Low Resolution Solution Structure Research Articles

Analysis of Plasmodium falciparum myosin B ATPase activity and structure in complex with the calmodulin-like domain of its light chain MLC-B

Myosin B (MyoB) is a class 14 myosin expressed in all invasive stages of the malaria parasite, Plasmodium falciparum. It is not associated with the glideosome complex that drives motility and invasion of host cells. During red blood cell invasion, MyoB remains at the apical tip of the merozoite but is no longer observed once invasion is completed. MyoB is not essential for parasite survival, but when it is knocked out, merozoites are delayed in the initial stages of red blood cell invasion, giving rise to a growth defect that correlates with reduced invasion success. Therefore, further characterization is needed to understand how MyoB contributes to parasite invasion. Here, we have expressed and purified functional MyoB with the help of parasite-specific chaperones Hsp90 and Unc45, characterized its binding to actin and its known light chain MLC-B using biochemical and biophysical methods and determined its low-resolution structure in solution using small angle X-ray scattering. In addition to MLC-B, we found that four other putative regulatory light chains bind to the MyoB IQ2 motif invitro. The purified recombinant MyoB adopted the overall shape of a myosin, exhibited actin-activated ATPase activity, and moved actin filaments invitro. Additionally, we determined that the ADP release rate was faster than the ATP turnover number, and thus, does not appear to be rate limiting. This, together with the observed high affinity to actin and the specific localization of MyoB, may point toward a role in tethering and/or force sensing during early stages of invasion.

Structural and mechanistic insights into Mycothiol Disulphide Reductase and the Mycoredoxin-1-alkylhydroperoxide reductase E assembly of Mycobacterium tuberculosis

Mycobacteria employ a versatile machinery of the mycothiol-dependent system, containing the proteins mycothiol disulfide reductase (Mtr), the oxido-reductase Mycoredoxin-1 (Mrx-1) and the alkyl-hydroperoxide subunit E (AhpE). The mycothiol-dependent protein ensemble regulates the balance of oxidized-reduced mycothiol, to ensure a reductive intracellular environment for optimal functioning of its proteins even upon exposure to oxidative stress. Here, we determined the first low-resolution solution structure of Mycobacterium tuberculosis Mtr (MtMtr) derived from small-angle X-ray scattering data, which provides insight into its dimeric state. The solution shape reveals the two NADPH-binding domains inside the dimeric MtMtr in different conformations. NMR-titration shows that the MtMtr-MtMrx-1 interaction is characterized by a fast exchange regime and critical residues involved in the protein-protein interaction were identified. Using NMR spectroscopy and docking studies, the epitopes of MtMrx-1 and MtAhpE interaction are described, shedding new light into the interaction interface and mechanism of action. Finally, the essential residue of MtMrx-1 identified in the interaction with MtMtr and MtAhpE form a platform for structure-guided drug design against the versatile enzyme machinery of the mycothiol-dependent system inside M. tuberculosis.

Low resolution solution structure of an enzymatic active AhpC10:AhpF2 ensemble of the Escherichia coli Alkyl hydroperoxide Reductase.

The ability of bacteria to combat oxidative stress is imperative for their survival. The Alkyl hydroperoxide Reductase (AhpR) system, composed of the AhpC and AhpF proteins, is one of the dominant antioxidant defense systems required for scavenging hydrogen peroxide and organic peroxide. Therefore, it is necessary to understand the mechanism of the AhpR ensemble formation. In previous studies, we were able to elucidate conformational flexibility of Escherichia coli AhpF during the catalytic cycle and its binding site, the N-terminal domain (NTD), to AhpC. We proposed the novel binding and release mechanism of EcAhpC-AhpF, which is mediated by the well defined redox-state linked conformational changes associated with the C-terminal tail and active site regions of EcAhpC. Here, we have proceeded further to elucidate the solution structure of E. coli AhpC and the stable ensemble formation with EcAhpF using size-exclusion chromatography (SEC), dynamic light scattering (DLS) and small angle X-ray scattering (SAXS) techniques. The EcAhpC-AhpF complex structure with a stoichiometry of AhpC10:AhpF2 reveals that dimeric EcAhpF in its extended conformation enables the NTD disulphide centers to come in close proximity to the redox-active disulphide centers of EcAhpC, and provides an efficient electron transfer. Furthermore, the significance of the C-terminal tail of EcAhpC in ensemble formation is elucidated. SAXS data-based modeling revealed the flexible C-terminal tail of EcAhpC in solution, and its exposed nature, making it possible to contact the NTD of EcAhpF for stable complex formation.

Alternative Binding Modes Identified for Growth and Differentiation Factor-associated Serum Protein (GASP) Family Antagonism of Myostatin

Myostatin, a member of the TGF-β family of ligands, is a strong negative regulator of muscle growth. As such, it is a prime therapeutic target for muscle wasting disorders. Similar to other TGF-β family ligands, myostatin is neutralized by binding one of a number of structurally diverse antagonists. Included are the antagonists GASP-1 and GASP-2, which are unique in that they specifically antagonize myostatin. However, little is known from a structural standpoint describing the interactions of GASP antagonists with myostatin. Here, we present the First low resolution solution structure of myostatin-free and myostatin-bound states of GASP-1 and GASP-2. Our studies have revealed GASP-1, which is 100 times more potent than GASP-2, preferentially binds myostatin in an asymmetrical 1:1 complex, whereas GASP-2 binds in a symmetrical 2:1 complex. Additionally, C-terminal truncations of GASP-1 result in less potent myostatin inhibitors that form a 2:1 complex, suggesting that the C-terminal domains of GASP-1 are the primary mediators for asymmetric complex formation. Overall, this study provides a new perspective on TGF-β antagonism, where closely related antagonists can utilize different ligand-binding strategies.

Solution structure and biophysical characterization of the multifaceted signalling effector protein growth arrest specific-1.

BackgroundThe protein growth arrest specific-1 (GAS1) was discovered based on its ability to stop the cell cycle. During development it is involved in embryonic patterning, inhibits cell proliferation and mediates cell death, and has therefore been considered as a tumor suppressor. GAS1 is known to signal through two different cell membrane receptors: Rearranged during transformation (RET), and the sonic hedgehog receptor Patched-1. Sonic Hedgehog signalling is important in stem cell renewal and RET mediated signalling in neuronal survival. Disorders in both sonic hedgehog and RET signalling are connected to cancer progression. The neuroprotective effect of RET is controlled by glial cell-derived neurotrophic factor family ligands and glial cell-derived neurotrophic factor receptor alphas (GFRαs). Human Growth arrest specific-1 is a distant homolog of the GFRαs.ResultsWe have produced and purified recombinant human GAS1 protein, and confirmed that GAS1 is a monomer in solution by static light scattering and small angle X-ray scattering analysis. The low resolution solution structure reveals that GAS1 is more elongated and flexible than the GFRαs, and the homology modelling of the individual domains show that they differ from GFRαs by lacking the amino acids for neurotrophic factor binding. In addition, GAS1 has an extended loop in the N-terminal domain that is conserved in vertebrates after the divergence of fishes and amphibians.ConclusionsWe conclude that GAS1 most likely differs from GFRαs functionally, based on comparative structural analysis, while it is able to bind the extracellular part of RET in a neurotrophic factor independent manner, although with low affinity in solution. Our structural characterization indicates that GAS1 differs from GFRα’s significantly also in its conformation, which probably reflects the functional differences between GAS1 and the GFRαs.

Small-angle X-ray scattering to obtain models of multivalent lectin-glycan complexes.

Recent advances in small-angle X-ray scattering (SAXS) have led to the ability to model the glycans on glycoproteins and to obtain the low-resolution solution structures of complexes of lectins bound to multivalent glycan-presenting scaffolds. This progress in SAXS can respond to the increasing interest in the biological action of glycoproteins and lectins and in the design of multivalent glycan-based antagonists. Carbohydrates make up a significant part of the X-ray scattering content in SAXS and should be included in the model together with the protein, whose structure is most often based on a crystal structure or NMR ensemble, to give a far-improved fit with the experimental data. The modeling of the spatial positioning of glycans on proteins or in the architecture of lectin-glycan complexes delivers low-resolution structural information hitherto unmatched by any other method. SAXS data on the bacterial lectin FimH, strongly bound to heptyl α-D-mannose on a sevenfold derivatized β-cyclodextrin, permitted determination of the stoichiometry of the complex and the geometry of the lectin deposition on the multivalent β-cyclodextrin. The SAXS methods can be applied to larger complexes as the technique imposes no limit on the size of the macromolecular assembly in solution.

Small-angle X-ray Solution Scattering Study of the Multi-aminoacyl-tRNA Synthetase Complex Reveals an Elongated and Multi-armed particle

In animal cells, nine aminoacyl-tRNA synthetases are associated with the three auxiliary proteins p18, p38, and p43 to form a stable and conserved large multi-aminoacyl-tRNA synthetase complex (MARS), whose molecular mass has been proposed to be between 1.0 and 1.5 MDa. The complex acts as a molecular hub for coordinating protein synthesis and diverse regulatory signal pathways. Electron microscopy studies defined its low resolution molecular envelope as an overall rather compact, asymmetric triangular shape. Here, we have analyzed the composition and homogeneity of the native mammalian MARS isolated from rabbit liver and characterized its overall internal structure, size, and shape at low resolution by hydrodynamic methods and small-angle x-ray scattering in solution. Our data reveal that the MARS exhibits a much more elongated and multi-armed shape than expected from previous reports. The hydrodynamic and structural features of the MARS are large compared with other supramolecular assemblies involved in translation, including ribosome. The large dimensions and non-compact structural organization of MARS favor a large protein surface accessibility for all its components. This may be essential to allow structural rearrangements between the catalytic and cis-acting tRNA binding domains of the synthetases required for binding the bulky tRNA substrates. This non-compact architecture may also contribute to the spatiotemporal controlled release of some of its components, which participate in non-canonical functions after dissociation from the complex.

Three-dimensional single-particle imaging using angular correlations from X-ray laser data

Femtosecond X-ray pulses from X-ray free-electron laser sources make it feasible to conduct room-temperature solution scattering experiments far below molecular rotational diffusion timescales. Owing to the ultra-short duration of each snapshot in these fluctuation scattering experiments, the particles are effectively frozen in space during the X-ray exposure. In contrast to standard small-angle scattering experiments, the resulting scattering patterns are anisotropic. The intensity fluctuations observed in the diffraction images can be used to obtain structural information embedded in the average angular correlation of the Fourier transform of the scattering species, of which standard small-angle scattering data are a subset. The additional information contained in the data of these fluctuation scattering experiments can be used to determine the structure of macromolecules in solution without imposing symmetry or spatial restraints during model reconstruction, reducing ambiguities normally observed in solution scattering studies. In this communication, a method that utilizes fluctuation X-ray scattering data to determine low-resolution solution structures is presented. The method is validated with theoretical data calculated from several representative molecules and applied to the reconstruction of nanoparticles from experimental data collected at the Linac Coherent Light Source.

The solution structure of apo-iron regulatory protein 1

Iron is a cofactor for many proteins that are involved in essential metabolic processes. However, iron must be strictly regulated because it can react with oxygen to generate cytotoxic reactive oxygen intermediates. Iron regulatory protein 1 (IRP1) is a bi-functional protein that can act either as a post-transcriptional regulator of mRNAs containing iron responsive elements, or as a [4Fe–4S] cluster-containing cytosolic aconitase. Previous X-ray crystallography results show that IRP1 is in an open L-shape conformation when bound to IRE-RNAs, and in a globular conformation when it binds an iron–sulfur cluster. The structure of apo-IRP1 and the mechanism by which it transforms to either functional state is unknown. Therefore, small angle X-ray scattering was used to determine the low resolution solution structure of apo-IRP1 and to characterize its biophysical properties. These results show that apo-IRP1 has a radius of gyration (Rg) of 33.6±0.3Å, and a Dmax of 118±2Å. The ab initio and rigid-body modeling results show that apo-IRP1 is in an open conformation in solution, and the ensemble optimization results show that the molecules stay narrowly distributed about a Rg of 33–34Å. The open apo-IRP1 conformation seems optimal for subsequent conversion to either functional end state: RNA-binding, or cytosolic aconitase.

Low-resolution structure of the soluble domain GPAA1 (yGPAA170–247) of the glycosylphosphatidylinositol transamidase subunit GPAA1 from Saccharomyces cerevisiae

The GPI (glycosylphosphatidylinositol) transamidase complex catalyses the attachment of GPI anchors to eukaryotic proteins in the lumen of ER (endoplasmic reticulum). The Saccharomyces cerevisiae GPI transamidase complex consists of the subunits yPIG-K (Gpi8p), yPIG-S (Gpi17p), yPIG-T (Gpi16p), yPIG-U (CDC91/GAB1) and yGPAA1. We present the production of the two recombinant proteins yGPAA170–247 and yGPAA170–339 of the luminal domain of S. cerevisiae GPAA1, covering the amino acids 70–247 and 70–339 respectively. The secondary structural content of the stable and monodisperse yGPAA170–247 has been determined to be 28% α-helix and 27% β-sheet. SAXS (small-angle X-ray scattering) data showed that yGPAA170–247 has an Rg (radius of gyration) of 2.72±0.025 nm and Dmax (maximum dimension) of 9.14 nm. These data enabled the determination of the two domain low-resolution solution structure of yGPAA170–247. The large elliptical shape of yGPAA170–247 is connected via a short stalk to the smaller hook-like domain of 0.8 nm in length and 3.5 nm in width. The topological arrangement of yGPAA170–247 will be discussed together with the recently determined low-resolution structures of yPIG-K24–337 and yPIG-S38–467 from S. cerevisiae in the GPI transamidase complex.

Low Resolution Solution Structure of HAMLET and the Importance of Its Alpha-Domains in Tumoricidal Activity

HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) is the first member in a new family of protein-lipid complexes with broad tumoricidal activity. Elucidating the molecular structure and the domains crucial for HAMLET formation is fundamental for understanding its tumoricidal function. Here we present the low-resolution solution structure of the complex of oleic acid bound HAMLET, derived from small angle X-ray scattering data. HAMLET shows a two-domain conformation with a large globular domain and an extended part of about 2.22 nm in length and 1.29 nm width. The structure has been superimposed into the related crystallographic structure of human α-lactalbumin, revealing that the major part of α-lactalbumin accommodates well in the shape of HAMLET. However, the C-terminal residues from L105 to L123 of the crystal structure of the human α-lactalbumin do not fit well into the HAMLET structure, resulting in an extended conformation in HAMLET, proposed to be required to form the tumoricidal active HAMLET complex with oleic acid. Consistent with this low resolution structure, we identified biologically active peptide epitopes in the globular as well as the extended domains of HAMLET. Peptides covering the alpha1 and alpha2 domains of the protein triggered rapid ion fluxes in the presence of sodium oleate and were internalized by tumor cells, causing rapid and sustained changes in cell morphology. The alpha peptide-oleate bound forms also triggered tumor cell death with comparable efficiency as HAMLET. In addition, shorter peptides corresponding to those domains are biologically active. These findings provide novel insights into the structural prerequisites for the dramatic effects of HAMLET on tumor cells.

Solution structure of subunit γ (γ1-204) of the Mycobacterium tuberculosis F-ATP synthase and the unique loop of γ165-178, representing a novel TB drug target

Tuberculosis, caused by the strain Mycobacterium tuberculosis, is in focus of interest due to the emergence of multi- and extensive drug-resistant TB strains. The F(1)F(O) ATP synthase is one of the essential enzymes in energy requirement of both proliferating aerobic and hypoxic dormant stage of mycobacterium life cycle, and therefore a potential TB drug target. Subunit γ of F-ATP synthases plays an important role in coupling and catalysis via conformational transitions of its N- and C-termini as well as the bottom segment of the globular domain of γ, which is in close proximity to the rotating and ion-pumping c-ring. Here we describe the first production, purification and low resolution solution structure of subunit γ (γ(1-204), Mtγ(1-204)) of the M. tuberculosis F-ATP synthase. Mtγ(1-204) is a pear-like shaped protein with a molecular weight of 23 ± 2 kDa. Protein sequence analysis of Mtγ revealed differences in the amino acid composition to γ subunits from other sources, in particular the presence of a unique stretch of 13 amino acid residues (Mtγ(165-178)). NMR studies showed that Mtγ(165-178) forms a loop of polar residues. Mtγ(165-178) has been aligned at the bottom of the globular domain of the Escherichia coli subunit γ, being in close vicinity to the polar residues R41, Q42, E44 and Q46 (M. tuberculosis nomenclature) of the c-ring. The putative role(s) of Mtγ(165-178) in coupling and as a potential drug target are discussed.

Variations of subunit {varepsilon} of the Mycobacterium tuberculosis F1Fo ATP synthase and a novel model for mechanism of action of the tuberculosis drug TMC207.

The subunit ε of bacterial F(1)F(O) ATP synthases plays an important regulatory role in coupling and catalysis via conformational transitions of its C-terminal domain. Here we present the first low-resolution solution structure of ε of Mycobacterium tuberculosis (Mtε) F(1)F(O) ATP synthase and the nuclear magnetic resonance (NMR) structure of its C-terminal segment (Mtε(103-120)). Mtε is significantly shorter (61.6 Å) than forms of the subunit in other bacteria, reflecting a shorter C-terminal sequence, proposed to be important in coupling processes via the catalytic β subunit. The C-terminal segment displays an α-helical structure and a highly positive surface charge due to the presence of arginine residues. Using NMR spectroscopy, fluorescence spectroscopy, and mutagenesis, we demonstrate that the new tuberculosis (TB) drug candidate TMC207, proposed to bind to the proton translocating c-ring, also binds to Mtε. A model for the interaction of TMC207 with both ε and the c-ring is presented, suggesting that TMC207 forms a wedge between the two rotating subunits by interacting with the residues W15 and F50 of ε and the c-ring, respectively. T19 and R37 of ε provide the necessary polar interactions with the drug molecule. This new model of the mechanism of TMC207 provides the basis for the design of new drugs targeting the F(1)F(O) ATP synthase in M. tuberculosis.

Membrane Curvature Protein Exhibits Interdomain Flexibility and Binds a Small GTPase

The APPL1 and APPL2 proteins (APPL (adaptor protein, phosphotyrosine interaction, pleckstrin homology (PH) domain, and leucine zipper-containing protein)) are localized to their own endosomal subcompartment and interact with a wide range of proteins and small molecules at the cell surface and in the nucleus. They play important roles in signal transduction through their ability to act as Rab effectors. (Rabs are a family of Ras GTPases involved in membrane trafficking.) Both APPL1 and APPL2 comprise an N-terminal membrane-curving BAR (Bin-amphiphysin-Rvs) domain linked to a PH domain and a C-terminal phosphotyrosine-binding domain. The structure and interactions of APPL1 are well characterized, but little is known about APPL2. Here, we report the crystal structure and low resolution solution structure of the BARPH domains of APPL2. We identify a previously undetected hinge site for rotation between the two domains and speculate that this motion may regulate APPL2 functions. We also identified Rab binding partners of APPL2 and show that these differ from those of APPL1, suggesting that APPL-Rab interaction partners have co-evolved over time. Isothermal titration calorimetry data reveal the interaction between APPL2 and Rab31 has a K(d) of 140 nM. Together with other biophysical data, we conclude the stoichiometry of the complex is 2:2.

The low-resolution solution structure of Vibrio cholerae Hfq in complex with Qrr1 sRNA.

In Vibrio cholerae, the RNA binding protein and chaperone Hfq (VcHfq) facilitates the pairing of the quorum regulatory RNA (Qrr) small regulatory RNAs (sRNAs) to the 5′ untranslated regions of the mRNAs for a number of global regulators that modulate the expression of virulence genes. This Qrr-mediated sRNA circuit is an attractive antimicrobial target, but characterization at the molecular level is required for this to be realized. Here, we investigate the interactions between VcHfq and the Qrr sRNAs using a variety of biochemical and biophysical techniques. We show that the ring-shaped VcHfq hexamer binds the Qrrs with 1:1 stoichiometry through its proximal face, and the molecular envelope of the VcHfq-Qrr complex is experimentally determined from small angle scattering data to present the first structural glimpse of a Hfq-sRNA complex. This structure reveals that the VcHfq protein does not change shape on complex formation but the RNA does, suggesting that a chaperone role for VcHfq is a critical part of the VcHfq-Qrr interaction. Overall, these studies enhance our understanding of VcHfq-Qrr interactions.

Low-resolution solution structures of Munc18:Syntaxin protein complexes indicate an open binding mode driven by the Syntaxin N-peptide

When nerve cells communicate, vesicles from one neuron fuse with the presynaptic membrane releasing chemicals that signal to the next. Similarly, when insulin binds its receptor on adipocytes or muscle, glucose transporter-4 vesicles fuse with the cell membrane, allowing glucose to be imported. These essential processes require the interaction of SNARE proteins on vesicle and cell membranes, as well as the enigmatic protein Munc18 that binds the SNARE protein Syntaxin. Here, we show that in solution the neuronal protein Syntaxin1a interacts with Munc18-1 whether or not the Syntaxin1a N-peptide is present. Conversely, the adipocyte protein Syntaxin4 does not bind its partner Munc18c unless the N-peptide is present. Solution-scattering data for the Munc18-1:Syntaxin1a complex in the absence of the N-peptide indicates that this complex adopts the inhibitory closed binding mode, exemplified by a crystal structure of the complex. However, when the N-peptide is present, the solution-scattering data indicate both Syntaxin1a and Syntaxin4 adopt extended conformations in complexes with their respective Munc18 partners. The low-resolution solution structure of the open Munc18:Syntaxin binding mode was modeled using data from cross-linking/mass spectrometry, small-angle X-ray scattering, and small-angle neutron scattering with contrast variation, indicating significant differences in Munc18:Syntaxin interactions compared with the closed binding mode. Overall, our results indicate that the neuronal Munc18-1:Syntaxin1a proteins can adopt two alternate and functionally distinct binding modes, closed and open, depending on the presence of the N-peptide, whereas Munc18c:Syntaxin4 adopts only the open binding mode.

Solution structure of subunit a, a 104-363, of the Saccharomyces cerevisiae V-ATPase and the importance of its C-terminus in structure formation

The 95 kDa subunit a of eukaryotic V-ATPases consists of a C-terminal, ion-translocating part and an N-terminal cytosolic domain. The latter's N-terminal domain (~40 kDa) is described to bind in an acidification-dependent manner with cytohesin-2 (ARNO), giving the V-ATPase the putative function as pH-sensing receptor. Recently, the solution structure of the very N-terminal segment of the cytosolic N-terminal domain has been solved. Here we produced the N-terminal truncated form SCa₁₀₄₋₃₆₃ of the N-terminal domain (SCa₁₋₃₆₃) of the Saccharomyces cerevisiae V-ATPase and determined its low resolution solution structure, derived from SAXS data. SCa₁₀₄₋₃₆₃ shows an extended S-like conformation with a width of about 3.88 nm and a length of 11.4 nm. The structure has been superimposed into the 3D reconstruction of the related A₁A₀ ATP synthase from Pyrococcus furiosus, revealing that the SCa₁₀₄₋₃₆₃ fits well into the density of the collar structure of the enzyme complex. To understand the importance of the C-terminus of the protein SCa₁₋₃₆₃, and to determine the localization of the N- and C-termini in SCa₁₀₄₋₃₆₃, the C-terminal truncated form SCa₁₀₆₋₃₂₄ was produced and analyzed by SAXS. Comparison of the SCa₁₀₄₋₃₆₃ and SCa₁₀₆₋₃₂₄ shapes showed that the additional loop region in SCa₁₀₄₋₃₆₃ consists of the C-terminal residues. Whereas SCa₁₀₄₋₃₆₃ is monomeric in solution, SCa₁₀₆₋₃₂₄ forms a dimer, indicating the importance of the very C-terminus in structure formation. Finally, the solution structure of SCa₁₀₄₋₃₆₃ and SCa₁₀₆₋₃₂₄ will be discussed in terms of the topological arrangement of subunit a and cytoheisn-2 in V-ATPases.

Structural Basis for the 14-3-3 Protein-dependent Inhibition of the Regulator of G Protein Signaling 3 (RGS3) Function

Regulator of G protein signaling (RGS) proteins function as GTPase-activating proteins for the α-subunit of heterotrimeric G proteins. The function of certain RGS proteins is negatively regulated by 14-3-3 proteins, a family of highly conserved regulatory molecules expressed in all eukaryotes. In this study, we provide a structural mechanism for 14-3-3-dependent inhibition of RGS3-Gα interaction. We have used small angle x-ray scattering, hydrogen/deuterium exchange kinetics, and Förster resonance energy transfer measurements to determine the low-resolution solution structure of the 14-3-3ζ·RGS3 complex. The structure shows the RGS domain of RGS3 bound to the 14-3-3ζ dimer in an as-yet-unrecognized manner interacting with less conserved regions on the outer surface of the 14-3-3 dimer outside its central channel. Our results suggest that the 14-3-3 protein binding affects the structure of the Gα interaction portion of RGS3 as well as sterically blocks the interaction between the RGS domain and the Gα subunit of heterotrimeric G proteins.

Low-resolution structure in solution and amyloidogenesis of human cystatin C

Low-resolution structure in solution and amyloidogenesis of human cystatin C

Structural Characterization of the Multidomain Regulatory Protein Rv1364c from Mycobacterium tuberculosis

The open reading frame rv1364c of Mycobacterium tuberculosis, which regulates the stress-dependent σ factor, σ(F), has been analyzed structurally and functionally. Rv1364c contains domains with sequence similarity to the RsbP/RsbW/RsbV regulatory system of the stress-response σ factor of Bacillus subtilis. Rv1364c contains, sequentially, a PAS domain (which shows sequence similarity to the PAS domain of the B. subtilis RsbP protein), an active phosphatase domain, a kinase (anti-σ(F) like) domain anda C-terminal anti-σ(F) antagonist like domain. The crystal structures of two PAS domain constructs (at 2.3 and 1.6Å) and a phosphatase/kinase dual domain construct (at 2.6Å) are described. The PAS domain is shown to bind palmitic acid but to have 100 times greater affinity for palmitoleic acid. The full-length protein can exist in solution as both monomer and dimer. We speculate that a switch between monomer and dimer, possibly resulting from fatty acid binding,affects the accessibility of the serine of the C-terminal, anti-σ(F) antagonist domain for dephosphorylation by the phosphatase domain thus indirectly altering the availability of σ(F).