Development of sesbania mosaic virus nanoparticles for imaging.

The capsids of viruses have a high degree of symmetry. Therefore, virus nanoparticles (VNPs) can be programmed to display many imaging agents precisely. Plant VNPs are biocompatible, biodegradable and non-infectious to mammals. We have carried out bioconjugation of sesbania mosaic virus (SeMV), a well characterized plant virus, with fluorophores using reactive lysine-N-hydroxysuccinimide ester and cysteine-maleimide chemistries. Monitoring of cellular internalization of labelled SeMV nanoparticles (NPs) by confocal microscopy and flow cytometry showed that the particles have a natural preference for entry into MDA-MB-231 (breast cancer) cells, although they could also enter various other cell lines. The fluorescence of SeMV NPs labelled via the cysteines with Cy5.5 dye was found to be more stable and was detectable with greater sensitivity than that of particles labelled via the lysines with Alexa Fluor. Live-cell imaging using SeMV internally labelled with Cy5.5 showed that it could bind to MDA-MB-231 cells in less than 5 minutes and enter the cells within 15 minutes. The particles undergo endolysosomal degradation by 6 h as evidenced by their co-localization with LAMP-1. Far-western blot analysis with a HeLa cell membrane protein fraction showed that SeMV interacts with 54-, 35- and 33-kDa proteins, which were identified by mass spectrometry as vimentin, voltage-dependent anion-selective channel protein (VDAC1), and annexin A2 isoform 2 (ANXA2), respectively, suggesting that the particles may bind and enter the cell through these proteins. The results presented here demonstrate that the SeMV NPs provide a new platform technology that could be used to develop in vivo imaging and targeted drug delivery agents for cancer diagnosis and therapy.

Comparative structural and enzymatic studies on Salmonella typhimurium diaminopropionate ammonia lyase reveal its unique features.

Cellular metabolism of amino acids is controlled by a large number of pyridoxal 5'-phosphate (PLP) dependent enzymes. Diaminopropionate ammonia lyase (DAPAL), a fold type II PLP-dependent enzyme, degrades both the D and L forms of diaminopropionic acid (DAP) to pyruvate and ammonia. Earlier studies on the Escherichia coli DAPAL (EcDAPAL) had suggested that a disulfide bond located close to the active site may be crucial for maintaining the geometry of the substrate entry channel and the active site. In order to obtain further insights into the catalytic properties of DAPAL, structural and functional studies on Salmonella typhimurium DAPAL (StDAPAL) were initiated. The three-dimensional X-ray crystal structure of StDAPAL was determined at 2.5 Šresolution. As expected, the polypeptide fold and dimeric organization of StDAPAL is similar to those of EcDAPAL. A phosphate group was located in the active site of StDAPAL and expulsion of this phosphate is probably essential to bring Asp125 to a conformation suitable for proton abstraction from the substrate (D-DAP). The unique disulfide bond of EcDAPAL was absent in StDAPAL, although the enzyme displayed comparable catalytic activity. Site directed mutagenesis of the cysteine residues involved in disulfide bond formation in EcDAPAL followed by functional and biophysical studies further confirmed that the disulfide bond is not necessary either for substrate binding or for catalysis. The activity of StDAPAL but not EcDAPAL was enhanced by monovalent cations suggesting subtle differences in the active site geometries of these two closely related enzymes.

Crystal structure of a thiolase from Escherichia coli at 1.8†Šresolution.

Thiolases catalyze the Claisen condensation of two acetyl-CoA molecules to give acetoacetyl-CoA, as well as the reverse degradative reaction. Four genes coding for thiolases or thiolase-like proteins are found in the Escherichia coli genome. In this communication, the successful cloning, purification, crystallization and structure determination at 1.8 Šresolution of a homotetrameric E. coli thiolase are reported. The structure of E. coli thiolase co-crystallized with acetyl-CoA at 1.9 Šresolution is also reported. As observed in other tetrameric thiolases, the present E. coli thiolase is a dimer of two tight dimers and probably functions as a biodegradative enzyme. Comparison of the structure and biochemical properties of the E. coli enzyme with those of other well studied thiolases reveals certain novel features of this enzyme, such as the modification of a lysine in the dimeric interface, the possible oxidation of the catalytic Cys88 in the structure of the enzyme obtained in the presence of CoA and active-site hydration. The tetrameric enzyme also displays an interesting departure from exact 222 symmetry, which is probably related to the deformation of the tetramerization domain that stabilizes the oligomeric structure of the protein. The current study allows the identification of substrate-binding amino-acid residues and water networks at the active site and provides the structural framework required for understanding the biochemical properties as well as the physiological function of this E. coli thiolase.

Structural studies on chimeric Sesbania mosaic virus coat protein: Revisiting SeMV assembly.

The capsid protein (CP) of Sesbania mosaic virus (SeMV, a T=3 plant virus) consists of a disordered N-terminal R-domain and an ordered S-domain. Removal of the R-domain results in the formation of T=1 particles. In the current study, the R-domain was replaced with unrelated polypeptides of similar lengths: the B-domain of Staphylococcus aureus SpA, and SeMV encoded polypeptides P8 and P10. The chimeric proteins contained T=3 or larger virus-like particles (VLPs) and could not be crystallized. The presence of metal ions during purification resulted in a large number of heterogeneous nucleoprotein complexes. N∆65-B (R domain replaced with B domain) could also be purified in a dimeric form. Its crystal structure revealed T=1 particles devoid of metal ions and the B-domain was disordered. However, the B-domain was functional in N∆65-B VLPs, suggesting possible biotechnological applications. These studies illustrate the importance of N-terminal residues, metal ions and robustness of the assembly process.

Structural studies on tobacco streak virus coat protein: Insights into the pleomorphic nature of ilarviruses.

Tobacco streak virus (TSV), the type member of Ilarvirus genus, is a major plant pathogen. TSV purified from infected plants consists of a ss-RNA genome encapsidated in spheroidal particles with diameters of 27, 30 and 33nm constructed from multiple copies of a single species of coat protein (CP) subunits. Apart from protecting the viral genome, CPs of ilarviruses play several key roles in the life cycle of these viruses. Unlike the related bromo and cucumoviruses, ilarvirus particles are labile and pleomorphic, which has posed difficulties in their crystallization and structure determination. In the current study, a truncated TSV-CP was crystallized in two distinct forms and their structures were determined at resolutions of 2.4Å and 2.1Å, respectively. The core of TSV CP was found to possess the canonical ß-barrel jelly roll tertiary structure observed in several other viruses. Dimers of CP with swapped C-terminal arms (C-arm) were observed in both the crystal forms. The C-arm was found to be flexible and is likely to be responsible for the polymorphic and pleomorphic nature of TSV capsids. Consistent with this observation, mutations in the hinge region of the C-arm that reduce the flexibility resulted in the formation of more uniform particles. TSV CP was found to be structurally similar to that of Alfalfa mosaic virus (AMV) accounting for similar mechanism of genome activation in alfamo and ilar viruses. This communication represents the first report on the structure of the CP from an ilarvirus.

Structural and functional analysis of two universal stress proteins YdaA and YnaF from Salmonella typhimurium: possible roles in microbial stress tolerance.

In many organisms "Universal Stress Proteins" (USPs) are induced in response to a variety of environmental stresses. Here we report the structures of two USPs, YnaF and YdaA from Salmonella typhimurium determined at 1.8Å and 2.4Å resolutions, respectively. YnaF consists of a single USP domain and forms a tetrameric organization stabilized by interactions mediated through chloride ions. YdaA is a larger protein consisting of two tandem USP domains. Two protomers of YdaA associate to form a structure similar to the YnaF tetramer. YdaA showed ATPase activity and an ATP binding motif G-2X-G-9X-G(S/T/N) was found in its C-terminal domain. The residues corresponding to this motif were not conserved in YnaF although YnaF could bind ATP. However, unlike YdaA, YnaF did not hydrolyse ATP in vitro. Disruption of interactions mediated through chloride ions by selected mutations converted YnaF into an ATPase. Residues that might be important for ATP hydrolysis could be identified by comparing the active sites of native and mutant structures. Only the C-terminal domain of YdaA appears to be involved in ATP hydrolysis. The structurally similar N-terminal domain was found to bind a zinc ion near the segment equivalent to the phosphate binding loop of the C-terminal domain. Mass spectrometric analysis showed that YdaA might bind a ligand of approximate molecular weight 800daltons. Structural comparisons suggest that the ligand, probably related to an intermediate in lipid A biosynthesis, might bind at a site close to the zinc ion. Therefore, the N-terminal domain of YdaA binds zinc and might play a role in lipid metabolism. Thus, USPs appear to perform several distinct functions such as ATP hydrolysis, altering membrane properties and chloride sensing.

Crystal structures of SCP2-thiolases of Trypanosomatidae, human pathogens causing widespread tropical diseases: the importance for catalysis of the cysteine of the unique HDCF loop.

Thiolases are essential CoA-dependent enzymes in lipid metabolism. In the present study we report the crystal structures of trypanosomal and leishmanial SCP2 (sterol carrier protein, type-2)-thiolases. Trypanosomatidae cause various widespread devastating (sub)-tropical diseases, for which adequate treatment is lacking. The structures reveal the unique geometry of the active site of this poorly characterized subfamily of thiolases. The key catalytic residues of the classical thiolases are two cysteine residues, functioning as a nucleophile and an acid/base respectively. The latter cysteine residue is part of a CxG motif. Interestingly, this cysteine residue is not conserved in SCP2-thiolases. The structural comparisons now show that in SCP2-thiolases the catalytic acid/base is provided by the cysteine residue of the HDCF motif, which is unique for this thiolase subfamily. This HDCF cysteine residue is spatially equivalent to the CxG cysteine residue of classical thiolases. The HDCF cysteine residue is activated for acid/base catalysis by two main chain NH-atoms, instead of two water molecules, as present in the CxG active site. The structural results have been complemented with enzyme activity data, confirming the importance of the HDCF cysteine residue for catalysis. The data obtained suggest that these trypanosomatid SCP2-thiolases are biosynthetic thiolases. These findings provide promise for drug discovery as biosynthetic thiolases catalyse the first step of the sterol biosynthesis pathway that is essential in several of these parasites.

Crystal structure of Salmonella typhimurium 2-methylcitrate synthase: Insights on domain movement and substrate specificity.

2-Methylcitric acid (2-MCA) cycle is one of the well studied pathways for the utilization of propionate as a source of carbon and energy in bacteria such as Salmonella typhimurium and Escherichia coli. 2-Methylcitrate synthase (2-MCS) catalyzes the conversion of oxaloacetate and propionyl-CoA to 2-methylcitrate and CoA in the second step of 2-MCA cycle. Here, we report the X-ray crystal structure of S. typhimurium 2-MCS (StPrpC) at 2.4Å resolution and its functional characterization. StPrpC was found to utilize propionyl-CoA more efficiently than acetyl-CoA or butyryl-CoA. The polypeptide fold and the catalytic residues of StPrpC are conserved in citrate synthases (CSs) suggesting similarities in their functional mechanisms. In the triclinic P1 cell, StPrpC molecules were organized as decamers composed of five identical dimer units. In solution, StPrpC was in a dimeric form at low concentrations and was converted to larger oligomers at higher concentrations. CSs are usually dimeric proteins. In Gram-negative bacteria, a hexameric form, believed to be important for regulation of activity by NADH, is also observed. Structural comparisons with hexameric E. coli CS suggested that the key residues involved in NADH binding are not conserved in StPrpC. Structural comparison with the ligand free and bound states of CSs showed that StPrpC is in a nearly closed conformation despite the absence of bound ligands. It was found that the Tyr197 and Leu324 of StPrpC are structurally equivalent to the ligand binding residues His and Val, respectively, of CSs. These substitutions might determine the specificities for acyl-CoAs of these enzymes.

Detection of the protein dimers, multiple monomeric states and hydrated forms of Plasmodium falciparum triosephosphate isomerase in the gas phase.

Dimeric and monomeric forms of the enzyme triosephosphate isomerase (TIM) from Plasmodium falciparum (Pf) have been detected under conditions of nanoflow by electrospray mass spectrometry. The dimer (M = 55 663 Da) exhibits a narrow charge state distribution with intense peaks limited to values of 18(+) to 21(+), maximal intensity being observed for charge states 19(+) and 20(+). A monomeric species with a charge state distribution ranging from 11(+) to 16(+) is also observed, which may be assigned to folded dissociated subunits. Complete dimer dissociation results under normal electrospray condition. The effects of solution pH and source temperature have been investigated. The observation of four distinct charge state distributions which may be assigned to a dimer, folded monomer, partially folded monomer and unfolded monomer is reported. Circular dichromism and fluorescence studies of Pf TIM at low pH support the retention of substantial secondary and tertiary structures. Satellite peaks in mass spectra corresponding to hydrated species are also observed and isotope shift upon deuteration is demonstrated. The analysis of all available independent crystal structures of Pf TIM and TIMs from other organisms permits identification of structurally conserved water molecules. Hydration observed in the dimer and folded monomeric forms in the gas phase may correspond to these conserved sites.

Stacking interactions of W271 and H275 of SeMV serine protease with W43 of natively unfolded VPg confer catalytic activity to protease.

N-terminal serine protease domain of Sesbania mosaic virus polyprotein, requires fused VPg for its activity. W43 of VPg mediates aromatic stacking interactions (characterized by 230 nm positive CD peak) with protease. A stretch of aromatic residues (F269, W271, Y315, and Y319) exposed in the protease domain were mutated to identify the interacting partner of W43. W271A Protease-VPg mutant showed absence of cleavage activity both in vivo and in trans, with concomitant loss of the 230 nm CD peak. F269A Protease-VPg mutant was partially active. Mutations of the tyrosines did not result in loss of protease activity or the CD peak. Interestingly, H275, though not a part of the exposed aromatic stretch, was shown to be essential for protease activity and contributed significantly to the CD peak. Hence, we conclude that W271 and H275 of the protease domain mediate aromatic stacking interactions with W43 of VPg thereby rendering the protease active.

Crystal structures of Salmonella typhimurium propionate kinase and its complex with Ap4A: evidence for a novel Ap4A synthetic activity.

Propionate kinase catalyses the last step in the anaerobic breakdown of L-threonine to propionate in which propionyl phosphate and ADP are converted to propionate and ATP. Here we report the structures of propionate kinase (TdcD) in the native form as well as in complex with diadenosine 5',5'''-P1,P4-tetraphosphate (Ap4A) by X-ray crystallography. Structure of TdcD obtained after cocrystallization with ATP showed Ap4A bound to the active site pocket suggesting the presence of Ap4A synthetic activity in TdcD. Binding of Ap4A to the enzyme was confirmed by the structure determination of a TdcD-Ap4A complex obtained after cocrystallization of TdcD with commercially available Ap4A. Mass spectroscopic studies provided further evidence for the formation of Ap4A by propionate kinase in the presence of ATP. In the TdcD-Ap4A complex structure, Ap4A is present in an extended conformation with one adenosine moiety present in the nucleotide binding site and other in the proposed propionate binding site. These observations tend to support direct in-line transfer of phosphoryl group during the kinase reaction.

Crystal structure of the serine protease domain of Sesbania mosaic virus polyprotein and mutational analysis of residues forming the S1-binding pocket.

Sesbania mosaic virus (SeMV) polyprotein is processed by its N-terminal serine protease domain. The crystal structure of the protease domain was determined to a resolution of 2.4 A using multiple isomorphous replacement and anomalous scattering. The SeMV protease domain exhibited the characteristic trypsin fold and was found to be closer to cellular serine proteases than to other viral proteases. The residues of the S1-binding pocket, H298, T279 and N308 were mutated to alanine in the DeltaN70-Protease-VPg polyprotein, and the cis-cleavage activity was examined. The H298A and T279A mutants were inactive, while the N308A mutant was partially active, suggesting that the interactions of H298 and T279 with P1-glutamate are crucial for the E-T/S cleavage. A region of exposed aromatic amino acids, probably essential for interaction with VPg, was identified on the protease domain, and this interaction could play a major role in modulating the function of the protease.

Structural studies on recombinant T = 3 capsids of Sesbania mosaic virus coat protein mutants.

When expressed in Escherichia coli, the recombinant coat protein (rCP) of Sesbania mosaic virus (SeMV) was shown to self-assemble into T = 3 capsids encapsidating CP mRNA and 23S rRNA derived from the host. Expression of CP-P53A, in which a conserved proline at position 53 in the beta-annulus was substituted by alanine (CP-P53A), also produced similar capsids. Purified rCP and CP-P53A particles were crystallized and X-ray crystal structures of their mutant capsids were determined to resolutions of 3.6 and 4.1 A, respectively. As in the native viral CP, the CPs in these recombinant capsids adopt the jelly-roll beta-sandwich fold. The amino-terminal residues of the C subunits alone are ordered and form the beta-annulus structure at the quasi-sixfold axes. A characteristic bend in the beta-annulus remains unaffected in CP-P53A. The quasi-threefold interfaces of the capsids harbour calcium ions coordinated by ligands from the adjacent threefold-related subunits in a geometry that is analogous to that observed in the native capsid. Taken together with studies on deletion and substitution mutants of SeMV CP, these results suggest the possibility that the beta-annulus and nucleic acid-mediated interactions may be less important for the assembly of sobemoviruses than previously envisaged.

Crystal structures of ADP and AMPPNP-bound propionate kinase (TdcD) from Salmonella typhimurium: comparison with members of acetate and sugar kinase/heat shock cognate 70/actin superfamily.

Recently, it has been shown that l-threonine can be catabolized non-oxidatively to propionate via 2-ketobutyrate. Propionate kinase (TdcD; EC 2.7.2.-) catalyses the last step of this metabolic process by enabling the conversion of propionyl phosphate and ADP to propionate and ATP. To provide insights into the substrate-binding pocket and catalytic mechanism of TdcD, the crystal structures of the enzyme from Salmonella typhimurium in complex with ADP and AMPPNP have been determined to resolutions of 2.2A and 2.3A, respectively, by molecular replacement using Methanosarcina thermophila acetate kinase (MAK; EC 2.7.2.1). Propionate kinase, like acetate kinase, contains a fold with the topology betabetabetaalphabetaalphabetaalpha, identical with that of glycerol kinase, hexokinase, heat shock cognaten 70 (Hsc70) and actin, the superfamily of phosphotransferases. The structure consists of two domains with the active site contained in a cleft at the domain interface. Examination of the active site pocket revealed a plausible structural rationale for the greater specificity of the enzyme towards propionate than acetate. This was further confirmed by kinetic studies with the purified enzyme, which showed about ten times lower K(m) for propionate (2.3 mM) than for acetate (26.9 mM). Comparison of TdcD complex structures with those of acetate and sugar kinase/Hsc70/actin obtained with different ligands has permitted the identification of catalytically essential residues involved in substrate binding and catalysis, and points to both structural and mechanistic similarities. In the well-characterized members of this superfamily, ATP phosphoryl transfer or hydrolysis is coupled to a large conformational change in which the two domains close around the active site cleft. The significant amino acid sequence similarity between TdcD and MAK has facilitated study of domain movement, which indicates that the conformation assumed by the two domains in the nucleotide-bound structure of TdcD may represent an intermediate point in the pathway of domain closure.