SAXS and the Working Protein

In this issue of Structure, Davies et al., 2005 present shape reconstructions for the molecular motor p97 using small angle X-ray scattering (SAXS) and offer insights into how ATP consumption is coupled to cyclical domain motions. This work emphasizes the emerging potential of SAXS for visualizing the workings of biological machines in solution

Molecular mechanism of activation of the immunoregulatory amidase NAAA

Palmitoylethanolamide is a bioactive lipid that strongly alleviates pain and inflammation in animal models and in humans. Its signaling activity is terminated through degradation by N-acylethanolamine acid amidase (NAAA), a cysteine hydrolase expressed at high levels in immune cells. Pharmacological inhibitors of NAAA activity exert profound analgesic and antiinflammatory effects in rodent models, pointing to this protein as a potential target for therapeutic drug discovery. To facilitate these efforts and to better understand the molecular mechanism of action of NAAA, we determined crystal structures of this enzyme in various activation states and in complex with several ligands, including both a covalent and a reversible inhibitor. Self-proteolysis exposes the otherwise buried active site of NAAA to allow catalysis. Formation of a stable substrate- or inhibitor-binding site appears to be conformationally coupled to the interaction of a pair of hydrophobic helices in the enzyme with lipid membranes, resulting in the creation of a linear hydrophobic cavity near the active site that accommodates the ligand's acyl chain

Molecular models should not be published without the corresponding atomic coordinates.

In PNAS, Romero et al. (1) present models of how glucocerebrosidase (GCase) interacts with saposin C (SAPC) and membranes. Unfortunately, the authors do not publish representative atomic coordinates or molecular dynamics trajectories for their models, denying researchers the opportunity to scrutinize the data Romero et al. (1) use to draw their functional conclusions. Access to these data is an important issue for structural biologists (2), and the open release of experimentally determined structural data has been the accepted practice for many years (3). Indeed, Romero et al. rely on several such publically available structures to carry out their study.J.E.D. is supported by a Royal Society University Research Fellowship (UF100371). S.C.G. is supported by a Sir Henry Dale Fellowship co-funded by the Royal Society and Wellcome Trust (098406/Z/12/B)

Chronometry and formation pathways of gypsum using Electron Spin Resonance and Fourier Transform Infrared Spectroscopy

Gypsum is an authigenic precipitate that forms under periods of accentuated aridity and occurs widely in arid zones. However its use in quantitative paleoclimatology has been limited due to the absence of a method to determine the timing of its formation. We present here the results of a feasibility study that demonstrates that the timing of the formation event of gypsum can be estimated using Electron Spin Resonance (ESR) analysis. We used well documented samples from White Sands in New Mexico, USA, the Thar Desert, India and lakes in the Simpson Desert and Mallee Region, Australia and found that ESR ages could be obtained using radiation sensitive SO4-, SO3- radicals and a photobleachable signal O3-. ESR signals were consistent with control ages based on contextual information. These suggest that the dating signals (SO4-, SO3-) are stable over time scales >100 ka. We propose that this stability of the SO4- signals over geological time scales arises due to hydrogen bonding between the water proton and the SO4- radical and that the suitability of these radiation-induced radicals comes from their being a part of the host matrix. Further, ESR along with Fourier Transform Infrared (FT-IR) Spectroscopy methods additionally inform on the geochemical pathways for gypsum formation and help elucidate complex formation processes even in samples that appeared unambiguous gypsum precipitates. Thus, the presence of Hannebachite (CaSO3.1/2H2O) and Mn2+ in Thar and Australian samples suggested a reducing environment such that low valence sulfur reacted with CaCO3 to form hannebachite and eventually gypsum. The presence of sulfur, partially as sulfite in Thar gypsum samples suggested that redox cycles were mediated by microbial activity. Absence of these features in White Sands samples suggested oxic conditions during gypsum precipitation

X-ray crystallographic analysis of 1) the two N-terminal domains of epithelial cadherin and 2) C3d, a fragment of the complement protein C3

grantor: University of TorontoThis thesis will present two protein structure determinations by x-ray crystallography and a methodological development. The first structure is of the two extracellular N-terminal domains of E-cadherin (Ecad12), a developmentally regulated cell adhesion molecule that mediates Ca2+-dependent cell-cell contacts. The structure reveals a two-fold symmetric dimer, each molecule of which binds a contiguous array of three bridged calcium ions. The calcium ions serve not only to rigidify the molecule into a linear conformation, but they also promote dimerization. The Ecad12 structure provided the first atomic level view of the role of calcium in the cadherin mediated formation and maintenance of solid tissues. The second structure was that of C3d, a proteolytic fragment of the central complement component C3. Activation and covalent attachment of C3 to pathogens is the key step in complement-mediated host defense. Additionally, antigen bound C3d interacts with complement receptor 2 (CR2, CD21) on B cells and thereby contributes to the initiation of an acquired humoral response. The structure of C3d reveals an Ã-Ã barrel with the residues responsible for covalent attachment at one end of the barrel and an acidic pocket well-suited to interact with CR2, at the other end. The structure supports a model whereby the transition of native C3 to its functionally active state involves the disruption of a complementary domain interface. Finally, the methods chapter describes a novel set of programs that provide a means for combining phases from slightly non isomorphous sources and extending them to higher resolution. The process of freezing the Ecad12 crystals caused non-isomorphism between the various data sets. Conventional density averaging and phase combination between the data sets could not be performed because of the poor quality of the electron density maps from two of the data sets (in part due to missing data). Application of the procedure to the Ecad12 case transformed the initial low resolution/quality electron density map into an easily interpretable map that contained continuous density with higher resolution side-chain information.Ph.D

X-ray crystallographic analysis of 1) the two N-terminal domains of epithelial cadherin and 2) C3d, a fragment of the complement protein C3

grantor: University of TorontoThis thesis will present two protein structure determinations by x-ray crystallography and a methodological development. The first structure is of the two extracellular N-terminal domains of E-cadherin (Ecad12), a developmentally regulated cell adhesion molecule that mediates Ca2+-dependent cell-cell contacts. The structure reveals a two-fold symmetric dimer, each molecule of which binds a contiguous array of three bridged calcium ions. The calcium ions serve not only to rigidify the molecule into a linear conformation, but they also promote dimerization. The Ecad12 structure provided the first atomic level view of the role of calcium in the cadherin mediated formation and maintenance of solid tissues. The second structure was that of C3d, a proteolytic fragment of the central complement component C3. Activation and covalent attachment of C3 to pathogens is the key step in complement-mediated host defense. Additionally, antigen bound C3d interacts with complement receptor 2 (CR2, CD21) on B cells and thereby contributes to the initiation of an acquired humoral response. The structure of C3d reveals an Ã-Ã barrel with the residues responsible for covalent attachment at one end of the barrel and an acidic pocket well-suited to interact with CR2, at the other end. The structure supports a model whereby the transition of native C3 to its functionally active state involves the disruption of a complementary domain interface. Finally, the methods chapter describes a novel set of programs that provide a means for combining phases from slightly non isomorphous sources and extending them to higher resolution. The process of freezing the Ecad12 crystals caused non-isomorphism between the various data sets. Conventional density averaging and phase combination between the data sets could not be performed because of the poor quality of the electron density maps from two of the data sets (in part due to missing data). Application of the procedure to the Ecad12 case transformed the initial low resolution/quality electron density map into an easily interpretable map that contained continuous density with higher resolution side-chain information.Ph.D

SAXS and the Working Protein

In this issue of Structure, Davies et al., 2005 present shape reconstructions for the molecular motor p97 using small angle X-ray scattering (SAXS) and offer insights into how ATP consumption is coupled to cyclical domain motions. This work emphasizes the emerging potential of SAXS for visualizing the workings of biological machines in solution

Structural Characterization of Human Immunity-Related GTPase Family M (IRGM)

The Immunity-Related GTPase Family M protein (IRGM) is involved in regulating cellular autophagy. Cellular knockdown of IRGM was shown to allow RNA viruses to hijack the autophagic immune response. Additionally, recent genetic studies have shown that underexpression of IRGM is associated with the incidence of Crohn's disease and infection by Mycobacterium tuberculosis. IRGM is an interferon-induced GTPase with an evolutionary conserved P-loop. It is an effector of the interferon-gamma pathway, but, unlike its protein family members, is not directly activated by the pathway. Its mechanism of action has been proposed to occur by translocation of IRGM to the mitochondria through recognition of cardiolipin, and affecting mitochondrial fission to induce autophagy. This potential interaction with cardiolipin might indicate the presence of a unique GTPase recognition and activation fold within IRGM. Our goal is to determine the X-ray crystal structure of IRGM in an effort to understand its molecular role in normal and diseased states. Additionally, we seek to test its interaction with and mechanism of recognition to mitochondrial cardiolipin as well as other autophagy-inducing binding partners. Currently, we have managed to express human IRGM in bacterial cells and have purified it to homogeneity using affinity and size-exclusion chromatography. These findings will serve to elucidate the mechanism of action of IRGM. Crucially, we hope to gain an understanding of its contributing role to Crohn's disease and tuberculosis infection at the molecular level, potentially paving the way to structure-based drug design and therapeutic opportunities.</jats:p