Molecular Binding Sites Research Articles

Determination of Three-dimensional Structure and Residues of the Novel Tumor Suppressor AIMP3/p18 Required for the Interaction with ATM

Although AIMP3/p18 is normally associated with the multi-tRNA synthetase complex via its specific interaction with methionyl-tRNA synthetase, it also works as a tumor suppressor by interacting with ATM, the upstream kinase of p53. To understand the molecular interactions of AIMP3 and the mechanisms involved, we determined the crystal structure of AIMP3 at 2.0-angstroms resolution and identified its potential sites of interaction with ATM. AIMP3 contains two distinct domains linked by a 7-amino acid (Lys57-Ser63) peptide, which contains a 3(10) helix. The 56-amino acid N-terminal domain consists of two helices into which three antiparallel beta strands are inserted, and the 111-amino acid C-terminal domain contains a bundle of five helices (Thr64-Tyr152) followed by a coiled region (Pro153-Leu169). Structural analyses revealed homologous proteins such as yeast glutamyl-tRNA synthetase, Arc1p, EF1Bgamma, and glutathione S-transferase and suggested two potential molecular binding sites. Moreover, mutations at the C-terminal putative binding site abolished the interaction between AIMP3 and ATM and the ability of AIMP3 to activate p53. Thus, this work identified the two potential molecular interaction sites of AIMP3 and determined the residues critical for its tumor-suppressive activity through the interaction with ATM.

Synthesis, DNA binding and antitumor activities of some novel polymer–cobalt(III) complexes containing 1,10-phenanthroline ligand

The novel water-soluble polymer–cobalt(III) complex samples, cis-[Co(phen) 2(BPEI)Cl]Cl 2 · 4H 2O (phen = 1,10-phenanthroline, BPEI = branched polyethyleneimine), with different amounts of cobalt complex content in the polymer chain, were prepared by ligand substitution method in water–ethanol medium and characterized by Infra-red, UV–Vis, 1H NMR spectral and elemental analysis methods. The interaction of these polymer–cobalt(III)-phenanthroline complex samples with calf thymus DNA has been explored using electronic absorption spectroscopy, emission spectroscopy and gel electrophoresis techniques. The presence of multiple small size molecular binding sites, namely, the cobalt(III)–phenanthroline complex moieties, and free amino groups in a single big sized polymer molecule enhanced both the electrostatic and/or van der Waals interaction and partial intercalative bindings with calf thymus DNA. The antitumor activity of a sample of polymer–cobalt(III) complex was determined using HEp-2 cell line and different cell death indicator stains and MTT assay. Many of the cultured HEp-2 cells treated with this complex suffered loss of viability and death mostly through apoptosis as evidenced by the nuclear and cytoplasmic morphology.

Dopamine transporter comparative molecular modeling and binding site prediction using the LeuTAa leucine transporter as a template

Pharmacological and behavioral studies indicate that binding of cocaine and the amphetamines by the dopamine transporter (DAT) protein is principally responsible for initiating the euphoria and addiction associated with these drugs. The lack of an X-ray crystal structure for the DAT or any other member of the neurotransmitter:sodium symporter (NSS) family has hindered understanding of psychostimulant recognition at the atomic level; structural information has been obtained largely from mutagenesis and biophysical studies. The recent publication of a crystal structure for the bacterial leucine transporter LeuT(Aa), a distantly related NSS family homolog, provides for the first time a template for three-dimensional comparative modeling of NSS proteins. A novel computational modeling approach using the capabilities of the Molecular Operating Environment program MOE 2005.06 in conjunction with other comparative modeling servers generated the LeuT(Aa)-directed DAT model. Probable dopamine and amphetamine binding sites were identified within the DAT model using multiple docking approaches. Binding sites for the substrate ligands (dopamine and amphetamine) overlapped substantially with the analogous region of the LeuT(Aa) crystal structure for the substrate leucine. The docking predictions implicated DAT side chains known to be critical for high affinity ligand binding and suggest novel mutagenesis targets in elucidating discrete substrate and inhibitor binding sites. The DAT model may guide DAT ligand QSAR studies, and rational design of novel DAT-binding therapeutics.

Nano-Scale Dynamic Recognition Imaging on Vascular Endothelial Cells

Combination of high-resolution atomic force microscope topography imaging with single molecule force spectroscopy provides a unique possibility for the detection of specific molecular recognition events. The identification and localization of specific receptor binding sites on complex heterogeneous biosurfaces such as cells and membranes are of particular interest in this context. Here simultaneous topography and recognition imaging (TREC) was applied to gently fixed microvascular endothelial cells from mouse myocardium (MyEnd) to identify binding sites of vascular endothelial (VE)-cadherin, known to play a crucial role in calcium-dependent, homophilic cell-to-cell adhesion. TREC images were acquired with magnetically oscillating atomic-force microscope tips functionalized with a recombinant VE-cadherin-Fc cis-dimer. The recognition images revealed single molecular binding sites and prominent, irregularly shaped dark spots (domains) with sizes ranging from 10 to 100nm. These domains arose from a decrease of the oscillation amplitude during specific binding between active VE-cadherin cis-dimers. The VE-cadherin clusters were subsequently assigned to topography features. TREC represents an exquisite method to quickly obtain the local distribution of receptors on cellular surface with an unprecedented lateral resolution of 5nm.

Exploring Molecular Oxygen Pathways in Hansenula polymorpha Copper-containing Amine Oxidase

The accessibility of large substrates to buried enzymatic active sites is dependent upon the utilization of proteinaceous channels. The necessity of these channels in the case of small substrates is questionable because diffusion through the protein matrix is often assumed. Copper amine oxidases contain a buried protein-derived quinone cofactor and a mononuclear copper center that catalyze the conversion of two substrates, primary amines and molecular oxygen, to aldehydes and hydrogen peroxide, respectively. The nature of molecular oxygen migration to the active site in the enzyme from Hansenula polymorpha is explored using a combination of kinetic, x-ray crystallographic, and computational approaches. A crystal structure of H. polymorpha amine oxidase in complex with xenon gas, which serves as an experimental probe for molecular oxygen binding sites, reveals buried regions of the enzyme suitable for transient molecular oxygen occupation. Calculated O(2) free energy maps using copper amine oxidase crystal structures in the absence of xenon correspond well with later experimentally observed xenon sites in these systems, and allow the visualization of O(2) migration routes of differing probabilities within the protein matrix. Site-directed mutagenesis designed to block individual routes has little effect on overall k(cat)/K(m) (O(2)), supporting multiple dynamic pathways for molecular oxygen to reach the active site.

Key Role of Local Regulation in Chemosensing Revealed by a New Molecular Interaction-Based Modeling Method

The signaling network underlying eukaryotic chemosensing is a complex combination of receptor-mediated transmembrane signals, lipid modifications, protein translocations, and differential activation/deactivation of membrane-bound and cytosolic components. As such, it provides particularly interesting challenges for a combined computational and experimental analysis. We developed a novel detailed molecular signaling model that, when used to simulate the response to the attractant cyclic adenosine monophosphate (cAMP), made nontrivial predictions about Dictyostelium chemosensing. These predictions, including the unexpected existence of spatially asymmetrical, multiphasic, cyclic adenosine monophosphate–induced PTEN translocation and phosphatidylinositol-(3,4,5)P3 generation, were experimentally verified by quantitative single-cell microscopy leading us to propose significant modifications to the current standard model for chemoattractant-induced biochemical polarization in this organism. Key to this successful modeling effort was the use of “Simmune,” a new software package that supports the facile development and testing of detailed computational representations of cellular behavior. An intuitive interface allows user definition of complex signaling networks based on the definition of specific molecular binding site interactions and the subcellular localization of molecules. It automatically translates such inputs into spatially resolved simulations and dynamic graphical representations of the resulting signaling network that can be explored in a manner that closely parallels wet lab experimental procedures. These features of Simmune were critical to the model development and analysis presented here and are likely to be useful in the computational investigation of many aspects of cell biology.

Chemical Contribution to Surface-Enhanced Raman Scattering

We present a new mechanism for the chemical contribution to surface-enhanced Raman scattering (SERS). The theory considers the modulation of the polarizability of a metal nanocluster or a flat metal surface by the vibrational motion of an adsorbed molecule. The modulated polarization of the substrate coupled with the incident light will contribute to the Raman scattering enhancement. We show that for a metal cluster and for a flat metal surface this new chemical contribution may enhance the Raman scattering intensity by a factor of approximately 102 and approximately 104, respectively. The new SERS process is determined by the electric field parallel to the surface of the metal substrate at the molecular binding site.

Photorhabdus luminescens toxin‐induced permeability change in Manduca sexta and Tenebrio molitor midgut brush border membrane and in unilamellar phospholipid vesicle

Photorhabdus luminescens, a Gram-negative bacterium, secretes a protein toxin (PL toxin) that is toxic to insects. In this study, the effects of the PL toxin on large receptor-free unilamellar phospholipid vesicles (LUVs) of Manduca sexta and on brush border membrane vesicles (BBMVs) of M. sexta and Tenebrio molitor were examined. Cry1Ac served as a positive control in our experiments due to its known channel-forming activity on M. sexta. Voltage clamping assays with dissected midguts of M. sexta and T. molitor clearly showed that both Cry1Ac and PL toxin caused channel formation in the midguts, although channel formation was not detected for T. molitor midguts under Cry1Ac and it was less sensitive to PL toxin than to Cry1Ac for M. sexta midguts. Calcein release experiments showed that both toxins made LUVs (unilamellar lipid vesicles) permeable, and at some concentrations of the toxins such permeabilizing effects were pH-dependent. The lowest concentrations of PL toxin were more than 600-fold and 24-fold lower to induce BBMV permeability of T. molitor and M. sexta than those to induce calcein release from LUVs of M. sexta. These further support that PL toxin is responsible for channel formation in the larvae midguts. The lower concentration to induce permeability in BBMV than in LUV is, probably, attributable to that BBMV has PL toxin receptors that facilitate the toxin to induce permeabilization. Furthermore, our results indicate that the effects of PL toxin on BBMV permeability of M. sexta were not significantly influenced by Gal Nac, but those of Cry1Ac were. This implies that PL toxin and Cry1Ac might use different molecular binding sites in BBMV to cause channel formation.

Molecular Mechanisms and Binding Site Location for the Noncompetitive Antagonist Crystal Violet on Nicotinic Acetylcholine Receptors

We investigated the molecular mechanisms and the binding site location for the fluorophor crystal violet (CrV), a noncompetitive antagonist of the nicotinic acetylcholine receptor (AChR). To this end, radiolabeled competition binding, fluorescence spectroscopy, Schild-type analysis, patch-clamp recordings, and molecular dynamics approaches were used. The results indicate that (i) CrV interacts with the desensitized Torpedo AChR with higher affinity than with the resting state at several temperatures (5-37 degrees C); (ii) CrV-induced inhibition of the phencyclidine (PCP) analogue [(3)H]thienylcyclohexylpiperidine binding to the desensitized or resting AChR is mediated by a steric mechanism; (iii) tetracaine inhibits CrV binding to the resting AChR, probably by a steric mechanism; (iv) barbiturates modulate CrV binding to the resting AChR by an allosteric mechanism; (v) CrV itself induces AChR desensitization; (vi) CrV decreases the peak of macroscopic currents by acting on the resting AChR but without affecting the desensitization rate from the open state; and (vii) two tertiary amino groups from CrV may bind to the alpha1-Glu(262) residues (located at position 20') in the resting state. We conclude that the CrV binding site overlaps the PCP locus in the resting and desensitized state. The noncompetitive action of CrV may be explained by an allosteric mechanism in which the binding of CrV to the extracellular mouth of the resting receptor leads to an inhibition of channel opening. Binding of CrV probably increases desensitization of the resting channel and stabilizes the desensitized state.

Interactions Between Chemical Functionality and Nanoscale Surface Topography Impact Fibronectin Conformation and Neuronal Differentiation on Model Sol-gel Silica Substrates

ABSTRACTFunctional relationships between the biomaterial interface and extracellular matrix (ECM) proteins are intimately involved in cellular adhesion and function. Structural changes of ECM proteins upon adsorption to a surface alter the protein's biological activity by varying the availability of molecular binding sites. Recent work using native and organically modified sol-gel silica as a neuronal biointerface revealed that changes in surface nanotopography of bulk versus thin film materials result in dramatic differences in fibronectin structure, cell survival, and neuronal differentiation. In order to further investigate interactions between chemical functionality and surface topography, we evaluated the global conformation of human fibronectin adsorbed to seven different organically modified silica gels and thin films. Chemical functional groups were introduced into the materials either by altering the starting precursor or by doping with poly-l-lysine or polyethylenimine. Surface topography measurements by atomic force microscopy show that films have surface features less than 25 nm while bulk materials of the same precursor chemistry have features ranging from 50 – 100 nm in size. Fluorescence resonance energy transfer spectroscopy (FRET) revealed a strong interaction between surface topography and chemical functionality. Fibronectin remain globular on all bulk materials regardless of chemical modification. The same changes in precursors or dopant chemistry, however, induced changes in the conformation of fibronectin on the thin films. The differentiation of PC12 cells on the surface indicated a strong impact of the surface features and suggest a possible optimal fibronectin folding state.

An analysis of the class of gene regulatory functions implied by a biochemical model

Understanding the integrated behavior of genetic regulatory networks, in which genes regulate one another's activities via RNA and protein products, is emerging as a dominant problem in systems biology. One widely studied class of models of such networks includes genes whose expression values assume Boolean values (i.e., on or off). Design decisions in the development of Boolean network models of gene regulatory systems include the topology of the network (including the distribution of input- and output-connectivity) and the class of Boolean functions used by each gene (e.g., canalizing functions, post functions, etc.). For example, evidence from simulations suggests that biologically realistic dynamics can be produced by scale-free network topologies with canalizing Boolean functions. This work seeks further insights into the design of Boolean network models through the construction and analysis of a class of models that include more concrete biochemical mechanisms than the usual abstract model, including genes and gene products, dimerization, cis-binding sites, promoters and repressors. In this model, it is assumed that the system consists of N genes, with each gene producing one protein product. Proteins may form complexes such as dimers, trimers, etc. The model also includes cis-binding sites to which proteins may bind to form activators or repressors. Binding affinities are based on structural complementarity between proteins and binding sites, with molecular binding sites modeled by bit-strings. Biochemically plausible gene expression rules are used to derive a Boolean regulatory function for each gene in the system. The result is a network model in which both topological features and Boolean functions arise as emergent properties of the interactions of components at the biochemical level. A highly biased set of Boolean functions is observed in simulations of networks of various sizes, suggesting a new characterization of the subset of Boolean functions that are likely to appear in gene regulatory networks.

Obituary

Obituary

Matching of trastuzumab (Herceptin ®) epitope mimics onto the surface of Her-2/neu – a new method of epitope definition

As seen with the proto-oncogene Her-2/neu, antibodies targeting different parts of a receptor can have opposing effects. Depending on epitope specificity, in this case, tumor growth can be inhibited – but also enhanced. Therefore, the definition of molecular binding sites is of increasing importance in modern medicine. We here introduce a novel approach for binding site localization, utilizing information obtained by the phage display technique. This is a high throughput screening method for identification of peptide mimics, so called mimotopes, of any binding structure of interest. All target molecules whose structure is available in the RCSB Protein Data Bank can be scanned for mimotope matches on their surface. In this study, we present the matching results of five mimotopes defined for the epitope recognized by trastuzumab (Herceptin ®), a humanized monoclonal antibody inhibiting tumor growth, on Her-2/neu. The localization thus obtained corresponds to the known trastuzumab epitope. We therefore suggest the algorithm as a novel way of binding site definition, circumventing co-crystallization experiments.

Demonstration of Enantiomer Specificity of Proteins and Drugs

In addition to the traditional use of molecular models of tetrahedral asymmetric centers, the concept of chirality and its importance to protein–drug interactions is demonstrated in a guided classroom activity. A watchglass with three different colored Post-it Notes is set on a desktop or table surface to simulate a molecular binding site of a protein. As students build models of symmetric and asymmetric centers, the models are tested for binding to the watchglass "binding site". By relating the chiral or achiral models to various common or familiar drugs, students can easily understand the importance of chirality in protein–drug interactions. This approach can be easily adapted to the knowledge base of the student and can include concepts of prochirality, nomenclature, properties, and chemical separation schemes of enantiomers.See the Discussion on this Tested Demonstation.

Structural changes of fibronectin adsorbed to model surfaces probed by fluorescence resonance energy transfer.

Structural changes of proteins during adsorption to biomaterials affect the presentation of molecular binding sites and, ultimately, biomaterial performance. We have applied fluorescence resonance energy transfer (FRET) spectroscopy to study structural changes of the cell adhesion protein, fibronectin (Fn), following adsorption to model hydrophilic and hydrophobic surfaces. Fn was labeled with donor and acceptor fluorophores using two labeling schemes and intramolecular energy transfer was calibrated against measured structural changes of Fn in denaturing solutions. FRET was then applied to measure Fn's structure on surfaces. Based on FRET, Fn underwent greater extension of its dimer arms on hydrophilic glass than on hydrophobic fluoroalklysilane-derivatized glass (fluorosilane), and this extension was insensitive to molecular packing over a range of adsorption concentrations. Fn's conformation on glass better promoted cell attachment than on fluorosilane; the roles of both global structural changes (movements of modules) and local structural changes (disruption of secondary structure) on Fn's cell integrin binding activity are discussed. Based on previous FRET work, we compare Fn's conformations on these surfaces with its conformations in fibroblast culture. FRET is unique in allowing direct comparison of protein structure between biomaterial surfaces and cell culture.

Staphylococcus epidermidis–fibronectin binding and its inhibition by heparin

Staphylococcus epidermidis is able to adhere onto biomaterials and to cause implant infections. Recently, host matrix proteins, which in vivo cover the implants, have been indicated as substrates for adhesion by specific bacterial adhesins. Here, the binding of S. epidermidis to fibronectin, a main protein of the extracellular matrix, and the effect of heparin on this interaction were studied by dynamic force spectroscopy (DFS). Novelties are that S. epidermidis strains analysed by DFS were clinical isolates from prosthesis-associated infections, genotyped and phenotyped for their adhesion properties to fibronectin and examined as living cells. Thus, fibronectin-binding staphylococci adhered to the fibronectin-coated substratum and formed a continuous layer assuring their contact with the fibronectin-coated cantilever tip during the approach–retraction cycles of the DFS measurements. Results show that only a single molecular binding site of fibronectin is involved in the interaction with S. epidermidis, that it takes place at the domain near the C-terminus and that it is specifically inhibited by heparin.

Preorganization of molecular binding sites in designed diiron proteins.

De novo protein design provides an attractive approach to critically test the features that are required for metalloprotein structure and function. Previously we designed and crystallographically characterized an idealized dimeric model for the four-helix bundle class of diiron and dimanganese proteins [Dueferri 1 (DF1)]. Although the protein bound metal ions in the expected manner, access to its active site was blocked by large bulky hydrophobic residues. Subsequently, a substrate-access channel was introduced proximal to the metal-binding center, resulting in a protein with properties more closely resembling those of natural enzymes. Here we delineate the energetic and structural consequences associated with the introduction of these binding sites. To determine the extent to which the binding site was preorganized in the absence of metal ions, the apo structure of DF1 in solution was solved by NMR and compared with the crystal structure of the di-Zn(II) derivative. The overall fold of the apo protein was highly similar to that of the di-Zn(II) derivative, although there was a rotation of one of the helices. We also examined the thermodynamic consequences associated with building a small molecule-binding site within the protein. The protein exists in an equilibrium between folded dimers and unfolded monomers. DF1 is a highly stable protein (K(diss) = 0.001 fM), but the dissociation constant increases to 0.6 nM (deltadeltaG = 5.4 kcalmol monomer) as the active-site cavity is increased to accommodate small molecules.

Molecular dynamics investigation of an antibody binding site by the fluorescence–photochrome method

A combined fluorescence–photochrome approach was used for investigation of the molecular dynamics antiDNP antibody binding site and its cavity. A 4-( N-2,4-dinitrophenylamino)-4′-( N, N′-dimethylamino)stilbene (StDNP) fluorescence DNP analog was incorporated into the antibody binding site. This was followed by measurements of fluorescence and photochrome parameters such as the StDNP excitation and emission spectra, fluorescence lifetime, steady-state and time-resolved fluorescence polarization, kinetics of trans– cis and cis– trans photoisomerization, and fluorescence quenching by nitroxide radicals freely diffused in solution. In parallel, computational modeling studies on the location and dynamics of DNP/TEMPO spin-label (Ns lDNP) and StDNP guests within a model of the binding site were performed. When all the experimental evidence is considered (including data from the antibody X-ray study), one can conclude that wobbling of the Trp 91 L/Trp 96 H binding-site·bound-hapten moiety (StDNP), can be responsible for the label's nanosecond dynamics monitored by fluorescence polarization techniques. A similar conclusion may be reached as a result of data analysis on Ns lDNP mobility within the antibody binding site. The mobility of Trp 91 L and Trp 96 H moieties provides the induced fit needed for effective stacking and release of the DNP epitope. Analysis of the above-mentioned data allows one to explore the mechanism of the probe's movement within the binding site and enables one to discuss the local dynamics of the binding site region. The combined fluorescence–photochrome approach can be used for investigation of local medium molecular dynamics in the immediate vicinity of specific sites of proteins and nucleic acids, as well as for other biologically important structures and synthetic analogues.

A low temperature scanning tunneling microscope designed for imaging molecular adsorbates

We report on the development of a low temperature scanning tunneling microscope (STM) used for imaging molecular adsorbates. The microscope operates near 85 K and features in situ tip and sample exchange. Cooling is accomplished via a clamping mechanism that provides a direct mechanical link between a liquid nitrogen dewar and the STM stage. During imaging, the clamping mechanism is released, and the STM stage is isolated from vibrations using a dual spring suspension system that incorporates magnetic eddy current damping. Additional isolation is provided by uncoupling the liquid nitrogen dewar from the vacuum chamber. We have used this STM to image benzene and carbon monoxide coadsorbed on the Pd (1 1 1) surface. The resulting acquired images show both the ordered arrangement and internal structure of the benzene molecules. Depending on the dosing conditions, three different ordered overlayer structures were formed, only one of which has been previously reported. Future plans include using the low temperature imaging capability of the STM to observe molecular binding sites, orientation, and tilt, as well as dynamical processes on the surface, such as chemical reactions.

X-Ray Crystallographic Structures of Biologically Active Trishomocubanes of the Types Pentacyclo[5.4.0.02,6.03,10.05,9]undecylamines and 4-Azahexacyclo[5.4.1.02,6.03,10.05,9.08,11]dodecane

The structures of three trishomocubanes of types pentacyclo[5.4.0.0 2,6 .0 3,10 .0 5,9 ]undecylamines and 4-azahexacyclo[5.4.1.0 2,6 .0 3,10 .0 5,9 .0 8,11 ]dodecanes have been determined. These ligands have demonstrated high selectivity and affinity for the sigma binding site, a molecular binding site important in the study of neurological disorders. The three compounds reported are members of an extensive series of ligands under investigation in a search for more efficacious pharmaceuticals. Compound (3) crystallized in the monoclinic space group C2/c with a 15.821(8), b 22.407(10), c 11.032(6) Å, β 115.15(3)°, V 3540(3) Å 3 , Z 8, and was refined to an R value of 0.054 on 1161F. Compound (4) crystallized in the triclinic, space group P1, a 11.278(3), b 13.139(2), c 7.444(2) Å, α 104.54(1), β 107.85(2), γ 68.51(1)°, V 964.9(4) Å 3 , Z 2, and was refined to an R value of 0.061 on 1939F. Compound (6) crystallized in the monoclinic space group P2 1 /c, a 9.971(4), b 11.989(4), c 12.993(5) Å, β 108.68(3)°, V 1471(1) Å3 , Z 4, and was refined to an R value of 0.060 on 1303F. The results are in accord with the hypothesis that the spatial relationship between the amine group and the hydrophobic regions of the molecule are important determinants of binding at the sigma site.