Crystal structures of the copper and nickel complexes of RNase A: Metal-induced interprotein interactions and identification of a novel copper binding motif

We report the crystal structures of the copper and nickel complexes of RNase A. The overall topology of these two complexes is similar to that of other RNase A structures. However, there are significant differences in the mode of binding of copper and nickel. There are two copper ions per molecule of the protein, but there is only one nickel ion per molecule of the protein. Significant changes occur in the interprotein interactions as a result of differences in the coordinating groups at the common binding site around His-105. Consequently, the copper- and nickel-ion-bound dimers of RNase A act as nucleation sites for generating different crystal lattices for the two complexes. A second copper ion is present at an active site residue His-119 for which all the ligands are from one molecule of the protein. At this second site, His-119 adopts an inactive conformation (B) induced by the copper. We have identified a novel copper binding motif involving the α-amino group and the N-terminal residues.

Comparison of x-ray crystal structures of an acyl-enzyme intermediate of subtilisin Carlsberg formed in anhydrous acetonitrile and in water

The x-ray crystal structures of trans-cinnamoyl–subtilisin, an acyl-enzyme covalent intermediate of the serine protease subtilisin Carlsberg, have been determined to 2.2-Å resolution in anhydrous acetonitrile and in water. The cinnamoyl–subtilisin structures are virtually identical in the two solvents. In addition, their enzyme portions are nearly indistinguishable from previously determined structures of the free enzyme in acetonitrile and in water; thus, acylation in either aqueous or nonaqueous solvent causes no appreciable conformational changes. However, the locations of bound solvent molecules in the active site of the acyl- and free enzyme forms in acetonitrile and in water are distinct. Such differences in the active site solvation may contribute to the observed variations in enzymatic activities. On prolonged exposure to organic solvent or removal of interstitial solvent from the crystal lattice, the channels within enzyme crystals are shown to collapse, leading to a drop in the number of active sites accessible to the substrate. The mechanistic and preparative implications of our findings for enzymatic catalysis in organic solvents are discussed.

Comparative analyses of the secondary structures of synthetic and intracellular yeast MFA2 mRNAs

The overall folded (global) structure of mRNA may be critical to translation and turnover control mechanisms, but it has received little experimental attention. Presented here is a comparative analysis of the basic features of the global secondary structure of a synthetic mRNA and the same intracellular eukaryotic mRNA by dimethyl sulfate (DMS) structure probing. Synthetic MFA2 mRNA of Saccharomyces cerevisiae first was examined by using both enzymes and chemical reagents to determine single-stranded and hybridized regions; RNAs with and without a poly(A) tail were compared. A folding pattern was obtained with the aid of the mfold program package that identified the model that best satisfied the probing data. A long-range structural interaction involving the 5′ and 3′ untranslated regions and causing a juxtaposition of the ends of the RNA, was examined further by a useful technique involving oligo(dT)-cellulose chromatography and antisense oligonucleotides. DMS chemical probing of A and C nucleotides of intracellular MFA2 mRNA was then done. The modification data support a very similar intracellular structure. When low reactivity of A and C residues is found in the synthetic RNA, ≈70% of the same sites are relatively more resistant to DMS modification in vivo. A slightly higher sensitivity to DMS is found in vivo for some of the A and C nucleotides predicted to be hybridized from the synthetic structural model. With this small mRNA, the translation process and mRNA-binding proteins do not block DMS modifications, and all A and C nucleotides are modified the same or more strongly than with the synthetic RNA.

Attenuated T2 relaxation by mutual cancellation of dipole–dipole coupling and chemical shift anisotropy indicates an avenue to NMR structures of very large biological macromolecules in solution

Fast transverse relaxation of 1H, 15N, and 13C by dipole-dipole coupling (DD) and chemical shift anisotropy (CSA) modulated by rotational molecular motions has a dominant impact on the size limit for biomacromolecular structures that can be studied by NMR spectroscopy in solution. Transverse relaxation-optimized spectroscopy (TROSY) is an approach for suppression of transverse relaxation in multidimensional NMR experiments, which is based on constructive use of interference between DD coupling and CSA. For example, a TROSY-type two-dimensional 1H,15N-correlation experiment with a uniformly 15N-labeled protein in a DNA complex of molecular mass 17 kDa at a 1H frequency of 750 MHz showed that 15N relaxation during 15N chemical shift evolution and 1HN relaxation during signal acquisition both are significantly reduced by mutual compensation of the DD and CSA interactions. The reduction of the linewidths when compared with a conventional two-dimensional 1H,15N-correlation experiment was 60% and 40%, respectively, and the residual linewidths were 5 Hz for 15N and 15 Hz for 1HN at 4°C. Because the ratio of the DD and CSA relaxation rates is nearly independent of the molecular size, a similar percentagewise reduction of the overall transverse relaxation rates is expected for larger proteins. For a 15N-labeled protein of 150 kDa at 750 MHz and 20°C one predicts residual linewidths of 10 Hz for 15N and 45 Hz for 1HN, and for the corresponding uniformly 15N,2H-labeled protein the residual linewidths are predicted to be smaller than 5 Hz and 15 Hz, respectively. The TROSY principle should benefit a variety of multidimensional solution NMR experiments, especially with future use of yet somewhat higher polarizing magnetic fields than are presently available, and thus largely eliminate one of the key factors that limit work with larger molecules.

NMR structures of three single-residue variants of the human prion protein

The NMR structures of three single-amino acid variants of the C-terminal domain of the human prion protein, hPrP(121–230), are presented. In hPrP(M166V) and hPrP(R220K) the substitution is with the corresponding residue in murine PrP, and in hPrP(S170N) it is with the corresponding Syrian hamster residue. All three substitutions are in the surface region of the structure of the cellular form of PrP (PrPC) that is formed by the C-terminal part of helix 3, with residues 218–230, and a loop of residues 166–172. This molecular region shows high species variability and has been implicated in specific interactions with a so far not further characterized “protein X,” and it is related to the species barrier for transmission of prion diseases. As expected, the three variant hPrP(121–230) structures have the same global architecture as the previously determined wild-type bovine, human, murine, and Syrian hamster prion proteins, but with the present study two localized “conformational markers” could be related with single amino acid exchanges. These are the length and quality of definition of helix 3, and the NMR-observability of the residues in the loop 166–172. Poor definition of the C-terminal part of helix 3 is characteristic for murine PrP and has now been observed also for hPrP(R220K), and NMR observation of the complete loop 166–172 has so far been unique for Syrian hamster PrP and is now also documented for hPrP(S170N).

Crystal structures of bovine milk xanthine dehydrogenase and xanthine oxidase: Structure-based mechanism of conversion

Mammalian xanthine oxidoreductases, which catalyze the last two steps in the formation of urate, are synthesized as the dehydrogenase form xanthine dehydrogenase (XDH) but can be readily converted to the oxidase form xanthine oxidase (XO) by oxidation of sulfhydryl residues or by proteolysis. Here, we present the crystal structure of the dimeric (Mr, 290,000) bovine milk XDH at 2.1-Å resolution and XO at 2.5-Å resolution and describe the major changes that occur on the proteolytic transformation of XDH to the XO form. Each molecule is composed of an N-terminal 20-kDa domain containing two iron sulfur centers, a central 40-kDa flavin adenine dinucleotide domain, and a C-terminal 85-kDa molybdopterin-binding domain with the four redox centers aligned in an almost linear fashion. Cleavage of surface-exposed loops of XDH causes major structural rearrangement of another loop close to the flavin ring (Gln 423—Lys 433). This movement partially blocks access of the NAD substrate to the flavin adenine dinucleotide cofactor and changes the electrostatic environment of the active site, reflecting the switch of substrate specificity observed for the two forms of this enzyme.

Crystal structures of eukaryotic translation initiation factor 5A from Methanococcus jannaschii at 1.8 Å resolution

Eukaryotic translation initiation factor 5A (eIF-5A) is a ubiquitous protein found in all eukaryotic cells. The protein is closely associated with cell proliferation in the G1–S stage of the cell cycle. Recent findings show that the eIF-5A proteins are highly expressed in tumor cells and act as a cofactor of the Rev protein in HIV-1-infected cells. The mature eIF is the only protein known to have the unusual amino acid hypusine, a post-translationally modified lysine. The crystal structure of eIF-5A from Methanococcus jannaschii (MJ eIF-5A) has been determined at 1.9 Å and 1.8 Å resolution in two crystal forms by using the multiple isomorphous replacement method and the multiwavelength anomalous diffraction method for the first crystal form and the molecular replacement method for the second crystal form. The structure consists of two folding domains, one of which is similar to the oligonucleotide-binding domain found in the prokaryotic cold shock protein and the translation initiation factor IF1 despite the absence of any significant sequence similarities. The 12 highly conserved amino acid residues found among eIF-5As include the hypusine site and form a long protruding loop at one end of the elongated molecule.

Structural code for DNA recognition revealed in crystal structures of papillomavirus E2-DNA targets

Transcriptional regulation in papillomaviruses depends on sequence-specific binding of the regulatory protein E2 to several sites in the viral genome. Crystal structures of bovine papillomavirus E2 DNA targets reveal a conformational variant of B-DNA characterized by a roll-induced writhe and helical repeat of 10.5 bp per turn. A comparison between the free and the protein-bound DNA demonstrates that the intrinsic structure of the DNA regions contacted directly by the protein and the deformability of the DNA region that is not contacted by the protein are critical for sequence-specific protein/DNA recognition and hence for gene-regulatory signals in the viral system. We show that the selection of dinucleotide or longer segments with appropriate conformational characteristics, when positioned at correct intervals along the DNA helix, can constitute a structural code for DNA recognition by regulatory proteins. This structural code facilitates the formation of a complementary protein–DNA interface that can be further specified by hydrogen bonds and nonpolar interactions between the protein amino acids and the DNA bases.

Structures of two molluscan hemocyanin genes: Significance for gene evolution

We present here the description of genes coding for molluscan hemocyanins. Two distantly related mollusks, Haliotis tuberculata and Octopus dofleini, were studied. The typical architecture of a molluscan hemocyanin subunit, which is a string of seven or eight globular functional units (FUs, designated a to h, about 50 kDa each), is reflected by the gene organization: a series of eight structurally related coding regions in Haliotis, corresponding to FU-a to FU-h, with seven highly variable linker introns of 174 to 3,198 bp length (all in phase 1). In Octopus seven coding regions (FU-a to FU-g) are found, separated by phase 1 introns varying in length from 100 bp to 910 bp. Both genes exhibit typical signal (export) sequences, and in both cases these are interrupted by an additional intron. Each gene also contains an intron between signal peptide and FU-a and in the 3′ untranslated region. Of special relevance for evolutionary considerations are introns interrupting those regions that encode a discrete functional unit. We found that five of the eight FUs in Haliotis each are encoded by a single exon, whereas FU-f, FU-g, and FU-a are encoded by two, three and four exons, respectively. Similarly, in Octopus four of the FUs each correspond to an uninterrupted exon, whereas FU-b, FU-e, and FU-f each contain a single intron. Although the positioning of the introns between FUs is highly conserved in the two mollusks, the introns within FUs show no relationship either in location nor phase. It is proposed that the introns between FUs were generated as the eight-unit polypeptide evolved from a monomeric precursor, and that the internal introns have been added later. A hypothesis for evolution of the ring-like quaternary structure of molluscan hemocyanins is presented.

Crystallographic structures of the ligand-binding domains of the androgen receptor and its T877A mutant complexed with the natural agonist dihydrotestosterone

The structures of the ligand-binding domains (LBD) of the wild-type androgen receptor (AR) and the T877A mutant corresponding to that in LNCaP cells, both bound to dihydrotestosterone, have been refined at 2.0 Å resolution. In contrast to the homodimer seen in the retinoid-X receptor and estrogen receptor LBD structures, the AR LBD is monomeric, possibly because of the extended C terminus of AR, which lies in a groove at the dimerization interface. Binding of the natural ligand dihydrotestosterone by the mutant LBD involves interactions with the same residues as in the wild-type receptor, with the exception of the side chain of threonine 877, which is an alanine residue in the mutant. This structural difference in the binding pocket can explain the ability of the mutant AR found in LNCaP cells (T877A) to accommodate progesterone and other ligands that the wild-type receptor cannot.

Crystal structures of the vitamin D receptor complexed to superagonist 20-epi ligands

The crystal structures of the ligand-binding domain (LBD) of the vitamin D receptor complexed to 1α,25(OH)2D3 and the 20-epi analogs, MC1288 and KH1060, show that the protein conformation is identical, conferring a general character to the observation first made for retinoic acid receptor (RAR) that, for a given LBD, the agonist conformation is unique, the ligands adapting to the binding pocket. In all complexes, the A- to D-ring moieties of the ligands adopt the same conformation and form identical contacts with the protein. Differences are observed only for the 17β-aliphatic chains that adapt their conformation to anchor the 25-hydroxyl group to His-305 and His-397. The inverted geometry of the C20 methyl group induces different paths of the aliphatic chains. The ligands exhibit a low-energy conformation for MC1288 and a more strained conformation for the two others. KH1060 compensates this energy cost by additional contacts. Based on the present data, the explanation of the superagonist effect is to be found in higher stability and longer half-life of the active complex, thereby excluding different conformations of the ligand binding domain.

The structures of anthranilate synthase of Serratia marcescens crystallized in the presence of (i) its substrates, chorismate and glutamine, and a product, glutamate, and (ii) its end-product inhibitor, l-tryptophan

The crystal structure of anthranilate synthase (AS) from Serratia marcescens, a mesophilic bacterium, has been solved in the presence of its substrates, chorismate and glutamine, and one product, glutamate, at 1.95 Å, and with its bound feedback inhibitor, tryptophan, at 2.4 Å. In comparison with the AS structure from the hyperthermophile Sulfolobus solfataricus, the S. marcescens structure shows similar subunit structures but a markedly different oligomeric organization. One crystal form of the S. marcescens enzyme displays a bound pyruvate as well as a putative anthranilate (the nitrogen group is ambiguous) in the TrpE subunit. It also confirms the presence of a covalently bound glutamyl thioester intermediate in the TrpG subunit. The tryptophan-bound form reveals that the inhibitor binds at a site distinct from that of the substrate, chorismate. Bound tryptophan appears to prevent chorismate binding by a demonstrable conformational effect, and the structure reveals how occupancy of only one of the two feedback inhibition sites can immobilize the catalytic activity of both TrpE subunits. The presence of effectors in the structure provides a view of the locations of some of the amino acid residues in the active sites. Our findings are discussed in terms of the previously described AS structure of S. solfataricus, mutational data obtained from enteric bacteria, and the enzyme's mechanism of action.

An approach to three-dimensional structures of biomolecules by using single-molecule diffraction images

We describe an approach to the high-resolution three-dimensional structural determination of macromolecules that utilizes ultrashort, intense x-ray pulses to record diffraction data in combination with direct phase retrieval by the oversampling technique. It is shown that a simulated molecular diffraction pattern at 2.5-Å resolution accumulated from multiple copies of single rubisco biomolecules, each generated by a femtosecond-level x-ray free electron laser pulse, can be successfully phased and transformed into an accurate electron density map comparable to that obtained by more conventional methods. The phase problem is solved by using an iterative algorithm with a random phase set as an initial input. The convergence speed of the algorithm is reasonably fast, typically around a few hundred iterations. This approach and phasing method do not require any ab initio information about the molecule, do not require an extended ordered lattice array, and can tolerate high noise and some missing intensity data at the center of the diffraction pattern. With the prospects of the x-ray free electron lasers, this approach could provide a major new opportunity for the high-resolution three-dimensional structure determination of single biomolecules.

Comparative Analysis of the Regulation of Expression and Structures of Two Evolutionarily Divergent Genes for Δ1-Pyrroline-5-Carboxylate Synthetase from Tomato1

We isolated two tomato (Lycopersicon esculentum) cDNA clones, tomPRO1 and tomPRO2, specifying Δ1-pyrroline-5-carboxylate synthetase (P5CS), the first enzyme of proline (Pro) biosynthesis. tomPRO1 is unusual because it resembles prokaryotic polycistronic operons (M.G. García-Ríos, T. Fujita, P.C. LaRosa, R.D. Locy, J.M. Clithero, R.A. Bressan, L.N. Csonka [1997] Proc Natl Acad Sci USA 94: 8249–8254), whereas tomPRO2 encodes a full-length P5CS. We analyzed the accumulation of Pro and the tomPRO1 and tomPRO2 messages in response to NaCl stress and developmental signals. Treatment with 200 mm NaCl resulted in a >60-fold increase in Pro levels in roots and leaves. However, there was a <3-fold increase in the accumulation of the tomPRO2 message and no detectable induction in the level of the tomPRO1 message in response to NaCl stress. Although pollen contained approximately 100-fold higher levels of Pro than other plant tissues, there was no detectable increase in the level of either message in pollen. We conclude that transcriptional regulation of these genes for P5CS is probably not important for the osmotic or pollen-specific regulation of Pro synthesis in tomato. Using restriction fragment-length polymorphism mapping, we determined the locations of tomPRO1 and tomPRO2 loci in the tomato nuclear genome. Sequence comparison suggested that tomPRO1 is similar to prokaryotic P5CS loci, whereas tomPRO2 is closely related to other eukaryotic P5CS genes.

Family of MADS-Box Genes Expressed Early in Male and Female Reproductive Structures of Monterey Pine

Three MADS-box genes isolated from Monterey pine (Pinus radiata), PrMADS1, PrMADS2, and PrMADS3, are orthologs to members of the AGL2 and AGL6 gene subfamilies in Arabidopsis. These genes were expressed during early stages of pine shoot development in differentiating seed- and pollen-cone buds. Their transcripts were found within a group of cells that formed ovuliferous scale and microsporophyll primordia. Expression of PrMADS3 was also detected in a group of cells giving rise to needle primordia within differentiated vegetative buds, and in needle primordia.