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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.

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Available from: Bauke W. Dijkstra, Jul 29, 2015
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    • "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. "
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    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
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    • "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. "
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    • "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. "
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