Biomedical subjects

Miriam Eisenstein

Publications and source records attributed to Miriam Eisenstein.

12 recordsLinked to original sources

PubMed2003

Weighted geometric docking: incorporating external information in the rotation-translation scan.

Weighted geometric docking is a prediction algorithm that matches weighted molecular surfaces. Each molecule is represented by a grid of complex numbers, storing information about the shape of the molecule in the real part and weight information in the imaginary part. The weights are based on experimental biochemical and biophysical data or on theoretical analyses of amino acid conservation or correlation patterns in multiple-sequence alignments of homologous proteins. Only a few surface residues on either one or both molecules are weighted. In contrast to methods that use postscan filtering based on biochemical information, our method incorporates the external data in the rotation-translation search, producing a different set of docking solutions biased toward solutions in which the up-weighted residues are at the interface. Similarly, interactions involving specified residues can be impeded. The weighted geometric algorithm was applied to five systems for which regular geometric docking of the unbound molecules gave poor results. We obtained much better ranking of the nearly correct prediction and higher statistical significance when weighted geometric docking was used. The method was successful even when the weighted portion of the surface corresponded only partially and approximately to the binding site.

Algorithms↗

PubMed2003

Prediction of the unknown: inspiring experience with the CAPRI experiment.

We submitted predictions for all seven targets in the CAPRI experiment. For four targets, our submitted models included acceptable, medium accuracy predictions of the structures of the complexes, and for a fifth target we identified the location of the binding site of one of the molecules. We used a weighted-geometric docking algorithm in which contacts involving specified parts of the surfaces of either one or both molecules were up-weighted or down-weighted. The weights were based on available structural and biochemical data or on sequence analyses. The weighted-geometric docking proved very useful for five targets, improving the complementarity scores and the ranks of the nearly correct solutions, as well as their statistical significance. In addition, the weighted-geometric docking promoted formation of clusters of similar solutions, which include more accurate predictions.

Algorithms↗

PubMed2003

Antibody recognition of chiral surfaces. Enantiomorphous crystals of leucine-leucine-tyrosine.

Monoclonal antibodies were selected after immunization with crystals of the tripeptide l-leucine-l-leucine-l-tyrosine. They interact with the tripeptide crystals, but do not interact with the tripeptide molecule, with other crystalline surfaces, or with adsorbed protein. The interactions of two antibodies with crystals of l-Leu-l-Leu-l-Tyr and of its enantiomer d-Leu-d-Leu-d-Tyr were characterized in depth. Antibody 48E is stereoselective and enantioselective: it recognizes only the [011] faces of the l-Leu-l-Leu-l-Tyr crystals, and not the enantiomorphous [011] faces of d-Leu-d-Leu-d-Tyr crystals, or any other faces of either crystal. In contrast, antibody 602E is poorly stereoselective and is not enantioselective: it recognizes the crystals of both enantiomers, interacting with a number of different faces of each. The different recognition patterns are explained on the basis of the nature of the interactions and the structure of the interacting surfaces. Understanding this antibody specificity advances our general understanding of surface recognition and transfer of chiral information across biological interfaces.

Adjuvants, Immunologic↗

PubMed2003

Prediction of the structure of the complex between the 30S ribosomal subunit and colicin E3 via weighted-geometric docking.

Colicin E3 kills Escherichia coli cells by ribonucleolytic cleavage in the 16S rRNA. The cleavage occurs at the ribosomal decoding A-site between nucleotides A1493 and G1494. The breaking of this single phosphodiester bond results in a complete termination of protein biosynthesis leading to cell death. A model structure of the complex of the ribosomal subunit 30S and colicin E3 was constructed by means of a new weighted-geometric docking algorithm, in which interactions involving specified parts of the molecular surface can be up-weighted, allowing incorporation of experimental data in the docking search. Our model, together with available experimental data, predicts the role of the catalytic residues of colicin E3. In addition, it suggests that bound acidic immunity protein inhibits the enzymatic activity of colicin E3 by electrostatic repulsion of the negatively charged substrate.

Algorithms↗

PubMed2003

The conserved Glu-60 residue in Thermoanaerobacter brockii alcohol dehydrogenase is not essential for catalysis.

Glu-60 of the zinc-dependent Thermoanaerobacter brockii alcohol dehydrogenase (TbADH) is a strictly conserved residue in all members of the alcohol dehydrogenase (ADH) family. Unlike most other ADHs, the crystal structures of TbADH and its analogs, ADH from Clostridium beijerinckii (CbADH), exhibit a unique zinc coordination environment in which this conserved residue is directly coordinated to the catalytic zinc ion in the native form of the enzymes. To explore the role of Glu-60 in TbADH catalysis, we have replaced it by alanine (E60A-TbADH) and aspartate (E60D-TbADH). Steady-state kinetic measurements show that the catalytic efficiency of these mutants is only four- and eightfold, respectively, lower than that of wild-type TbADH. We applied X-ray absorption fine-structure (EXAFS) and near-UV circular dichroism to characterize the local environment around the catalytic zinc ion in the variant enzymes in their native, cofactor-bound, and inhibited forms. We show that the catalytic zinc site in the studied complexes of the variant enzymes exhibits minor changes relative to the analogous complexes of wild-type TbADH. These moderate changes in the kinetic parameters and in the zinc ion environment imply that the Glu-60 in TbADH does not remain bound to the catalytic zinc ion during catalysis. Furthermore, our results suggest that a water molecule replaces this residue during substrate turnover.

Absorptiometry, Photon↗

PubMed2002

The DAP-kinase family of proteins: study of a novel group of calcium-regulated death-promoting kinases.

DAP-kinase (DAPk) is a Ca(2+)/calmodulin (CaM)-regulated Ser/Thr kinase that functions as a positive mediator of programmed cell death. It associates with actin microfilament and has a unique multidomain structure. One of the substrates of DAPk was identified as myosin light chain (MLC), the phosphorylation of which mediates membrane blebbing. Four additional kinases have been identified based on the high homology of their catalytic domain to that of DAPk. Yet, they differ in the structure of their extracatalytic domains and in their intracellular localization. One member of this family, DRP-1, also shares with DAPk both the property of activation by Ca(2+)/CaM and a specific phosphorylation-based regulatory mechanism. The latter involves an inhibitory type of autophosphorylation on a conserved serine at position 308, in the CaM regulatory domains of these two kinases. This phosphorylation, which occurs in growing cells, restrains the death-promoting effects of these kinases, and is specifically removed upon exposure of cells to various apoptotic stimuli. The dephosphorylation at this site increases the binding and sensitivity of each of these two kinases to their common activator-CaM. In DAPk, the dephosphorylation of serine 308 also increases the Ca(2+)/CaM-independent substrate phosphorylation. In DPR-1, it also promotes the formation of homodimers necessary for its full activity. These results are consistent with a molecular model in which phosphorylation on serine 308 stabilizes a locked conformation of the CaM regulatory domain within the catalytic cleft and simultaneously also interferes with CaM binding. In DRP-1, it introduces an additional locking device by preventing homodimerization. We propose that this unique mechanism of autoinhibition, evolved to keep these death-promoting kinases silent in healthy cells and ensures their activation only in response to apoptotic signals.

Amino Acid Sequence↗

PubMed2002

The mechanism for acetylcholine receptor inhibition by alpha-neurotoxins and species-specific resistance to alpha-bungarotoxin revealed by NMR.

The structure of a peptide corresponding to residues 182-202 of the acetylcholine receptor alpha1 subunit in complex with alpha-bungarotoxin was solved using NMR spectroscopy. The peptide contains the complete sequence of the major determinant of AChR involved in alpha-bungarotoxin binding. One face of the long beta hairpin formed by the AChR peptide consists of exposed nonconserved residues, which interact extensively with the toxin. Mutations of these receptor residues confer resistance to the toxin. Conserved AChR residues form the opposite face of the beta hairpin, which creates the inner and partially hidden pocket for acetylcholine. An NMR-derived model for the receptor complex with two alpha-bungarotoxin molecules shows that this pocket is occupied by the conserved alpha-neurotoxin residue R36, which forms cation-pi interactions with both alphaW149 and gammaW55/deltaW57 of the receptor and mimics acetylcholine.

Amino Acid Sequence↗

PubMed2002

Interrelation between hydration and interheadgroup interaction in phospholipids.

The stability and the ionic conductivity of biological membranes and of lipid bilayers depend on their hydration. A small number of water molecules adhere strongly to the different residues of the lipid headgroups and are oriented by them. An additional number of water molecules adhere more weakly, preserving their freedom of rotation, but are essential for bestowing the thermodynamic properties of hydrated bilayers and of biological membranes. Around six water molecules are attached so strongly to the headgroups of different phospholipids (PL) that they are rendered unfreezable, or their freezing is extended over such a wide range of temperatures that it cannot be detected by differential scanning calorimetry (DSC). If cholesterol is added to the PL above the concentration at which phase separation of the cholesterol phase occurs, the number of unfreezable water molecules per PL increases, indicating that the PL molecules on the border line between the two phases attach nearly twice as many water molecules as those in the middle of the phase. The orientation of about seven or eight water molecules attached to PL headgroups (seven to phosphatidyl serine (PS)) can be detected by polarized FTIR. The dichroic ratio of the successively adhering water molecules to the headgroup of PS fluctuates between 2.6 and 2.9, with the cumulative value of about 2.8 for the seven water molecules adhering to the headgroup of PS. In addition, in this case, the number of water molecules oriented by PL molecule residues on the border line of the two phases is much larger ( approximately 13 for PS). Interaction between two opposite negatively charged layers containing PS approaching each other may lead, after correlated electrostatic attraction, to change in the conformation of the headgroups with concomitant dehydration. This process is enhanced by Ca(+) and by Li(+), but it may also occur with Na(+) and K(+) as counter-ions if the layers are mutually aligned. This process may be important in the fusion mechanism of biological membranes, and its molecular modeling has been carried out.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed2002

Electrostatics in protein-protein docking.

A novel geometric-electrostatic docking algorithm is presented, which tests and quantifies the electrostatic complementarity of the molecular surfaces together with the shape complementarity. We represent each molecule to be docked as a grid of complex numbers, storing information regarding the shape of the molecule in the real part and information regarding the electrostatic character of the molecule in the imaginary part. The electrostatic descriptors are derived from the electrostatic potential of the molecule. Thus, the electrostatic character of the molecule is represented as patches of positive, neutral, or negative values. The potential for each molecule is calculated only once and stored as potential spheres adequate for exhaustive rotation/translation scans. The geometric-electrostatic docking algorithm is applied to 17 systems, starting form the structures of the unbound molecules. The results-in terms of the complementarity scores of the nearly correct solutions, their ranking in the lists of sorted solutions, and their statistical uniqueness-are compared with those of geometric docking, showing that the inclusion of electrostatic complementarity in docking is very important, in particular in docking of unbound structures. Based on our results, we formulate several "good electrostatic docking rules": The geometric-electrostatic docking procedure is more successful than geometric docking when the potential patches are large and when the potential extends away from the molecular surface and protrudes into the solvent. In contrast, geometric docking is recommended when the electrostatic potential around the molecules to be docked appears homogeneous, that is, with a similar sign all around the molecule.

Algorithms↗

PubMed2002

Modulation of the hydrophobic domain of polymyxin B nonapeptide: effect on outer-membrane permeabilization and lipopolysaccharide neutralization.

Polymyxin B nonapeptide (PMBN), a cationic cyclic peptide derived from the antibacterial peptide polymyxin B, is capable of specifically increasing the permeability of the outer membrane (OM) of Gram-negative bacteria toward hydrophobic antibiotics. In this study, we evaluated the contribution of the hydrophobic segment of PMBN (i.e., D-Phe(5)-Leu(6)) to this activity. Accordingly, we synthesized four analogs of PMBN by replacing D-Phe(5) with either with D-Trp or D-Tyr and Leu(6) with Phe or Ala and evaluated their ability to bind cell-free lipopolysaccharide (LPS) and increase bacterial OM permeability. Compared with PMBN, [D-Tyr(5)]PMBN and [Ala(6)]PMBN possessed reduced LPS affinity (IC(50) = 2.5, 25, and 12 microM, respectively) and significantly reduced OM permeability and LPS neutralization activity. [Phe(6)]PMBN exhibited rather similar affinity to cell-free LPS (IC(50) = 5 microM) and the same OM permeability capacity as PMBN. However, [D-Trp(5)]PMBN, despite its similar affinity to cell-free LPS (IC(50) = 4 microM), had moderately reduced OM permeability capacity. These results demonstrate the significant role of the PMBN hydrophobic segment in promoting biological activity.

Anti-Bacterial Agents↗

PubMedDate not supplied

On the interaction of colicin E3 with the ribosome.

Colicin E3 is a protein that kills Escherichia coli cells by a process that involves binding to a surface receptor, entering the cell and inactivating its protein biosynthetic machinery. Colicin E3 kills cells by a catalytic mechanism of a specific ribonucleolytic cleavage in 16S rRNA at the ribosomal decoding A-site between A1493 and G1494 (E. coli numbering system). The breaking of this single phosphodiester bond results in a complete cessation of protein biosynthesis and cell death. The inactive E517Q mutant of the catalytic domain of colicin E3 binds to 30S ribosomal subunits of Thermus thermophilus, as demonstrated by an immunoblotting assay. A model structure of the complex of the ribosomal subunit 30S and colicin E3, obtained via docking, explains the role of the catalytic residues, suggests a catalytic mechanism and provides insight into the specificity of the reaction. Furthermore, the model structure suggests that the inhibitory action of bound immunity is due to charge repulsion of this acidic protein by the negatively charged rRNA backbone

Bacterial Proteins↗

PubMedDate not supplied

The regulation of death-associated protein (DAP) kinase in apoptosis.

DAP-kinase is a calcium/calmodulin (Ca2+/CaM) serine/threonine kinase which positively mediates programmed cell death in a variety of cell systems. The kinase is localized to the actin microfilament and has a unique, multidomain structure consisting of ankyrin repeats and a death domain. One of the substrates of DAP-kinase was identified as myosin light chain (MLC), the phosphorylation of which mediates membrane blebbing. Another arm in its mode of action leads to the formation of autophagic vesicles. Recent work addressed its mode of regulation and identified a mechanism which restrains its apoptotic function in growing cells and enables its activation during cell death. It involves an inhibitory type of autophosphorylation on serine 308 within the CaM regulatory domain. This negative phosphorylation takes place in growing cells and is strongly reduced upon their exposure to the apoptotic stimulus of C6-ceramide. The substitution of serine 308 to alanine, which mimics the ceramide-induced dephosphorylation at this site, increases Ca2+/CaM-independent substrate phosphorylation, as well as binding and overall sensitivity of the kinase to CaM. At the cellular level, it strongly enhances the death-promoting activity of the kinase. These results are consistent with a molecular model in which phosphorylation on serine 308 stabilizes a locked conformation of the CaM regulatory domain within the catalytic cleft and, simultaneously, also interferes with CaM binding. We propose that this unique mechanism of auto-inhibition evolved to impose a locking device which keeps DAP-kinase silent in healthy cells and ensures its activation only in response to apoptotic signals.

Amino Acid Substitution↗