Structural investigations of the active‐site mutant Asn156Ala of outer membrane phospholipase A: Function of the Asn–His interaction in the catalytic triad

Snijder, H.J.; Eerde, J.H. Van; Kingma, R.L.; Kalk, K.H.; Dekker, Niek; Egmond, Maarten R.; Dijkstra, Bauke W.

doi:10.1110/ps.17701

Cited by 13 publications

(8 citation statements)

References 56 publications

(78 reference statements)

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“…4), nearby aromatic residues form stacking interactions between the sugar rings and the aromatic rings. The same intimate interaction can be observed in a number of other membrane protein structures (21,22). Alkyl glycoside headgroups seem to mimic phosphatidyl head groups in their well known interactions with tryptophan and tyrosine (19,23).…”

Section: Conserved Sites Of Detergent and Lipid Interactionsupporting

confidence: 61%

Identification of conserved lipid/detergent-binding sites in a high-resolution structure of the membrane protein cytochrome c oxidase

Qin

Hiser

Mulichak

et al. 2006

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

284

385

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Well ordered reproducible crystals of cytochrome c oxidase (CcO) from Rhodobacter sphaeroides yield a previously unreported structure at 2.0 Å resolution that contains the two catalytic subunits and a number of alkyl chains of lipids and detergents. Comparison with crystal structures of other bacterial and mammalian CcOs reveals that the positions occupied by native membrane lipids and detergent substitutes are highly conserved, along with amino acid residues in their vicinity, suggesting a more prevalent and specific role of lipid in membrane protein structure than often envisioned. Well defined detergent head groups (maltose) are found associated with aromatic residues in a manner similar to phospholipid head groups, likely contributing to the success of alkyl glycoside detergents in supporting membrane protein activity and crystallizability. Other significant features of this structure include the following: finding of a previously unreported crystal contact mediated by cadmium and an engineered histidine tag; documentation of the unique His-Tyr covalent linkage close to the active site; remarkable conservation of a chain of waters in one proton pathway (D-path); and discovery of an inhibitory cadmium-binding site at the entrance to another proton path (K-path). These observations provide important insight into CcO structure and mechanism, as well as the significance of bound lipid in membrane proteins.cadmium binding ͉ membrane protein structure ͉ proton-conducting water chains C ytochrome c oxidase (CcO) is the terminal enzyme in the electron transfer chain in eukaryotes and many bacteria. It provides the final electron sink by accepting electrons from cytochrome c and reducing oxygen to water (1). The energy generated from this reaction is used to translocate protons across the membrane against the membrane potential and pH gradient (2). The proton gradient so formed is then used to make ATP, a universal energy source. CcO is an intrinsic membrane protein with varying numbers of subunits from 3 in some bacteria to 13 in mammalian mitochondria. However, only subunits I and II, which contain the redox active heme and metal centers and are highly conserved among different species, are required for electron transfer and proton pumping activities. Other subunits, particularly the highly conserved subunit III, likely play key roles in stabilizing and regulating the enzyme activity and energy metabolism in general.Over the past decade, several x-ray crystal structures of aa 3 -type CcOs from bovine heart mitochondria and bacteria have been determined (3-8). However, the mechanism of vectorial translocation of protons by CcO remains to be solved (9). It is now recognized that water molecules play a critical role in facilitating and controlling proton transfer (10). Thus, highresolution crystal structures of different redox states and key mutants with defined waters are necessary to elucidate the energy conservation process. However, achieving a reproducible high-resolution structure of membrane proteins remains a ...

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Section: Conserved Sites Of Detergent and Lipid Interactionsupporting

confidence: 61%

Identification of conserved lipid/detergent-binding sites in a high-resolution structure of the membrane protein cytochrome c oxidase

Qin

Hiser

Mulichak

et al. 2006

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

284

385

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“…structural fold. However, a major difference is the nature of LmClpP1's unusual Asn-His-Ser catalytic triad (39). Our mutational and structural experiments emphasize that this triad represents a key regulatory element that keeps LmClpP1 heptameric and inactive in absence of LmClpP2.…”

Section: Discussionmentioning

confidence: 81%

Structural and functional insights into caseinolytic proteases reveal an unprecedented regulation principle of their catalytic triad

Zeiler

List

Alte

et al. 2013

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

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Caseinolytic proteases (ClpPs) are large oligomeric protein complexes that contribute to cell homeostasis as well as virulence regulation in bacteria. Although most organisms possess a single ClpP protein, some organisms encode two or more ClpP isoforms. Here, we elucidated the crystal structures of ClpP1 and ClpP2 from pathogenic Listeria monocytogenes and observe an unprecedented regulation principle by the catalytic triad. Whereas L. monocytogenes (Lm)ClpP2 is both structurally and functionally similar to previously studied tetradecameric ClpP proteins from Escherichia coli and Staphylococcus aureus , heptameric LmClpP1 features an asparagine in its catalytic triad. Mutation of this asparagine to aspartate increased the reactivity of the active site and led to the assembly of a tetradecameric complex. We analyzed the heterooligomeric complex of LmClpP1 and LmClpP2 via coexpression and subsequent labeling studies with natural product-derived probes. Notably, the LmClpP1 peptidase activity is stimulated 75-fold in the complex providing insights into heterooligomerization as a regulatory mechanism. Collectively, our data point toward different preferences for substrates and inhibitors of the two ClpP enzymes and highlight their structural and functional characteristics.

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“…An intricate hydrogen-bonding network occludes the central cavity of OMPLA. The three active site residues, Asn156, His142, and Ser144, are organized on the exterior of the β-barrel in a catalytic triad that normally associates with the inert glucosamine backbone of lipid A in the outer leaflet [121,122]. The nucleophilic Ser144 of OMPLA can be irreversibly sulfonylated by the palmitate analog hexadecanesulfonyl fluoride.…”

Section: The Phospholipase Omplamentioning

confidence: 99%

Structural biology of membrane-intrinsic β-barrel enzymes: Sentinels of the bacterial outer membrane

Bishop

2008

Biochimica et Biophysica Acta (BBA) - Biomembranes

113

111

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The outer membranes of Gram-negative bacteria are replete with integral membrane proteins that exhibit antiparallel beta-barrel structures, but very few of these proteins function as enzymes. In Escherichia coli, only three beta-barrel enzymes are known to exist in the outer membrane; these are the phospholipase OMPLA, the protease OmpT, and the phospholipidColon, two colonslipid A palmitoyltransferase PagP, all of which have been characterized at the structural level. Structural details have also emerged for the outer membrane beta-barrel enzyme PagL, a lipid A 3-O-deacylase from Pseudomonas aeruginosa. Lipid A can be further modified in the outer membrane by two beta-barrel enzymes of unknown structure; namely, the Salmonella enterica 3'-acyloxyacyl hydrolase LpxR, and the Rhizobium leguminosarum oxidase LpxQ, which employs O(2) to convert the proximal glucosamine unit of lipid A into 2-aminogluconate. Structural biology now indicates how beta-barrel enzymes can function as sentinels that remain dormant when the outer membrane permeability barrier is intact. Host immune defenses and antibiotics that perturb this barrier can directly trigger beta-barrel enzymes in the outer membrane. The ensuing adaptive responses occur instantaneously and rapidly outpace other signal transduction mechanisms that similarly function to restore the outer membrane permeability barrier.

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Structural investigations of the active‐site mutant Asn156Ala of outer membrane phospholipase A: Function of the Asn–His interaction in the catalytic triad

Cited by 13 publications

References 56 publications

Identification of conserved lipid/detergent-binding sites in a high-resolution structure of the membrane protein cytochrome c oxidase

Identification of conserved lipid/detergent-binding sites in a high-resolution structure of the membrane protein cytochrome c oxidase

Structural and functional insights into caseinolytic proteases reveal an unprecedented regulation principle of their catalytic triad

Structural biology of membrane-intrinsic β-barrel enzymes: Sentinels of the bacterial outer membrane

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