Folding Of Outer Membrane Proteins Research Articles

Loss of Outer Membrane Proteins without Inhibition of Lipid Export in an Escherichia coli YaeT Mutant

Escherichia coli yaeT encodes an essential, conserved outer membrane (OM) protein that is an ortholog of Neisseria meningitidis Omp85. Conflicting data with N. meningitidis indicate that Omp85 functions either in assembly of OM proteins or in export of OM lipids. The role of YaeT in E. coli was investigated with a new temperature-sensitive mutant harboring nine amino acid substitutions. The mutant stops growing after 60 min at 44 degrees C. After 30 min at 44 degrees C, incorporation of [35S]methionine into newly synthesized OM proteins is selectively inhibited. Synthesis and export of OM phospholipids and lipopolysaccharide are not impaired. OM protein levels are low, even at 30 degrees C, and the buoyant density of the OM is correspondingly lower. By Western blotting, we show that levels of the major OM protein OmpA are lower in the mutant in whole cells, membranes, and the growth medium. SecA functions as a multicopy suppressor of the temperature-sensitive phenotype and partially restores OM proteins. Our data are consistent with a critical role for YaeT in OM protein assembly in E. coli.

Interactions between folding factors and bacterial outer membrane proteins

The outer membrane is the first line of contact between Gram-negative bacteria and their external environment. Embedded in the outer membrane are integral outer membrane proteins (OMPs) that perform a diverse range of tasks. OMPs are synthesized in the cytoplasm and are translocated across the inner membrane and probably diffuse through the periplasm before they are inserted into the outer membrane in a folded and biologically active form. Passage through the periplasm presents a number of challenges, due to the hydrophobic nature of the OMPs and the choice of membranes into which they can insert. Recently, a number of periplasmic proteins and one OMP have been shown to play a role in OMP biogenesis. In this review, we describe what is known about these folding factors and how they function in a biological context. In particular, we focus on how they interact with the OMPs at the molecular level and present a comprehensive overview of data relating to a possible effect on OMP folding yield and kinetics. Furthermore, we discuss the role of lipo-chaperones, i.e. lipopolysaccharide and phospholipids, in OMP folding. Important advances have clearly been made in the field, but much work remains to be done, particularly in terms of describing the biophysical basis for the chaperone-OMP interactions which so intricately regulate OMP biogenesis.

The Periplasmic Chaperone SurA Exploits Two Features Characteristic of Integral Outer Membrane Proteins for Selective Substrate Recognition

The Escherichia coli periplasmic chaperone and peptidyl-prolyl isomerase (PPIase) SurA facilitates the maturation of outer membrane porins. Although the PPIase activity exhibited by one of its two parvulin-like domains is dispensable for this function, the chaperone activity residing in the non-PPIase regions of SurA, a sizable N-terminal domain and a short C-terminal tail, is essential. Unlike most cytoplasmic chaperones SurA is selective for particular substrates and recognizes outer membrane porins synthesized in vitro much more efficiently than other proteins. Thus, SurA may be specialized for the maturation of outer membrane proteins. We have characterized the substrate specificity of SurA based on its natural, biologically relevant substrates by screening cellulose-bound peptide libraries representing outer membrane proteins. We show that two features are critical for peptide binding by SurA: specific patterns of aromatic residues and the orientation of their side chains, which are found more frequently in integral outer membrane proteins than in other proteins. For the first time this sufficiently explains the capability of SurA to discriminate between outer membrane protein and non-outer membrane protein folding intermediates. Furthermore, peptide binding by SurA requires neither an active PPIase domain nor the presence of proline, indicating that the observed substrate specificity relates to the chaperone function of SurA. Finally, we show that SurA is capable of associating with the outer membrane. Together, our data support a model in which SurA is specialized to interact with non-native periplasmic outer membrane protein folding intermediates and to assist in their maturation from early to late outer membrane-associated steps.

Crystallographic Structure of SurA, a Molecular Chaperone that Facilitates Folding of Outer Membrane Porins

The SurA protein facilitates correct folding of outer membrane proteins in gram-negative bacteria. The sequence of Escherichia coli SurA presents four segments, two of which are peptidyl-prolyl isomerases (PPIases); the crystal structure reveals an asymmetric dumbbell, in which the amino-terminal, carboxy-terminal, and first PPIase segments of the sequence form a core structural module, and the second PPIase segment is a satellite domain tethered ∼30 Å from this module. The core module, which is implicated in membrane protein folding, has a novel fold that includes an extended crevice. Crystal contacts show that peptides bind within the crevice, suggesting a model for chaperone activity whereby segments of polypeptide may be repetitively sequestered and released during the membrane protein-folding process.

Periplasmic stress and ECF sigma factors.

Envelope stress responses play important physiological roles in a variety of processes, including protein folding, cell wall biosynthesis, and pathogenesis. Many of these responses are controlled by extracytoplasmic function (ECF) sigma factors that respond to external signals by means of a membrane-localized anti-sigma factor. One of the best-characterized, ECF-regulated responses is the sigma(E) envelope stress response of Escherichia coli. The sigma(E) pathway ensures proper assembly of outer-membrane proteins (OMP) by controlling expression of genes involved in OMP folding and degradation in response to envelope stresses that disrupt these processes. Prevailing evidence suggests that, in E. coli, a second envelope stress response controlled by the Cpx two-component system ensures proper pilus assembly. The sensor kinase CpxA recognizes misfolded periplasmic proteins, such as those generated during pilus assembly, and transduces this signal to the response regulator CpxR through conserved phosphotransfer reactions. Phosphorylated CpxR activates transcription of periplasmic factors necessary for pilus assembly.

The σ E and Cpx regulatory pathways: Overlapping but distinct envelope stress responses

The Cpx and sigmaE extracytoplasmic stress responses sense and respond to misfolded proteins in the bacterial envelope. Recent studies have highlighted differences between these regulatory pathways in terms of activating signals, mechanisms of signal transduction and the nature of the responses. Cumulatively, the findings suggest distinct physiological roles for these partially overlapping envelope stress responses. The sigmaE pathway is essential for survival and is primarily responsible for monitoring and responding to alterations in outer membrane protein folding. Mounting evidence suggests that the Cpx regulon may have been adapted to ensure properly timed expression and assembly of adhesive organelles.

A new heat-shock gene, ppiD, encodes a peptidyl-prolyl isomerase required for folding of outer membrane proteins in Escherichia coli.

We have identified a new folding catalyst, PpiD, in the periplasm of Escherichia coli. The gene encoding PpiD was isolated as a multicopy suppressor of surA, a mutation which severely impairs the folding of outer membrane proteins (OMPs). The ppiD gene was also identified based on its ability to be transcribed by the two-component system CpxR-CpxA. PpiD was purified to homogeneity and shown to have peptidyl-prolyl isomerase (PPIase) activity in vitro. The protein is anchored to the inner membrane via a single transmembrane segment, and its catalytic domain faces the periplasm. In addition, we have identified by site-directed mutagenesis some of the residues essential for its PPIase activity. A null mutation in ppiD leads to an overall reduction in the level and folding of OMPs and to the induction of the periplasmic stress response. The combination of ppiD and surA null mutations is lethal. This is the first time two periplasmic folding catalysts have been shown to be essential. Another unique aspect of PpiD is that its gene is regulated by both the Cpx two-component system and the sigma32 heat shock factor, known to regulate the expression of cytoplasmic chaperones.

New components of protein folding in extracytoplasmic compartments of Escherichia coli SurA, FkpA and Skp/OmpH.

A global search for extracytoplasmic folding catalysts in Escherichia coli was undertaken using different genetic systems that produce unstable or misfolded proteins in the periplasm. The extent of misfolding was monitored by the increased activity of the sigma E regulon that is specifically induced by misfolded proteins in the periplasm. Using multicopy libraries, we cloned two genes, surA and fkpA, that decreased the sigma E-dependent response constitutively induced by misfolded proteins. According to their sequences and their biochemical activities, SurA and FkpA belong to two different peptidyl prolyl isomerase (PPI) families. Interestingly, surA was also selected as a multicopy suppressor of a defined htrM (rfaD) null mutation. Such mutants produce a defective lipopolysaccharide that is unable to protect outer membrane proteins from degradation during folding. The SurA multicopy suppression effect in htrM (rfaD) mutant bacteria was directly associated with its ability to catalyse the folding of outer membrane proteins immediately after export. Finally, Tn10 insertions were isolated, which led to an increased activity of the sigma E regulon. Such insertions were mapped to the dsb genes encoding catalysts of the protein disulphide isomerase (PDI) family, as well as to the surA, fkpA and ompH/skp genes. We propose that these three proteins (SurA, FkpA and OmpH/Skp) play an active role either as folding catalysts or as chaperones in extracytoplasmic compartments.

Molecular characterization of the structural gene for the lactoferrin receptor of the meningococcal strain H44/76.

The meningococcal lactoferrin receptor is a promising vaccine candidate since it seems to be antigenically rather stable. Monoclonal antibodies against this protein reacted with more than 50% of the strains tested. To gain further insight in its variability, the IbpA gene from strain H44/76, encoding this protein, was cloned and sequenced. This strain does not cross-react with monoclonal antibodies that recognize LbpA of strain BNCV. The deduced amino acid sequence was found to be 95% homologous to the previously established sequence of LbpA of strain BNCV. A topology model was proposed for LbpA, making use of the sequence comparisons and some general rules for the folding of outer membrane proteins. The protein is supposed to traverse the membrane 26 times in a β-sheet conformation. The epitope recognized by the monoclonals was mapped and found to reside in the largest predicted surface-exposed loop. No iron-regulation of LbpA expression was found in E. coli, probably because IbpA is located in an operon, the promoter of which was not cloned. Upstream of IbpA, a part of an open reading frame was found. Whereas the LbpA protein shows homology to the transferrin-binding protein 1 (Tbp1), the putative protein encoded by the open reading frame upstream of Ibp A shows extensive homology to Tbp2, suggesting that iron-acquisition from lactoferrin, like from transferrin, requires two specific proteins in the outer membrane. The upstream open reading frame is tentatively designated Ibp B.