Structure of the crystalline complex between ribonuclease A and D(pA)4.

Crystals of a complex formed between ribonuclease A and d(pA)4 were grown and their structure determined by a combination of multiple isomorphous replacement (MIR) and molecular replacement techniques. The known structure of ribonuclease A in the correct orientation in the unit cell yielded a conventional crystallographic R factor of 0.32 at 2.8 A resolution when refined as a rigid body. Difference Fourier syntheses permitted determination of the disposition of the DNA in the unit cell. Refinement of both protein and DNA by constrained-restrained least squares procedures resulted in an R factor of 0.22 at 2.5 A resolution. The structure of the crystalline complex is comprised of four ordered oligomers of d(pA)4 associated with each molecule of RNAse. If the sites of interaction between protein and d(pA)4 fragments are mapped on the surface of the protein, they describe an essentially continuous path into and through the active site, across the surface of the enzyme and finally into the basic amino acid cluster on the opposite side of the protein.     

The crystal structure of ribonuclease B at 2.5-A resolution

The glycosylated form of bovine pancreatic ribonuclease, RNase B, was crystallized from polyethylene glycol 4000 at low ionic strength in space group C2 with unit cell dimensions of a = 101.81 A, b = 33.36 A, c = 73.60 A, and beta = 90.4 degrees. The crystals, which contained two independent molecules of RNase B as the asymmetric unit, were solved by a combination of multiple isomorphous replacement and molecular replacement approaches. The structures of the two molecules were refined to 2.5-A resolution and a conventional R factor of 0.22 using a constrained-restrained least squares procedure (CORELS). Complexes were also investigated of RNase B plus ruthenium pentaamine and between RNase B and a substrate analogue iodouridine. The polypeptide backbones of the two molecules of RNase B in the asymmetric unit were found to be statistically identical and their differences from RNase A to be statistically insignificant. The carbohydrate chains of both molecules extended into solvent cavities in the crystal lattice and appear to be disordered for the most part. The oligosaccharides appear to exert no influence on the structure of the protein. Iodouridine was observed to bind identically in the pyrimidine site of both RNase B molecules and in a way apparently the same as that previously observed for RNase A. Ruthenium pentaamine bound at histidine 105 of both RNase B molecules in the asymmetric unit, but at a number of secondary sites as well. An array of bound ions was observed by Fo-Fc difference Fourier syntheses. These ions were proximal to lysine and arginine residues at the surface of the proteins while a pair of strong ion binding sites were seen to fall exactly in the active site clefts of both RNase B molecules in the asymmetric unit.     

5'-modified agonist and antagonist (2'-5')(A)n analogues: synthesis and biological activity

Two 5'-modified (2'-5')(A)4 oligomers with an increased resistance to phosphatase degradation were synthesized and evaluated for their ability to develop an antiviral response when introduced into intact cells by microinjection or by chemical conjugation to poly(L-lysine). The enzymatic synthesis of 5'-gamma-phosphorothioate and beta,gamma-difluoromethylene (2'-5')(A)4 from adenosine 5'-O-(3-thiotriphosphate) and adenosine beta,gamma-difluoromethylenetriphosphate by (2'-5')-oligoadenylate synthetase is described. The isolation and characterization of these (2'-5')(A)4 analogues were achieved by high-performance liquid chromatography. The structures of 5'-modified tetramers were corroborated by enzyme digestion. These two 5'-modified tetramers compete as efficiently as natural (2'-5')(A)4 for the binding of a radiolabeled (2'-5')(A)4 probe to ribonuclease (RNase) L. Nevertheless, at the opposite to 5'-gamma-phosphorothioate (2'-5')(A)4, beta,gamma-difluoromethylene (2'-5')(A)4 failed to induce an antiviral response after microinjection in HeLa cells. In addition, it behaves as an antagonist of RNase L as demonstrated by its ability to inhibit the antiviral properties of 5'-gamma-phosphorothioate (2'-5')(A)4 when both are microinjected in HeLa cells. The increased metabolic stability of 5'-gamma-phosphorothioate (2'-5')(A)4 as compared to that of (2'-5')(A)4 was first demonstrated in cell-free extracts and then confirmed in intact cells after introduction in the form of a conjugate to poly(L-lysine). Indeed, 5'-gamma-phosphorothioate (2'-5')(A)4-poly(L-lysine) conjugate induces protein synthesis inhibition and characteristic ribosomal RNA cleavages for longer times than unmodified (2'-5')(A)4-poly(L-lysine) in the same cell system.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antitumor activity and other biological actions of oligomers of ribonuclease A

Dimers, trimers, and tetramers of bovine ribonuclease A, obtained by lyophilization of the enzyme from 40% acetic acid solutions, were purified and isolated by cation exchange chromatography. The two conformers constituting each aggregated species were assayed for their antitumor, aspermatogenic, or embryotoxic activities in comparison with monomeric RNase A and bovine seminal RNase, which is dimeric in nature. The antitumor action was tested in vitro on ML-2 (human myeloid leukemia) and HL-60 (human myeloid cell line) cells and in vivo on the growth of human non-pigmented melanoma (line UB900518) transplanted subcutaneously in nude mice. RNase A oligomers display a definite antitumor activity that increases as a function of the size of the oligomers. On ML-2 and HL-60 cells, dimers and trimers generally show a lower activity than bovine seminal RNase; the activity of tetramers, instead, is similar to or higher than that of the seminal enzyme. The growth of human melanoma in nude mice is inhibited by RNase A oligomers in the order dimers < trimers < tetramers. The action of the two tetramers is very strong, blocking almost completely the growth of melanoma. RNase A dimers, trimers, and tetramers display aspermatogenic effects similar to those of bovine seminal RNase, but, contrarily, they do not show any embryotoxic activity.     

Crystal structure disposition of thymidylic acid tetramer in complex with ribonuclease A.

The crystal structure of ribonuclease A with bound thymidylic acid tetramer is reported at 2.5-A resolution. The diffusion of the tetramer into native orthorhombic crystals of the ribonuclease allows for the formation of a structurally stable complex where the single-stranded nucleic acid enters and leaves the enzyme's catalytic region in a persistent 5'-3' direction. The binding of the tetramer to the enzyme's surface is facilitated and mediated by electrostatic interactions between basic protein residues and nucleotide phosphates. Two pyrimidine nucleotides are bound to the enzyme's active site in a manner similar to that observed for other complexes between ribonuclease A and nucleic acid oligomers.     

Mechanisms of degradation of 2′-5′ oligoadenylates

We have studied the mechanisms of breakdown of 2'-5' oligoadenylates. We monitored the time-courses of degradation of ppp(A2'p5')nA (dimer to tetramer) and of 5'OH-(A2'p5')nA (dimer to pentamer) in unfractionated L1210 cell extract. The 5' triphosphorylated 2'-5' oligoadenylates are converted by a phosphatase activity. However, 2'-5' oligoadenylates are degraded mainly by phosphodiesterase activity which splits the 2'-5' phosphodiester bond sequentially at the 2' end to yield 5' AMP and one-unit-shorter oligomers. The nonlinear least-squares curve-fitting program CONSAM was used to fit these kinetics and to determine the degradation rate constant of each oligomer. Trimers and tetramers, whether 5' triphosphorylated or not, are degraded at the same rate, whereas 5' triphosphorylated dimer is rapidly hydrolyzed and 5'-OH dimer is the most stable oligomer. The interaction between degradation enzymes and the substrate strongly depends on the presence of a 5' phosphate group in the vicinity of the phosphodiester bond to be hydrolyzed; indeed, when this 5' phosphate group is present, as in pp/pA2'p5'A/or A2'/p5'A2'p5'A/, affinity is high and maximal velocity is low. Such a degradation pattern can control the concentration of 2'-5' oligoadenylates active on RNAse L either by limiting their synthesis (5' triphosphorylated dimer is the primer necessary for the formation of longer oligomers) and/or by converting them into inhibitory (e.g., monophosphorylated trimer) or inactive (e.g., nonphosphorylated oligomers) molecules.     

The three-dimensional structure of diaminopimelate decarboxylase from Mycobacterium tuberculosis reveals a tetrameric enzyme organisation

The three-dimensional structure of the enzyme diaminopimelate decarboxylase from Mycobacterium tuberculosis has been determined in a new crystal form and refined to a resolution of 2.33 Å. The monoclinic crystals contain one tetramer exhibiting D₂-symmetry in the asymmetric unit. The tetramer exhibits a donut-like structure with a hollow interior. All four active sites are accessible only from the interior of the tetrameric assembly. Small-angle X-ray scattering indicates that in solution the predominant oligomeric species of the protein is a dimer, but also that higher oligomers exist at higher protein concentrations. The observed scattering data are best explained by assuming a dimer–tetramer equilibrium with about 7% tetramers present in solution. Consequently, at the elevated protein concentrations in the crowded environment inside the cell the observed tetramer may constitute the biologically relevant functional unit of the enzyme.     

The binding of pentaammineruthenium (III) to RNase B and RNase A+d(pA)4 in the crystalline state

The binding of pentaammineruthenium (III) to ribonuclease A and B both free and complexed with d(pA)₄ has been examined in the crystalline state through the application of X-ray diffraction and difference Fourier techniques. In crystals of native RNase B, the reagent was observed to have many binding sites, some entirely electrostatic in nature and others consistent with coordination to histidine residues. The primary histidine in the latter case was 105 with 119 also partially substituted. In crystals of RNase A+d(pA)₄ complex only a single, extremely strong site of substitution was observed, and this was 2.4 Å from the native position of the imidazole ring of histidine 105. Thus, the results of these X-ray diffraction studies appear to be quite consistent with the findings of earlier NMR studies and with the results obtained in crystals of the gene 5 DNA binding protein.     

Interplay among processing and degradative enzymes and a precursor ribonucleic acid in the selective maturation and maintenance of ribonucleic acid molecules

In order to understand why the first tRNA (tRNAGln) in the T4 tRNA gene cluster is not produced when T4 infects an RNase III- mutant of Escherichia coli, RNA metabolism was analyzed in RNase III- RNase P- (rnc, rnp) cells infected with bacteriophage T4. After such an infection a new dimeric precursor RNA molecule of tRNAGln and tRNALeu has been identified and analyzed. This molecule is structurally very similar to K band RNA that accumulates in rnc+ rnp strains. It is four nucleotides shorter than K RNA at the 5' end. This molecule like K RNA contains two RNase P processing sites at the 5' ends of each tRNA. Both sites are accessible to RNase P. However, while in the K RNA the site at the 5' end of tRNALeu (the site in the middle of the substrate) is more efficiently cleaved than the other site, this differential is even increased in the Ks (K like) molecule. This difference is sufficiently large that in vivo in the RNase III- strain the smaller precursor of tRNAGln is degraded rather than being matured to tRNAGln by RNase P. This information contributes to the elucidation of the key role of RNase III in the processing of T4 tRNA. It shows the dependence of RNase P activity at the 5' end of tRNAGln on a correct and specific cleavage by RNase III at a position six nucleotides proximal to the RNase P site, and it explains why in the absence of RNase III the first tRNA in the T4 tRNA cluster, tRNAGln, does not accumulate.     

Oligomerization of ribonuclease A under reducing conditions

By lyophilization from 40% acetic acid solutions, bovine ribonuclease A forms well characterized, three-dimensional domain-swapped oligomers: dimers, trimers, tetramers, and higher order multimers. Each oligomeric species consists of at least two conformers. Identical oligomers also form by thermally-inducing the oligomerization of highly concentrated RNase A dissolved in fluids endowed with various denaturing power. Now, our question is: which might the influence of a reducing agent be on RNase A oligomerization, i.e., of conditions that decrease the stability of the protein and increase the mobility of its swapping domains? To address this question, we carried out experiments of RNase A oligomerization in the presence of increasing concentrations of dithiothreitol (DTT) under the two experimental conditions mentioned above. Results indicate that RNase A oligomers similar to those previously known form anyhow, but with a change of their relative proportions. The amounts of dimers and trimers decrease by increasing the concentration of DTT, while the yields of two tetramers remarkably increase. Moreover, in the presence of DTT RNase A forms labile and probably unstructured aggregates that can possibly drive the protein towards precipitation when the reducing agent's concentration increases. Taken together, these results point out once again (i) the important role of the 3D domain swapping mechanism in protein oligomerization, and (ii) the importance of the native structure of RNase A (and of proteins in general) in preventing an uncontrolled aggregation and precipitation in a reducing and highly crowded environment like that existing in a living cell.     

Modification of an arginine residue of a base-nonspecific ribonuclease from Aspergillus saitoi.

1. A base-nonspecific ribonuclease from Aspergillus saitoi [RNase Ms, EC 3.1.4.23; molecular weight, 12,500] was modified with phenylglyoxal (PG) and 1,2-cyclohexanedione (CHD) in order to determine whether a single arginine residue was involved in the active site of the enzyme. 2. RNase Ms was inactivated by both PG and CHD with concomitant loss of one arginine residue. A competitive inhibitor of RNase Ms, 2',(3')-AMP, protected the enzyme from inactivation by PG. These findings strongly suggest that one arginine residue is involved in the active site of RNase Ms. 3. Difference CD spectra were measured at pH 5.5 for the binding of 2'-AMP and adenosine to native RNase Ms and the CHD- and PG-modified enzyme derivatives to determine the association constants. The arginine modification brought about a marked decrease in the binding affinity of 2'-AMP for the enzyme, but only a slight decrease for adenosine, suggesting that the arginine residue had interacted with the phosphate groups of the substrate.     

Does domain swapping improve the stability of RNase A?

Self-assembling complexes have potential as novel supramolecular biomaterials but domain swapped complexes have yet to investigated in this capacity. Bovine ribonuclease A (RNase A) is a useful model protein as it is able to form a range of three dimensional domain swapped structures, including dimers, trimers and tetramers that have similar catalytic ability. However, little work has been carried out investigating the physical characteristics of these complexes. In an effort to characterise the strength of these oligomeric interactions, analytical ultracentrifugation was carried out to measure the dissociation of higher order complexes, using fluorescent tags to test for dissociation at very low concentrations. Results of this work suggest that the oligomers form a very tight complex, with no evidence of dissociation down to 250 pM. RNase A oligomers also had similar thermal stability to that of monomeric enzyme, suggesting that the main limiting factor in RNase A stability is the tertiary, rather than quaternary structure. Following thermal unfolding of RNase A, the protein refolded upon cooling, but returned to the monomeric state. This latter result may limit the potential of domain swapping as a means of material assembly.     

Area of 16S ribonucleic acid at or near the interface between 30S and 50S ribosomes of Escherichia coli.

To determine the region of 16S ribonucleic acid (RNA) at the interface between 30 and 50S ribosomes of Escherichia coli, 30 and 70S ribosomes were treated with T1 ribonuclease (RNase). The accessibility of 16S RNA in the 5' half of the molecule is the same in 30 and 70S ribosomes. The interaction with 50S ribosomes decreases the sensitivity to T1 RNase of an area in the middle of 16S RNA. A large area near the 3' end of 16S RNA is completely protected in 70S ribosomes. The RNA near the 3' end of the molecule and an area of RNA in the middle of the molecule appear to be at the interface between 30 and 50S ribosomes. One site in 16S RNA, 13 to 15 nucleotides from the 3' end, normally inaccessible to T1 RNase in 30S ribosomes, becomes accessible to T1 RNase in 70S ribosomes. This indicates a conformational change at the 3' end of 16S RNA when 30S ribosomes are associated with 50S ribosomes.     

A site in a tRNA precursor that can be processed by the whole RNase P enzyme but not by the RNA alone

A precursor molecule for 10 Sb RNA, the RNA moiety of the RNA processing enzyme RNase P, was purified, characterized for enzymatic activity, and compared to 10 Sb RNA and to RNase P. In these studies the K RNA, a dimeric precursor of tRNAGln-tRNALeu, coded by bacteriophage T4, was used as a substrate. This precursor contains two RNase P cleavage sites, one at each 5' end of the two tRNAs. The precursor 10 Sb and 10 Sb RNAs have the capacity to cleave the precursor tRNA molecule but only at the 5' end of tRNALeu, not at the 5' end of tRNAGln. Even when a substrate was prepared that contained only one site for RNase P (the one next to tRNAGln), this substrate was not cleaved by the RNA alone while the whole enzyme was effective in processing this substrate. The possible function of the protein of RNase P in the enzymatic reaction is discussed.     

Alternative function of a protein kinase homology domain in 2', 5'-oligoadenylate dependent RNase L.

RNase L is the 2',5'-oligoadenylate (2-5A)-dependent endoribonuclease that functions in interferon action and apoptosis. One of the intriguing, albeit unexplained, features of RNase L is its significant homology to protein kinases. Despite the homology, however, no protein kinase activity was detected during activation and RNA cleavage reactions with human RNase L. Similarly, the kinase plus ribonuclease domains of RNase L produced no detectable protein kinase activity in contrast to the phosphorylation obtained with homologous domains of the related kinase and endoribonuclease, yeast IRE1p. In addition, neither ATP nor pA(2'p5'A)3was hydrolyzed by RNase L. To further investigate the function of the kinase homology in RNase L, the conserved lysine at residue 392 in protein kinase-like domain II was replaced with an arginine residue. The resulting mutant, RNase LK392R, showed >100-fold decreases in 2-5A-dependent ribonuclease activity without reducing 2-5A- or RNA-binding activities. The greatly reduced activity of RNase LK392Rwas correlated to a defect in the ability of RNase L to dimerize. These results demonstrate a critical role for lysine 392 in the activation and dimerization of RNase L, thus suggesting that these two activities are intimately linked.     

Refined crystal structure of deoxyhemoglobin S. II. Molecular interactions in the crystal

The refined crystal structure of deoxyhemoglobin S (Padlan, E. A., and Love, W. E. (1985) J. Biol. Chem. 260, 8272-8279) was used to analyze in detail the molecular interactions between hemoglobin tetramers in the crystal. The analysis confirms the close similarity and also the nonequivalence of the molecular interactions involving the two independent tetramers in the asymmetric unit of the crystal. The residue at the site of the hemoglobin S mutation, beta 6, is intimately involved in the lateral contacts between adjacent molecules. The molecular contacts in the crystals of deoxyhemoglobin S, deoxyhemoglobin A, and deoxyhemoglobin F were compared; some contacts involve the same regions of the molecule although the details of the interactions are very different. The effect of introducing an R state tetramer into the deoxyhemoglobin S strands was investigated using the known structure of carbon monoxyhemoglobin A. It was found that substituting a molecule of carbon monoxyhemoglobin A for one of the deoxyhemoglobin S tetramers results in extensive molecular interpenetration.     

Productive and nonproductive binding to ribonuclease A: X-ray structure of two complexes with uridylyl(2',5')guanosine

Guanine-containing mono- and dinucleotides bind to the active site of ribonuclease A in a nonproductive mode (retro-binding) (Aguilar CF, Thomas PJ, Mills A, Moss DS, Palmer RA. 1992. J Mol Biol 224:265-267). Guanine binds to the highly specific pyrimidine site by forming hydrogen bonds with Thr45 and with the sulfate anion located in the P1 site. To investigate the influence of the anion present in the P1 site on retro-binding, we determined the structure of two new complexes of RNase A with uridylyl(2',5')guanosine obtained by soaking two different forms of pre-grown RNase A crystals. In one case, RNase A was crystallized without removing the sulfate anion strongly bound to the active site; in the other, the protein was first equilibrated with a basic solution to displace the anion from the P1 site. The X-ray structures of the complexes with and without sulfate in P1 were refined using diffraction data up to 1.8 A (R-factor 0.192) and 2.0 A (R-factor 0.178), respectively. The binding mode of the substrate analogue to the protein differs markedly in the two complexes. When the sulfate is located in P1, we observe retro-binding; whereas when the anion is removed from the active site, the uridine is productively bound at the B1 site. In the productive complex, the electron density is very well defined for the uridine moiety, whereas the downstream guanine is disordered. This finding indicates that the interactions of guanine in the B2 site are rather weak and that this site is essentially adenine preferring. In this crystal form, there are two molecules per asymmetric unit, and due to crystal packing, only the active site of one molecule is accessible to the ligand. Thus, in the same crystal we have a ligand-bound and a ligand-free RNase A molecule. The comparison of these two structures furnishes a detailed and reliable picture of the structural alterations induced by the binding of the substrate. These results provide structural information to support the hypotheses on the role of RNase A active site residues that have recently emerged from site-directed mutagenesis studies.     

Three-dimensional structure of ribonuclease T1 complexed with an isosteric phosphonate substrate analogue of GpU: alternate substrate binding modes and catalysis

The X-ray crystal structure of a complex between ribonuclease T1 and guanylyl(3'-6')-6'-deoxyhomouridine (GpcU) has been determined at 2. 0 A resolution. This ligand is an isosteric analogue of the minimal RNA substrate, guanylyl(3'-5')uridine (GpU), where a methylene is substituted for the uridine 5'-oxygen atom. Two protein molecules are part of the asymmetric unit and both have a GpcU bound at the active site in the same manner. The protein-protein interface reveals an extended aromatic stack involving both guanines and three enzyme phenolic groups. A third GpcU has its guanine moiety stacked on His92 at the active site on enzyme molecule A and interacts with GpcU on molecule B in a neighboring unit via hydrogen bonding between uridine ribose 2'- and 3'-OH groups. None of the uridine moieties of the three GpcU molecules in the asymmetric unit interacts directly with the protein. GpcU-active-site interactions involve extensive hydrogen bonding of the guanine moiety at the primary recognition site and of the guanosine 2'-hydroxyl group with His40 and Glu58. On the other hand, the phosphonate group is weakly bound only by a single hydrogen bond with Tyr38, unlike ligand phosphate groups of other substrate analogues and 3'-GMP, which hydrogen-bonded with three additional active-site residues. Hydrogen bonding of the guanylyl 2'-OH group and the phosphonate moiety is essentially the same as that recently observed for a novel structure of a RNase T1-3'-GMP complex obtained immediately after in situ hydrolysis of exo-(Sp)-guanosine 2',3'-cyclophosphorothioate [Zegers et al. (1998) Nature Struct. Biol. 5, 280-283]. It is likely that GpcU at the active site represents a nonproductive binding mode for GpU [Steyaert, J., and Engleborghs (1995) Eur. J. Biochem. 233, 140-144]. The results suggest that the active site of ribonuclease T1 is adapted for optimal tight binding of both the guanylyl 2'-OH and phosphate groups (of GpU) only in the transition state for catalytic transesterification, which is stabilized by adjacent binding of the leaving nucleoside (U) group.