Always on the bright side: the climbing mechanism of Galium aparine

Galium aparine is a herbaceous climbing plant that attaches to host plants mainly via its leaves, which are covered by hooked trichomes. Although such hooks are found on both leaf surfaces, the leaves of G. aparine are mainly positioned upon the leaves of supporting plants and rarely beneath. In order to understand the mechanism underlying this observation, we have studied structural and mechanical properties of single leaf hooks, frictional properties of leaf surfaces, turgor pressure in different leaf tissues and bending properties of the leaves in different directions. Abaxial and adaxial leaf hooks differ significantly in orientation, distribution, structure and mechanical properties. In accordance with these differences, friction properties of leaves depend on the direction of the applied force and differ significantly between both leaf surfaces. This results in a ratchet mechanism. Abaxial leaf hooks provide strong attachment upon the leaves of adjacent plants, whereas adaxial hooks cause a gliding-off from the underside of the leaves of host plants. Thus, the leaves of G. aparine can function as attachment organs, and simultaneously orient themselves advantageously for their photosynthetic function. Further adaptations in turgor pressure or concerning an anisotropy of the flexural stiffness of the leaves have not been found.     

Role for a conserved structural motif in assembly of a class I aminoacyl-tRNA synthetase active site

The catalytic domains of class I aminoacyl-tRNA synthetases are built around a conserved Rossmann nucleotide binding fold, with additional polypeptide domains responsible for tRNA binding or hydrolytic editing of misacylated substrates. Structural comparisons identified a conserved motif bridging the catalytic and anticodon binding domains of class Ia and Ib enzymes. This stem contact fold (SCF) has been proposed to globally orient each enzyme's cognate tRNA by interacting with the inner corner of the L-shaped tRNA. Despite the structural similarity of the SCF among class Ia/Ib enzymes, the sequence conservation is low. We replaced amino acids of the MetRS SCF with portions of the structurally similar glutaminyl-tRNA synthetase (GlnRS) motif or with alanine residues. Chimeric variants retained significant tRNA methionylation activity, indicating that structural integrity of the helix-turn-strand-helix motif contributes more to tRNA aminoacylation than does amino acid identity. In contrast, chimeras were significantly reduced in methionyl adenylate synthesis, suggesting a role for the SCF in formation of a structured active site domain. A highly conserved aspartic acid within the MetRS SCF is proposed to make an electrostatic interaction with an active site lysine; these residues were replaced with alanines or conservative substitutions. Both methionyl adenylate formation and methionine transfer were impaired, and activity was not significantly recovered by making the compensatory double substitution.     

M42 aminopeptidase catalytic site: the structural and functional role of a strictly conserved aspartate residue

The M42 aminopeptidases are a family of dinuclear aminopeptidases widely distributed in Prokaryotes. They are potentially associated to the proteasome, achieving complete peptide destruction. Their most peculiar characteristic is their quaternary structure, a tetrahedron-shaped particle made of twelve subunits. The catalytic site of M42 aminopeptidases is defined by seven conserved residues. Five of them are involved in metal ion binding which is important to maintain both the activity and the oligomeric state. The sixth conserved residue, a glutamate, is the catalytic base deprotonating the water molecule during peptide bond hydrolysis. The seventh residue is an aspartate whose function remains poorly understood. This aspartate residue, however, must have a critical role as it is strictly conserved in all MH clan enzymes. It forms some kind of catalytic triad with the histidine residue and the metal ion of the M2 binding site. We assess its role in TmPep1050, an M42 aminopeptidase of Thermotoga maritima, through a mutational approach. Asp-62 was substituted with alanine, asparagine, or glutamate residue. The Asp-62 substitutions completely abolished TmPep1050 activity and impeded dodecamer formation. They also interfered with metal ion binding as only one cobalt ion is bound per subunit instead of two. The structure of Asp62Ala variant was solved at 1.5 Å showing how the substitution has an impact on the active site fold. We propose a structural role for Asp-62, helping to stabilize a crucial loop in the active site and to position correctly the catalytic base and a metal ion ligand of the M1 site.     

Formation of a type I collagen RNA dimer by intermolecular base-pairing of a conserved sequence around the translation initiation site.

A symmetrical sequence around the translation initiation site of several collagen genes is highly conserved. Deletions in this sequence increase translational efficiency of an alpha 2(I) collagen - CAT chimeric gene after DNA transfection of NIH 3T3 fibroblasts (Schmidt, Rossi and de Crombrugghe, submitted). The secondary structure, predicted by the sequence of this segment, was shown to exist in solution in 200 mM NaCl at 37 degrees C. Cell-free translation of the corresponding RNA using a reticulocyte lysate is inhibited 2 to 4-fold by preincubation with a 0.5 M NaCl extract of an NIH 3T3 ribosomal eluate. Cell-free translation of two mutant RNAs, with partial deletions of the conserved sequence, is not inhibited by such preincubation. This inhibition is not due to degradation of the RNA and requires a protein component of the ribosomal eluate, which, however, is not required after the preincubation step. Preincubation of the RNA with the ribosomal eluate from NIH 3T3 fibroblasts causes the reversible formation of an intermolecular dimer in which the conserved symmetrical sequences hybridize to each other. This results in an increase in the degree of secondary structure of the conserved segment around the translation initiation site. We speculate that translational efficiency could be modulated by influencing the equilibrium between monomer and dimer.     

Symmetry at the active site of the ribosome: structural and functional implications

The sizable symmetrical region, comprising 180 ribosomal RNA nucleotides, which has been identified in and around the peptidyl transferase center (PTC) in crystal structures of eubacterial and archaeal large ribosomal subunits, indicates its universality, confirms that the ribosome is a ribozyme and evokes the suggestion that the PTC evolved by gene fusion. The symmetrical region can act as a center that coordinates amino acid polymerization by transferring intra-ribosomal signals between remote functional locations, as it connects, directly or through its extensions, the PTC, the three tRNA sites, the tunnel entrance, and the regions hosting elongation factors. Significant deviations from the overall symmetry stabilize the entire region and can be correlated with the shaping and guiding of the motion of the tRNA 3'-end from the A- into the P-site. The linkage between the elaborate PTC architecture and the spatial arrangements of the tRNA 3'-ends revealed the rotatory mechanism that integrates peptide bond formation, translocation within the PTC and nascent protein entrance into the exit tunnel. The positional catalysis exerted by the ribosome places the reactants in stereochemistry close to the intermediate state and facilitates the catalytic contribution of the P-site tRNA 2'-hydroxyl.     

Dissection of the structural and functional role of a conserved hydration site in RNase T1

The reoccurrence of water molecules in crystal structures of RNase T1 was investigated. Five waters were found to be invariant in RNase T1 as well as in six other related fungal RNases. The structural, dynamical, and functional characteristics of one of these conserved hydration sites (WAT1) were analyzed by protein engineering, X-ray crystallography, and (17)O and 2H nuclear magnetic relaxation dispersion (NMRD). The position of WAT1 and its surrounding hydrogen bond network are unaffected by deletions of two neighboring side chains. In the mutant Thr93Gln, the Gln93N epsilon2 nitrogen replaces WAT1 and participates in a similar hydrogen bond network involving Cys6, Asn9, Asp76, and Thr91. The ability of WAT1 to form four hydrogen bonds may explain why evolution has preserved a water molecule, rather than a side-chain atom, at the center of this intricate hydrogen bond network. Comparison of the (17)O NMRD profiles from wild-type and Thr93Gln RNase T1 yield a mean residence time of 7 ns at 27 degrees C and an orientational order parameter of 0.45. The effects of mutations around WAT1 on the kinetic parameters of RNase T1 are small but significant and probably relate to the dynamics of the active site.     

Matrix metalloproteinase‐3 in the central nervous system: a look on the bright side

Matrix metalloproteinases (MMPs) are a large family of proteases involved in many cell‐matrix and cell‐cell signalling processes through activation, inactivation or release of extracellular matrix (ECM) and non‐ECM molecules, such as growth factors and receptors. Uncontrolled MMP activities underlie the pathophysiology of many disorders. Also matrix metalloproteinase‐3 (MMP‐3) or stromelysin‐1 contributes to several pathologies, such as cancer, asthma and rheumatoid arthritis, and has also been associated with neurodegenerative diseases like Alzheimer's disease, Parkinson's disease and multiple sclerosis. However, based on defined MMP spatiotemporal expression patterns, the identification of novel candidate molecular targets and in vitro and in vivo studies, a beneficial role for MMPs in CNS physiology and recovery is emerging. The main purpose of this review is to shed light on the recently identified roles of MMP‐3 in normal brain development and in plasticity and regeneration after CNS injury and disease. As such, MMP‐3 is correlated with neuronal migration and neurite outgrowth and guidance in the developing CNS and contributes to synaptic plasticity and learning in the adult CNS. Moreover, a strict spatiotemporal MMP‐3 up‐regulation in the injured or diseased CNS might support remyelination and neuroprotection, as well as genesis and migration of stem cells in the damaged brain.     

Catalytic and structural role of a conserved active site histidine in berberine bridge enzyme

Berberine bridge enzyme (BBE) is a paradigm for the class of bicovalently flavinylated oxidases, which catalyzes the oxidative cyclization of (S)-reticuline to (S)-scoulerine. His174 was identified as an important active site residue because of its role in the stabilization of the reduced state of the flavin cofactor. It is also strictly conserved in the family of BBE-like oxidases. Here, we present a detailed biochemical and structural characterization of a His174Ala variant supporting its importance during catalysis and for the structural organization of the active site. Substantial changes in all kinetic parameters and a decrease in midpoint potential were observed for the BBE His174Ala variant protein. Moreover, the crystal structure of the BBE His174Ala variant showed significant structural rearrangements compared to wild-type enzyme. On the basis of our findings, we propose that His174 is part of a hydrogen bonding network that stabilizes the negative charge at the N1-C2═O locus via interaction with the hydroxyl group at C2' of the ribityl side chain of the flavin cofactor. Hence, replacement of this residue with alanine reduces the stabilizing effect for the transiently formed negative charge and results in drastically decreased kinetic parameters as well as a lower midpoint redox potential.     

A structural analysis of the group II intron active site and implications for the spliceosome

Group II introns are self-splicing, mobile genetic elements that have fundamentally influenced the organization of terrestrial genomes. These large ribozymes remain important for gene expression in almost all forms of bacteria and eukaryotes and they are believed to share a common ancestry with the eukaryotic spliceosome that is required for processing all nuclear pre-mRNAs. The three-dimensional structure of a group IIC intron was recently determined by X-ray crystallography, making it possible to visualize the active site and the elaborate network of tertiary interactions that stabilize the molecule. Here we describe the molecular features of the active site in detail and evaluate their correspondence with prior biochemical, genetic, and phylogenetic analyses on group II introns. In addition, we evaluate the structural significance of RNA motifs within the intron core, such as the major-groove triple helix and the domain 5 bulge. Having combined what is known about the group II intron core, we then compare it with known structural features of U6 snRNA in the eukaryotic spliceosome. This analysis leads to a set of predictions for the molecular structure of the spliceosomal active site.     

MutaProt: a web interface for structural analysis of point mutations

A web-based tool, termed 'MutaProt', is described which analyses pairs of PDB files whose members differ in one, or two, amino acids. MutaProt examines the micro environment surrounding the exchanged residue(s) and can be searched by specifying a PDB ID, keywords, or any pair of amino acids. Detailed information about accessibility of the exchanged residue(s) and its atomic contacts are provided based on CSU software (Sobolev et al., Bioinformatics, 15, 327-332, 1999). An interactive 3D presentation of the superimposed regions around the mutation(s) is included. MutaProt is updated weekly.     

Preferential partitioning of melittin into the air/water interface: structural and thermodynamic implications.

The membrane active agent melittin has been investigated with regard to the formation of a Langmuir monolayer and the accordingly induced surface activities. We show that in spite of its considerable solubility in an aqueous medium, this peptide nevertheless largely accumulates in the air/water interface unless the lateral pressure is raised beyond a certain threshold value depending on the pH in the subphase. The true surface concentrations have been determined by means of a recently developed novel method based on thermodynamic principles. It affords an access to the partitioning equilibrium between the surface and subphase domains, provided the latter surrounding is not excessively preferred. In the present case this approach was used to derive quantitative information on the pertinent interfacial structure and thermodynamics. In particular, the apparent molecular area and the Gibbs energy of mutual interaction in the monolayer could be evaluated as a function of the applied surface pressure. The data suggest the existence of two structural conversions in the course of an increasing lateral compression. The surface-associated peptide accordingly assumes three different states of successively reduced area requirements, supposedly owing to an orientational transition involving a straightening up of a helical conformation. This conclusion is corroborated by surface potential measurements reflecting corresponding changes of the effective dipole moment perpendicular to the surface.     

Multivariate data analysis in empirical research. A look on the bright side

The interpretive benefits of employing multivariate analysis methods on experimental data with more than one dependent variable are described heuristically and illustrated on a set of data from a simply designed experiment in physiological psychology. Multivariate analysis of variance (MANOVA) is performed on the 9 dependent variables contained in the sample data and on the four composites derived from a principal components analysis (PCA) of the variability of the nine. A linear discriminant analysis (LDA) is conducted following both MANOVA results, and 5 methods of determining the "important" dependent variables in the experimental-control group difference are presented and discussed in terms of the data at hand.     

Models of the cooperative mechanism for Rho effector recognition: implications for RhoA-mediated effector activation

Activated GTPases of the Rho family regulate a spectrum of functionally diverse downstream effectors, initiating a network of signal transduction pathways by interaction and activation of effector proteins. Although effectors are defined as proteins that selectively bind the GTP-bound state of the small GTPases, there have been also several indications for a nucleotide-independent binding mode. By characterizing the molecular mechanism of RhoA interaction with its effectors, we have determined the equilibrium dissociation constants of several Rho-binding domains of three different effector proteins (Rhotekin, ROCKI/ROK beta/p160ROCK, PRK1/PKNalpha where ROK is RhoA-binding kinase) for both RhoA.GDP and RhoA.GTP using fluorescence spectroscopy. In addition, we have identified two novel Rho-interacting domains in ROCKI, which bind RhoA with high affinity but not Cdc42 or Rac1. Our results, together with recent structural data, support the notion of multiple effector-binding sites in RhoA and strongly indicate a cooperative binding mechanism for PRK1 and ROCKI that may be the molecular basis of Rho-mediated effector activation.     

Microtubule-kinesin interface mutants reveal a site critical for communication

Strict coordination of the two motor domains of kinesin is required for driving the processive movement of organelles along microtubules. Glutamate 164 of the kinesin heavy chain was shown to be critical for kinesin function through in vivo genetics in Drosophila melanogaster. The mutant motor E164K exhibited reduced steady-state ATPase activity and higher affinity for both ATP and microtubules. Moreover, an alanine substitution at this position (E164A) caused similar defects. It became stalled on the microtubule and was unable to bind and hydrolyze ATP at the second motor domain. Glu(164), which has been conserved through evolution, is located at the motor-microtubule interface close to key residues on helix alpha12 of beta-tubulin. We explored further the contributions of Glu(164) to motor function using several site-directed mutant proteins: E164K, E164N, E164D, E164Q, and D165A. The results indicate that the microtubule-E164K complex can only bind and hydrolyze one ATP. ATP with increased salt was able to dissociate a population of E164K motors from the microtubule but could not dissociate E164A. We tested the basis of the stabilized microtubule interaction with E164K, E164N, and E164A. The results provide new insights about the motor-microtubule interface and the pathway of communication for processive motility.     

PIER: protein interface recognition for structural proteomics

Recent advances in structural proteomics call for development of fast and reliable automatic methods for prediction of functional surfaces of proteins with known three-dimensional structure, including binding sites for known and unknown protein partners as well as oligomerization interfaces. Despite significant progress the problem is still far from being solved. Most existing methods rely, at least partially, on evolutionary information from multiple sequence alignments projected on protein surface. The common drawback of such methods is their limited applicability to the proteins with a sparse set of sequential homologs, as well as inability to detect interfaces in evolutionary variable regions. In this study, the authors developed an improved method for predicting interfaces from a single protein structure, which is based on local statistical properties of the protein surface derived at the level of atomic groups. The proposed Protein IntErface Recognition (PIER) method achieved the overall precision of 60% at the recall threshold of 50% at the residue level on a diverse benchmark of 490 homodimeric, 62 heterodimeric, and 196 transient interfaces (compared with 25% precision at 50% recall expected from random residue function assignment). For 70% of proteins in the benchmark, the binding patch residues were successfully detected with precision exceeding 50% at 50% recall. The calculation only took seconds for an average 300-residue protein. The authors demonstrated that adding the evolutionary conservation signal only marginally influenced the overall prediction performance on the benchmark; moreover, for certain classes of proteins, using this signal actually resulted in a deteriorated prediction. Thorough benchmarking using other datasets from literature showed that PIER yielded improved performance as compared with several alignment-free or alignment-dependent predictions. The accuracy, efficiency, and dependence on structure alone make PIER a suitable tool for automated high-throughput annotation of protein structures emerging from structural proteomics projects.