Improving protein crystal quality by selective removal of a Ca<sup>2+</sup>-dependent membrane-insertion loop

Neau, David; Gilbert, Nathaniel C.; Bartlett, Sue G.; Dassey, Adam J.; Newcomer, Marcia E.

doi:10.1107/s1744309107050993

Cited by 15 publications

(17 citation statements)

References 26 publications

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“…In addition, a deletion mutant that lacks a putative membrane insertion loop in the C2-like domain does not display Ca 2+ -stimulated activity in a membrane-based assay, but has wild type activity in the absence of membranes. Furthermore, in conditions in which the wild-type enzyme is found in the membrane fraction, the LOX deletion mutant is soluble (15). In an effort to determine how the presence of AOS might impact the membrane binding function of the 8 R -LOX C2-like domain, we monitored Ca 2+ -dependent membrane targeting of FP, 8 R -LOX, and HD by FRET and ultracentrifugation assays.…”

Section: Resultsmentioning

confidence: 99%

“…A double mutant in which amino acids in two of the Ca 2+ -sites are mutated does not bind membranes, but has wild-type activity in a membrane-free assay (11). In addition, a deletion mutant lacking a putative C2-like domain membrane-insertion loop common to 8 R - and 5-LOX (but absent in 15-LOX) has wild type activity in a membrane-free assay, but is impaired with respect to membrane binding: enzyme activity is not stimulated by Ca 2+ when assayed in the presence of liposomes (15). In the context of the naturally occurring fusion protein, the C2-like domain is flanked by the N-terminal AOS domain and the C-terminal LOX catalytic domain.…”

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confidence: 99%

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A Covalent Linker Allows for Membrane Targeting of an Oxylipin Biosynthetic Complex

Gilbert

Niebuhr²,

Tsuruta³

et al. 2008

Biochemistry

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A naturally occurring bi-functional protein from Plexaura homomalla links sequential catalytic activities in an oxylipin biosynthetic pathway. The C-terminal lipoxygenase (LOX) portion of the molecule catalyzes the transformation of arachidonic acid (AA) to the corresponding 8R-hydroperoxide, and the N-terminal allene oxide synthase (AOS) domain promotes the conversion of the hydroperoxide intermediate to the product allene oxide (AO). Small angle X-ray scattering data indicate that in the absence of a covalent linkage the two catalytic domains that transform AA to AO associate to form a complex that recapitulates the structure of the bi-functional protein. The SAXS data also support a model for LOX and AOS domain orientation in the fusion protein inferred from a low resolution crystal structure. However, results of membrane binding experiments indicate that covalent linkage of the domains is required for Ca 2+ -dependent membrane targeting of the sequential activities, despite the non-covalent domain association. Furthermore, membrane targeting is accompanied by a conformational change as monitored by specific proteolysis of the linker that joins the AOS and LOX domains. Our data are consistent with a model in which Ca 2+ -dependent membrane binding relieves the non-covalent interactions between the AOS and LOX domains and suggests that the C2-like domain of LOX mediates both protein-protein and protein-membrane interactions. KeywordsEicosanoids; lipoxygenase; allene oxide synthase; bi-functional enzymes; C2-domains; Calciumdependent membrane binding; arachidonic acid; protein-protein interactions; Small angle X-ray scattering (SAXS); X-ray crystallographyThe ability of the cell to respond to its environment is dependent upon effective coordination of metabolic pathways. For those pathways that involve the biosynthesis of the arachidonic acid (AA) -derived lipid mediators such as the leukotrienes, prostaglandins, and thromboxanes, their coordination may be exceptionally challenging in terms of substrate acquisition and specificity. The hydrophobic substrates partition into the membrane phase, and active sites that recognize bulky hydrophobic compounds might be inherently promiscuous. Furthermore, the highly reactive fatty acid hydroperoxide intermediates promote cellular oxidative damage if their metabolism is not stringently regulated. Both compartmentalization of enzymes and organization of multi-enzyme complexes are thought to provide cellular mechanisms for "traffic control" in pathways for the synthesis of these potent signaling molecules (1-3). In order to understand how coordination of biosynthetic pathways is achieved in the context of *Author to whom correspondence should be addressed: Department of Biological Sciences, Louisiana State University, Baton Rouge, LA, 70803, Tel : (225) Fax: (225) NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript cell trafficking, and specifically how facilitated transfer of intermediates between active sites might be a means to regulate p...

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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

A Covalent Linker Allows for Membrane Targeting of an Oxylipin Biosynthetic Complex

Gilbert

Niebuhr²,

Tsuruta³

et al. 2008

Biochemistry

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“…Substitution of the membrane insertion loops was based on a similar approach with the Plexaura homomalla enzyme, which shares both these amino acids and Ca 2+ -binding residues with 5-LOX in the amino terminal membrane-binding domain (14). The substitution of KKK with ENL in this context led to a ~3°C increase in the melting temperature of the enzyme (Fig 1C).…”

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The Structure of Human 5-Lipoxygenase

Gilbert

Bartlett

Waight

et al. 2011

Science

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317

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The synthesis of both pro-inflammatory leukotrienes and anti-inflammatory lipoxins requires the enzyme 5-lipoxygenase (5-LOX). 5-LOX activity is short-lived, apparently in part due to an intrinsic instability of the enzyme. We identified a 5-LOX-specific destabilizing sequence that is involved in orienting the carboxy-terminus which binds the catalytic iron. Herein we report the crystal structure at 2.4 Å resolution of human 5-LOX stabilized by replacement of this sequence.

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“…For example, the variant used in the successful crystallization of the HIV gp120 envelope glycoprotein had two flexible loops which were replaced with Gly-Ala-Gly linkages to obtain a crystallizable variant [119,120]. In the case of 8R-lipoxygenase, the replacement of a flexible Ca 2+ -dependent membrane insertion loop, consisting of five amino acids, with a Gly-Ser dipeptide resulted in crystals that diffracted to a resolution 1 Å higher than the wild-type protein [121]. An interesting variation of this approach was introduced for the preparation of crystals of the β-subunit of the signal recognition particle receptor.…”

Section: Target Protein Modification For Enhanced Crystallizabilitymentioning

confidence: 99%

The “Sticky Patch” Model of Crystallization and Modification of Proteins for Enhanced Crystallizability

Derewenda

Godzik

2017

Methods in Molecular Biology

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Crystallization of macromolecules has long been perceived as a stochastic process, which cannot be predicted or controlled. This is consistent with another popular notion that the interactions of molecules within the crystal, i.e. crystal contacts, are essentially random and devoid of specific physicochemical features. In contrast, functionally relevant surfaces, such as oligomerization interfaces and specific protein-protein interaction sites, are under evolutionary pressures so their amino acid composition, structure and topology are distinct. However, current theoretical and experimental studies are significantly changing our understanding of the nature of crystallization. The increasingly popular ‘sticky patch’ model, derived from soft matter physics, describes crystallization as a process driven by interactions between select, specific surface patches, with properties thermodynamically favorable for cohesive interactions. Independent support for this model comes from various sources including structural studies and bioinformatics. Proteins that are recalcitrant to crystallization can be modified for enhanced crystallizability through chemical or mutational modification of their surface to effectively engineer ‘sticky patches’ which would drive crystallization. Here, we discuss the current state of knowledge of the relationship between the microscopic properties of the target macromolecule and its crystallizability, focusing on the ‘sticky patch’ model. We discuss state-of-art in silico methods that evaluate the propensity of a given target protein to form crystals based on these relationships, with the objective to design of variants with modified molecular surface properties and enhanced crystallization propensity. We illustrate this discussion with specific cases where these approaches allowed to generate crystals suitable for structural analysis.

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Improving protein crystal quality by selective removal of a Ca²⁺-dependent membrane-insertion loop

Cited by 15 publications

References 26 publications

A Covalent Linker Allows for Membrane Targeting of an Oxylipin Biosynthetic Complex

A Covalent Linker Allows for Membrane Targeting of an Oxylipin Biosynthetic Complex

The Structure of Human 5-Lipoxygenase

The “Sticky Patch” Model of Crystallization and Modification of Proteins for Enhanced Crystallizability

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Improving protein crystal quality by selective removal of a Ca2+-dependent membrane-insertion loop

Cited by 15 publications

References 26 publications

A Covalent Linker Allows for Membrane Targeting of an Oxylipin Biosynthetic Complex

A Covalent Linker Allows for Membrane Targeting of an Oxylipin Biosynthetic Complex

The Structure of Human 5-Lipoxygenase

The “Sticky Patch” Model of Crystallization and Modification of Proteins for Enhanced Crystallizability

Contact Info

Product

Resources

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Improving protein crystal quality by selective removal of a Ca²⁺-dependent membrane-insertion loop