Crystallization and preliminary X‐ray analysis of outer membrane phospholipase A from<i>Escherichia coli</i>

Blaauw, Mieke; Dekker, Niek; Verheij, Hubertus M.; Kalk, Kor H.; Dijkstra, Bauke W.

doi:10.1016/0014-5793(95)01002-v

Cited by 15 publications

(7 citation statements)

References 22 publications

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“…Crystals of monomeric OMPLA were grown by mixing protein solution (30 mg/ml OMPLA, 10 mM succinate buffer, pH 6.5, 1.5% (w/v) 1-O-n-octyl-b-D Dglucopyranoside, 1 mM NaN 3 ) in a 3:2 ratio with a reservoir solution containing 25-29% (v/v) 2-methyl-2,4-pentanediol (MPD), 1 mM CaCl 2 , and 0.1 M bis-Tris, pH 5.9-6.0 (Blaauw et al, 1995). Rounds of macro seeding were necessary to obtain crystals of sufficient dimensions (typically 1:3 Â 0:4 Â 0:2 mm 3 ).…”

Section: Crystallisation and Soakingmentioning

confidence: 99%

Detergent organisation in crystals of monomeric outer membrane phospholipase A

Snijder¹,

Timmins²,

Kalk³

et al. 2003

Journal of Structural Biology

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The structure of the detergent in crystals of outer membrane phospholipase A (OMPLA) has been determined using neutron diffraction contrast variation. Large crystals were soaked in stabilising solutions, each containing a different H 2 O=D 2 O contrast. From the neutron diffraction at five contrasts, the 12 A A resolution structure of the detergent micelle around the protein molecule was determined. The hydrophobic b-barrel surfaces of the protein molecules are covered by rings of detergent. These detergent belts are fused to neighbouring detergent rings forming a continuous three-dimensional network throughout the crystal. The thickness of the detergent layer around the protein varies from 7-20 A A. The enzymeÕs active site is positioned just outside the hydrophobic detergent zone and is thus in a proper location to catalyse the hydrolysis of phospholipids in a natural membrane. Although the dimerisation face of OMPLA is covered with detergent, the detergent density is weak near the exposed polar patch, suggesting that burying this patch in the enzymeÕs dimer interface may be energetically favourable. Furthermore, these results indicate a crucial role for detergent coalescence during crystal formation and contribute to the understanding of membrane protein crystallisation.

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Section: Crystallisation and Soakingmentioning

confidence: 99%

Detergent organisation in crystals of monomeric outer membrane phospholipase A

Snijder¹,

Timmins²,

Kalk³

et al. 2003

Journal of Structural Biology

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“…36 Crystallisation of the monomeric form of OMPLA was performed as reported by Blaauw et al, 37 except that macro-seeding was used. Crystals were transferred to a stabilising mother liquor containing 27-29 % Calcium Binding to Outer Membrane Phospholipase A 1.5 % (w/v) b-OG, 0.3-2.0 mM SmCl 3 and 0.1 M Bis-Tris buffer at pH 5.9.…”

Section: Structure Determinationmentioning

confidence: 99%

Structural investigations of calcium binding and its role in activity and activation of outer membrane phospholipase A from Escherichia coli

Snijder

Kingma

Kalk

et al. 2001

Journal of Molecular Biology

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Outer membrane phospholipase A (OMPLA) is an integral membrane enzyme that catalyses the hydrolysis of phospholipids. Enzymatic activity is regulated by reversible dimerisation and calcium-binding. We have investigated the role of calcium by X-ray crystallography. In monomeric OMPLA, one calcium ion binds between two external loops (L3L4 site) at 10 A Ê from the active site. After dimerisation, a new calcium-binding site (catalytic site) is formed at the dimer interface in the active site of each molecule at 6 A Ê from the L3L4 calcium site. The close spacing and the difference in calcium af®nity of both sites suggests that the L3L4 site may function as a storage site for a calcium ion, which relocates to the catalytic site upon dimerisation. A sequence alignment demonstrates conservation of the catalytic calcium site but evolutionary variation of the L3L4 site. The residues in the dimer interface are conserved as well, suggesting that all outer membrane phospholipases require dimerisation and calcium in the catalytic site for activity. For this family of phospholipases, we have characterised a consensus sequence motif (YTQ-X n -G-X 2 -H-X-SNG) that contains conserved residues involved in dimerisation and catalysis.

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“…A ␤-barrel structure has been proposed for OMPLA (8), analogous to the outer membrane porins, of which the x-ray structures have been solved (9 -11). Recently, we succeeded in the overproduction, in vitro refolding, and subsequent purification of the enzyme on a large scale (12), which allowed its crystallization (13).…”

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confidence: 99%

Dimerization Regulates the Enzymatic Activity of Escherichia coli Outer Membrane Phospholipase A

Dekker

Tommassen

Lustig³

et al. 1997

Journal of Biological Chemistry

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106

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The outer membrane phospholipase A (OMPLA) of Escherichia coli is present in a dormant state in the cell envelope. The enzyme is activated by various processes, which have in common that they perturb the outer membrane. Kinetic experiments, chemical cross-linking, and analytical ultracentrifugation were carried out with purified, detergent-solubilized OMPLA to understand the underlying mechanism that results in activation. Under conditions in which the enzyme displayed full activity, OMPLA was dimeric. High detergent concentrations or very dilute protein concentrations resulted in low specific activity of the enzyme, and under those conditions the enzyme was monomeric. The cofactor Ca 2؉ was required for dimerization. Covalent modification of the active site serine with hexadecylsulfonylfluoride resulted in stabilization of the dimeric form and a loss of the absolute calcium requirement for dimerization. The results of these experiments provide evidence for dimerization as the molecular mechanism by which the enzymatic activity of OMPLA is regulated. This dimerization probably plays a role in vivo as well. Data from chemical cross-linking on whole cells indicate that OMPLA is present in the outer membrane as a monomer and that activation of the enzyme induces dimerization concurrent with the appearance of enzymatic activity.Outer membrane phospholipase A (OMPLA 1 ; also known as detergent-resistant phospholipase A or PldA protein) is one of the few enzymes present in the outer membrane of Gramnegative bacteria. OMPLA hydrolyzes the acyl ester bonds in (phospho)lipids and has Ca 2ϩ as an essential cofactor (1, 2). Initially, the Escherichia coli enzyme was purified and characterized (3), and its structural gene, designated pldA, was subsequently cloned and overexpressed (4 -7). Recently, Brok et al. (8) reported the cloning of the pldA genes of several other Enterobacteriaceae species. Comparison of the OMPLA amino acid sequences revealed a high degree of homology within this family, but no clear homology exists with sequences of watersoluble (phospho)lipases. A ␤-barrel structure has been proposed for OMPLA (8), analogous to the outer membrane porins, of which the x-ray structures have been solved (9 -11). Recently, we succeeded in the overproduction, in vitro refolding, and subsequent purification of the enzyme on a large scale (12), which allowed its crystallization (13).Although OMPLA is embedded in its own substrate in the cell envelope, no enzymatic activity can be detected in normally growing cells (14,15). Because OMPLA is expressed constitutively, genetic regulation cannot explain the absence of enzymatic activity. Moreover, the expressed protein is correctly transported to and inserted into the outer membrane, where it resides in a dormant state. Activity is induced by various processes that perturb the membrane, such as phage-induced lysis (16), temperature shock (4), and colicin secretion (17, 18). Similar results have been reported in vitro after reconstitution of OMPLA in lipid vesicles (19). Memb...

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Crystallization and preliminary X‐ray analysis of outer membrane phospholipase A fromEscherichia coli

Cited by 15 publications

References 22 publications

Detergent organisation in crystals of monomeric outer membrane phospholipase A

Detergent organisation in crystals of monomeric outer membrane phospholipase A

Structural investigations of calcium binding and its role in activity and activation of outer membrane phospholipase A from Escherichia coli

Dimerization Regulates the Enzymatic Activity of Escherichia coli Outer Membrane Phospholipase A

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