Newly observed binding mode in pancreatic ribonuclease.

Dinucleotides containing guanine, when soaked into crystals of bovine pancreatic ribonuclease, have been found to bind in an unexpected manner, quite unlike interpretations of earlier X-ray diffraction studies. This finding has prompted a reexamination of three mononucleotide-RNase complexes from this laboratory resulting in a re-interpretation of the complex that involved a guanine mononucleotide.     

Crystal structures of two mutants that have implications for the folding of bovine pancreatic ribonuclease A.

The Tyr92-Pro93 peptide group of bovine pancreatic ribonuclease A (RNase A) exists in the cis conformation in the native state. From unfolding/refolding kinetic studies of the disulfide-intact wild-type protein and of a variant in which Pro93 had been replaced by Ala, it had been suggested that the Tyr92-Ala93 peptide group also exists in the cis conformation in the native state. Here, we report the crystal structure of the P93A variant. Although there is disorder in the region of residues 92 and 93, the best structural model contains a cis peptide at this position, lending support to the results of the kinetics experiments. We also report the crystal structure of the C[40, 95]A variant, which is an analog of the major rate-determining three-disulfide intermediate in the oxidative folding of RNase A, missing the 40-95 disulfide bond. As had been detected by NMR spectroscopy, the crystal structure of this analog shows disorder in the region surrounding the missing disulfide. However, the global chain fold of the remainder of the protein, including the disulfide bond between Cys65 and Cys72, appears to be unaffected by the mutation.     

The kinetics of pancreatic ribonuclease reaction with alkaline and acidic forms of poly A

Summary The RNase hydrolysis of random-coil (alkaline form) poly A follows biphasic kinetics at low salt concentrations. However, its resistance to RNase increases with the ionic strength. Helical (acidic form) poly A is also susceptible to RNase but its hydrolysis follows first-order kinetics, and its resistance increases as the pH is lowered. These conformation-dependent kinetics of poly A hydrolysis are similar to those obtained in the hydrolysis of cellular RNA and reovirus double-stranded RNA.     

The primary structure of muskrat pancreatic ribonuclease.

Pancreatic ribonuclease from muskrat (Ondatra zibethica) was isolated and its amino acid sequence was determined from tryptic digests of the performic acid-oxidized and the reduced and aminoethylated enzyme. The peptides have been positioned in the sequence by homology with other ribonucleases. This could be done unambiguously for all peptides except Arg-Arg (tentative position 32-33) and Ser-Arg (tentative position 75-76). The amino acid sequences of the peptides were determined by the dansyl-Edman method, with the exception of residues 23-25 and 99-102, which were positioned by homology. The enzyme differs in 38 positions from the enzyme from rat and in 31-42 positions from other mammalian pancreatic ribonucleases, while rat ribonuclease differs at 44-52 positions from the other enzymes. These data point to a common ancestry of the enzymes from muskrat and rat and an increased evolution rate of rat ribonuclease after divergence of the ancestors of both species. Muskrat ribonuclease contains no carbohydrate, although the enzyme possesses a recognition site for carbohydrate attachment in the sequence Asn-Val-Thr (62-64).     

Crystal structure of the BIR1 domain of XIAP in two crystal forms

X-linked inhibitor of apoptosis (XIAP) is a potent negative regulator of apoptosis. It also plays a role in BMP signaling, TGF-beta signaling, and copper homeostasis. Previous structural studies have shown that the baculoviral IAP repeat (BIR2 and BIR3) domains of XIAP interact with the IAP-binding-motifs (IBM) in several apoptosis proteins such as Smac and caspase-9 via the conserved IBM-binding groove. Here, we report the crystal structure in two crystal forms of the BIR1 domain of XIAP, which does not possess this IBM-binding groove and cannot interact with Smac or caspase-9. Instead, the BIR1 domain forms a conserved dimer through the region corresponding to the IBM-binding groove. Structural and sequence analyses suggest that this dimerization of BIR1 in XIAP may be conserved in other IAP family members such as cIAP1 and cIAP2 and may be important for the action of XIAP in TGF-beta and BMP signaling and the action of cIAP1 and cIAP2 in TNF receptor signaling.     

Crystallization of mouse pancreatic ribonuclease

Mouse pancreatic ribonuclease has been crystallized in a form suitable for X-ray structure determination. The crystals grown from solutions of 2-methyl-2,4-pentanediol diffract to high resolution and belong to the hexagonal space group P6(1) (P6(5)) with unit cells dimensions a = b = 64.44 A, c = 53.91 A, y = 120 degrees and V = 1.94 x 10(5) A3 (1 A = 0.1 nm). There are six molecules per unit cell (1 molecule/asymmetric unit), and Vm = 2.3 A3/dalton.     

Effect of crystal packing environment on conformation of the DNA duplex. Molecular structure of the A-DNA octamer d(G-T-G-T-A-C-A-C) in two crystal forms

The structure of the octamer d(G-T-G-T-A-C-A-C) was determined in two different crystal forms, tetragonal P4(3)2(1)2 and hexagonal P6(1)22. Although in both forms the octamer adopts an A-DNA structure, there are significant conformational differences between them. In particular, the P-05' and the C5'-C4' bonds of the middle adenine (A5) residue exhibit a distorted trans-trans conformation in the tetragonal form, while they adopt the standard gauche-, gauche+ conformation in the hexagonal form. These differences can be correlated with certain features of the crystal packing interactions in the two forms. Furthermore, a comparison of the structures of various A-DNA octamers reveals that the A-form can be divided into two subclasses such that the hexagonal structures have helical and base pair parameters that fall closer to fiber A-DNA values, while in the tetragonal structures these parameters deviate more from fiber A-DNA. These results indicate that environment plays a major role in determining DNA conformation.     

Multiple forms of ribonuclease H from rat liver cytosol.

Three forms (termed I, II, and III) of ribonuclease H (RNase H) [EC 3.1.4.34] activity are present in rat liver cytosol. These enzymes degrade RNA specifically in RNA-DNA hybrid structures. They were eluted at 0 M, 0.25 M, and 0.5 M KCl in phosphocellulose chromatography, and were further purified by using blue Sepharose. They are further distinguished from one another by their ionic requirements, optimal pH, molecular weights, sedimentation coefficients, and sensitivity to the -SH reagent, p-chloromercuribenzoate, although I and III have similar characteristics. They liberate a mixture of oligonucleotides with 5'-phosphate and 3'-hydroxyl termini.     

Interaction of human pancreatic ribonuclease with human ribonuclease inhibitor. Generation of inhibitor-resistant cytotoxic variants

Mammalian ribonucleases interact very strongly with the intracellular ribonuclease inhibitor (RI). Eukaryotic cells exposed to mammalian ribonucleases are protected from their cytotoxic action by the intracellular inhibition of ribonucleases by RI. Human pancreatic ribonuclease (HPR) is structurally and functionally very similar to bovine RNase A and interacts with human RI with a high affinity. In the current study, we have investigated the involvement of Lys-7, Gln-11, Asn-71, Asn-88, Gly-89, Ser-90, and Glu-111 in HPR in its interaction with human ribonuclease inhibitor. These contact residues were mutated either individually or in combination to generate mutants K7A, Q11A, N71A, E111A, N88R, G89R, S90R, K7A/E111A, Q11A/E111A, N71A/E111A, K7A/N71A/E111A, Q11A/N71A/E111A, and K7A/Q11A/N71A/E111A. Out of these, eight mutants, K7A, Q11A, N71A, S90R, E111A, Q11A/E111A, N71A/E111A, and K7A/N71A/E111A, showed an ability to evade RI more than the wild type HPR, with the triple mutant K7A/N71A/E111A having the maximum RI resistance. As a result, these variants exhibited higher cytotoxic activity than wild type HPR. The mutation of Gly-89 in HPR produced no change in the sensitivity of HPR for RI, whereas it has been reported that mutating the equivalent residue Gly-88 in RNase A yielded a variant with increased RI resistance and cytotoxicity. Hence, despite its considerable homology with RNase A, HPR shows differences in its interaction with RI. We demonstrate that interaction between human pancreatic ribonuclease and RI can be disrupted by mutating residues that are involved in HPR-RI binding. The inhibitor-resistant cytotoxic HPR mutants should be useful in developing therapeutic molecules.     

Crystal structure analyses of uncomplexed ecotin in two crystal forms: implications for its function and stability.

Ecotin, a homodimeric protein composed of 142 residue subunits, is a novel serine protease inhibitor present in Escherichia coli. Its thermostability and acid stability, as well as broad specificity toward proteases, make it an interesting protein for structural characterization. Its structure in the uncomplexed state, determined for two different crystalline environments, allows a structural comparison of the free inhibitor with that in complex with trypsin. Although there is no gross structural rearrangement of ecotin when binding trypsin, the loops involved in binding trypsin show relatively large shifts in atomic positions. The inherent flexibility of the loops and the highly nonglobular shape are the two features essential for its inhibitory function. An insight into the understanding of the structural basis of thermostability and acid stability of ecotin is also provided by the present structure.     

The molecular evolution of pancreatic ribonuclease

Summary The primary structures of pancreatic ribonucleases from 26 species (18 artiodactyls, horse, whale, 5 rodents and turtle) are known. Several species contain identical ribonucleases (cow/bison; sheep/goat), other species show polymorphism (arabian camel) or the presence of two structural gene loci (guinea pig pancreas contains two ribonucleases that differ at 31 positions). 26 different sequences (including the ribonuclease from bovine seminal plasma which is paralogous to the pancreatic ribonucleases) were used to construct a most parsimonious tree. A second tree that most closely approximates current biological opinion requires 402 whereas the most parsimonious tree requires 389 nucleotide substitutions. The artiodactyl part of the most parsimonious tree conforms quite well with the biological one of this order, except for the position of the giraffe which is placed with the pronghorn. Other parts of the most parsimonious tree agree less with the biological tree, probably as a result of the occurrence of many parallel and back substitutions. Bovine seminal ribonuclease was found to be the result of a gene duplication which occurred before the divergence of the true ruminants, but after the divergence of this group from the cameloids.The evolutionary rate of ribonuclease was found to be 390, 3.0 and 11 nucleotide substitutions per 10⁹ yrs per ribonuclease gene, codon and covarion respectively. However, there is much variation in evolutionary rate in different taxa. Values ranging from about 100 (in the bovidae) to about 700 (in the rodents) nucleotide substitutions per 10⁹ yrs per gene were found.A method for counting parallel and back mutations is presented. The 389 nucleotide substitutions in the most parsimonious tree occur at 88 codon positions; 154 of them are the result of parallel and back mutations. Parallel evolution to a similar structure, including the presence of 2 sites with carbohydrate, was demonstrated in an extensive region at the surface of pig and guinea pig ribonuclease B. The presence of carbohydrate probably is important in a number of species. A correlation between the presence of heavily glycosidated ribonucleases and coecal digestion was observed. Hypothetical sequences of ancestral ungulate ribonucleases contain many recognition sites for carbohydrate attachment; this suggests that herbivores with coecal digestion might have preceded the true ruminants in mammalian evolution.     

Proline isomerization in bovine pancreatic ribonuclease A. 2. Folding conditions

The kinetics of cis-trans isomerization of individual X-Pro peptide groups is used to study the backbone dynamics of bovine pancreatic ribonuclease A (RNase A). We previously developed and validated a fluorescence method for monitoring the cis-trans isomerization of the Tyr92-Pro93 and Asn113-Pro114 peptide groups of RNase A under unfolding conditions [Juminaga, D., Wedemeyer, W. J., and Scheraga, H. A. (1998) Biochemistry 37, 11614-11620]. The essence of this method is to introduce a fluorescent residue (Tyr or Trp) in a position adjacent to the isomerizing proline (if one is not already present) and to eliminate the fluorescence of other such residues adjacent to prolines by mutating them to phenylalanine. Here, we extend this method to observe the cis-trans isomerization of these peptide groups under folding conditions using two site-directed mutants (Y92F and Y115F) of RNase A. Both isomerizations decelerate with increasing concentrations of GdnHCl, with nearly identical m values (1.11 and 1.19 M(-1), respectively) and extrapolated zero-GdnHCl time constants (42 and 32 s, respectively); by contrast, under unfolding conditions, the cis-trans isomerizations of both Pro93 and Pro114 are independent of GdnHCl concentration. Remarkably, the isomerization rates under folding conditions at GdnHCl concentrations above 1 M are significantly slower than those measured under unfolding conditions. The temperature dependence of the Pro114 isomerization under folding conditions is also unusual; whereas Pro93 exhibits an activation energy typical of proline isomerization (19.4 kcal/mol), Pro114 exhibits a sharply reduced activation energy of 5.7 kcal/mol. A structurally plausible model accounts for these results and, in particular, shows that folding conditions strongly accelerate the cis-trans isomerization of both peptide groups to their native cis conformation, suggesting the presence of flickering local structure in their beta-hairpins.     

Location of the antigenic determinants of bovine pancreatic ribonuclease.

Four antigenic regions of native bovine pancreatic ribonuclease have been located by using antibodies that react specifically with segments 1--13, 31--79, and 80--124. These antibodies were purified by affinity chromatography on columns to which these peptide segments were bound. Analysis of precipitin curves indicates that there are at least three antigenic determinants to which antibody molecules can bind simultaneously in the presence of excess antibodies. Analysis of binding data, however, for each purified specific antibody preparation, carried out by the method of Berzofsky et al. [Berzofsky, J. A., Curd, J. G., & Schechter, A. N. (1976) Biochemistry, 15, 2113], leads to an estimate of four for the number of antigenic determinants in ribonuclease; this estimate had also been made earlier by Stelos et al. [Stelos, P., Fothergill, J. E., & Singer, S. J. (1960) J. Am. Chem. Soc. 82, 6034]. We find that one determinant is associated with each of segments 1--13 and 80--124 and two with segment 31--79. No antigenic activity could be detected for segment 14--29 either in native ribonuclease or in the free fragment. These conclusions are based on (1) the use of specific peptides to isolate purified antibodies by affinity chromatography, (2) immunoprecipitation of an antigenic peptide from the peptic digest of ribonuclease, (3) competitive inhibition studies with various peptide and protein fragments [cyanogen bromide fragments 1--13, 31--79, and 80--124, the tryptic peptides 40--61 and 105--224, S-peptide, S-protein, and des(121--124)-RNase], and (4) comparison and evaluation of the published effects on antigenicity of chemical and enzymatic modifications and changes in sequence among homologous ribonucleases. These approaches provide evidence that the four antigenic determinants are localized around the alpha-helical portion of segment 1--10, somewhere in segment 40--61, at the beta bend in segment 63--75, and either at the beta bend or beta sheet in segment 87--104 of native ribonuclease.     

Mechanism of glutathione regeneration of reduced pancreatic ribonuclease a.

A comparative study was made of the kinetics of glutathione regeneration of reduced pancreatic ribonuclease A, as determined by circular dishroism, sulfhydryl oxidation and the kinetics of reactivation. Four sulfhydryls were reozidizedeactivation. Four sulfhydryls were reoxidized prior to any large circular dichroic changes or recovery of enzymatic activity. The helical and beta segments in ribonuclease were shown to reform at approximately the same rate. The results are discussed in terms of a regeneration mechanism for ribonuclease involving (1) nucleation, (2) polyeptide backbone refolding, and (3) reshuffling of incorrectly paired disulfide bonds.