Structural Analysis and Molecular Model of a Self-Incompatibility RNase from Wild Tomato1

Self-incompatibility RNases (S-RNases) are an allelic series of style glycoproteins associated with rejection of self-pollen in solanaceous plants. The nucleotide sequences of S-RNase alleles from several genera have been determined, but the structure of the gene products has only been described for those from Nicotiana alata. We report on the N-glycan structures and the disulfide bonding of the S3-RNase from wild tomato (Lycopersicon peruvianum) and use this and other information to construct a model of this molecule. The S3-RNase has a single N-glycosylation site (Asn-28) to which one of three N-glycans is attached. S3-RNase has seven Cys residues; six are involved in disulfide linkages (Cys-16-Cys-21, Cys-46-Cys-91, and Cys-166-Cys-177), and one has a free thiol group (Cys-150). The disulfide-bonding pattern is consistent with that observed in RNase Rh, a related RNase for which radiographic-crystallographic information is available. A molecular model of the S3-RNase shows that four of the most variable regions of the S-RNases are clustered on one surface of the molecule. This is discussed in the context of recent experiments that set out to determine the regions of the S-RNase important for recognition during the self-incompatibility response.

A model for the structure of the P domains in the subtilisin-like prohormone convertases

The proprotein convertases are a family of at least seven calcium-dependent endoproteases that process a wide variety of precursor proteins in the secretory pathway. All members of this family possess an N-terminal proregion, a subtilisin-like catalytic module, and an additional downstream well-conserved region of ≈150 amino acid residues, the P domain, which is not found in any other subtilase. The pro and catalytic domains cannot be expressed in the absence of the P domains; their thermodynamic instability may be attributable to the presence of large numbers of negatively charged Glu and Asp side chains in the substrate binding region for recognition of multibasic residue cleavage sites. Based on secondary structure predictions, we here propose that the P domains consist of 8-stranded β-barrels with well-organized inner hydrophobic cores, and therefore are independently folded components of the proprotein convertases. We hypothesize further that the P domains are integrated through strong hydrophobic interactions with the catalytic domains, conferring structural stability and regulating the properties and activity of the convertases. A molecular model of these interdomain interactions is proposed in this report.

Molecular mechanism of use-dependent calcium channel block by phenylalkylamines: Role of inactivation

The role of channel inactivation in the molecular mechanism of calcium (Ca2+) channel block by phenylalkylamines (PAA) was analyzed by designing mutant Ca2+ channels that carry the high affinity determinants of the PAA receptor site [Hockerman, G. H., Johnson, B. D., Scheuer, T., and Catterall, W. A. (1995) J. Biol. Chem. 270, 22119–22122] but inactivate at different rates. Use-dependent block by PAAs was studied after expressing the mutant Ca2+ channels in Xenopus oocytes. Substitution of single putative pore-orientated amino acids in segment IIIS6 by alanine (F-1499-A, F-1500-A, F-1510-A, I-1514-A, and F-1515-A) gradually slowed channel inactivation and simultaneously reduced inhibition of barium currents (IBa) by (−)D600 upon depolarization by 100 ms steps at 0.1 Hz. This apparent reduction in drug sensitivity was only evident if test pulses were applied at a low frequency of 0.1 Hz and almost disappeared at the frequency of 1 Hz. (−)D600 slowed IBa recovery after maintained membrane depolarization (1–3 sec) to a comparable extent in all channel constructs. A drug-induced delay in the onset of IBa recovery from inactivation suggests that PAAs promote the transition to a deep inactivated channel conformation. These findings indicate that apparent PAA sensitivity of Ca2+ channels is not only defined by drug interaction with its receptor site but also crucially dependent on intrinsic gating properties of the channel molecule. A molecular model for PAA-Ca2+ channel interaction that accounts for the relationship between drug induced inactivation and channel block by PAA is proposed.

Characterization of a Low-Molecular-Weight Glutenin Subunit Gene from Bread Wheat and the Corresponding Protein That Represents a Major Subunit of the Glutenin Polymer1

Both high- and low-molecular-weight glutenin subunits (LMW-GS) play the major role in determining the viscoelastic properties of wheat (Triticum aestivum L.) flour. To date there has been no clear correspondence between the amino acid sequences of LMW-GS derived from DNA sequencing and those of actual LMW-GS present in the endosperm. We have characterized a particular LMW-GS from hexaploid bread wheat, a major component of the glutenin polymer, which we call the 42K LMW-GS, and have isolated and sequenced the putative corresponding gene. Extensive amino acid sequences obtained directly for this 42K LMW-GS indicate correspondence between this protein and the putative corresponding gene. This subunit did not show a cysteine (Cys) at position 5, in contrast to what has frequently been reported for nucleotide-based sequences of LMW-GS. This Cys has been replaced by one occurring in the repeated-sequence domain, leaving the total number of Cys residues in the molecule the same as in various other LMW-GS. On the basis of the deduced amino acid sequence and literature-based assignment of disulfide linkages, a computer-generated molecular model of the 42K subunit was constructed.

An information theory model of hydrophobic interactions.

A molecular model of poorly understood hydrophobic effects is heuristically developed using the methods of information theory. Because primitive hydrophobic effects can be tied to the probability of observing a molecular-sized cavity in the solvent, the probability distribution of the number of solvent centers in a cavity volume is modeled on the basis of the two moments available from the density and radial distribution of oxygen atoms in liquid water. The modeled distribution then yields the probability that no solvent centers are found in the cavity volume. This model is shown to account quantitatively for the central hydrophobic phenomena of cavity formation and association of inert gas solutes. The connection of information theory to statistical thermodynamics provides a basis for clarification of hydrophobic effects. The simplicity and flexibility of the approach suggest that it should permit applications to conformational equilibria of nonpolar solutes and hydrophobic residues in biopolymers.

Mutational analysis and molecular modeling of the nonapeptide hormone binding domains of the [Arg8]vasotocin receptor.

To identify determinants that form nonapeptide hormone binding domains of the white sucker Catostomus commersoni [Arg8]vasotocin receptor, chimeric constructs encoding parts of the vasotocin receptor and parts of the isotocin receptor have been analyzed by [(3,5-3H)Tyr2, Arg8]vasotocin binding to membranes of human embryonic kidney cells previously transfected with the different cDNA constructs and by functional expression studies in Xenopus laevis oocytes injected with mutant cRNAs. The results indicate that the N terminus and a region spanning the second extracellular loop and its flanking transmembrane segments, which contains a number of amino acid residues that are conserved throughout the nonapeptide receptor family, contribute to the affinity of the receptor for its ligand. Nonapeptide selectivity, however, is mainly defined by transmembrane region VI and the third extracellular loop. These results are complemented by a molecular model of the vasotocin receptor obtained by aligning its sequence with those of other G-protein coupled receptors as well as that of bacteriorhodopsin. The model indicates that amino acid residues of transmembrane regions II-VII that are located close to the extracellular surface also contribute to the binding of vasotocin.

Fine specificity of the antibody response to myelin basic protein in the central nervous system in multiple sclerosis: the minimal B-cell epitope and a model of its features.

T cells, B cells, and antibody are found in the white matter of the central nervous system in multiple sclerosis. The epitope center for the antibody response to human myelin basic protein (MBP) fits precisely the minimal epitope Pro85-Val-Val-His-Phe-Phe-Lys-Asn-Ile-Val-Thr-Pro96 for that reported for HLA DR2b (DRB1*1501)-restricted T cells that recognize MBP [Wucherpfenning, K.W., Sette, A., Southwood, S., Oseroff, C., Matsui, M., Strominger, J. & Hafler, D. (1994) J. Exp. Med. 179, 279-290], and overlaps with the reported DR2a-restricted epitope for T cells reactive to MBP [Martin, R., Howell, M. D., Jaraquemada, D., Furlage, M., Richert, J., Brostoff, S., Long, E. O., McFarlin, D. E. & McFarland, H. F. (1991) J. Exp. Med. 173, 19-24]. We describe a molecular model of this epitope.

The C9 methyl group of retinal interacts with glycine-121 in rhodopsin

The visual pigment rhodopsin is a prototypical G protein-coupled receptor. These receptors have seven transmembrane helices and are activated by specific receptor–ligand interactions. Rhodopsin is unusual in that its retinal prosthetic group serves as an antagonist in the dark in the 11-cis conformation but is rapidly converted to an agonist on photochemical cis to trans isomerization. Receptor–ligand interactions in rhodopsin were studied in the light and dark by regenerating site-directed opsin mutants with synthetic retinal analogues. A progressive decrease in light-dependent transducin activity was observed when a mutant opsin with a replacement of Gly121 was regenerated with 11-cis-retinal analogues bearing progressively larger R groups (methyl, ethyl, propyl) at the C9 position of the polyene chain. A progressive decrease in light activity was also observed as a function of increasing size of the residue at position 121 for both the 11-cis-9-ethyl- and the 11-cis-9-propylretinal pigments. In contrast, a striking increase of receptor activity in the dark—i.e., without chromophore isomerization—was observed when the molecular volume at either position 121 of opsin or C9 of retinal was increased. The ability of bulky replacements at either position to hinder ligand incorporation and to activate rhodopsin in the dark suggests a direct interaction between these two sites. A molecular model of the retinal-binding site of rhodopsin is proposed that illustrates the specific interaction between Gly121 and the C9 methyl group of 11-cis-retinal. Steric interactions in this region of rhodopsin are consistent with the proposal that movement of transmembrane helices 3 and 6 is concomitant with receptor activation.

Identification, retinoid binding, and x-ray analysis of a human retinol-binding protein

Two cellular retinol-binding proteins (CRBP I and II) with distinct tissue distributions and retinoid-binding properties have been recognized thus far in mammals. Here, we report the identification of a human retinol-binding protein resembling type I (55.6% identity) and type II (49.6% identity) CRBPs, but with a unique H residue in the retinoid-binding site and a distinctively different tissue distribution. Additionally, this binding protein (CRBP III) exhibits a remarkable sequence identity (62.2%) with the recently identified ι-crystallin/CRBP of the diurnal gecko Lygodactylus picturatus [Werten, P. J. L., Röll, B., van Alten, D. M. F. & de Jong, W. W. (2000) Proc. Natl. Acad. Sci. USA 97, 3282–3287 (First Published March 21, 2000; 10.1073/pnas.050500597)]. CRBP III and all-trans-retinol form a complex (Kd ≈ 60 nM), the absorption spectrum of which is characterized by the peculiar fine structure typical of the spectra of holo-CRBP I and II. As revealed by a 2.3-Å x-ray molecular model of apo-CRBP III, the amino acid residues that line the retinol-binding site in CRBP I and II are positioned nearly identically in the structure of CRBP III. At variance with the human CRBP I and II mRNAs, which are most abundant in ovary and intestine, respectively, the CRBP III mRNA is expressed at the highest levels in kidney and liver thus suggesting a prominent role for human CRBP III as an intracellular mediator of retinol metabolism in these tissues.

Symmetry in chemistry from the hydrogen atom to proteins

The last 2 decades have seen discoveries in highly excited states of atoms and molecules of phenomena that are qualitatively different from the “planetary” model of the atom, and the near-rigid model of molecules, characteristic of these systems in their low-energy states. A unified view is emerging in terms of approximate dynamical symmetry principles. Highly excited states of two-electron atoms display “molecular” behavior of a nonrigid linear structure undergoing collective rotation and vibration. Highly excited states of molecules described in the “standard molecular model” display normal mode couplings, which induce bifurcations on the route to molecular chaos. New approaches such as rigid–nonrigid correlation, vibrons, and quantum groups suggest a unified view of collective electronic motion in atoms and nuclear motion in molecules.

Dual-function regulators: the cAMP receptor protein and the CytR regulator can act either to repress or to activate transcription depending on the context.

Studies of gene regulation have revealed that several transcriptional regulators can switch between activator and repressor depending upon both the promoter and the cellular context. A relatively simple prokaryotic example is illustrated by the Escherichia coli CytR regulon. In this system, the cAMP receptor protein (CRP) assists the binding of RNA polymerase as well as a specific negative regulator, CytR. Thus, CRP functions either as an activator or as a corepressor. Here we show that, depending on promoter architecture, the CRP/CytR nucleoprotein complex has opposite effects on transcription. When acting from a site close to the DNA target for RNA polymerase, CytR interacts with CRP to repress transcription, whereas an interaction with CRP from appropriately positioned upstream binding sites can result in formation of a huge preinitiation complex and transcriptional activation. Based on recent results about CRP-mediated regulation of transcription initiation and the finding that CRP possesses discrete surface-exposed patches for protein-protein interaction with RNA polymerase and CytR, a molecular model for this dual regulation is discussed.

Crystal structure of Pseudomonas aeruginosa catabolic ornithine transcarbamoylase at 3.0-A resolution: a different oligomeric organization in the transcarbamoylase family.

The crystal structure of the Glu-105-->Gly mutant of catabolic ornithine transcarbamoylase (OTCase; carbamoyl phosphate + L-ornithine = orthophosphate + L-citrulline, EC 2.1.3.3) from Pseudomonas aeruginosa has been determined at 3.0-A resolution. This mutant is blocked in the active R (relaxed) state. The structure was solved by the molecular replacement method, starting from a crude molecular model built from a trimer of the catalytic subunit of another transcarbamoylase, the extensively studied aspartate transcarbamoylase (ATCase) from Escherichia coli. This model was used to generate initial low-resolution phases at 8-A resolution, which were extended to 3-A by noncrystallographic symmetry averaging. Four phase extensions were required to obtain an electron density map of very high quality from which the final model was built. The structure, including 4020 residues, has been refined to 3-A, and the current crystallographic R value is 0.216. No solvent molecules have been added to the model. The catabolic OTCase is a dodecamer composed of four trimers organized in a tetrahedral manner. Each monomer is composed of two domains. The carbamoyl phosphate binding domain shows a strong structural homology with the equivalent ATCase part. In contrast, the other domain, mainly implicated in the binding of the second substrate (ornithine for OTCase and aspartate for ATCase) is poorly conserved. The quaternary structures of these two allosteric transcarbamoylases are quite divergent: the E. coli ATCase has pseudo-32 point-group symmetry, with six catalytic and six regulatory chains; the catabolic OTCase has 23 point-group symmetry and only catalytic chains. However, both enzymes display homotropic and heterotropic cooperativity.

The CuA center of cytochrome-c oxidase: electronic structure and spectra of models compared to the properties of CuA domains.

The electronic structure and spectrum of several models of the binuclear metal site in soluble CuA domains of cytochrome-c oxidase have been calculated by the use of an extended version of the complete neglect of differential overlap/spectroscopic method. The experimental spectra have two strong transitions of nearly equal intensity around 500 nm and a near-IR transition close to 800 nm. The model that best reproduces these features consists of a dimer of two blue (type 1) copper centers, in which each Cu atom replaces the missing imidazole on the other Cu atom. Thus, both Cu atoms have one cysteine sulfur atom and one imidazole nitrogen atom as ligands, and there are no bridging ligands but a direct Cu-Cu bond. According to the calculations, the two strong bands in the visible region originate from exciton coupling of the dipoles of the two copper monomers, and the near-IR band is a charge-transfer transition between the two Cu atoms. The known amino acid sequence has been used to construct a molecular model of the CuA site by the use of a template and energy minimization. In this model, the two ligand cysteine residues are in one turn of an alpha-helix, whereas one ligand histidine is in a loop following this helix and the other one is in a beta-strand.

Erythrocyte membrane vesiculation: Model for the molecular mechanism of protein sorting

Budding and vesiculation of erythrocyte membranes occurs by a process involving an uncoupling of the membrane skeleton from the lipid bilayer. Vesicle formation provides an important means whereby protein sorting and trafficking can occur. To understand the mechanism of sorting at the molecular level, we have developed a micropipette technique to quantify the redistribution of fluorescently labeled erythrocyte membrane components during mechanically induced membrane deformation and vesiculation. Our previous studies indicated that the spectrin-based membrane skeleton deforms elastically, producing a constant density gradient during deformation. Our current studies showed that during vesiculation the skeleton did not fragment but rather retracted to the cell body, resulting in a vesicle completely depleted of skeleton. These local changes in skeletal density regulated the sorting of nonskeletal membrane components. Highly mobile membrane components, phosphatidylethanolamine- and glycosylphosphatidylinositol-linked CD59 with no specific skeletal association were enriched in the vesicle. In contrast, two components with known specific skeletal association, band 3 and glycophorin A, were differentially depleted in vesicles. Increasing the skeletal association of glycophorin A by liganding its extrafacial domain reduced the fraction partitioning to the vesicle. We conclude that this technique of bilayer/skeleton uncoupling provides a means with which to study protein sorting driven by changes in local skeletal density. Moreover, it is the interaction of particular membrane components with the spectrin-based skeleton that determines molecular partitioning during protein sorting.

Molecular mechanism of transmembrane signaling by the aspartate receptor: a model.

The aspartate receptor of bacterial chemotaxis is representative of a large class of membrane-spanning receptors found in prokaryotic and eukaryotic organisms. These receptors, which regulate histidine kinase pathways and possess two putative transmembrane helices per subunit, appear to control a wide variety of cellular processes. The best characterized subgroup of the two-helix receptor class is the homologous family of chemosensory receptors from Escherichia coli and Salmonella typhimurium, including the aspartate receptor. This receptor binds aspartate, an attractant, in the periplasmic compartment and undergoes an intramolecular, transmembrane conformational change, thereby modulating the autophosphorylation rate of a bound histidine kinase in the cytoplasm. Here, we analyze recent results from x-ray crystallographic, solution 19F NMR, and engineered disulfide studies probing the aspartate-induced structural change within the periplasmic and transmembrane regions of the receptor. Together, these approaches provide evidence that aspartate binding triggers a "swinging-piston" displacement of the second membrane-spanning helix, which is proposed to communicate the signal across the bilayer.