Structural evidence for dimerization-regulated activation of an integral membrane phospholipase

Laboratory of Biophysical Chemistry, BIOSON Research Institute, University of Groningen, The Netherlands.
Nature (Impact Factor: 42.35). 11/1999; 401(6754):717-21. DOI: 10.1038/44890
Source: PubMed

ABSTRACT Dimerization is a biological regulatory mechanism employed by both soluble and membrane proteins. However, there are few structural data on the factors that govern dimerization of membrane proteins. Outer membrane phospholipase A (OMPLA) is an integral membrane enzyme which participates in secretion of colicins in Escherichia coli. In Campilobacter and Helicobacter pylori strains, OMPLA is implied in virulence. Its activity is regulated by reversible dimerization. Here we report X-ray structures of monomeric and dimeric OMPLA from E. coli. Dimer interactions occur almost exclusively in the apolar membrane-embedded parts, with two hydrogen bonds within the hydrophobic membrane area being key interactions. Dimerization results in functional oxyanion holes and substrate-binding pockets, which are absent in monomeric OMPLA. These results provide a detailed view of activation by dimerization of a membrane protein.

Download full-text


Available from: Bauke W. Dijkstra, Jul 29, 2015
  • Source
    • "The fact that the truncated OmpA-1-276 and OmpA-188-276 were also able to form dimers suggests that in the context of the dimer, the soluble domains are not integrating into this proposed 16-stranded pore. Dimerization as a mechanism to regulate an enzymatic role is also plausible, as seen for the dimeric OmpA (OmpLA) (Snijder et al., 1999). OmpA by contrast has no known enzymatic activity. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The transmembrane domain of the outer membrane protein A (OmpA) from Escherichia coli is an excellent model for structural and folding studies of β-barrel membrane proteins. However, full-length OmpA resists crystallographic efforts, and the link between its function and tertiary structure remains controversial. Here we use site-directed mutagenesis and mass spectrometry of different constructs of OmpA, released in the gas phase from detergent micelles, to define the minimal region encompassing the C-terminal dimer interface. Combining knowledge of the location of the dimeric interface with molecular modeling and ion mobility data allows us to propose a low-resolution model for the full-length OmpA dimer. Our model of the dimer is in remarkable agreement with experimental ion mobility data, with none of the unfolding or collapse observed for full-length monomeric OmpA, implying that dimer formation stabilizes the overall structure and prevents collapse of the flexible linker that connects the two domains.
    Structure 04/2014; 22(5). DOI:10.1016/j.str.2014.03.004 · 6.79 Impact Factor
  • Source
    • "It is shown that the protein consists of 12 anti-parallel betastrands . OMPLA is Ca 2? dependent; in the presence of Ca 2? ions, it dimerizes and transforms into the active state (Snijder et al. 1999; Stanley et al. 2006). The catalytic center of the enzyme is built by the triad Asn156–His142– Ser144. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Phospholipase A of the bacterial outer membrane (OMPLA) is a β-barrel membrane protein which is activated under various stress conditions. The current study examines interaction of inhibitors of eucaryotic phospholipases A2-palmitoyl trifluoromethyl ketone (PACOCF3) and aristolochic acid (AA)-with OMPLA and considers a possible involvement of the enzyme in the Ca(2+)-dependent permeabilization of the outer membrane of Escherichia coli. Using the method of molecular docking, it has been predicted that PACOCF3 and AA bind to OMPLA at the same site and with the same affinity as the OMPLA inhibitors, hexadecanesulfonylfluoride and bromophenacyl bromide, and the substrate of the enzyme palmitoyl oleoyl phosphatidylethanolamine. It has also been shown that PACOCF3, AA, and bromophenacyl bromide inhibit the Ca(2+)-induced temperature-dependent changes in the permeability of the bacterial membrane for the fluorescent probe propidium iodide and suppressed the transformation of E. coli cells with plasmid DNA induced by Ca(2+) and heat shock. The cell viability was not affected by the eucaryotic phospholipases A2 inhibitors. The study discusses a possible involvement of OMPLA in the mechanisms of bacterial transmembrane transport based on the permeabilization of the bacterial outer membrane.
    Journal of Membrane Biology 01/2014; 247(3). DOI:10.1007/s00232-014-9633-4 · 2.17 Impact Factor
  • Source
    • "The tryptophan fluorescence spectrum for folded OmpLA has a wavelength position of maximum emission (λ max ) at 336 nm with our spectrofluorometer set-up. That value is consistent with the majority of OmpLA's nine tryptophans being embedded in the apolar bilayer or protein interior in the folded state, which is the result we expected based on OmpLA's known three-dimensional folded structure [19] [20]. The unfolded form of OmpLA demonstrates a lower quantum yield than the folded form and displays an emission spectrum with a λ max of 352 nm. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The free energy of unfolding of a membrane protein from lipids into water (ΔG(o)(w,l)) describes its equilibrium thermodynamic stability. Knowing this parameter gives insight into a membrane protein's sequence-structure-energy relationships. However, there are few measures of membrane protein stability because of the technical difficulties associated with unfolded and partially folded states. Here, we describe the experimental process that allowed us to measure the ΔG(o)(w,l) of the outer membrane phospholipase A into large unilamellar vesicles (LUVs) of 1,2-dilauroyl-sn-glycero-3-phosphocholine. To arrive at this reversible folding condition, we screened a large number of experimental variables: temperature, incubation time, salt concentration, pH, lipid composition and liposome morphology. The principal challenge we encountered under most conditions was hysteresis between folding and unfolding titrations. A second factor that compromised reversible folding was the observation that a fraction of the protein population tended to aggregate. We found that hysteresis could be completely eliminated on a feasible timescale by conducting experiments at acidic pH, by the slow dilution of the protein in the initial titration setup and by utilizing a low concentration of a detergent as a temporary "holdase" to solubilize the protein upon its initial dilution into folding conditions. We confirmed that the detergent did not disrupt the LUVs using fluorescence emission of lipid-sensitive dyes and light scattering. The results of our parameter search should be generally useful for efforts to measure ΔG(o)(w,l) for other membrane proteins.
    Journal of Molecular Biology 08/2011; 413(2):484-94. DOI:10.1016/j.jmb.2011.08.041 · 4.33 Impact Factor
Show more