Crystal structure of the pseudoenzyme PDX1.2 in complex with its cognate enzyme PDX1.3: a total eclipse

Pseudoenzymes have burst into the limelight recently as they provide another dimension to regulation of cellular protein activity. In the eudicot plant lineage, the pseudoenzyme PDX1.2 and its cognate enzyme PDX1.3 interact to regulate vitamin B6 biosynthesis. This partnership is important for plant fitness during environmental stress, in particular heat stress. PDX1.2 increases the catalytic activity of PDX1.3, with an overall increase in vitamin B6 biosynthesis. However, the mechanism by which this is achieved is not known. In this study, the Arabidopsis thaliana PDX1.2–PDX1.3 complex was crystallized in the absence and presence of ligands, and attempts were made to solve the X-ray structures. Three PDX1.2–PDX1.3 complex structures are presented: the PDX1.2–PDX1.3 complex as isolated, PDX1.2–PDX1.3-intermediate (in the presence of substrates) and a catalytically inactive complex, PDX1.2–PDX1.3-K97A. Data were also collected from a crystal of a selenomethionine-substituted complex, PDX1.2–PDX1.3-SeMet. In all cases the protein complexes assemble as dodecamers, similar to the recently reported individual PDX1.3 homomer. Intriguingly, the crystals of the protein complex are statistically disordered owing to the high degree of structural similarity of the individual PDX1 proteins, such that the resulting configuration is a composite of both proteins. Despite the differential methionine content, selenomethionine substitution of the PDX1.2–PDX1.3 complex did not resolve the problem. Furthermore, a comparison of the catalytically competent complex with a noncatalytic complex did not facilitate the resolution of the individual proteins. Interestingly, another catalytic lysine in PDX1.3 (Lys165) that pivots between the two active sites in PDX1 (P1 and P2), and the corresponding glutamine (Gln169) in PDX1.2, point towards P1, which is distinctive to the initial priming for catalytic action. This state was previously only observed upon trapping PDX1.3 in a catalytically operational state, as Lys165 points towards P2 in the resting state. Overall, the study shows that the integration of PDX1.2 into a heteromeric dodecamer assembly with PDX1.3 does not cause a major structural deviation from the overall architecture of the homomeric complex. Nonetheless, the structure of the PDX1.2–PDX1.3 complex highlights enhanced flexibility in key catalytic regions for the initial steps of vitamin B6 biosynthesis. This report highlights what may be an intrinsic limitation of X-ray crystallography in the structural investigation of pseudoenzymes.

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Integrative x-ray structure and molecular modeling for the rationalization of procaspase-8 inhibitor potency and selectivity

10.1101/721951 ◽

2019 ◽

Author(s):

Janice H. Xu ◽

Jerome Eberhardt ◽

Brianna Hill-Payne ◽

Gonzalo E. González-Páez ◽

José Omar Castellón ◽

...

Keyword(s):

Molecular Modeling ◽

Small Molecules ◽

Small Molecule ◽

Conformational Changes ◽

Active Sites ◽

Structural Similarity ◽

X Ray ◽

X Ray Crystallography ◽

Molecule Inhibitor ◽

High Structural Similarity

AbstractCaspases are a critical class of proteases involved in regulating programmed cell death and other biological processes. Selective inhibitors of individual caspases, however, are lacking, due in large part to the high structural similarity found in the active sites of these enzymes. We recently discovered a small-molecule inhibitor, 63-R, that covalently binds the zymogen, or inactive precursor (pro-form), of caspase-8, but not other caspases, pointing to an untapped potential of procaspases as targets for chemical probes. Realizing this goal would benefit from a structural understanding of how small molecules bind to and inhibit caspase zymogens. There have, however, been very few reported procaspase structures. Here, we employ x-ray crystallography to elucidate a procaspase-8 crystal structure in complex with 63-R, which reveals large conformational changes in active-site loops that accommodate the intramolecular cleavage events required for protease activation. Combining these structural insights with molecular modeling and mutagenesis-based biochemical assays, we elucidate key interactions required for 63-R inhibition of procaspase-8. Our findings inform the mechanism of caspase activation and its disruption by small molecules, and, more generally, have implications for the development of small molecule inhibitors and/or activators that target alternative (e.g., inactive precursor) protein states to ultimately expand the druggable proteome.

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Three-dimensional crystallization of membrane proteins

Quarterly Reviews of Biophysics ◽

10.1017/s0033583500004625 ◽

1988 ◽

Vol 21 (4) ◽

pp. 429-477 ◽

Cited By ~ 156

Author(s):

W. Kühlbrandt

Keyword(s):

High Resolution ◽

Membrane Proteins ◽

Membrane Protein ◽

Protein Complexes ◽

Three Dimensional ◽

Biological Macromolecules ◽

X Ray ◽

X Ray Crystallography ◽

Membrane Protein Complexes ◽

Essential Prerequisite

As recently as 10 years ago, the prospect of solving the structure of any membrane protein by X-ray crystallography seemed remote. Since then, the threedimensional (3-D) structures of two membrane protein complexes, the bacterial photosynthetic reaction centres of Rhodopseudomonas viridis (Deisenhofer et al. 1984, 1985) and of Rhodobacter sphaeroides (Allen et al. 1986, 1987 a, 6; Chang et al. 1986) have been determined at high resolution. This astonishing progress would not have been possible without the pioneering work of Michel and Garavito who first succeeded in growing 3-D crystals of the membrane proteins bacteriorhodopsin (Michel & Oesterhelt, 1980) and matrix porin (Garavito & Rosenbusch, 1980). X-ray crystallography is still the only routine method for determining the 3-D structures of biological macromolecules at high resolution and well-ordered 3-D crystals of sufficient size are the essential prerequisite.

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Characterization of Aptamer-Protein Complexes by X-ray Crystallography and Alternative Approaches

International Journal of Molecular Sciences ◽

10.3390/ijms130810537 ◽

2012 ◽

Vol 13 (8) ◽

pp. 10537-10552 ◽

Cited By ~ 24

Author(s):

Vincent J. B. Ruigrok ◽

Mark Levisson ◽

Johan Hekelaar ◽

Hauke Smidt ◽

Bauke W. Dijkstra ◽

...

Keyword(s):

Protein Complexes ◽

X Ray ◽

X Ray Crystallography ◽

Alternative Approaches

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Cryo-Electron Microscopy: Moving Beyond X-Ray Crystal Structures for Drug Receptors and Drug Development

The Annual Review of Pharmacology and Toxicology ◽

10.1146/annurev-pharmtox-010919-023545 ◽

2020 ◽

Vol 60 (1) ◽

pp. 51-71 ◽

Cited By ~ 19

Author(s):

Javier García-Nafría ◽

Christopher G. Tate

Keyword(s):

Membrane Proteins ◽

G Protein ◽

Rational Design ◽

Protein Complexes ◽

Host Cells ◽

X Ray ◽

X Ray Crystallography ◽

Receptor Modulation ◽

Lipid Environment ◽

Drug Receptors

Electron cryo-microscopy (cryo-EM) has revolutionized structure determination of membrane proteins and holds great potential for structure-based drug discovery. Here we discuss the potential of cryo-EM in the rational design of therapeutics for membrane proteins compared to X-ray crystallography. We also detail recent progress in the field of drug receptors, focusing on cryo-EM of two protein families with established therapeutic value, the γ-aminobutyric acid A receptors (GABAARs) and G protein–coupled receptors (GPCRs). GABAARs are pentameric ion channels, and cryo-EM structures of physiological heteromeric receptors in a lipid environment have uncovered the molecular basis of receptor modulation by drugs such as diazepam. The structures of ten GPCR–G protein complexes from three different classes of GPCRs have now been determined by cryo-EM. These structures give detailed insights into molecular interactions with drugs, GPCR–G protein selectivity, how accessory membrane proteins alter receptor–ligand pharmacology, and the mechanism by which HIV uses GPCRs to enter host cells.

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Oriented overgrowth of acicular maghemite crystals on quartz

Clay Minerals ◽

10.1180/claymin.1988.023.4.03 ◽

1988 ◽

Vol 23 (4) ◽

pp. 357-365 ◽

Cited By ~ 5

Author(s):

M. M. Abreu ◽

M. O. Figueiredo ◽

J. C. Waerenborgh ◽

J. M. P. Cabral

Keyword(s):

Electron Microscopy ◽

Infrared Absorption ◽

Active Sites ◽

Structural Similarity ◽

Chemical Model ◽

X Ray Diffraction ◽

Absorption Data ◽

Oriented Growth ◽

X Ray ◽

Southern Portugal

AbstractMaghemite with acicular morphology occurs as an oriented growth on fresh quartz surfaces in the magnetic sandy fraction of the B horizon of a Typic Rhodoxeralf from southern Portugal. The maghemite was characterized by Mössbauer spectroscopy, X-ray diffraction and electron microscopy (scanning and transmission), and on the basis of a structural similarity between quartz and maghemite, a mechanism has been proposed for the formation of the oriented overgrowth. Correlation with an interpretative chemical model is discussed, assuming active sites over quartz surfaces. Infrared absorption data agree with the proposed models.

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New insights into the catalytic active-site structure of multicopper oxidases

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s1399004713033051 ◽

2014 ◽

Vol 70 (3) ◽

pp. 772-779 ◽

Cited By ~ 16

Author(s):

Hirofumi Komori ◽

Ryosuke Sugiyama ◽

Kunishige Kataoka ◽

Kentaro Miyazaki ◽

Yoshiki Higuchi ◽

...

Keyword(s):

Electron Density ◽

Active Sites ◽

Catalytic Mechanism ◽

Reduction Process ◽

Multicopper Oxidases ◽

Site Structure ◽

X Ray ◽

X Ray Crystallography ◽

Catalytic Active Site ◽

Hydrated Electrons

Structural models determined by X-ray crystallography play a central role in understanding the catalytic mechanism of enzymes. However, X-ray radiation generates hydrated electrons that can cause significant damage to the active sites of metalloenzymes. In the present study, crystal structures of the multicopper oxidases (MCOs) CueO fromEscherichia coliand laccase from a metagenome were determined. Diffraction data were obtained from a single crystal under low to high X-ray dose conditions. At low levels of X-ray exposure, unambiguous electron density for an O atom was observed inside the trinuclear copper centre (TNC) in both MCOs. The gradual reduction of copper by hydrated electrons monitored by measurement of the Cu K-edge X-ray absorption spectra led to the disappearance of the electron density for the O atom. In addition, the size of the copper triangle was enlarged by a two-step shift in the location of the type III coppers owing to reduction. Further, binding of O2to the TNC after its full reduction was observed in the case of the laccase. Based on these novel structural findings, the diverse resting structures of the MCOs and their four-electron O2-reduction process are discussed.

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Charting Hydrogen Bond Anisotropy

10.26434/chemrxiv.8865944 ◽

2019 ◽

Author(s):

Diogo Santos-Martins ◽

Stefano Forli

Keyword(s):

Hydrogen Bond ◽

Chemical Biology ◽

Solid Phase ◽

Electronic Configuration ◽

Interaction Energies ◽

X Ray ◽

X Ray Crystallography ◽

Chemical Groups ◽

High Degree ◽

Context Free

<div>Hydrogen bond (HB) is an essential interaction in countless phenomena, and regulates the chemistry of life. HBs are characterized by two main features, strength and directionality, with a high degree of heterogeneity across different chemical groups. These characteristics are dependent on the electronic configuration of the atoms involved in the interaction, which, in turn, is influenced strongly by the molecular environment where they are found. Studies based on the analysis of HB in solid phase, such as X-ray crystallography, suffer from significant biases due to the packing forces. These will tend to better describe strong HBs at the expenses of weak ones, which are either distorted or under represented. Using quantum mechanics (QM), we calculated interaction energies for about a hundred acceptor and donors, in a rigorously defined set of geometries. We performed about 180,000 independent QM calculations, covering all relevant angular components, and mapping strength and directionality in a context free from external biases, with both single-site and cooperative HBs. We show that by quantifying directionality, there is not correlation with strength, and therefore these two components need to be addressed separately. Results demonstrate that there are very strong HB acceptors (e.g.,DMSO) with nearly isotropic interactions, and weak ones (e.g.,thioacetone) with a sharp directional profile. Similarly, groups can have comparable directional propensity, but be very distant in the strength spectrum (e.g., thioacetone and pyridine). These findings have implications for biophysics and molecular recognition, providing new insight for chemical biology, protein engineering, and drug design. The results require rethinking the way directionality is described, with implications for the thermodynamics of HB.</div>

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A basic introduction to single particles cryo-electron microscopy

AIMS Biophysics ◽

10.3934/biophy.2022002 ◽

2021 ◽

Vol 9 (1) ◽

pp. 5-20

Author(s):

Vittoria Raimondi ◽

◽

Alessandro Grinzato ◽

◽

Keyword(s):

Electron Microscopy ◽

Structural Biology ◽

Protein Complexes ◽

Computational Power ◽

Single Particles ◽

X Ray ◽

X Ray Crystallography ◽

Cryo Electron Microscopy ◽

First Choice ◽

New Generation

<abstract> <p>In the last years, cryogenic-electron microscopy (cryo-EM) underwent the most impressive improvement compared to other techniques used in structural biology, such as X-ray crystallography and NMR. Electron microscopy was invented nearly one century ago but, up to the beginning of the last decades, the 3D maps produced through this technique were poorly detailed, justifying the term “blobbology” to appeal to cryo-EM. Recently, thanks to a new generation of microscopes and detectors, more efficient algorithms, and easier access to computational power, single particles cryo-EM can routinely produce 3D structures at resolutions comparable to those obtained with X-ray crystallography. However, unlike X-ray crystallography, which needs crystallized proteins, cryo-EM exploits purified samples in solution, allowing the study of proteins and protein complexes that are hard or even impossible to crystallize. For these reasons, single-particle cryo-EM is often the first choice of structural biologists today. Nevertheless, before starting a cryo-EM experiment, many drawbacks and limitations must be considered. Moreover, in practice, the process between the purified sample and the final structure could be trickier than initially expected. Based on these observations, this review aims to offer an overview of the principal technical aspects and setups to be considered while planning and performing a cryo-EM experiment.</p> </abstract>

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Atomic resolution studies of carbonic anhydrase II

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s0907444910006554 ◽

2010 ◽

Vol 66 (5) ◽

pp. 616-627 ◽

Cited By ~ 18

Author(s):

Craig A. Behnke ◽

Isolde Le Trong ◽

Jeff W. Godden ◽

Ethan A. Merritt ◽

David C. Teller ◽

...

Keyword(s):

Carbonic Anhydrase ◽

Structural Similarity ◽

Atomic Displacement ◽

Atomic Resolution ◽

Protein Surfaces ◽

X Ray ◽

Density Maps ◽

Structural Details ◽

Atomic Displacement Parameters ◽

The Individual

Carbonic anhydrase has been well studied structurally and functionally owing to its importance in respiration. A large number of X-ray crystallographic structures of carbonic anhydrase and its inhibitor complexes have been determined, some at atomic resolution. Structure determination of a sulfonamide-containing inhibitor complex has been carried out and the structure was refined at 0.9 Å resolution with anisotropic atomic displacement parameters to anRvalue of 0.141. The structure is similar to those of other carbonic anhydrase complexes, with the inhibitor providing a fourth nonprotein ligand to the active-site zinc. Comparison of this structure with 13 other atomic resolution (higher than 1.25 Å) isomorphous carbonic anhydrase structures provides a view of the structural similarity and variability in a series of crystal structures. At the center of the protein the structures superpose very well. The metal complexes superpose (with only two exceptions) with standard deviations of 0.01 Å in some zinc–protein and zinc–ligand bond lengths. In contrast, regions of structural variability are found on the protein surface, possibly owing to flexibility and disorder in the individual structures, differences in the chemical and crystalline environments or the different approaches used by different investigators to model weak or complicated electron-density maps. These findings suggest that care must be taken in interpreting structural details on protein surfaces on the basis of individual X-ray structures, even if atomic resolution data are available.

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Syntheses and Redox Chemistry of Antiaromatic Core-Modified Isophlorinoids

Synlett ◽

10.1055/s-0037-1609907 ◽

2018 ◽

Vol 29 (18) ◽

pp. 2362-2371 ◽

Cited By ~ 1

Author(s):

Venkataramanarao Anand ◽

Santosh Panchal ◽

Baddigam Reddy

Keyword(s):

Noncovalent Interactions ◽

Chemical Properties ◽

Structural Similarity ◽

Redox Chemistry ◽

Spectroscopic Techniques ◽

Ambient Conditions ◽

X Ray Diffraction ◽

X Ray ◽

X Ray Crystallography ◽

Stable Core

During the attempted total synthesis of chlorophyll, Woodward hypothesized the formation of tetrapyrrolic 20π isophlorin as a transient antiaromatic intermediate which provides a plausible template to synthesize stable antiaromatic molecules. Despite its structural similarity with the 18π aromatic porphyrin, it significantly differs in its electronic and chemical properties. However, due to its unstable nature under ambient conditions it immediately gets oxidized to stable 18π aromatic porphyrin. Similar macrocyclic structures with β-substituted heterocycles, such as furan/thiophene/selenophene, have been synthesized, which undergo facile oxidation to yield the 18π porphyrin dication. Attempts to synthesize stable tetrapyrrolic isophlorin and its metal complex have remained unaccomplished till date. Strategies to synthesize stable core-modified 20π isophlorin and its confused isophlorin derivative have met with considerable success. Predominantly, they are synthesized by replacing three or four pyrroles with furan/thiophene. The 20π systems either with four furan units or with a ‘pair’ of furan and thiophene units were sufficiently stable enough to resist oxidation towards the corresponding porphyrin dication. The 20π isophlorins displayed noncovalent interactions with the curved π surface of fullerene which predominantly rises due to van der Waals attraction between the dissimilar π systems. This antiaromatic isophlorin-fullerene complexes were obtained and successfully characterized by single-crystal X-ray diffraction studies. Replacing only three pyrroles by furan rings yielded the first stable pyrrole derivative of antiaromatic 20π isophlorin, which can be reversibly oxidized to 18π aromatic porphyrin without deprotonating the inner pyrrole NH. In addition, replacing all the pyrrole units of N-confused porphyrin with thiophene yielded the first derivative of confused isophlorin. Further, its two-electron oxidation led to the formation of 18π aromatic cation with enhanced aromaticity. The structure and electronic properties of the oxidized and neutral species were unambiguously determined from a combination of spectroscopic techniques, X-ray crystallography, and computational methods. These studies reveal that antiaromatic systems like isophlorin and its confused isophlorin derivative can be stabilized under ambient conditions and they offer potential to explore the chemistry of 4nπ systems. This Account focuses of recent advances in 20π antiaromatic isophlorins, confused isophlorins, and nocorroles along with their redox chemistry. 1 Introduction2 Stable 20π Isophlorins2.1 Furan-/Thiophene-Based Isophlorins2.2 Pyrrole Isophlorins3 Confused Isophlorins3.1 Isophlorin-Fullerene3.2 meso-meso-Bridged Tetraoxaisophlorin Dimer4. Norcorroles5. Conclusion and Outlook

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