X-ray refinement significantly underestimates the level of microscopic heterogeneity in biomolecular crystals

Kuzmanic, Antonija; Pannu, Navraj S.; Žagrović, Bojan

doi:10.1038/ncomms4220

Cited by 116 publications

(123 citation statements)

References 74 publications

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“…Because the latter codependence of activity could be rationalized by a dynamic functional role of these residues, we turned to the highresolution X-ray data (13,15) deposited in the Protein Data Bank (PDB; www.rcsb.org) (16) to provide evidence of their dynamic motion within the crystal structure. Several recent studies have shown that the information present in such data is often underestimated and that it is possible to refine multiple conformations of residues simultaneously, each with individually refined occupancy and B factors, without overfitting the data (17)(18)(19)(20)(21)(22). By such sampling of low-level electron density, discrete, "hidden" conformations are revealed, facilitating a more quantitative representation of dynamic…”

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confidence: 99%

A dynamic Asp–Arg interaction is essential for catalysis in microsomal prostaglandin E ₂ synthase

Brock

Hámberg

Balagunaseelan

et al. 2016

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

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Microsomal prostaglandin E 2 synthase type 1 (mPGES-1) is responsible for the formation of the potent lipid mediator prostaglandin E 2 under proinflammatory conditions, and this enzyme has received considerable attention as a drug target. Recently, a high-resolution crystal structure of human mPGES-1 was presented, with Ser-127 being proposed as the hydrogen-bond donor stabilizing thiolate anion formation within the cofactor, glutathione (GSH). We have combined site-directed mutagenesis and activity assays with a structural dynamics analysis to probe the functional roles of such putative catalytic residues. We found that Ser-127 is not required for activity, whereas an interaction between Arg-126 and Asp-49 is essential for catalysis. We postulate that both residues, in addition to a crystallographic water, serve critical roles within the enzymatic mechanism. After characterizing the size or charge conservative mutations Arg-126-Gln, Asp-49-Asn, and Arg-126-Lys, we inferred that a crystallographic water acts as a general base during GSH thiolate formation, stabilized by interaction with Arg-126, which is itself modulated by its respective interaction with Asp-49. We subsequently found hidden conformational ensembles within the crystal structure that correlate well with our biochemical data. The resulting contact signaling network connects Asp-49 to distal residues involved in GSH binding and is ligand dependent. Our work has broad implications for development of efficient mPGES-1 inhibitors, potential anti-inflammatory and anticancer agents.is an abundant lipid mediator that signals via four receptors (EP1-4) to induce an array of important biological actions in physiology as well as pathophysiology (1). Under proinflammatory conditions, biosynthesis of PGE 2 proceeds from arachidonic acid, which is converted to the unstable endoperoxide PGH 2 by cyclooxygenase type 2 (COX-2). PGH 2 is further isomerized into PGE 2 by microsomal PGE synthase type 1 (mPGES-1) (2, 3). mPGES-1 is encoded by PTGES and is up-regulated by mitogens and cytokines in a pathway that is functionally coupled to COX-2 (2, 4). Because of its key role in PGE 2 synthesis, mPGES-1 has attracted attention as a potential drug target in the areas of inflammation, pain, fever, and cancer (5).mPGES-1 is a member of the MAPEG (Membrane-Associated Proteins in Eicosanoid and Glutathione metabolism) superfamily of enzymes (6), which also includes two key proteins in the leukotriene (LT) cascade, viz. 5-lipoxygenase activating protein and LT C 4 synthase (LTC4S). All MAPEG members are integral, homotrimeric membrane proteins, and structural information on this family has been scarce. However, significant progress has recently been made in this area with several high-resolution structures being solved by X-ray crystallography (7-9). In particular, the crystal structures of human LTC4S provided detailed structural information, including an arginine residue that was later shown to activate the glutathione (GSH) thiolate (10, 11). We have previously proposed ...

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A dynamic Asp–Arg interaction is essential for catalysis in microsomal prostaglandin E ₂ synthase

Brock

Hámberg

Balagunaseelan

et al. 2016

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

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“…Many methods have emerged for using Bragg data to model conformational diversity in protein crystals (6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17). The development of these methods has been important as conformational diversity can lead to inaccuracies in protein structure models (9,(18)(19)(20). A key limitation of using the Bragg data, however, is that different models of conformational diversity can yield the same mean electron density.…”

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confidence: 99%

Conformational dynamics of a crystalline protein from microsecond-scale molecular dynamics simulations and diffuse X-ray scattering

Wall

Benschoten

Sauter

et al. 2014

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

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X-ray diffraction from protein crystals includes both sharply peaked Bragg reflections and diffuse intensity between the peaks. The information in Bragg scattering is limited to what is available in the mean electron density. The diffuse scattering arises from correlations in the electron density variations and therefore contains information about collective motions in proteins. Previous studies using molecular-dynamics (MD) simulations to model diffuse scattering have been hindered by insufficient sampling of the conformational ensemble. To overcome this issue, we have performed a 1.1-μs MD simulation of crystalline staphylococcal nuclease, providing 100-fold more sampling than previous studies. This simulation enables reproducible calculations of the diffuse intensity and predicts functionally important motions, including transitions among at least eight metastable states with different active-site geometries. The total diffuse intensity calculated using the MD model is highly correlated with the experimental data. In particular, there is excellent agreement for the isotropic component of the diffuse intensity, and substantial but weaker agreement for the anisotropic component. Decomposition of the MD model into protein and solvent components indicates that protein-solvent interactions contribute substantially to the overall diffuse intensity. We conclude that diffuse scattering can be used to validate predictions from MD simulations and can provide information to improve MD models of protein motions.diffuse scattering | protein crystallography | molecular-dynamics simulation | protein dynamics | staphylococcal nuclease P roteins explore many conformations while carrying out their functions in biological systems (1-3). X-ray crystallography is the dominant source of information about protein structure; however, crystal structure models usually consist of just a single major conformation and at most a small portion of the model as alternate conformations. Crystal structures therefore are missing many details about the underlying conformational ensemble (4).Proteins assembled in crystalline arrays, like proteins in solution, exhibit rich conformational diversity (4) and often can perform their native functions (5). Many methods have emerged for using Bragg data to model conformational diversity in protein crystals (6-17). The development of these methods has been important as conformational diversity can lead to inaccuracies in protein structure models (9,(18)(19)(20). A key limitation of using the Bragg data, however, is that different models of conformational diversity can yield the same mean electron density.Whereas the Bragg scattering only contains information about the mean electron density, diffuse scattering (diffraction resulting in intensity between the Bragg peaks) is sensitive to spatial correlations in electron density variations (21-28) and therefore contains information about the way that atomic positions vary together in protein crystals. Because models that yield the same mean electron density c...

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“…It has been suggested that the extent of atomic motions is significantly underestimated by B-factor refinement [59], especially when distinct alternate conformations are sampled [60]. It becomes increasingly clear that even with high-resolution X-ray diffraction data (and even more so with low-resolution data), a single model might often not be sufficient to describe the experimental data, due to conformational heterogeneity and dynamics [61,62 ,63,64].…”

Section: Uncertainty Error and Dynamicsmentioning

confidence: 99%

Hybrid methods for macromolecular structure determination: experiment with expectations

Schröder

2015

Current Opinion in Structural Biology

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X-ray refinement significantly underestimates the level of microscopic heterogeneity in biomolecular crystals

Cited by 116 publications

References 74 publications

A dynamic Asp–Arg interaction is essential for catalysis in microsomal prostaglandin E ₂ synthase

A dynamic Asp–Arg interaction is essential for catalysis in microsomal prostaglandin E ₂ synthase

Conformational dynamics of a crystalline protein from microsecond-scale molecular dynamics simulations and diffuse X-ray scattering

Hybrid methods for macromolecular structure determination: experiment with expectations

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X-ray refinement significantly underestimates the level of microscopic heterogeneity in biomolecular crystals

Cited by 116 publications

References 74 publications

A dynamic Asp–Arg interaction is essential for catalysis in microsomal prostaglandin E 2 synthase

A dynamic Asp–Arg interaction is essential for catalysis in microsomal prostaglandin E 2 synthase

Conformational dynamics of a crystalline protein from microsecond-scale molecular dynamics simulations and diffuse X-ray scattering

Hybrid methods for macromolecular structure determination: experiment with expectations

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A dynamic Asp–Arg interaction is essential for catalysis in microsomal prostaglandin E ₂ synthase

A dynamic Asp–Arg interaction is essential for catalysis in microsomal prostaglandin E ₂ synthase