunits are less well defined. Some components seem to be involved in the recognition and utilization of glycosylation sites in specific glycoproteins. Indeed, mutations in the subunit paralogs N33/Tusc3 and IAP do not yield the pleiotropic phenotypes typical for CDG type I but specifically result in nonsyndromic mental retardation, suggesting that the oxidoreductase activity of these subunits is required for glycosylation of a subset of proteins essential for brain developmen

Circularly permuted variants of the green fluorescent protein

AbstractFolding of the green fluorescent protein (GFP) from Aequorea victoria is characterized by autocatalytic formation of its p-hydroxybenzylideneimidazolidone chromophore, which is located in the center of an 11-stranded β-barrel. We have analyzed the in vivo folding of 20 circularly permuted variants of GFP and find a relatively low tolerance towards disruption of the polypeptide chain by introduction of new termini. All permuted variants with termini in strands of the β-barrel and about half of the variants with termini in loops lost the ability to form the chromophore. The thermal stability of the permuted GFPs with intact chromophore is very similar to that of the wild-type, indicating that chromophore-side chain interactions strongly contribute to the extraordinary stability of GFP

Struktur, Assemblierung und Stabilität von Typ-1-Pili

Type 1 pili are extracellular, supramolecular protein complexes required for the attachment of pathogenic E. coli strains to host cells. Hundreds to thousands of protein subunits are assembled within minutes in vivo and form filaments with unique kinetic stability against dissociation. Here, we review recent work on the structure, assembly mechanism and potential technical applications derived from this exciting biological syste

The Escherichia coli glycophage display system

We describe a phage display technique that allows the production and selective enrichment of phages that display an N-glycoprotein (glycophages). We applied glycophage display to select functional glycosylation sequons from a pool of randomized acceptor sequences. Our system provides a genetic platform to study and engineer different steps in the pathway of bacterial N-linked protein glycosylatio

A metabolite binding protein moonlights as a bile- responsive chaperone

Bile salts are secreted into the gastrointestinal tract to aid in the absorption of lipids. In addition, bile salts show potent antimicrobial activity in part by mediating bacterial protein unfolding and aggregation. Here, using a protein folding sensor, we made the surprising discovery that the Escherichia coli periplasmic glycerol- 3- phosphate (G3P)- binding protein UgpB can serve, in the absence of its substrate, as a potent molecular chaperone that exhibits anti- aggregation activity against bile salt- induced protein aggregation. The substrate G3P, which is known to accumulate in the later compartments of the digestive system, triggers a functional switch between UgpB’s activity as a molecular chaperone and its activity as a G3P transporter. A UgpB mutant unable to bind G3P is constitutively active as a chaperone, and its crystal structure shows that it contains a deep surface groove absent in the G3P- bound wild- type UgpB. Our work illustrates how evolution may be able to convert threats into signals that first activate and then inactivate a chaperone at the protein level in a manner that bypasses the need for ATP.SynopsisThe periplasmic glycerol- 3- phosphate binding protein, UgpB, was found to have dual functions, as a metabolite binding protein and as a bile- responsive molecular chaperone. Stomach- acid induced stripping of its glycerol- 3- phosphate substrate functions as a switch that activates the chaperone activity of UgpB.A tripartite periplasmic protein folding sensor and Tn- Seq uncover UgpB as a new chaperone.UgpB prevents bile- induced protein aggregation when in its G3P- free form.Stomach acid- induced G3P stripping activates UgpB chaperone function.Crystal structure of a G3P- nonbinding variant of UgpB reveals opening of a deep surface groove when compared to the structure of G3P- bound wild- type UgpB.A periplasmic folding sensor reveals a mechanism by which stomach acid- induced G3P stripping remodels UgpB into a chaperone that prevents bile- induced bacterial protein aggregation.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/163430/6/embj2019104231.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/163430/5/embj2019104231-sup-0002-EVFigs.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/163430/4/embj2019104231-sup-0006-SDataFig3.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/163430/3/embj2019104231.reviewer_comments.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/163430/2/embj2019104231_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/163430/1/embj2019104231-sup-0005-SDataFig2.pd

Structural and functional characterization of the oxidoreductase a-DsbA1 from wolbachia pipientis

The &alpha;-proteobacterium Wolbachia pipientis is a highly successful intracellular endosymbiont of invertebrates that manipulates its host\u27s reproductive biology to facilitate its own maternal transmission. The fastidious nature of Wolbachia and the lack of genetic transformation have hampered analysis of the molecular basis of these manipulations. Structure determination of key Wolbachia proteins will enable the development of inhibitors for chemical genetics studies. Wolbachia encodes a homologue (&alpha;-DsbA1) of the Escherichia coli dithiol oxidase enzyme EcDsbA, essential for the oxidative folding of many exported proteins. We found that the active-site cysteine pair of Wolbachia &alpha;-DsbA1 has the most reducing redox potential of any characterized DsbA. In addition, Wolbachia &alpha;-DsbA1 possesses a second disulfide that is highly conserved in &alpha;-proteobacterial DsbAs but not in other DsbAs. The &alpha;-DsbA1 structure lacks the characteristic hydrophobic features of EcDsbA, and the protein neither complements EcDsbA deletion mutants in E. coli nor interacts with EcDsbB, the redox partner of EcDsbA. The surface characteristics and redox profile of &alpha;-DsbA1 indicate that it probably plays a specialized oxidative folding role with a narrow substrate specificity. This first report of a Wolbachia protein structure provides the basis for future chemical genetics studies.<br /

Cloning, expression, purification and characterization of a DsbA-like protein from Wolbachia pipientis

Wolbachia pipientis are obligate endosymbionts that infect a wide range of insect and other arthropod species. They act as reproductive parasites by manipulating the host reproduction machinery to enhance their own transmission. This unusual phenotype is thought to be a consequence of the actions of secreted Wolbachia proteins that are likely to contain disulfide bonds to stabilize the protein structure. In bacteria, the introduction or isomerization of disulfide bonds in proteins is catalyzed by Dsb proteins. The Wolbachia genome encodes two proteins, a-DsbA1 and a-DsbA2, that might catalyze these steps. In this work we focussed on the 234 residue protein a-DsbA1; the gene was cloned and expressed in Escherichia coli, the protein was purified and its identity confirmed by mass spectrometry. The sequence identity of a-DsbA1 for both dithiol oxidants(E. coli DsbA, 12%) and disulfide isomerases(E. coli DsbC, 14%) is similar. We therefore sought to establish whether a-DsbA1 is an oxidant or an isomerase based on functional activity. The purified a-DsbA1 was active in an oxidoreductase assay but had little isomerase activity, indicating that a-DsbA1 is DsbA-like rather than DsbC-like. This work represents the first successful example of the characterization of a recombinant Wolbachia protein. Purified a-DsbA1 will now be used in further functional studies to identify protein substrates that could help explain the molecular basis for the unusual Wolbachia phenotypes, and in structural studies to explore its relationship to other disulfide oxidoreductase proteins. Copyright © 2008 Elsevier In

Staphylococcus aureus DsbA does not have a destabilizing disulfide: A new paradigm for bacterial oxidative folding

In Gram-negative bacteria, the introduction of disulfide bonds into folding proteins occurs in the periplasm and is catalyzed by donation of an energetically unstable disulfide from DsbA, which is subsequently re-oxidized through interaction with DsbB. Gram-positive bacteria lack a classic periplasm but nonetheless encode Dsb-like proteins. Staphylococcus aureus encodes just one Dsb protein, a DsbA, and no DsbB. Here we report the crystal structure of S. aureus DsbA (SaDsbA), which incorporates a thioredoxin fold with an inserted helical domain, like its Escherichia coli counterpart EcDsbA, but it lacks the characteristic hydrophobic patch and has a truncated binding groove near the active site. These findings suggest that SaDsbA has a different substrate specificity than EcDsbA. Thermodynamic studies indicate that the oxidized and reduced forms of SaDsbA are energetically equivalent, in contrast to the energetically unstable disulfide form of EcDsbA. Further, the partial complementation of EcDsbA by SaDsbA is independent of EcDsbB and biochemical assays show that SaDsbA does not interact with EcDsbB. The identical stabilities of oxidized and reduced SaDsbA may facilitate direct re-oxidation of the protein by extracellular oxidants, without the need for DsbB

Protein Folding and Assembly

One of the central dogmas in biochemistry is the view that the biologically active, three-dimensional structure of a protein is unique and exclusively determined by its amino acid sequence, and that the active conformation of a protein represents its state of lowest free energy in aqueous solution. Despite a large number of novel experiments supporting this view, including an exponentially increasing number of solved three-dimensional protein structures, it is still impossible to predict the tertiary structure of a protein from knowledge of its amino acid sequence alone.Towards the goal of identifying general principles underlying the mechanism of protein folding in vitro and in vivo, we are pursuing several projects that are briefly described in this article: (1) Circular permutation of proteins as a tool to study protein folding, (2) Catalysis of disulfide bond formation during protein folding, (3) Assembly of adhesive type 1 pili from Escherichia coli strains, and (4) Structure and folding of the mammalian prion protein