Marta Jiménez

Solution structure of gamma 1-H and gamma 1-P thionins from barley and wheat endosperm determined by 1H-NMR: a structural motif common to toxic arthropod proteins.

The complete assignment of the proton NMR spectra of the homologous gamma 1-hordothionin and gamma 1-purothionin (47 amino acids, 4 disulfide bridges) from barley and wheat, respectively, has been performed by two-dimensional sequence-specific methods. A total of 299 proton-proton distance constraints for gamma 1-H and 285 for gamma 1-P derived from NOESY spectra have been used to calculate the three-dimensional solution structures. Initial structures have been generated by distance geometry methods and further refined by dynamical simulated annealing calculations. Both proteins show identical secondary and tertiary structure with a well-defined triple-stranded antiparallel beta-sheet (residues 1-6, 31-34, and 39-47), an alpha-helix (residues 16-28), and the corresponding connecting loops. Three disulfide bridges are located in the hydrophobic core holding together the alpha-helix and the beta-sheet and forming a cysteine-stabilized alpha-helical (CSH) motif. Moreover, a clustering of positive charges is observed on the face of the beta-sheet opposite to the helix. The three-dimensional structures of the gamma-thionins differ remarkably from plant alpha- and beta-thionins and crambin. However, they show a higher structural analogy with scorpion toxins and insect defensins which also present the CSH motif.

1H NMR and CD evidence of the folding of the isolated ribonuclease 50-61 fragment

In our search for potential folding intermediates we have prepared and characterized the fragment of RNase A corresponding to residues 50–61. Proton chemical shift variations with temperature, addition of stabilizing (TFE) or denaturing agents (urea) provide a strong experimental basis for concluding that in aqueous solution this RNase fragment forms an α‐helix structure similar to that in the intact RNase A crystal. This conclusion lends strong support to the idea that elements of secondary structure (mainly α‐helices) can be formed in the absence of tertiary interactions and act as nucleation centers in the protein folding process.

Structural Basis of the Regulatory Mechanism of the Plant Cipk Family of Protein Kinases Controlling Ion Homeostasis and Abiotic Stress

Significance The transport of ions through the plant cell membrane establishes the key physicochemical parameters for cell function. Stress situations such as those created by soil salinity or low potassium conditions alter the ion transport across the membrane producing dramatic changes in the cell turgor, the membrane potential, and the intracellular pH and concentrations of toxic cations such as sodium and lithium. As a consequence, fundamental metabolic routes are inhibited. The CIPK family of 26 protein kinases regulates the function of several ion transporters at the cell membrane to restore ion homeostasis under stress situations. Our analyses provide an explanation on how the CIPKs are differentially activated to coordinate the adequate cell response to a particular stress. Plant cells have developed specific protective molecular machinery against environmental stresses. The family of CBL-interacting protein kinases (CIPK) and their interacting activators, the calcium sensors calcineurin B-like (CBLs), work together to decode calcium signals elicited by stress situations. The molecular basis of biological activation of CIPKs relies on the calcium-dependent interaction of a self-inhibitory NAF motif with a particular CBL, the phosphorylation of the activation loop by upstream kinases, and the subsequent phosphorylation of the CBL by the CIPK. We present the crystal structures of the NAF-truncated and pseudophosphorylated kinase domains of CIPK23 and CIPK24/SOS2. In addition, we provide biochemical data showing that although CIPK23 is intrinsically inactive and requires an external stimulation, CIPK24/SOS2 displays basal activity. This data correlates well with the observed conformation of the respective activation loops: Although the loop of CIPK23 is folded into a well-ordered structure that blocks the active site access to substrates, the loop of CIPK24/SOS2 protrudes out of the active site and allows catalysis. These structures together with biochemical and biophysical data show that CIPK kinase activity necessarily requires the coordinated releases of the activation loop from the active site and of the NAF motif from the nucleotide-binding site. Taken all together, we postulate the basis for a conserved calcium-dependent NAF-mediated regulation of CIPKs and a variable regulation by upstream kinases.

Modulating glycosidase degradation and lectin recognition of gold glyconanoparticles

Glyconanoparticles (GNPs) are water-soluble carbohydrate-functionalized gold nanoclusters with a promising potential to serve as versatile tools in studies ranging from basic chemical glycobiology to clinical applications. In this paper we evaluate the influence of ligand density and presentation on the recognition by protein receptors by examining the interaction of lactose-functionalized GNPs with two different galactose-specific carbohydrate-binding proteins: an enzyme, Escherichia coli beta-galactosidase, and a lectin, Viscum album agglutinin. The results suggest that the proper selection of ligand densities and spacers in GNP functionalization is an important requisite to match the topological requirements of the target receptor while escaping glycosidase degradation.

BINDING OF MANNOSE-6-PHOSPHATE AND HEPARIN BY BOAR SEMINAL PLASMA PSP-II, A MEMBER OF THE SPERMADHESIN PROTEIN FAMILY

PSP‐I/PSP‐II, a heterodimer of glycosylated spermadhesins, is the major component of boar seminal plasma. Similarly to other spermadhesins, the PSP‐II subunit is a lectin which displays heparin‐ and zona pellucida glycoprotein‐binding activities. We have investigated the ligand binding capabilities of the heterodimer and the isolated subunits using several polysaccharides, glycoproteins, and phospholipids. PSP‐II binds the sulfated polysaccharides heparin and fucoidan in a dose‐dependent and seemingly‐specific manner. In addition, PSP‐II binds oligosaccharides containing exposed mannose‐6‐phosphate monoester groups and the binding is selectively inhibited by mannose‐6‐phosphate and glucose‐6‐phosphate. Inhibition experiments indicate that binding of PSP‐II to sulfated polysaccharides and mannose‐6‐phosphate‐containing oligosaccharides involves distinct but possibly overlapping binding sites. Heterodimer formation with PSP‐I abolishes both the heparin and the mannose‐6‐phosphate binding capabilities, suggesting that the corresponding sites may be located at the dimer interface. Using the crystal structure of PSP‐I/PSP‐II heterodimer as a template, we have explored possible binding sites which satisfy the observed binding characteristics. In the proposed models, PSP‐II Arg43 appears to play a pivotal role in both heparin‐ and mannose‐6‐phosphate‐complexation as well as in heterodimer formation.

NMR investigations of protein–carbohydrate interactions: insights into the topology of the bound conformation of a lactose isomer and β-galactosyl xyloses to mistletoe lectin and galectin-1

A hallmark of oligosaccharides is their often limited spatial flexibility, allowing them to access a distinct set of conformers in solution. Viewing each individual or even the complete ensemble of conformations as potential binding partner(s) for lectins in protein-carbohydrate interactions, it is pertinent to address the question on the characteristics of bound state conformation(s) in solution. Also, it is possible that entering the lectins binding site distorts the low-energy topology of a glycosidic linkage. As a step to delineate the strategy of ligand selection for galactosides, a common physiological docking point, we have performed a NMR study on two non-homologous lectins showing identical monosaccharide specificity. Thus, the conformation of lactose analogues bound to bovine heart galectin-1 and to mistletoe lectin in solution has been determined by transferred nuclear Overhauser effect measurements. It is demonstrated that the lectins select the syn conformation of lactose and various structural analogues (Galbeta(1-->4)Xyl, Galbeta(1-->3)Xyl, Galbeta(1-->2)Xyl, and Galbeta(1-->3)Glc) from the ensemble of presented conformations. No evidence for conformational distortion was obtained. Docking of the analogues to the modeled binding sites furnishes explanations, in structural terms, for exclusive recognition of the syn conformer despite the non-homologous design of the binding sites.

Domain versatility in plant AB-toxins: Evidence for a local, pH-dependent rearrangement in the 2γ lectin site of the mistletoe lectin by applying ligand derivatives and modelling

Mistletoe lectin is a potent biohazard. Lectin activity in the toxic dimer primarily originates from the 2γ‐subdomain (Tyr‐site) of the B‐subunit. Crystallographic information on lectin–sugar complexes is available only at acidic pH, where lectin activity is low. Thus, we mapped ligand‐binding properties including comparison to ricins Tyr‐site at neutral pH. Using these results and molecular dynamics simulations, a local conformational change was rendered likely. The obtained structural information is valuable for the design of potent inhibitors.

Nuclear Overhauser effects in aqueous solution as dynamic probes in short linear peptides.

The possibility of obtaining interresidue NOEs from short linear peptides in aqueous solution has been investigated from an experimental point of view using peptides of various lengths (namely GGRA, LHRH and RNase S‐peptide). It is shown that, provided that long (8̃00 ms) NOESY mixing times are used, complete sets of sequential αN NOEs are obtainable. From the intensities and signs of the observed NOEs, the relative mobilities of different parts of the polypeptide chain can be determined.

Structural Basis for Membrane Anchorage of Viral ϕ29 DNA during Replication

Prokaryotic DNA replication is compartmentalized at the cellular membrane. Functional and biochemical studies showed that the Bacillus subtilis phage ϕ29-encoded membrane protein p16.7 is directly involved in the organization of membrane-associated viral DNA replication. The structure of the functional domain of p16.7 in complex with DNA, presented here, reveals the multimerization mode of the protein and provides insights in the organization of the phage genome at the membrane of the infected cell.