Investigating the Interfacial Binding of Bacterial Phosphatidylinositol-Specific Phospholipase C

Wehbi, Hania; Feng, Jianwen; Kolbeck, James; Ananthanarayanan, Bharath; Cho, Wonhwa; Roberts, Mary F.

doi:10.1021/bi034195+

Cited by 22 publications

(89 citation statements)

References 28 publications

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“…Tyr 57 , a surface residue that is not situated at the putative dimer or membrane interface, was included as a control. As shown in Table 1, all Cys mutants but W242C exhibited nK d values (for POPC small unilamellar vesicles) comparable with the 64 Ϯ 3 M previously obtained for recombinant PI-PLC (29). Previous work showed that W242A lost significant affinity for PC with an apparent dissociation constant of 8600 Ϯ 600 M (20).…”

Section: Resultssupporting

confidence: 77%

“…Removal of these two tryptophan residues, singly or together, dramatically increases the apparent K D (or nK d in a Langmuir adsorption model for binding (29)) for partitioning of the enzyme onto PC vesicles, and the magnitude of the change is consistent with the change in free energy for removing a group that partitions into the PC membrane. X-ray crystallography was used to investigate protein structural changes that might be associated with the W47A/W242A mutation and to determine whether such changes might occur in the active site, resulting in a lower k cat .…”

Section: Discussionmentioning

confidence: 63%

“…The filtrate, containing free enzyme, was lyophilized and analyzed by SDS-PAGE. Binding was analyzed using a Langmuir adsorption isotherm (29,30), where n is the number of lipid molecules in the surface to which each enzyme molecule is assumed to bind, and K d is the dissociation constant for the binding site composed of n lipids. The product "nK d " provides an apparent dissociation constant for comparison of the different PI-PLC proteins.…”

Section: Popc Vesicle Bindingmentioning

confidence: 99%

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Dimer Structure of an Interfacially Impaired Phosphatidylinositol-specific Phospholipase C

Shao

Shi

Wehbi

et al. 2007

Journal of Biological Chemistry

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The crystal structure of the W47A/W242A mutant of phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis has been solved to 1.8 Å resolution. The W47A/ W242A mutant is an interfacially challenged enzyme, and it has been proposed that one or both tryptophan side chains serve as membrane interfacial anchors (Feng, J., Wehbi, H., and Roberts, M. F. (2002) J. Biol. Chem. 277, 19867-19875). The crystal structure supports this hypothesis. Relative to the crystal structure of the closely related (97% identity) wild-type PI-PLC from Bacillus cereus, significant conformational differences occur at the membrane-binding interfacial region rather than the active site. The Trp 3 Ala mutations not only remove the membrane-partitioning aromatic side chains but also perturb the conformations of the so-called helix B and rim loop regions, both of which are implicated in interfacial binding. The crystal structure also reveals a homodimer, the first such observation for a bacterial PI-PLC, with pseudo-2-fold symmetry. The symmetric dimer interface is stabilized by hydrophobic and hydrogen-bonding interactions, contributed primarily by a central swath of aromatic residues arranged in a quasiherringbone pattern. Evidence that interfacially active wild-type PI-PLC enzymes may dimerize in the presence of phosphatidylcholine vesicles is provided by fluorescence quenching of PI-PLC mutants with pyrene-labeled cysteine residues. The combined data suggest that wild-type PI-PLC can form similar homodimers, anchored to the interface by the tryptophan and neighboring membranepartitioning residues.Phosphatidylinositol-specific phospholipase C (PI-PLC) 2 enzymes catalyze the specific cleavage of the sn-3-phosphodiester bond in phosphatidylinositol (PI) and phosphoinositides. PI hydrolysis occurs in two sequential reactions (1-3): (i) an intramolecular phosphotransferase reaction at a phospholipid/ aggregate surface to produce diacylglycerol and water-soluble 1,2-cyclic inositol phosphate (cIP) (or cIP x in the case of a phosphoinositide substrate), followed by (ii) a phosphodiesterase reaction where water-soluble cIP is hydrolyzed to inositol-1-phosphate. Since their substrate is presented in an interface, their activity can often be modulated by the composition of the interface. Eukaryotic PI-PLCs, as critical components of phosphatidylinositol-mediated signaling pathways (4, 5), have multiple domains for regulation of enzyme activity both by regulating partitioning to membranes and by allosteric effects on catalysis, and these are often hard to separate. However, the much smaller bacterial PI-PLC enzymes are secreted, single domain proteins whose crystal structures (6 -8) resemble the catalytic domain of PLC␦ 1 , the only mammalian PI-PLC for which there is a structure (9 -11). The molecular topology is that of a distorted TIM barrel. Around the barrel rim, there are regions where hydrophobic side chains are exposed to solvent. The structural similarity of the bacterial PI-PLC to the mammalian catalytic domain and...

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Section: Resultssupporting

confidence: 77%

Section: Discussionmentioning

confidence: 63%

Section: Popc Vesicle Bindingmentioning

confidence: 99%

See 1 more Smart Citation

Dimer Structure of an Interfacially Impaired Phosphatidylinositol-specific Phospholipase C

Shao

Shi

Wehbi

et al. 2007

Journal of Biological Chemistry

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“…SUVs of the anionic phospholipid dioleoylphosphatidylglycerol (used as the substrate analog) with different mole fractions of 1-palmitoyl-2-oleoyl-PC were prepared by sonication. FCS data were analyzed as previously described (23,26 (27) to separate vesicle bound and free protein, with quantification of free protein with the Bio-Rad DC protein assay, was used to compare the fraction of other related protein variants bound to 1 mM PG/PC (1:4) in 50 mM MES, pH 6.5, with 140 mM salt added.…”

mentioning

confidence: 99%

The Cation-π Box Is a Specific Phosphatidylcholine Membrane Targeting Motif

Cheng

Goldstein

Gershenson

et al. 2013

Journal of Biological Chemistry

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“…Yet such knowledge is likely to be very useful in understanding how proteins and assemblies interact with this interfacial region. For example, there is indirect evidence that the peripheral membrane protein phosphatidylinositol-specific phospholipase inserts a tryptophan residue into a phosphatidylcholine membrane surface, which in turn activates the enzyme toward its substrates, but how this happens in detail is unknown (3,4).…”

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confidence: 99%

Phospholipid bilayer surface configuration probed quantitatively by ³¹ P field-cycling NMR

Roberts

Redfield

2004

Proc. Natl. Acad. Sci. U.S.A.

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175

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31 P relaxation of the diester phosphate of phospholipids in unilamellar vesicles has been studied from 0.004 to 11.7 T. Relaxation at very low fields, below 0.1 T, shows a rate increase that reflects a residual dipolar interaction with neighboring protons, probably dominated by the glycerol C3 protons. This interaction is not fully averaged by faster motion such as rotational diffusion perpendicular to the membrane surface. The remaining dipolar interaction, modulated by overall rotational diffusion of the vesicle and lateral diffusion of the lipid molecules, is responsible for the very low-field relaxation. These measurements yield a good estimate of the time-average angle between the membrane surface and the vector connecting the phosphorus to the glycerol C3 dynamics ͉ membranes ͉ phosphorus M uch remains to be known about the details of the configuration and dynamics of the phosphodiester region of phospholipid bilayer membranes, despite a large body of work (1, 2). Yet such knowledge is likely to be very useful in understanding how proteins and assemblies interact with this interfacial region. For example, there is indirect evidence that the peripheral membrane protein phosphatidylinositol-specific phospholipase inserts a tryptophan residue into a phosphatidylcholine membrane surface, which in turn activates the enzyme toward its substrates, but how this happens in detail is unknown (3, 4).The lack of structural information for phospholipid polar and interfacial moieties results from the difficulty in obtaining crystals and making other ordered structures that are necessary for most structural techniques. Even when available, the resemblance of such ordered structures to membranes functioning in vivo can be questioned. Modern methods of NMR spectroscopy are generally difficult to apply to this problem, mainly because of the slow rate of tumbling of molecules in reasonable analogs of biological membranes such as vesicles. Most NMR studies ( 1 H, 13 C, and 2 H) of membranes have focused on acyl chain dynamics (5-7). Phosphorus-31 NMR, which would appear ideally suited for obtaining information on the phosphodiester linkage conformation and dynamics, has seen limited use, because 31 P resonances in phospholipid aggregates exhibit a large linewidth due to their chemical-shift anisotropy (CSA) [although this property had made this nucleus very useful in characterizing the phase behavior of phospholipid bilayers (5)]. 31 P chemical shifts in phosphodiesters embedded in membranes do reflect orientation of chemical bonds relative to the membrane surface but not in a very usefully specific way. Structures deduced from other NMR data suffer from unknown dynamic averaging effects. Computer simulations show great promise to eventually give completely detailed information (8, 9) but still need to be validated with quantitative experiments.As a contribution to this problem, we have estimated the angle PH between the vector connecting the phosphorus to its nearest protons and the vector perpendicular to the membrane sur...

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Investigating the Interfacial Binding of Bacterial Phosphatidylinositol-Specific Phospholipase C

Cited by 22 publications

References 28 publications

Dimer Structure of an Interfacially Impaired Phosphatidylinositol-specific Phospholipase C

Dimer Structure of an Interfacially Impaired Phosphatidylinositol-specific Phospholipase C

The Cation-π Box Is a Specific Phosphatidylcholine Membrane Targeting Motif

Phospholipid bilayer surface configuration probed quantitatively by ³¹ P field-cycling NMR

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Investigating the Interfacial Binding of Bacterial Phosphatidylinositol-Specific Phospholipase C

Cited by 22 publications

References 28 publications

Dimer Structure of an Interfacially Impaired Phosphatidylinositol-specific Phospholipase C

Dimer Structure of an Interfacially Impaired Phosphatidylinositol-specific Phospholipase C

The Cation-π Box Is a Specific Phosphatidylcholine Membrane Targeting Motif

Phospholipid bilayer surface configuration probed quantitatively by 31 P field-cycling NMR

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Product

Resources

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Phospholipid bilayer surface configuration probed quantitatively by ³¹ P field-cycling NMR