Comparing in vitro, in situ, and in vivo experimental data in a three-dimensional model of mammalian cochlear mechanics

Normal mammalian hearing is refined by amplification of the motion of the cochlear partition. This partition, comprising the organ of Corti sandwiched between the basilar and tectorial membranes, contains the outer hair cells that are thought to drive this amplification process. Force generation by outer hair cells has been studied extensively in vitro and in situ, but, to understand cochlear amplification fully, it is necessary to characterize the role played by each of the components of the cochlear partition in vivo. Observations of cochlear partition motion in vivo are severely restricted by its inaccessibility and sensitivity to surgical trauma, so, for the present study, a computer model has been used to simulate the operation of the cochlea under different experimental conditions. In this model, which uniquely retains much of the three-dimensional complexity of the real cochlea, the motions of the basilar and tectorial membranes are fundamentally different during in situ- and in vivo-like conditions. Furthermore, enhanced outer hair cell force generation in vitro leads paradoxically to a decrease in the gain of the cochlear amplifier during sound stimulation to the model in vivo. These results suggest that it is not possible to extrapolate directly from experimental observations made in vitro and in situ to the normal operation of the intact organ in vivo.

Three-dimensional model of the potyviral genome-linked protein.

The full sequence of the genome-linked viral protein (VPg) cistron located in the central part of potato virus Y (common strain) genome has been identified. The VPg gene codes for a protein of 188 amino acids, with significant homology to other known potyviral VPg polypeptides. A three-dimensional model structure of VPg is proposed on the basis of similarity of hydrophobic-hydrophilic residue distribution to the sequence of malate dehydrogenase of known crystal structure. The 5' end of the viral RNA can be fitted to interact with the protein through the exposed hydroxyl group of Tyr-64, in agreement with experimental data. The complex favors stereochemically the formation of a phosphodiester bond [5'-(O4-tyrosylphospho)adenylate] typical for representatives of picornavirus-like viruses. The chemical mechanisms of viral RNA binding to VPg are discussed on the basis of the model structure of protein-RNA complex.

Three-dimensional modeling of and ligand docking to vitamin D receptor ligand binding domain

The ligand binding domain of the human vitamin D receptor (VDR) was modeled based on the crystal structure of the retinoic acid receptor. The ligand binding pocket of our VDR model is spacious at the helix 11 site and confined at the β-turn site. The ligand 1α,25-dihydroxyvitamin D3 was assumed to be anchored in the ligand binding pocket with its side chain heading to helix 11 (site 2) and the A-ring toward the β-turn (site 1). Three residues forming hydrogen bonds with the functionally important 1α- and 25-hydroxyl groups of 1α,25-dihydroxyvitamin D3 were identified and confirmed by mutational analysis: the 1α-hydroxyl group is forming pincer-type hydrogen bonds with S237 and R274 and the 25-hydroxyl group is interacting with H397. Docking potential for various ligands to the VDR model was examined, and the results are in good agreement with our previous three-dimensional structure-function theory.

Three-dimensional structure of poliovirus receptor bound to poliovirus

Poliovirus initiates infection by binding to its cellular receptor (Pvr). We have studied this interaction by using cryoelectron microscopy to determine the structure, at 21-Å resolution, of poliovirus complexed with a soluble form of its receptor (sPvr). This density map aided construction of a homology-based model of sPvr and, in conjunction with the known crystal structure of the virus, allowed delineation of the binding site. The virion does not change significantly in structure on binding sPvr in short incubations at 4°C. We infer that the binding configuration visualized represents the initial interaction that is followed by structural changes in the virion as infection proceeds. sPvr is segmented into three well-defined Ig-like domains. The two domains closest to the virion (domains 1 and 2) are aligned and rigidly connected, whereas domain 3 diverges at an angle of ≈60°. Two nodules of density on domain 2 are identified as glycosylation sites. Domain 1 penetrates the “canyon” that surrounds the 5-fold protrusion on the capsid surface, and its binding site involves all three major capsid proteins. The inferred pattern of virus–sPvr interactions accounts for most mutations that affect the binding of Pvr to poliovirus.

The two-dimensional IR nonlinear spectroscopy of a cyclic penta-peptide in relation to its three-dimensional structure

A form of two-dimensional (2D) vibrational spectroscopy, which uses two ultrafast IR laser pulses, is used to examine the structure of a cyclic penta-peptide in solution. Spectrally resolved cross peaks occur in the off-diagonal region of the 2D IR spectrum of the amide I region, analogous to those in 2D NMR spectroscopy. These cross peaks measure the coupling between the different amide groups in the structure. Their intensities and polarizations relate directly to the three-dimensional structure of the peptide. With the help of a model coupling Hamiltonian, supplemented by density functional calculations, the spectra of this penta-peptide can be regenerated from the known solution phase structure. This 2D-IR measurement, with an intrinsic time resolution of less than 1 ps, could be used in all time regimes of interest in biology.

Photoactive yellow protein: A structural prototype for the three-dimensional fold of the PAS domain superfamily

PAS domains are found in diverse proteins throughout all three kingdoms of life, where they apparently function in sensing and signal transduction. Although a wealth of useful sequence and functional information has become recently available, these data have not been integrated into a three-dimensional (3D) framework. The very early evolutionary development and diverse functions of PAS domains have made sequence analysis and modeling of this protein superfamily challenging. Limited sequence similarities between the ∼50-residue PAS repeats and one region of the bacterial blue-light photosensor photoactive yellow protein (PYP), for which ground-state and light-activated crystallographic structures have been determined to high resolution, originally were identified in sequence searches using consensus sequence probes from PAS-containing proteins. Here, we found that by changing a few residues particular to PYP function, the modified PYP sequence probe also could select PAS protein sequences. By mapping a typical ∼150-residue PAS domain sequence onto the entire crystallographic structure of PYP, we show that the PAS sequence similarities and differences are consistent with a shared 3D fold (the PAS/PYP module) with obvious potential for a ligand-binding cavity. Thus, PYP appears to prototypically exhibit all the major structural and functional features characteristic of the PAS domain superfamily: the shared PAS/PYP modular domain fold of ∼125–150 residues, a sensor function often linked to ligand or cofactor (chromophore) binding, and signal transduction capability governed by heterodimeric assembly (to the downstream partner of PYP). This 3D PAS/PYP module provides a structural model to guide experimental testing of hypotheses regarding ligand-binding, dimerization, and signal transduction.

The Three-Dimensional Structure of Aspergillus niger Pectin Lyase B at 1.7-Å Resolution1

The three-dimensional structure of Aspergillus niger pectin lyase B (PLB) has been determined by crystallographic techniques at a resolution of 1.7 Å. The model, with all 359 amino acids and 339 water molecules, refines to a final crystallographic R factor of 16.5%. The polypeptide backbone folds into a large right-handed cylinder, termed a parallel β helix. Loops of various sizes and conformations protrude from the central helix and probably confer function. The largest loop of 53 residues folds into a small domain consisting of three antiparallel β strands, one turn of an α helix, and one turn of a 310 helix. By comparison with the structure of Erwinia chrysanthemi pectate lyase C (PelC), the primary sequence alignment between the pectate and pectin lyase subfamilies has been corrected and the active site region for the pectin lyases deduced. The substrate-binding site in PLB is considerably less hydrophilic than the comparable PelC region and consists of an extensive network of highly conserved Trp and His residues. The PLB structure provides an atomic explanation for the lack of a catalytic requirement for Ca2+ in the pectin lyase family, in contrast to that found in the pectate lyase enzymes. Surprisingly, however, the PLB site analogous to the Ca2+ site in PelC is filled with a positive charge provided by a conserved Arg in the pectin lyases. The significance of the finding with regard to the enzymatic mechanism is discussed.

Faithful representation of similarities among three-dimensional shapes in human vision.

Efficient and reliable classification of visual stimuli requires that their representations reside a low-dimensional and, therefore, computationally manageable feature space. We investigated the ability of the human visual system to derive such representations from the sensory input-a highly nontrivial task, given the million or so dimensions of the visual signal at its entry point to the cortex. In a series of experiments, subjects were presented with sets of parametrically defined shapes; the points in the common high-dimensional parameter space corresponding to the individual shapes formed regular planar (two-dimensional) patterns such as a triangle, a square, etc. We then used multidimensional scaling to arrange the shapes in planar configurations, dictated by their experimentally determined perceived similarities. The resulting configurations closely resembled the original arrangements of the stimuli in the parameter space. This achievement of the human visual system was replicated by a computational model derived from a theory of object representation in the brain, according to which similarities between objects, and not the geometry of each object, need to be faithfully represented.

Three-dimensional structure of human protein kinase C interacting protein 1, a member of the HIT family of proteins.

The three-dimensional structure of protein kinase C interacting protein 1 (PKCI-1) has been solved to high resolution by x-ray crystallography using single isomorphous replacement with anomalous scattering. The gene encoding human PKCI-1 was cloned from a cDNA library by using a partial sequence obtained from interactions identified in the yeast two-hybrid system between PKCI-1 and the regulatory domain of protein kinase C-beta. The PKCI-1 protein was expressed in Pichia pastoris as a dimer of two 13.7-kDa polypeptides. PKCI-1 is a member of the HIT family of proteins, shown by sequence identity to be conserved in a broad range of organisms including mycoplasma, plants, and humans. Despite the ubiquity of this protein sequence in nature, no distinct function has been shown for the protein product in vitro or in vivo. The PKCI-1 protomer has an alpha+beta meander fold containing a five-stranded antiparallel sheet and two helices. Two protomers come together to form a 10-stranded antiparallel sheet with extensive contacts between a helix and carboxy terminal amino acids of a protomer with the corresponding amino acids in the other protomer. PKCI-1 has been shown to interact specifically with zinc. The three-dimensional structure has been solved in the presence and absence of zinc and in two crystal forms. The structure of human PKCI-1 provides a model of this family of proteins which suggests a stable fold conserved throughout nature.

Three-dimensional tracking of motile bacteria near a solid planar surface.

Knowing how motile bacteria move near and along a solid surface is crucial to understanding such diverse phenomena as the migration of infectious bacteria along a catheter, biofilm growth, and the movement of bacteria through the pore spaces of saturated soil, a critical step in the in situ bioremediation of contaminated aquifers. In this study, a tracking microscope is used to record the three-dimensional motion of Escherichia coli near a planar glass surface. Data from the tracking microscope are analyzed to quantify the effects of bacteria-surface interactions on the swimming behavior of bacteria. The speed of cells approaching the surface is found to decrease in agreement with the mathematical model of Ramia et al. [Ramia, M., Tullock, D. L. & Phan-Tien, N. (1993) Biophys J. 65,755-778], which represents the bacteria as spheres with a single polar flagellum rotating at a constant rate. The tendency of cells to swim adjacent to the surface is shown in computer-generated reproductions of cell traces. The attractive interaction potential between the cells and the solid surface is offered as one of several possible explanations for this tendency.

Three-dimensional scroll waves of cAMP could direct cell movement and gene expression in Dictyostelium slugs.

Complex three-dimensional waves of excitation can explain the observed cell movement pattern in Dictyostelium slugs. Here we show that these three-dimensional waves can be produced by a realistic model for the cAMP relay system [Martiel, J. L. & Goldbeter, A. (1987) Biophys J. 52, 807-828]. The conversion of scroll waves in the prestalk zone of the slug into planar wave fronts in the prespore zone can result from a smaller fraction of relaying cells in the prespore zone. Further, we show that the cAMP concentrations to which cells in a slug are exposed over time display a simple pattern, despite the complex spatial geometry of the waves. This cAMP distribution agrees well with observed patterns of cAMP-regulated cell type-specific gene expression. The core of the spiral, which is a region of low cAMP concentration, might direct expression of stalk-specific genes during culmination.

Three-dimensional structure of NADPH–cytochrome P450 reductase: Prototype for FMN- and FAD-containing enzymes

Microsomal NADPH–cytochrome P450 reductase (CPR) is one of only two mammalian enzymes known to contain both FAD and FMN, the other being nitric-oxide synthase. CPR is a membrane-bound protein and catalyzes electron transfer from NADPH to all known microsomal cytochromes P450. The structure of rat liver CPR, expressed in Escherichia coli and solubilized by limited trypsinolysis, has been determined by x-ray crystallography at 2.6 Å resolution. The molecule is composed of four structural domains: (from the N- to C- termini) the FMN-binding domain, the connecting domain, and the FAD- and NADPH-binding domains. The FMN-binding domain is similar to the structure of flavodoxin, whereas the two C-terminal dinucleotide-binding domains are similar to those of ferredoxin–NADP+ reductase (FNR). The connecting domain, situated between the FMN-binding and FNR-like domains, is responsible for the relative orientation of the other domains, ensuring the proper alignment of the two flavins necessary for efficient electron transfer. The two flavin isoalloxazine rings are juxtaposed, with the closest distance between them being about 4 Å. The bowl-shaped surface near the FMN-binding site is likely the docking site of cytochrome c and the physiological redox partners, including cytochromes P450 and b5 and heme oxygenase.

The primary fibrin polymerization pocket: Three-dimensional structure of a 30-kDa C-terminal γ chain fragment complexed with the peptide Gly-Pro-Arg-Pro

After vascular injury, a cascade of serine protease activations leads to the conversion of the soluble fibrinogen molecule into fibrin. The fibrin monomers then polymerize spontaneously and noncovalently to form a fibrin gel. The primary interaction of this polymerization reaction is between the newly exposed N-terminal Gly-Pro-Arg sequence of the α chain of one fibrin molecule and the C-terminal region of a γ chain of an adjacent fibrin(ogen) molecule. In this report, the polymerization pocket has been identified by determining the crystal structure of a 30-kDa C-terminal fragment of the fibrin(ogen) γ chain complexed with the peptide Gly-Pro-Arg-Pro. This peptide mimics the N terminus of the α chain of fibrin. The conformational change in the protein upon binding the peptide is subtle, with electrostatic interactions primarily mediating the association. This is consistent with biophysical experiments carried out over the last 50 years on this fundamental polymerization reaction.

Twist grain boundaries in three-dimensional lamellar Turing structures

Steady spatial self-organization of three-dimensional chemical reaction-diffusion systems is discussed with the emphasis put on the possible defects that may alter the Turing patterns. It is shown that one of the stable defects of a three-dimensional lamellar Turing structure is a twist grain boundary embedding a Scherk minimal surface.

Reciprocal interactions between β1-integrin and epidermal growth factor receptor in three-dimensional basement membrane breast cultures: A different perspective in epithelial biology

Anchorage and growth factor independence are cardinal features of the transformed phenotype. Although it is logical that the two pathways must be coregulated in normal tissues to maintain homeostasis, this has not been demonstrated directly. We showed previously that down-modulation of β1-integrin signaling reverted the malignant behavior of a human breast tumor cell line (T4–2) derived from phenotypically normal cells (HMT-3522) and led to growth arrest in a three-dimensional (3D) basement membrane assay in which the cells formed tissue-like acini (14). Here, we show that there is a bidirectional cross-modulation of β1-integrin and epidermal growth factor receptor (EGFR) signaling via the mitogen-activated protein kinase (MAPK) pathway. The reciprocal modulation does not occur in monolayer (2D) cultures. Antibody-mediated inhibition of either of these receptors in the tumor cells, or inhibition of MAPK kinase, induced a concomitant down-regulation of both receptors, followed by growth-arrest and restoration of normal breast tissue morphogenesis. Cross-modulation and tissue morphogenesis were associated with attenuation of EGF-induced transient MAPK activation. To specifically test EGFR and β1-integrin interdependency, EGFR was overexpressed in nonmalignant cells, leading to disruption of morphogenesis and a compensatory up-regulation of β1-integrin expression, again only in 3D. Our results indicate that when breast cells are spatially organized as a result of contact with basement membrane, the signaling pathways become coupled and bidirectional. They further explain why breast cells fail to differentiate in monolayer cultures in which these events are mostly uncoupled. Moreover, in a subset of tumor cells in which these pathways are misregulated but functional, the cells could be “normalized” by manipulating either pathway.