Jan Tommassen

Carboxy-terminal phenylalanine is essential for the correct assembly of a bacterial outer membrane protein.

Bacterial outer membrane proteins are supposed to span the membrane repeatedly, mostly in the form of amphipathic beta-sheets. The last ten C-terminal amino acid residues of PhoE protein are supposed to form such a membrane-spanning segment. Deletion of this segment completely prevents incorporation into the outer membrane. Comparison of the last ten amino acid residues of other outer membrane proteins from different Gram-negative bacteria revealed the presence of a potential amphipathic beta-sheet with hydrophobic residues at positions 1 (Phe), 3 (preferentially Tyr), 5, 7 and 9 from the C terminus, in the vast majority of these proteins. Since such sequences were not detected at the C termini of periplasmic proteins, it appears to be possible to discriminate between the majority of outer membrane proteins and periplasmic proteins on the basis of sequence data. The highly conserved phenylalanine at the C termini of outer membrane proteins suggests an important function for this amino acid in assembly into the outer membrane. Site-directed mutagenesis was applied to study the role of the C-terminal Phe in PhoE protein assembly. All mutant proteins were correctly incorporated into the outer membrane to some extent, but the efficiency of the process was severely affected. It appears that both the hydrophobicity and the aromatic nature of Phe are of importance.

Structure of the translocator domain of a bacterial autotransporter

Autotransporters are virulence‐related proteins of Gram‐negative bacteria that are secreted via an outer‐membrane‐based C‐terminal extension, the translocator domain. This domain supposedly is sufficient for the transport of the N‐terminal passenger domain across the outer membrane. We present here the crystal structure of the in vitro‐folded translocator domain of the autotransporter NalP from Neisseria meningitidis, which reveals a 12‐stranded β‐barrel with a hydrophilic pore of 10 × 12.5 Å that is filled by an N‐terminal α‐helix. The domain has pore activity in vivo and in vitro. Our data are consistent with the model of passenger‐domain transport through the hydrophilic channel within the β‐barrel, and inconsistent with a model for transport through a central channel formed by an oligomer of translocator domains. However, the dimensions of the pore imply translocation of the secreted domain in an unfolded form. An alternative model, possibly covering the transport of folded domains, is that passenger‐domain transport involves the Omp85 complex, the machinery required for membrane insertion of outer‐membrane proteins, on which autotransporters are dependent.

Assembly factor Omp85 recognizes its outer membrane protein substrates by a species-specific C-terminal motif.

Integral β-barrel proteins are found in the outer membranes of Gram-negative bacteria, mitochondria, and chloroplasts. The assembly of these proteins requires a proteinaceous apparatus of which Omp85 is an evolutionary conserved central component. To study its molecular mechanism, we have produced Omp85 from Escherichia coli in inclusion bodies and refolded it in vitro. The interaction of Omp85 with its substrate proteins was studied in lipid-bilayer experiments, where it formed channels. The properties of these channels were affected upon addition of unfolded outer-membrane proteins (OMPs) or synthetic peptides corresponding to their C-terminal signature sequences. The interaction exhibited species specificity, explaining the inefficient assembly of OMPs from Neisseria in E. coli. Accordingly, the in vivo assembly of the neisserial porin PorA into the E. coli outer membrane was accomplished after adapting its signature sequence. These results demonstrate that the Omp85 assembly machinery recognizes OMPs by virtue of their C-terminal signature sequence.

The outer membrane component, YscC, of the Yop secretion machinery of Yersinia enterocolitica forms a ring‐shaped multimeric complex

The YscC protein of Yersinia enterocolitica is essential for the secretion of anti‐host factors, called Yops, into the extracellular environment. It belongs to a family of outer membrane proteins, collectively designated secretins, that participate in a variety of transport processes. YscC has been shown to exist as a stable oligomeric complex in the outer membrane. The production of the YscC complex is regulated by temperature and is reduced in strains carrying mutations in the yscN‐U operon or in the virG gene. The VirG lipoprotein was shown to be required for efficient targeting of the complex to the outer membrane. Electron microscopy revealed that purified YscC complexes form ring‐shaped structures of ≈20 nm with an apparent central pore. Because of the architecture of the multimer, YscC appears to represent a novel type of channel‐forming proteins in the bacterial outer membrane.

Formation of oligomeric rings by XcpQ and PilQ, which are involved in protein transport across the outer membrane of Pseudomonas aeruginosa

Pseudomonas aeruginosa is able to translocate proteins across both membranes of the cell envelope. Many of these proteins are transported via the type II secretion pathway and adopt their tertiary conformation in the periplasm, which implies the presence of a large transport channel in the outer membrane. The outer membrane protein, XcpQ, which is involved in transport of folded proteins across the outer membrane of P. aeruginosa, was purified as a highly stable homomultimer. Insertion and deletion mutagenesis of xcpQ revealed that the C‐terminal part of XcpQ is sufficient for the formation of the multimer. However, linker insertions in the N‐terminal part can disturb complex formation completely. Furthermore, complex formation is strictly correlated with lethality, caused by overexpression of xcpQ. Electron microscopic evaluation of the XcpQ multimers revealed large, ring‐shaped structures with an apparent central cavity of 95 Å. Purified PilQ, a homologue of XcpQ involved in the biogenesis of type IV pili, formed similar structures. However, the apparent cavity formed by PilQ was somewhat smaller, 53 Å. The size of this cavity could allow for the transport of intact type IV pili.

Protein secretion in Pseudomonas aeruginosa: characterization of seven xcp genes and processing of secretory apparatus components by prepilin peptidase

The xcp genes are required for the secretion of most extracellular proteins by Pseudomonas aeruginosa. The products of these genes are essential for the transport of exoproteins across the outer membrane after they have reached the periptasm via a signal sequence‐dependent pathway. To date, analysis of three xcp genes has suggested the conservation of this secretion pathway in many Gram‐negative bacteria. Furthermore, the xcpA gene was shown to be identical to pilD, which encodes a peptidase involved in the processing of fimbrial (pili) subunits, suggesting a connection between pili biogenesis and protein secretion. Here the nucleotide sequences of seven other xcp genes, designated xcpR to ‐X, are presented. The N termini of four of the encoded Xcp proteins display similarity to the N‐termini of type IV pili, suggesting that XcpA is involved in the processing of these Xcp proteins. This could indeed be demonstrated in vivo. Furthermore, two other proteins, XcpR and XcpS, show similarity to the PilB and PilC proteins required for fimbriae assembly. Since XcpR and PilB display a canonical nucleotide‐binding site, ATP hydrolysis may provide energy for both systems.

Involvement of stress protein PspA (phage shock protein A) of Escherichia coli in maintenance of the protonmotive force under stress conditions.

The expression of specific PhoE mutant proteins leads to induction of the expression of the psp operon of Escherichia coli and the export of various plasmid‐encoded precursors is retarded in a pspA mutant strain. Here, we have investigated the specific role of various Psp proteins in the export process. PspB and PspC are both inner membrane proteins that are involved in the regulation of the transcription of the psp operon. Precursor PhoE translocation was retarded in a pspB mutant strain to a similar extent as in a pspA mutant strain. The reduced translocation efficiencies in the various psp mutants could be complemented by expression of PspA from a plasmid, indicating that only PspA is required for efficient translocation. Mutant prePhoE proteins that can be translocated independently of the deltamu H+ appeared to translocate equally efficiently in a wild‐type and in a pspA mutant strain. Furthermore, quantitative in vivo determination of the deltamu H+ showed that it specifically decreased in a pspA mutant strain upon expression of plasmid‐encoded (mutant) prePhoE protein. Apparently, the translocation defects observed in a psp mutant strain are caused by a decrease of the delta mu H+ and PspA functions by maintaining the delta mu H+ under these conditions.

Solid-State NMR Spectroscopy on Cellular Preparations Enhanced by Dynamic Nuclear Polarization†

Solid-state NMR (ssNMR) spectroscopy offers increasing possibilities to study complex biomolecules at the atomic level. An important target area concerns membrane-associated proteins, which can be investigated by ssNMR methods after reconstitution in synthetic bilayers. While such preparations allow examination of functional aspects of the protein of interest, the influence of the native cellular environment on protein structure and function cannot be monitored. Very recently, we introduced a general approach aimed at determining complex molecular structures, including integral membrane proteins, in their native cellular environment by ssNMR under magic-angle-spinning (MAS) conditions. Using dedicated sample-preparation routes, we demonstrated that high-resolution ssNMR spectra can be obtained on uniformly C,N-labeled preparations of Escherichia coli whole cells (WC) and cell envelopes (CE). Both CE and WC morphology are preserved under standard ssNMR experimental conditions and the corresponding C and N crosspolarization (CP-MAS) spectra are invariant over time. However, with increasing levels of molecular complexity, especially in the case of WC preparations, spectroscopic sensitivity becomes a critical factor. In recent years, dynamic nuclear polarization (DNP) has developed into a routine tool to increase the sensitivity of multidimensional ssNMR. DNP enhancements of up to 148fold have been obtained on micro/nanocrystalline biomolecular samples, including an amyloidogenic peptide and a deuterated protein, 6] while enhancements between 18and 46fold have been reported for membrane-embedded polypeptides, purple membrane preparations, and bacteriophages. Here, we investigated the use of DNP to conduct ssNMR studies on C,N-labeled preparations of E. coli WC overproducing the integral outer membrane protein PagL. In Figure 1, we compared C and N CP-MAS spectra of uniformly C,N-labeled WC with the CE isolated from PagL-overproducing E. coli cells, recorded in the presence and absence of microwave irradiation. At higher temperatures (271 K), ssNMR spectra of the E. coli CE had previously revealed atomic details of PagL as well as endogenous membrane-associated macromolecules, including the major lipoprotein Lpp and non-proteinaceous components such as lipopolysaccharides (LPS), peptidoglycans (PG), and phospholipids. Under low-temperature (LT) DNP conditions, we observed significant DNP enhancement factors for both preparations in spectral regions characteristic for protein signals (aliphatic C resonances: d = 50–55 ppm, amide N backbone and side-chain resonances at about 120 and 80–30 ppm) as well as for C signals of endogenous

A Neisserial autotransporter NalP modulating the processing of other autotransporters

Autotransporters constitute a relatively simple secretion system in Gram‐negative bacteria, depending for their translocation across the outer membrane only on a C‐terminal translocator domain. We have studied a novel autotransporter serine protease, designated NalP, from Neisseria meningitidis strain H44/76, featuring a lipoprotein motif at the signal sequence cleavage site. Indeed, lipidation of NalP could be demonstrated, but the secreted 70 kDa domain of NalP lacked the lipid‐moiety as a result of additional N‐terminal processing. A nalP mutant showed a drastically altered profile of secreted proteins. Mass‐spectrometric analysis of tryptic fragments identified the autotransporters IgA protease and App, a homologue of the adhesin Hap of Haemophilus influenzae, as the major secreted proteins. Two forms of both of these proteins were found in the culture supernatant of the wild‐type strain, whereas only the lower molecular‐weight forms predominated in the culture supernatant of the nalP mutant. The serine‐protease active site of NalP was required for the modulation of the processing of these autotransporters. We propose that, apart from the autoproteolytic processing, NalP can process App and IgA protease and hypothesize that this function of NalP could contribute to the virulence of the organism.

Cellular solid-state nuclear magnetic resonance spectroscopy

Decrypting the structure, function, and molecular interactions of complex molecular machines in their cellular context and at atomic resolution is of prime importance for understanding fundamental physiological processes. Nuclear magnetic resonance is a well-established imaging method that can visualize cellular entities at the micrometer scale and can be used to obtain 3D atomic structures under in vitro conditions. Here, we introduce a solid-state NMR approach that provides atomic level insights into cell-associated molecular components. By combining dedicated protein production and labeling schemes with tailored solid-state NMR pulse methods, we obtained structural information of a recombinant integral membrane protein and the major endogenous molecular components in a bacterial environment. Our approach permits studying entire cellular compartments as well as cell-associated proteins at the same time and at atomic resolution.