Structural Characterization of Intramolecular Hg2+ Transfer between Flexibly Linked Domains of Mercuric Ion Reductase

The dynamics of macromolecular conformations are critical to the action of cellular networks. Solution X-ray scattering studies, in combination with macromolecular X-ray crystallography (MX) and nuclear magnetic resonance (NMR), strive to determine complete and accurate states of macromolecules, providing novel insights describing allosteric mechanisms, supramolecular complexes, and dynamic molecular machines. This review addresses theoretical and practical concepts, concerns, and considerations for using these techniques in conjunction with computational methods to productively combine solution-scattering data with high-resolution structures. I discuss the principal means of direct identification of macromolecular flexibility from SAXS data followed by critical concerns about the methods used to calculate theoretical SAXS profiles from high-resolution structures. The SAXS profile is a direct interrogation of the thermodynamic ensemble and techniques such as, for example, minimal ensemble search (MES), enhance interpretation of SAXS experiments by describing the SAXS profiles as population-weighted thermodynamic ensembles. I discuss recent developments in computational techniques used for conformational sampling, and how these techniques provide a basis for assessing the level of the flexibility within a sample. Although these approaches sacrifice atomic detail, the knowledge gained from ensemble analysis is often appropriate for developing hypotheses and guiding biochemical experiments. Examples of the use of SAXS and combined approaches with X-ray crystallography, NMR, and computational methods to characterize dynamic assemblies are presented.

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“…Inset shows the schematic representation of mutMerA and S–S-mutMerA. Data were adapted from Johs et al (2011)…”

Section: Modeling Of the Conformational Spacementioning

confidence: 99%

Validation of macromolecular flexibility in solution by small-angle X-ray scattering (SAXS)

2012

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“…CueR accomplishes his task taking advantage on a couple of Cys on a loop between a long helix and a 2-turn helix spaced by 7 amino acidic residues, which traps Ag(I), Cu(I) and Au(I) between the two sulfurs arranged in a linear geometry [16]. The evolutionarily unrelated flavoenzyme mercuric ion reductase [MerA, 17], which protects cells from mercury(II), also presents the C(X) n C motif. This enzyme is able to bind and to reduce mercuric ion between the two cysteines of the same motif at the FAD binding site, which is comprised in a secondary structure region analogous to CueR, i.e.…”

Section: The Geometries Of Gold–cys Complexesmentioning

confidence: 99%

On the mechanism and rate of gold incorporation into thiol-dependent flavoreductases

Saccoccia

Angelucci

Boumis

et al. 2012

Journal of Inorganic Biochemistry

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NADPH-dependent flavoreductases are important drug targets. During their enzymatic cycle thiolates and selenolates that have high affinity for transition metals are generated. Auranofin (AF), a gold-containing compound, is classified by the World Health Organization as an antirheumatic agent and it is indicated as the scaffold for the development of new anticancer and antiparasitic drugs. AF inhibits selenocysteine-containing flavoreductases (thioredoxin reductase and thioredoxin glutathione reductase) more effectively than non Se-containing ones (glutathione reductase); this preference has been ascribed to the high affinity of selenium for gold. We solved the 3D structure of the Se-containing Thioredoxin Glutathione Reductase from the human parasite Schistosoma mansoni complexed with Au and our results challenge this view: we believe that the relative velocity of the reaction rather than the relative affinity, depends on the presence of Sec residues, which appear to dictate AF selectivity.

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“…Small angle x-ray scattering (SAXS) and NMR studies combined with molecular dynamics simulations has become an increasingly powerful tool to understand the conformational ensemble properties of proteins in solution [19], [20], [21], [22], [23], [24], [25], [26]. Using a combination of SAXS and structure-based models (SBM), or SAXS-SBM, we showed that apo Csk is comprised of an ensemble of extended and compact conformations in solution, rather than adopting a single conformational form as depicted by the X-ray structure [27].…”

Section: Introductionmentioning

confidence: 99%

Substrate-Specific Reorganization of the Conformational Ensemble of CSK Implicates Novel Modes of Kinase Function

et al. 2012

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Protein kinases use ATP as a phosphoryl donor for the posttranslational modification of signaling targets. It is generally thought that the binding of this nucleotide induces conformational changes leading to closed, more compact forms of the kinase domain that ideally orient active-site residues for efficient catalysis. The kinase domain is oftentimes flanked by additional ligand binding domains that up- or down-regulate catalytic function. C-terminal Src kinase (Csk) is a multidomain tyrosine kinase that is up-regulated by N-terminal SH2 and SH3 domains. Although the X-ray structure of Csk suggests the enzyme is compact, X-ray scattering studies indicate that the enzyme possesses both compact and open conformational forms in solution. Here, we investigated whether interactions with the ATP analog AMP-PNP and ADP can shift the conformational ensemble of Csk in solution using a combination of small angle x-ray scattering and molecular dynamics simulations. We find that binding of AMP-PNP shifts the ensemble towards more extended rather than more compact conformations. Binding of ADP further shifts the ensemble towards extended conformations, including highly extended conformations not adopted by the apo protein, nor by the AMP-PNP bound protein. These ensembles indicate that any compaction of the kinase domain induced by nucleotide binding does not extend to the overall multi-domain architecture. Instead, assembly of an ATP-bound kinase domain generates further extended forms of Csk that may have relevance for kinase scaffolding and Src regulation in the cell.

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Structural Characterization of Intramolecular Hg2+ Transfer between Flexibly Linked Domains of Mercuric Ion Reductase

Cited by 27 publications

References 80 publications

Validation of macromolecular flexibility in solution by small-angle X-ray scattering (SAXS)

Validation of macromolecular flexibility in solution by small-angle X-ray scattering (SAXS)

On the mechanism and rate of gold incorporation into thiol-dependent flavoreductases

Substrate-Specific Reorganization of the Conformational Ensemble of CSK Implicates Novel Modes of Kinase Function

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