Two conformational states in the crystal structure of the Homo sapiens cytoplasmic ribosomal decoding A site

The decoding A site of the small ribosomal subunit is an RNA molecular switch, which monitors codon–anticodon interactions to guarantee translation fidelity. We have solved the crystal structure of an RNA fragment containing two Homo sapiens cytoplasmic A sites. Each of the two A sites presents a different conformational state. In one state, adenines A1492 and A1493 are fully bulged-out with C1409 forming a wobble-like pair to A1491. In the second state, adenines A1492 and A1493 form non-Watson–Crick pairs with C1409 and G1408, respectively while A1491 bulges out. The first state of the eukaryotic A site is, thus, basically the same as in the bacterial A site with bulging A1492 and A1493. It is the state used for recognition of the codon/anticodon complex. On the contrary, the second state of the H.sapiens cytoplasmic A site is drastically different from any of those observed for the bacterial A site without bulging A1492 and A1493.     

Three-dimensional structures of the mammalian multidrug resistance P-glycoprotein demonstrate major conformational changes in the transmembrane domains upon nucleotide binding

P-glycoprotein is an ATP-binding cassette transporter that is associated with multidrug resistance and the failure of chemotherapy in human patients. We have previously shown, based on two-dimensional projection maps, that P-glycoprotein undergoes conformational changes upon binding of nucleotide to the intracellular nucleotide binding domains. Here we present the three-dimensional structures of P-glycoprotein in the presence and absence of nucleotide, at a resolution limit of approximately 2 nm, determined by electron crystallography of negatively stained crystals. The data reveal a major reorganization of the transmembrane domains throughout the entire depth of the membrane upon binding of nucleotide. In the absence of nucleotide, the two transmembrane domains form a single barrel 5-6 nm in diameter and about 5 nm deep with a central pore that is open to the extracellular surface and spans much of the membrane depth. Upon binding nucleotide, the transmembrane domains reorganize into three compact domains that are each 2-3 nm in diameter and 5-6 nm deep. This reorganization opens the central pore along its length in a manner that could allow access of hydrophobic drugs (transport substrates) directly from the lipid bilayer to the central pore of the transporter.     

Acetylcholinesterase in motion: visualizing conformational changes in crystal structures by a morphing procedure

In order to visualize and appreciate conformational changes between homologous three-dimensional (3D) protein structures or protein/inhibitor complexes, we have developed a user-friendly morphing procedure. It enabled us to detect coordinated conformational changes not easily discernible by analytic methods or by comparison of static images. This procedure was applied to comparison of native Torpedo californica acetylcholinesterase and of complexes with reversible inhibitors and conjugates with covalent inhibitors. It was likewise shown to be valuable for the visualization of conformational differences between acetylcholinesterases from different species. The procedure involves generation, in Cartesian space, of 25 interpolated intermediate structures between the initial and final 3D structures, which then serve as the individual frames in a QuickTime movie.     

Hysteresis and conformational changes in ribosomal ribonucleic acid

Both rat liver and Escherichia coli rRNA in 0.1m-sodium chloride were titrated with acid or alkali over the range pH3-7 at approx. 0 degrees C. rRNA did not bind acid reversibly and hysteresis was observed, i.e. the plot of acid bound to rRNA against pH had the form of a loop showing that the amount of acid bound at a particular pH depended on the direction of the titration. Although the boundary curves were reproducibly followed on titration from pH7 to 3 and from pH3 to 7, points within the loop were ;scanned', e.g. by titration from pH7 to a point in the range pH3-4 followed by titration with alkali to pH7. It is inferred that the ;lag' in the release of certain bound protons is at least 1 pH unit, that at least about 9-15% of the titratable groups (adenine and cytosine residues) that are involved in this process and that the free energy dissipated in completing a cycle is approx. 4.2kJ/mol (1kcal/mol) of nucleotide involved in hysteresis. The interpretation of the ;scanning' curves was illustrated by means of a cycle of possible changes in the conformation of a hypothetical nucleotide sequence that allows formation of poly(A).poly(AalphaH(+))-like regions in acidic solutions. It is also inferred that the extent of ;hysteresis' might depend on the primary nucleotide sequence of rRNA as well as on secondary structure.     

A comparison of the crystal structures of eukaryotic and bacterial SSU ribosomal RNAs reveals common structural features in the hypervariable regions

While the majority of the ribosomal RNA structure is conserved in the three major domains of life--archaea, bacteria, and eukaryotes, specific regions of the rRNA structure are unique to at least one of these three primary forms of life. In particular, the comparative secondary structure for the eukaryotic SSU rRNA contains several regions that are different from the analogous regions in the bacteria. Our detailed analysis of two recently determined eukaryotic 40S ribosomal crystal structures, Tetrahymena thermophila and Saccharomyces cerevisiae, and the comparison of these results with the bacterial Thermus thermophilus 30S ribosomal crystal structure: (1) revealed that the vast majority of the comparative structure model for the eukaryotic SSU rRNA is substantiated, including the secondary structure that is similar to both bacteria and archaea as well as specific for the eukaryotes, (2) resolved the secondary structure for regions of the eukaryotic SSU rRNA that were not determined with comparative methods, (3) identified eukaryotic helices that are equivalent to the bacterial helices in several of the hypervariable regions, (4) revealed that, while the coaxially stacked compound helix in the 540 region in the central domain maintains the constant length of 10 base pairs, its two constituent helices contain 5+5 bp rather than the 6+4 bp predicted with comparative analysis of archaeal and eukaryotic SSU rRNAs.     

Ligand-induced conformational changes in the crystal structures of Pneumocystis carinii dihydrofolate reductase complexes with folate and NADP+

Structural data from two independent crystal forms (P212121 and P21) of the folate (FA) binary complex and from the ternary complex with the oxidized coenzyme, NADP+, and recombinant Pneumocystis carinii dihydrofolate reductase (pcDHFR) refined to an average of 2.15 A resolution, show the first evidence of ligand-induced conformational changes in the structure of pcDHFR. These data are also compared with the crystal structure of the ternary complex of methotrexate (MTX) with NADPH and pcDHFR in the monoclinic lattice with data to 2.5 A resolution. Comparison of the data for the FA binary complex of pcDHFR with those for the ternary structures reveals significant differences, with a >7 A movement of the loop region near residue 23 that results in a new "flap-open" position for the binary complex, and a "closed" position in the ternary complexes, similar to that reported for Escherichia coli (ec) DHFR complexes. In the orthorhombic lattice for the binary FA pcDHFR complex, there is also an unwinding of a short helical region near residue 47 that places hydrophobic residues Phe-46 and Phe-49 toward the outer surface, a conformation that is stabilized by intermolecular packing contacts. The pyrophosphate moiety of NADP+ in the ternary folate pcDHFR complexes shows significant differences in conformation compared with that observed in the MTX-NADPH-pcDHFR ternary complex. Additionally, comparison of the conformations among these four pcDHFR structures reveals evidence for subdomain movement that correlates with cofactor binding states. The larger binding site access in the new "flap-open" loop 23 conformation of the binary FA complex is consistent with the rapid release of cofactor from the product complex during catalysis as well as the more rapid release of substrate product from the binary complex as a result of the weaker contacts of the closed loop 23 conformation, compared to ecDHFR.     

Evaluating conformational changes in protein structures binding RNA

Many protein-RNA recognition events are known to exhibit conformational changes from qualitative observations of individual complexes. However, a quantitative estimation of conformational changes is required if protein-RNA docking and template-based methods for RNA binding site prediction are to be developed. This study presents the first quantitative evaluation of conformational changes that occur when proteins bind RNA. The analysis of twelve RNA-binding proteins in the bound and unbound states using error-scaled difference distance matrices is presented. The binding site residues are mapped to each structure, and the conformational changes that affect these residues are evaluated. Of the twelve proteins four exhibit greater movements in nonbinding site residues, and a further four show the greatest movements in binding site residues. The remaining four proteins display no significant conformational change. When interface residues are found to be in conformationally variable regions of the protein they are typically seen to move less than 2 A between the bound and unbound conformations. The current data indicate that conformational changes in the binding site residues of RNA binding proteins may not be as significant as previously suggested, but a larger data set is required before wider conclusions may be drawn. The implications of the observed conformational changes for protein function prediction are discussed.     

Redox-dependent conformational changes in eukaryotic cytochromes revealed by paramagnetic NMR spectroscopy

Cytochrome c (Cc) is a soluble electron carrier protein, transferring reducing equivalents between Cc reductase and Cc oxidase in eukaryotes. In this work, we assessed the structural differences between reduced and oxidized Cc in solution by paramagnetic NMR spectroscopy. First, we have obtained nearly-complete backbone NMR resonance assignments for iso-1-yeast Cc and horse Cc in both oxidation states. These were further used to derive pseudocontact shifts (PCSs) arising from the paramagnetic haem group. Then, an extensive dataset comprising over 450 measured PCSs and high-resolution X-ray and solution NMR structures of both proteins were used to define the anisotropic magnetic susceptibility tensor, Δχ. For most nuclei, the PCSs back-calculated from the Δχ tensor are in excellent agreement with the experimental PCS values. However, several contiguous stretches—clustered around G41, N52, and A81—exhibit large deviations both in yeast and horse Cc. This behaviour is indicative of redox-dependent structural changes, the extent of which is likely conserved in the protein family. We propose that the observed discrepancies arise from the changes in protein dynamics and discuss possible functional implications.     

Detection of cation-specific conformational changes in ribosomal RNA by gel electrophoresis

Electrophoresis of ribosomal RNA in polyacrylamide-agarose composite gels separates 16S and 23S species into multiple bands. These bands of RNA represent multiple conformational forms of the molecules as judged by oligonucleotide analysis of the 16S RNA. Gel electrophoresis was used to test for cation-specific conformational changes in ribosomal RNA. Relative to magnesium-equilibrated RNA, barium ion and putrescine induced alterations in the electrophoretic behavior of ribosomal RNA while calcium ion produced no change. Exchange of a critical level of bound magnesium ion for barium or putrescine was necessary for these changes to take place. The alterations in electrophoretic behavior were unaffected by simply restoring magnesium ion, but in addition required heating for reversal. We suggest that these conformational changes are a result of interaction at a specific class of cation binding sites previously observed with intact ribosomes.     

Two crystal structures of the leupeptin-trypsin complex.

Three-dimensional structures of trypsin with the reversible inhibitor leupeptin have been determined in two different crystal forms. The first structure was determined at 1.7 A resolution with R-factor = 17.7% in the trigonal crystal space group P3(1)21, with unit cell dimensions of a = b = 55.62 A, c = 110.51 A. The second structure was determined at a resolution of 1.8 A with R-factor = 17.5% in the orthorhombic space group P2(1)2(1)2(1), with unit cell dimensions of a = 63.69 A, b = 69.37 A, c = 63.01 A. The overall protein structure is very similar in both crystal forms, with RMS difference for main-chain atoms of 0.27 A. The leupeptin backbone forms four hydrogen bonds with trypsin and a fifth hydrogen bond interaction is mediated by a water molecule. The aldehyde carbonyl of leupeptin forms a covalent bond of 1.42 A length with side-chain oxygen of Ser-195 in the active site. The reaction of trypsin with leupeptin proceeds through the formation of stable tetrahedral complex in which the hemiacetal oxygen atom is pointing out of the oxyanion hole and forming a hydrogen bond with His-57.     

Trimetrexate: molecular structures and conformational similarities in two crystal forms

The structure of the non-classical quinazoline antifolate trimetrexate (TMQ) has been determined in two crystal forms, TMQ acetate monohydrate, and hydrated TMQ free base. Trimetrexate has an extended conformation in both structures, and the quinazoline and phenyl rings are mutually perpendicular. Protonation occurs at N1 in the acetate salt. The TMQ conformation is similar to corresponding parts of quinespar, the only other quinazoline antifolate structurally determined, and the hydrated strontium salt of methotrexate.     

Detection of cation-specific conformational changes in ribosomal RNA by gel electrophoresis.

Electrophoresis of ribosomal RNA in polyacrylamide-agrose composite gels separates 16S and 23S species into multiple bands. These bands of RNA represent multiple conformational forms of the molecules as judged by oligonucleotide analysis of the 16S RNA. Gel elctrophoresis was used to test for cation-specific conformational changes in ribosomal RNA. Relative to magnesium-equilibrated RNA, barium ion and putrescine induced alterations in the electrophoretic behavior of ribosomal RNA while calcium ion produced no change. Exchange of a critical level of bound magnesium ion for barium or putrescine was necessary for these changes to take place. The alterations in electrophoretic behavior were unaffected by simply restoring magnesium ion, but in addition required heating for reversal. We suggest that these conformational changes are a result of interaction at a specific class of cation binding sites previously observed with intact ribosomes.     

New structures of allosteric proteins revealing remarkable conformational changes

New three-dimensional structures of allosteric proteins reveal they have a flexible architecture that is instrumental to the regulation of protein function. Highlights are the structures of GroEL, pyruvate kinase, -3-phosphoglycerate dehydrogenase and the acetylcholine receptor. Furthermore, significant progress in understanding the nature of the intermediates involved in an allosteric reaction has been achieved through recent spectroscopic and crystallographic studies on haemoglobin.     

Control of the transmembrane phospholipid distribution in eukaryotic cells by aminophospholipid translocase

The aminophospholipid translocase is a plasma membrane Mg2(+)-ATPase which selectively pumps the aminophospholipids (phosphatidylserine and phosphatidylethanolamine) from the outer to the inner monolayer in eukaryotic cells and is predominantly responsible for the asymmetric phospholipid distribution of the plasma membrane. Similar ATP-dependent transport of phospholipid takes place in some organelles such as chromaffin granules. On the other hand, the phospholipid flippase of rat liver endoplasmic reticulum does not require ATP and has a low lipid specificity. The biological implications of these phospholipid flippases are discussed.     

Protein-induced conformational changes in 16 S ribosomal RNA during the initial assembly steps of the Escherichia coli 30 S ribosomal subunit

The mechanism of 16 S ribosomal RNA folding into its compact form in the native 30 S ribosomal subunit of Escherichia coli was studied by scanning transmission electron microscopy and circular dichroism spectroscopy. This approach made it possible to visualize and quantitatively analyze the conformational changes induced in 16 S rRNA under various ionic conditions and to characterize the interactions of ribosomal proteins S4, S8, S15, S20, S17 and S7, the six proteins known to bind to 16 S rRNA in the initial assembly steps. 16 S rRNA and the reconstituted RNA-protein core particles were characterized by their mass, morphology, radii of gyration (RG), and the extent and stability of 16 S rRNA secondary structure. The stepwise binding of S4, S8 and S15 led to a corresponding increase of mass and was accompanied by increased folding of 16 S rRNA in the core particles, as evident from the electron micrographs and from the decrease of RG values from 114 A and 91 A. Although the binding of S20, S17 and S7 continued the trend of mass increase, the RG values of these core particles showed a variable trend. While there was a slight increase in the RG value of the S20 core particles to 94 A, the RG value remained unchanged (94 A) with the further addition of S17. With subsequent addition of S7 to the core particles, the RG values showed an increase to 108 A. Association with S7 led to the formation of a globular mass cluster with a diameter of about 115 A and a mass of about 300 kDa. The rest of the mass (about 330 kDa) remained loosely coiled, giving the core particle a "medusa-like" appearance. Morphology of the 16 S rRNA and 16 S rRNA-protein core particles, even those with all six proteins, does not resemble the native 30 S subunit, contrary to what has been reported by others. The circular dichroism spectra of the 16 S rRNA-protein complexes and of free 16 S rRNA indicate a similarity of RNA secondary structure in the core particles with the first four proteins, S4, S8, S15, S20. The circular dichroism melting profiles of these core particles show only insignificant variations, implying no obvious changes in the distribution or the stability of the helical segments of 16 S rRNA. However, subsequent binding of proteins S17 and S7 affected both the extent and the thermal stability of 16 S rRNA secondary structure.(ABSTRACT TRUNCATED AT 400 WORDS)     

Studies on the sites expressing evolutionary changes in the structure of eukaryotic 5S ribosomal RNA

Summary We have determined the complete sequences of 5S rRNAs from a lamprey (Lampetra reissneri), a lancelet (Branchiostoma belcheri), silkworms (Philosamia cynthia ricini, Bombyx mori, Antheraea pernyi), and a silkworm hybrid (artificially fertilized hybrid species ofPhilosamia cynthia ricini x Bombyx mori ), as well as those of cotton seeds (Gossypium hirsutum L.). Having compared more than 170 eukaryotic 5S rRNAs of which seven sequences have been determined by our group as mentioned above, we have found that the evolutionary sites that exist at special locations in these structures are closely related to the evolution of eukaryotes. The changes proceed step by step in an orderly way, i.e., the change in nucleotide residues of the evolutionary sites depends on the order of the evolution of the species and shows group-specific patterns.     

The structures of four macrolide antibiotics bound to the large ribosomal subunit

Crystal structures of the Haloarcula marismortui large ribosomal subunit complexed with the 16-membered macrolide antibiotics carbomycin A, spiramycin, and tylosin and a 15-membered macrolide, azithromycin, show that they bind in the polypeptide exit tunnel adjacent to the peptidyl transferase center. Their location suggests that they inhibit protein synthesis by blocking the egress of nascent polypeptides. The saccharide branch attached to C5 of the lactone rings extends toward the peptidyl transferase center, and the isobutyrate extension of the carbomycin A disaccharide overlaps the A-site. Unexpectedly, a reversible covalent bond forms between the ethylaldehyde substituent at the C6 position of the 16-membered macrolides and the N6 of A2103 (A2062, E. coli). Mutations in 23S rRNA that result in clinical resistance render the binding site less complementary to macrolides.