Volkensin from Adenia volkensii Harms (kilyambiti plant), a type 2 ribosome-inactivating protein.

Volkensin, a type 2 ribosome-inactivating protein from the roots of Adenia volkensii Harms (kilyambiti plant) was characterized both at the protein and nucleotide level by direct amino acid sequencing and cloning of the gene encoding the protein. Gene sequence analysis revealed that volkensin is encoded by a 1569-bp ORF (523 amino acid residues) without introns, with an internal linker sequence of 45 bp. Differences in residues present at several sequence positions (reproduced after repeated protein sequence analyses), with respect to the gene sequence, suggest several isoforms for the volkensin A-chain. Based on the crystallographic coordinates of ricin, which shares a high sequence identity with volkensin, a molecular model of volkensin was obtained. The 3D model suggests that the amino acid residues of the active site of the ricin A-chain are conserved at identical spatial positions, including Ser203, a novel amino acid residue found to be conserved in all known ribosome-inactivating proteins. The sugar binding site 1 of the ricin B-chain is also conserved in the volkensin B-chain, whilst in binding site 2, His246 replaces Tyr248. Native volkensin contains two free cysteinyl residues out of 14 derived from the gene sequence, thus suggesting a further disulphide bridge in the B chain, in addition to the inter- and intrachain disulphide bond pattern common to other type 2 ribosome-inactivating proteins.     

Cloning and expression of the B chain of volkensin, type 2 ribosome inactivating protein from Adenia volkensii harms: co-folding with the A chain for heterodimer reconstitution

Type 2 ribosome inactivating proteins (RIPs) include some potent plant toxins, among which ricin from Ricinus communis and abrin from Abrus precatorius seeds, have been known for more than a century. Two other type 2 RIPs belong to this class of proteins, both isolated from plants of the same family (Passifloraceae), modeccin and volkensin, from Adenia digitata and Adenia volkensii roots, respectively. Volkensin is probably the most potent plant toxin known, with an LD50 for rats of 50-60 ng/kg. Here we report the cloning, expression and renaturation of recombinant volkensin B chain. Furthermore, starting from separately expressed A and B chains, a co-association procedure was set-up, leading to in vitro heterodimeric volkensin reconstitution. The recombinant heterodimer was characterized by N-terminal sequence analysis and its hemagglutinating activity assessed. In parallel, we have explored the carbohydrate-binding properties of native volkensin with the aim to correlate toxin-specific properties (i.e., axonal transport along neurons) to lectin's sugar-binding preferences.     

Topology of the PhoR protein of Escherichia coli and functional analysis of internal deletion mutants

The PhoR protein of Escherichia coli K-12 belongs to a family of structurally related sensor-kinases that regulate responses to environmental stimuli. These proteins are often located in the inner membrane with two membrane-spanning segments that are separated by a periplasmic domain, which is supposed to sense the environmental stimuli. However, the hydrophobicity plot of PhoR suggests a somewhat different topology in which a large periplasmic domain is lacking and an extended cytoplasmic domain is present besides the kinase domain. In protease-accessibility experiments and by using phoR-phoA gene fusions, the topology of PhoR was investigated and the absence of a large periplasmic domain was confirmed. Furthermore, the function of the extended cytoptasmic domain was studied by creating internal deletions. The mutations in this domain resulted in a constitutive expression of the pho regulon, indicating that the mutant PhoR proteins are locked in their kinase function. We propose that this extended cytoplasmic domain functions by sensing an internal signal that represses the kinase function of the PhoR protein.     

Properties of volkensin, a toxic lectin from Adenia volkensii

Volkensin, a highly toxic protein from the roots of Adenia volkensii (kilyambiti, kinoria), was purified by affinity chromatography on acid-treated Sepharose 6B. The toxin is a glycoprotein (Mr 62,000, neutral sugar content 5.74%) consisting of an A subunit (Mr 29,000) and of a B subunit (Mr 36,000) linked by disulfide and noncovalent bond(s). The amino acid, amino sugar, and neutral sugar composition of the protein were determined. Volkensin is a galactose-specific lectin and is a potent inhibitor of eukaryotic protein synthesis in whole cells as well as in a cell-free system (a rabbit reticulocyte lysate). The inhibitory and the lectin activities are functions of the A and B subunits, respectively. Volkensin can be included amongst the ricin-like toxins and resembles most closely modeccin, the toxin of Adenia digitata.     

A Novel Topology of Proline-rich Transmembrane Protein 2 (PRRT2): HINTS FOR AN INTRACELLULAR FUNCTION AT THE SYNAPSE*

Proline-rich transmembrane protein 2 (PRRT2) has been identified as the single causative gene for a group of paroxysmal syndromes of infancy, including epilepsy, paroxysmal movement disorders, and migraine. On the basis of topology predictions, PRRT2 has been assigned to the recently characterized family of Dispanins, whose members share the two-transmembrane domain topology with a large N terminus and short C terminus oriented toward the outside of the cell. Because PRRT2 plays a role at the synapse, it is important to confirm the exact orientation of its N and C termini with respect to the plasma membrane to get clues regarding its possible function. Using a combination of different experimental approaches, including live immunolabeling, immunogold electron microscopy, surface biotinylation and computational modeling, we demonstrate a novel topology for this protein. PRRT2 is a type II transmembrane protein in which only the second hydrophobic segment spans the plasma membrane, whereas the first one is associated with the internal surface of the membrane and forms a helix-loop-helix structure without crossing it. Most importantly, the large proline-rich N-terminal domain is not exposed to the extracellular space but is localized intracellularly, and only the short C terminus is extracellular (N_cyt/C_exo topology). Accordingly, we show that PRRT2 interacts with the Src homology 3 domain-bearing protein Intersectin 1, an intracellular protein involved in synaptic vesicle cycling. These findings will contribute to the clarification of the role of PRRT2 at the synapse and the understanding of pathogenic mechanisms on the basis of PRRT2-related neurological disorders.     

Crystal structure of the Melampsora lini effector AvrP reveals insights into a possible nuclear function and recognition by the flax disease resistance protein P

The effector protein AvrP is secreted by the flax rust fungal pathogen (Melampsora lini) and recognized specifically by the flax (Linum usitatissimum) P disease resistance protein, leading to effector‐triggered immunity. To investigate the biological function of this effector and the mechanisms of specific recognition by the P resistance protein, we determined the crystal structure of AvrP. The structure reveals an elongated zinc‐finger‐like structure with a novel interleaved zinc‐binding topology. The residues responsible for zinc binding are conserved in AvrP effector variants and mutations of these motifs result in a loss of P‐mediated recognition. The first zinc‐coordinating region of the structure displays a positively charged surface and shows some limited similarities to nucleic acid‐binding and chromatin‐associated proteins. We show that the majority of the AvrP protein accumulates in the plant nucleus when transiently expressed in Nicotiana benthamiana cells, suggesting a nuclear pathogenic function. Polymorphic residues in AvrP and its allelic variants map to the protein surface and could be associated with differences in recognition specificity. Several point mutations of residues on the non‐conserved surface patch result in a loss of recognition by P, suggesting that these residues are required for recognition.     

Crystal stuctures of MglB‐2 (TP0684), a topologically variant d‐glucose‐binding protein from Treponema pallidum,reveal a ligand‐induced conformational change

Previously, we determined the crystal structure of apo‐TpMglB‐2, a d ‐glucose‐binding component of a putative ABC transporter from the syphilis spirochete Treponema pallidum. The protein had an unusual topology for this class of proteins, raising the question of whether the d ‐glucose‐binding mode would be different in TpMglB‐2. Here, we present the crystal structures of a variant of TpMglB‐2 with and without d ‐glucose bound. The structures demonstrate that, despite its aberrant topology, the protein undergoes conformational changes and binds d ‐glucose similarly to other Mgl‐type proteins, likely facilitating d ‐glucose uptake in T. pallidum.     

Native secondary structure topology has near minimum contact energy among all possible geometrically constrained topologies

Secondary structure topology in this article refers to the order and the direction of the secondary structures, such as helices and strands, with respect to the protein sequence. Even when the locations of the secondary structure Cα atoms are known, there are still (N!2^N)(M!2^M) different possible topologies for a protein with N helices and M strands. This work explored the question if the native topology is likely to be identified among a large set of all possible geometrically constrained topologies through an evaluation of the residue contact energy formed by the secondary structures, instead of the entire chain. We developed a contact pair specific and distance specific multiwell function based on the statistical characterization of the side chain distances of 413 proteins in the Protein Data Bank. The multiwell function has specific parameters to each of the 210 pairs of residue contacts. We illustrated a general mathematical method to extend a single well function to a multiwell function to represent the statistical data. We have performed a mutation analysis using 50 proteins to generate all the possible geometrically constrained topologies of the secondary structures. The result shows that the native topology is within the top 25% of the list ranked by the effective contact energies of the secondary structures for all the 50 proteins, and is within the top 5% for 34 proteins. As an application, the method was used to derive the structure of the skeletons from a low resolution density map that can be obtained through electron cryomicroscopy. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Effect of ribosome-inactivating proteins on ribosomes from Tetrahymena pyriformis and Acanthamoeba castellanii

The effect of ribosome-inactivating proteins type 1 (single-chain) and type 2 (two-chain, toxins) on polyphenylalanine polymerization by Tetrahymena pyriformis and Acanthamoeba castellanii ribosomes has been studied. The reaction catalysed by tetrahymena ribosomes was inhibited by two ribosome-inactivating proteins type 1 (dianthin 32 and, less effectively, momordin) whereas the reaction catalysed by amoeba ribosomes was inhibited, in a decreasing order of activity, by three ribosome-inactivating proteins type 1 (dianthin 32, saporin 6 and bryodin) and by two toxins (abrin and volkensin).     

Molecular cloning and phylogenetic analysis of cereal type II metacaspase cDNA from wheat

A new cereal type II metacaspase full-length cDNA from wheat (Triticum aestivum L.) leaves, TaeMCAII, was for the first time successfully amplified and sequenced. The full-length sequence of the TaeMCAII cDNA of 1 551 bp contains a 1 218 bp open reading frame. The deduced protein encoded by the TaeMCAII cDNA consists of 405 amino acids with a calculated molecular mass of 44 kDa and an isoelectric point of 5.29. In response to wounding or heat shock, a similar sequence of ultrastructural events including the tonoplast rupture, chromatin condensation, degradation of chloroplasts and disappearance of cytoplasm and organelles were observed using transmission electron microscopy. As the observed changes in TaeMCAII mRNA level did not occur to be statistically significant wounding-induced programmed cell death (PCD) seems to be metacaspase-independent pathway. Interestingly, in PCD caused by a heat-shock treatment, the level of TaeMCAII mRNA remained unaltered until 48 h after the stress what suggests that TaeMCAII participates in later stages of PCD triggered by heat-shock. Phylogenetic analysis enabled to classify TaeMCAII as a type II metacaspase. Finally, homology modelling of the putative three-dimensional structure of the TaeMCAII protein and a topology analysis of its probable active site were performed.     

Characterization of inner membrane protein YciB in Escherichia coli: YciB interacts with cell elongation and division proteins

The function of inner membrane protein YciB in Escherichia coli has not been identified. In this study, the membrane topology of the protein that contains five transmembrane domains was clarified. YciB was found to interact with various proteins involved in cell elongation and cell division using a bacterial two‐hybrid system. It was also found that the deletion mutant of yciB is susceptible to the low osmolarity. These observations together with previous reports indicate that YciB is involved in synthesis of the cell envelope by interacting with cell elongation and cell division complexes.     

Structures of domains I and IV from YbbR are representative of a widely distributed protein family

YbbR domains are widespread throughout Eubacteria and are expressed as monomeric units, linked in tandem repeats or cotranslated with other domains. Although the precise role of these domains remains undefined, the location of the multiple YbbR domain‐encoding ybbR gene in the Bacillus subtilis glmM operon and its previous identification as a substrate for a surfactin‐type phosphopantetheinyl transferase suggests a role in cell growth, division, and virulence. To further characterize the YbbR domains, structures of two of the four domains (I and IV) from the YbbR‐like protein of Desulfitobacterium hafniense Y51 were solved by solution nuclear magnetic resonance and X‐ray crystallography. The structures show the domains to have nearly identical topologies despite a low amino acid identity (23%). The topology is dominated by β‐strands, roughly following a “figure 8” pattern with some strands coiling around the domain perimeter and others crossing the center. A similar topology is found in the C‐terminal domain of two stress‐responsive bacterial ribosomal proteins, TL5 and L25. Based on these models, a structurally guided amino acid alignment identifies features of the YbbR domains that are not evident from naïve amino acid sequence alignments. A structurally conserved cis‐proline (cis‐Pro) residue was identified in both domains, though the local structure in the immediate vicinities surrounding this residue differed between the two models. The conservation and location of this cis‐Pro, plus anchoring Val residues, suggest this motif may be significant to protein function.     

Effects of ribosome-inactivating proteins on Escherichia coli and Agrobacterium tumefaciens translation systems.

The effects of 30 type 1 and of 2 (ricin and volkensin) type 2 ribosome-inactivating proteins (RIPs) on Escherichia coli and Agrobacterium tumefaciens cell-free translation systems were compared with the effects on a rabbit reticulocyte translation system. The depurinating activity of RIPs on E. coli ribosomes was also evaluated. Only six type 1 RIPs inhibited endogenous mRNA-directed translational activity of E. coli lysates, with submicromolar 50% inhibitory concentrations. Four RIPs had similar activities on poly(U)-directed phenylalanine polymerization by E. coli ribosomes, and three RIPs inhibited poly(U)-directed polyphenylalanine synthesis by A. tumefaciens ribosomes, with submicromolar 50% inhibitory concentrations.     

The ER luminal C‐terminus of AtSec62 is critical for male fertility and plant growth in Arabidopsis thaliana

Protein translocation into the endoplasmic reticulum (ER) occurs either co‐ or post‐translationally through the Sec translocation system. The Arabidopsis Sec post‐translocon is composed of the protein‐conducting Sec61 complex, the chaperone‐docking protein AtTPR7, the J‐domain‐containing proteins AtERdj2A/B and the yet uncharacterized AtSec62. Yeast Sec62p is suggested to mainly function in post‐translational translocation, whereas mammalian Sec62 also interacts with ribosomes. In Arabidopsis, loss of AtSec62 leads to impaired growth and drastically reduced male fertility indicating the importance of AtSec62 in protein translocation and subsequent secretion in male gametophyte development. Moreover, AtSec62 seems to be divergent in function as compared with yeast Sec62p, since we were not able to complement the thermosensitive yeast mutant sec62‐ts. Interestingly, AtSec62 has an additional third transmembrane domain in contrast to its yeast and mammalian counterparts resulting in an altered topology with the C‐terminus facing the ER lumen instead of the cytosol. In addition, the AtSec62 C‐terminus has proven to be indispensable for AtSec62 function, since a construct lacking the C‐terminal region was not able to rescue the mutant phenotype in Arabidopsis. We thus propose that Sec62 acquired a unique topology and function in protein translocation into the ER in plants.     

Phosphoproteomic identification and phylogenetic analysis of ribosomal P-proteins in Populus dormant terminal buds

To better understand the role that reversible phosphorylation plays in woody plant ribosomal P-protein function, we initiated a phosphoproteomic investigation of P-proteins from Populus dormant terminal buds. Using gel-free (in-solution) protein digestion and phosphopeptide enrichment combined with a nanoUPLC–ESI–MS/MS strategy, we identified six phosphorylation sites on eight P-proteins from Populus dormant terminal buds. Among these, six Ser sites and one Thr site were identified in the highly conserved C-terminal region of eight P-proteins of various P-protein subfamilies, including two P0, two P1, three P2 and one P3 protein. Among these, the Thr site was shown to be novel and has not been identified in any other organisms. Sequence analysis indicated that the phosphothreonine sites identified in the C-terminus of Ptr RPP2A exclusively occurred in woody species of Populus, etc. The identified phosphopeptides shared a common phosphorylation motif of (S/T)XX(D/E) and may be phosphorylated in vivo by casein kinase 2 as suggested by using Scansite analysis. Furthermore, phylogenetic analysis suggested that divergence of P2 also occurred in Populus, including type I and type II. To the best of our knowledge, this is the first systematic phosphoproteomic and phylogenetic analysis of P-proteins in woody plants, the results of which will provide a wealth of resources for future understanding and unraveling of the regulatory mechanisms of Populus P-protein phosphorylation during the maintenance of dormancy.     

Glucose kinase of Streptomyces coelicolor A3(2): large-scale purification and biochemical analysis

Glucose kinase of Streptomyces coelicolor A3(2) is essential for glucose utilisation and is required for carbon catabolite repression (CCR) exerted through glucose and other carbon sources. The protein belongs to the ROK-family, which comprises bacterial sugar kinases and regulators. To better understand glucose kinase function, we have monitored the cellular activity and demonstrated that the choice of carbon sources did not significantly change the synthesis and activity of the enzyme. The DNA sequence of the Streptomyces lividans glucose kinase gene glkA was determined. The predicted gene product of 317 amino acids was found to be identical to S. coelicolor glucose kinase, suggesting a similar role for this protein in both organisms. A procedure was developed to produce pure histidine-tagged glucose kinase with a yield of approximately 10 mg/l culture. The protein was stable for several weeks and was used to raise polyclonal antibodies. Purified glucose kinase was used to explore protein-protein interaction by surface plasmon resonance. The experiments revealed the existence of a binding activity present in S. coelicolor cell extracts. This indicated that glucose kinase may interact with (an)other factor(s), most likely of protein nature. A possible cross-talk with proteins of the phosphotransferase system, which are involved in carbon catabolite repression in other bacteria, was investigated.     

A novel KCNQ4 pore-region mutation (p.G296S) causes deafness by impairing cell-surface channel expression

Mutations in the potassium channel gene KCNQ4 underlie DFNA2, a subtype of autosomal dominant progressive, high-frequency hearing loss. Based on a phenotype-guided mutational screening we have identified a novel mutation c.886G>A, leading to the p.G296S substitution in the pore region of KCNQ4 channel. The possible impact of this mutation on total KCNQ4 protein expression, relative surface expression and channel function was investigated. When the G296S mutant was expressed in Xenopus oocytes, electrophysiological recordings did not show voltage-activated K⁺ currents. The p.G296S mutation impaired KCNQ4 channel activity in two manners. It greatly reduced surface expression and, secondarily, abolished channel function. The deficient expression at the cell surface membrane was further confirmed in non-permeabilized NIH-3T3 cells transfected with the mutant KCNQ4 tagged with the hemagglutinin epitope in the extracellular S1–S2 linker. Co-expression of mutant and wild type KCNQ4 in oocytes was performed to mimic the heterozygous condition of the p.G296S mutation in the patients. The results showed that the G296S mutant exerts a strong dominant-negative effect on potassium currents by reducing the wild type KCNQ4 channel expression at the cell surface. This is the first study to identify a trafficking-dependent dominant mechanism for the loss of KCNQ4 channel function in DFNA2.     

The phosphatase domains of LAR, CD45, and PTP1B: structural correlations with peptide-based inhibitors.

PTP1B is a cytosolic protein tyrosine phosphatase that is a regulator of the kinase activity of the insulin receptor; the two protein tyrosine phosphatases LAR and CD45 are receptor type phosphatases crucially important to cell function. LAR also is involved in regulation of the insulin receptor while CD45 is critical for T-cell activation. Although LAR and CD45 are both transmembrane phosphatases, these enzymes manifest their phosphatase activity through a catalytic cytosolic domain. We have utilized X-ray coordinates of related phosphatases (RPTPalpha and RPTmu) and comparative protein modeling to obtain molecular models of the D1 catalytic domains of CD45 and LAR. The models were tested using established protocols and found to be comparable to low resolution X-ray structures. The structure obtained for LAR was compared with the recently reported X-ray structure. Both the CD45-D1 and LAR-D1 structures were then compared to and contrasted with PTP1B. The active site of pockets of the three enzymes were found to be very uniform in structure and charge distribution. Also, the gross surface topology around the active site was found to be somewhat similar for the 3 phosphatases. However, there were significant differences in surface topology, and, more importantly, large changes in surface charge distribution. The differences between the surface features of these enzymes provide an explanation for the selectivity of inhibition by a number of peptides.     

Transmembrane topology of the NuoL,M and N subunits of NADH:quinone oxidoreductase and their homologues among membrane-bound hydrogenases and bona fide antiporters

Nicotinamide adenine dinucleotide-reduced form (NADH):quinone oxidoreductase (respiratory Complex I), F420H2 oxidoreductase and complex, membrane-bound NiFe-hydrogenase contain protein subunits homologous to a certain type of bona fide antiporters. In Complex I, these polypeptides (NuoL/ND5, NuoM/ND4, NuoN/ND2) are most likely core components of the proton pumping mechanism, and it is thus important to learn more about their structure and function. In this work, we have determined the transmembrane topology of one such polypeptide, and built a 2D structural model of the protein valid for all the homologous polypeptides. The experimentally determined transmembrane topology was different from that predicted by majority vote hydrophobicity analyses of members of the superfamily. A detailed phylogenetic analysis of a large set of primary sequences shed light on the functional relatedness of these polypeptides.