Probing the sweet determinants of brazzein: wild-type brazzein and a tasteless variant, brazzein-ins(R18a-I18b), exhibit different pH-dependent NMR chemical shifts

Brazzein is a small, intensely sweet protein. As a probe of the functional properties of its solvent-exposed loop, two residues (Arg-Ile) were inserted between Leu18 and Ala19 of brazzein. Psychophysical testing demonstrated that this mutant is totally tasteless. NMR chemical shift mapping of differences between this mutant and brazzein indicated that residues affected by the insertion are localized to the mutated loop, the region of the single alpha-helix, and around the Cys16-Cys37 disulfide bond. Residues unaffected by this mutation included those near the C-terminus and in the loop connecting the alpha-helix and the second beta-strand. In particular, several residues of brazzein previously shown to be essential for its sweetness (His31, Arg33, Glu41, Arg43, Asp50, and Tyr54) exhibited negligible chemical shift changes. Moreover, the pH dependence of the chemical shifts of His31, Glu41, Asp50, and Tyr54 were unaltered by the insertion. The insertion led to large chemical shift and pKa perturbation of Glu36, a residue shown previously to be important for brazzein's sweetness. These results serve to refine the known sweetness determinants of brazzein and lend further support to the idea that the protein interacts with a sweet-taste receptor through a multi-site interaction mechanism, as has been postulated for brazzein and other sweet proteins (monellin and thaumatin).     

Electrostatic interactions in leucine zippers: thermodynamic analysis of the contributions of Glu and His residues and the effect of mutating salt bridges

Electrostatic interactions play a complex role in stabilizing proteins. Here, we present a rigorous thermodynamic analysis of the contribution of individual Glu and His residues to the relative pH-dependent stability of the designed disulfide-linked leucine zipper AB(SS). The contribution of an ionized side-chain to the pH-dependent stability is related to the shift of the pK(a) induced by folding of the coiled coil structure. pK(a)(F) values of ten Glu and two His side-chains in folded AB(SS) and the corresponding pK(a)(U) values in unfolded peptides with partial sequences of AB(SS) were determined by 1H NMR spectroscopy: of four Glu residues not involved in ion pairing, two are destabilizing (-5.6 kJ mol(-1)) and two are interacting with the positive alpha-helix dipoles and are thus stabilizing (+3.8 kJ mol(-1)) in charged form. The two His residues positioned in the C-terminal moiety of AB(SS) interact with the negative alpha-helix dipoles resulting in net stabilization of the coiled coil conformation carrying charged His (-2.6 kJ mol(-1)). Of the six Glu residues involved in inter-helical salt bridges, three are destabilizing and three are stabilizing in charged form, the net contribution of salt-bridged Glu side-chains being destabilizing (-1.1 kJ mol(-1)). The sum of the individual contributions of protonated Glu and His to the higher stability of AB(SS) at acidic pH (-5.4 kJ mol(-1)) agrees with the difference in stability determined by thermal unfolding at pH 8 and pH 2 (-5.3 kJ mol(-1)). To confirm salt bridge formation, the positive charge of the basic partner residue of one stabilizing and one destabilizing Glu was removed by isosteric mutations (Lys-->norleucine, Arg-->norvaline). Both mutations destabilize the coiled coil conformation at neutral pH and increase the pK(a) of the formerly ion-paired Glu side-chain, verifying the formation of a salt bridge even in the case where a charged side-chain is destabilizing. Because removing charges by a double mutation cycle mainly discloses the immediate charge-charge effect, mutational analysis tends to overestimate the overall energetic contribution of salt bridges to protein stability.     

Sequence and structure patterns in proteins from an analysis of the shortest helices: implications for helix nucleation

The shortest helices (three-length 3(10) and four-length alpha), most abundant among helices of different lengths, have been analyzed from a database of protein structures. A characteristic feature of three-length 3(10)-helices is the shifted backbone conformation for the C-terminal residue (phi,psi angles: -95 degrees,0 degrees ), compared to the rest of the helix (-62 degrees,-24 degrees ). The deviation can be attributed to the release of electrostatic repulsion between the carbonyl oxygen atoms at the two C-terminal residues and further stabilization (due to a more linear geometry) of an intrahelical hydrogen bond. A consequence of this non-canonical C-terminal backbone conformation can be a potential origin of helix kinks when a 3(10)-helix is sequence-contiguous at the alpha-helix N-terminal. An analysis of hydrogen bonding, as well as hydrophobic interactions in the shortest helices shows that capping interactions, some of them not observed for longer helices, dominate at the N termini. Further, consideration of the distribution of amino acid residues indicates that the shortest helices resemble the N-terminal end of alpha-helices rather than the C terminus, implying that the folding of helices may be initiated at the N-terminal end, which does not get propagated in the case of the shortest helices. Finally, pairwise comparison of beta-turns and the shortest helices, based on correlation matrices of site-specific amino acid composition, and the relative abundance of these short secondary structural elements, leads to a helix nucleation scheme that considers the formation of an isolated beta-turn (and not an alpha-turn) as the helix nucleation step, with shortest 3(10)-helices as intermediates between the shortest alpha-helix and the beta-turn. Our results ascribe an important role played by shortest 3(10)-helices in proteins with important structural and folding implications.     

Removal of N-terminal methionine from recombinant proteins by engineered E. coli methionine aminopeptidase

The removal of N-terminal translation initiator Met by methionine aminopeptidase (MetAP) is often crucial for the function and stability of proteins. On the basis of crystal structure and sequence alignment of MetAPs, we have engineered Escherichia coli MetAP by the mutation of three residues, Y168G, M206T, Q233G, in the substrate-binding pocket. Our engineered MetAPs are able to remove the Met from bulky or acidic penultimate residues, such as Met, His, Asp, Asn, Glu, Gln, Leu, Ile, Tyr, and Trp, as well as from small residues. The penultimate residue, the second residue after Met, was further removed if the antepenultimate residue, the third residue after Met, was small. By the coexpression of engineered MetAP in E. coli through the same or a separate vector, we have successfully produced recombinant proteins possessing an innate N terminus, such as onconase, an antitumor ribonuclease from the frog Rana pipiens. The N-terminal pyroglutamate of recombinant onconase is critical for its structural integrity, catalytic activity, and cyto-toxicity. On the basis of N-terminal sequence information in the protein database, 85%-90% of recombinant proteins should be produced in authentic form by our engineered MetAPs.     

The N-terminal alpha-helix of pancreatic phospholipase A2 determines productive-mode orientation of the enzyme at the membrane surface

Phospholipase A(2) (PLA(2)) hydrolyzes glycerophospholipids to free fatty acid and lyso-phospholipid, which serve as precursors for the biosynthesis of eicosanoids and other lipid-derived mediators of inflammation and allergy. PLA(2) activity strongly increases upon binding to the surface of aggregated phospholipid. The N-terminal approximately ten residue alpha-helix of certain PLA(2) isoforms plays important roles in the interfacial activation of the enzyme by providing residues for membrane binding of PLA(2) and by contributing to the formation of the substrate-binding pocket. However, the relative contributions of the N-terminal alpha-helix and the rest of the protein in membrane binding of PLA(2) and its productive-mode orientation at the membrane surface are not well understood. Here we use a variety of biophysical approaches to determine the role of the N-terminal helix in membrane binding strength, orientation, and activity of human pancreatic PLA(2). While the full-length PLA(2) binds to membranes with a defined orientation, an engineered PLA(2) fragment DeltaN10 that lacks the N-terminal ten residues binds to membranes with weaker affinity and at random orientation, and exhibits approximately 100-fold lower enzymatic activity compared to the full-length PLA(2), indicating the key role of the N terminus in PLA(2) function. The results of polarized infrared spectroscopic experiments permit determination of the orientation of membrane-bound PLA(2) and identification of its interfacial binding surface. Moreover, the full-length PLA(2) demonstrates increased conformational flexibility in solution and is stabilized upon membrane binding, while the DeltaN10 fragment is more rigid than the full-length PLA(2) both in free and membrane-bound states. Our results suggest that the N-terminal alpha-helix supports the activation of PLA(2) by (a) enhancing the membrane binding strength, (b) facilitating a productive-mode orientation of PLA(2) at the membrane surface, and (c) conferring conformational integrity and plasticity to the enzyme.     

Crystal structure and structure-based mutational analyses of RNase HIII from Bacillus stearothermophilus: a new type 2 RNase H with TBP-like substrate-binding domain at the N terminus

Ribonuclease HIII (Bst-RNase HIII) from the moderate thermophile Bacillus stearothermophilus is a type 2 RNase H but shows poor amino acid sequence identity with another type 2 RNase H, RNase HII. It is composed of 310 amino acid residues and acts as a monomer. Bst-RNase HIII has a large N-terminal extension with unknown function and a unique active-site motif (DEDE), both of which are characteristics common to RNases HIII. To understand the role of these N-terminal extension and active-site residues, the crystal structure of Bst-RNase HIII was determined in both metal-free and metal-bound forms at 2.1-2.6 angstroms resolutions. According to these structures, Bst-RNase HIII consists of the N-terminal domain and C-terminal RNase H domain. The structures of the N and C-terminal domains were similar to those of TATA-box binding proteins and archaeal RNases HII, respectively. The steric configurations of the four conserved active-site residues were very similar to those of other type 1 and type 2 RNases H. Single Mn and Mg ions were coordinated with Asp97, Glu98, and Asp202, which correspond to Asp10, Glu48, and Asp70 of Escherichia coli RNase HI, respectively. The mutational studies indicated that the replacement of either one of these residues with Ala resulted in a great reduction of the enzymatic activity. Overproduction, purification, and characterization of the Bst-RNase HIII derivatives with N and/or C-terminal truncations indicated that the N-terminal domain and C-terminal helix are involved in substrate binding, but the former contributes to substrate binding more greatly than the latter.     

Stable proline box motif at the N-terminal end of alpha-helices.

We describe a novel N-terminal alpha-helix local motif that involves three hydrophobic residues and a Pro residue (Pro-box motif). Database analysis shows that when Pro is the N-cap of an alpha-helix the distribution of amino acids in adjacent positions changes dramatically with respect to the average distribution in an alpha-helix, but not when Pro is at position N1. N-cap Pro residues are usually associated to Ile and Leu, at position N', Val at position N3 and a hydrophobic residue (h) at position N4. The side chain of the N-cap Pro packs against Val, while the hydrophobic residues at positions N' and N4 make favorable interactions. To analyze the role of this putative motif (sequence fingerprint hPXXhh), we have synthesized a series of peptides and analyzed them by circular dichroism (CD) and NMR. We find that this motif is formed in peptides, and that the accompanying hydrophobic interactions contribute up to 1.2 kcal/mol to helix stability. The fact that some of the residues in this fingerprint are not good N-cap and helix formers results in a small overall stabilization of the alpha-helix with respect to other peptides having Gly as the N-cap and Ala at N3 and N4. This suggests that the Pro-box motif will not specially contribute to protein stability but to the specificity of its fold. In fact, 80% of the sequences that contain the fingerprint sequence in the protein database are adopting the described structural motif, and in none of them is the helix extended to place Pro at the more favorable N1 position.     

Evidence that three histidine residues of a base non-specific and adenylic acid preferential ribonuclease from Rhizopus niveus are involved in the catalytic function.

In order to study the structure-function relationship of an RNase T2 family enzyme, RNase Rh, from Rhizopus niveus, we investigated the roles of three histidine residues by means of site-specific mutagenesis. One of the three histidine residues of RNase RNAP Rh produced in Saccharomyces cerevisiae by recombinant DNA technology was substituted to a phenylalanine or alanine residue. A Phe or Ala mutant enzyme at His46 or His109 showed less than 0.03%, but a mutant enzyme at His104 showed 0.54% of the enzymatic activity of the wild-type enzyme with RNA as a substrate. Similar results were obtained, when ApU was used as a substrate. The binding constant of a Phe mutant enzyme at His46 or His109 towards 2'-AMP decreased twofold, but that at His104 decreased more markedly. Therefore, we assumed that these three histidine residues are components of the active site of RNase Rh, that His104 contributes to some extent to the binding and less to the catalysis, and that the other two histidine residues and one carboxyl group not yet identified are probably involved in the catalysis. We assigned the C-2 proton resonances of His46, His104, and His109 by comparison of the 1H-NMR spectra of the three mutant enzymes containing Phe in place of His with that of the native enzyme, and also determined the individual pKa values for His46 and His104 to be 6.70 and 5.94. His109 was not titrated in a regular way, but the apparent pKa value was estimated to be around 6.3. The fact that addition of 2'-AMP caused a greater effect on the chemical shift of His104 in the 1NMR spectra as compared with those of the other histidine residues, may support the idea described above on the role of His104.     

NMR solution structure of the mitochondrial F1beta presequence from Nicotiana plumbaginifolia

We have isolated, characterized and determined the three-dimensional NMR solution structure of the presequence of ATPsynthase F1beta subunit from Nicotiana plumbaginifolia. A general method for purification of presequences is presented. The method is based on overexpression of a mutant precursor containing a methionine residue introduced at the processing site, followed by CNBr-cleavage and purification of the presequence on a cation-exchange column. The F1beta presequence, 53 amino acid residues long, retained its native properties as evidenced by inhibition of in vitro mitochondrial import and processing at micromolar concentrations. CD spectroscopy revealed that the F1beta presequence formed an alpha-helical structure in membrane mimetic environments such as SDS and DPC micelles (approximately 50% alpha-helix), and in acidic phospholipid bicelles (approximately 60% alpha-helix). The NMR solution structure of the F1beta presequence in SDS micelles was determined on the basis of 518 distance and 21 torsion angle constraints. The structure was found to contain two helices, an N-terminal amphipathic alpha-helix (residues 4-15) and a C-terminal alpha-helix (residues 43-53), separated by a largely unstructured 27 residue long internal domain. The N-terminal amphipathic alpha-helix forms the putative Tom20 receptor binding site, whereas the C-terminal alpha-helix is located upstream of the mitochondrial processing peptidase cleavage site.     

Effect of the N3 residue on the stability of the alpha-helix

N3 is the third position from the N terminus in the alpha-helix with helical backbone dihedral angles. All 20 amino acids have been placed in the N3 position of a synthetic helical peptide (CH(3)CO-[AAX AAAAKAAAAKAGY]-NH(2)) and the helix content measured by circular dichroism spectroscopy at 273 K. The dependence of peptide helicity on N3 residue identity has been used to determine a free energy scale by analysis with a modified Lifson-Roig helix coil theory that includes a parameter for the N3 energy (n3). The most stabilizing residues at N3 in rank order are Ala, Glu, Met/Ile, Leu, Lys, Ser, Gln, Thr, Tyr, Phe, Asp, His, and Trp. Free energies for the most destabilizing residues (Cys, Gly, Asn, Arg, and Pro) could not be fitted. The results correlate with N1, N2, and helix interior energies and not at all with N-cap preferences. This completes our work on studying the structural and energetic preferences of the amino acids for the N-terminal positions of the alpha-helix. These results can be used to rationally modify protein stability, help design helices, and improve prediction of helix location and stability.     

Enzymatic and structural characterisation of amphinase, a novel cytotoxic ribonuclease from Rana pipiens oocytes

Besides Onconase (ONC) and its V11/N20/R103-variant, oocytes of the Northern Leopard frog (Rana pipiens) contain another homologue of ribonuclease A, which we named Amphinase (Amph). Four variants (Amph-1-4) were isolated and sequenced, each 114 amino acid residues in length and N-glycosylated at two positions. Sequence identities (a) among the variants and (b) versus ONC are 86.8-99.1% and 38.2-40.0%, respectively. When compared with other amphibian ribonucleases, a typical pattern of cysteine residues is evident but the N-terminal pyroglutamate residue is replaced by a six-residue extension. Amph variants have relatively weak ribonucleolytic activity that is insensitive to human ribonuclease inhibitor protein (RI). Values of k(cat)/K(M) with hypersensitive fluorogenic substrates are 10(4) and 10(2)-fold lower than the maximum values exhibited by ribonuclease A and ONC, respectively, and there is little cytosine/uracil or adenine/guanine discrimination at the B(1) or B(2) subsites, respectively. Amph variants have cytotoxic activity toward A-253 carcinoma cells that requires intact ribonucleolytic activity. The glycan component has little or no influence over single-stranded RNA cleavage, RI evasion or cytotoxicity. The crystal structures of natural and recombinant Amph-2 (determined at 1.8 and 1.9 A resolution, respectively) reveal that the N terminus is unlikely to play a catalytic role (but an unusual alpha2-beta1 loop may do so) and the B(2) subsite is rudimentary. At the active site, structural features that may contribute to the enzyme's low ribonucleolytic activity are the fixture of Lys14 in an obstructive position, the accompanying ejection of Lys42, and a lack of constraints on the conformations of Lys42 and His107.     

Novel beta-barrel fold in the nuclear magnetic resonance structure of the replicase nonstructural protein 1 from the severe acute respiratory syndrome coronavirus

The nonstructural protein 1 (nsp1) of the severe acute respiratory syndrome coronavirus has 179 residues and is the N-terminal cleavage product of the viral replicase polyprotein that mediates RNA replication and processing. The specific function of nsp1 is not known. Here we report the nuclear magnetic resonance structure of the nsp1 segment from residue 13 to 128, which represents a novel alpha/beta-fold formed by a mixed parallel/antiparallel six-stranded beta-barrel, an alpha-helix covering one opening of the barrel, and a 3(10)-helix alongside the barrel. We further characterized the full-length 179-residue protein and show that the polypeptide segments of residues 1 to 12 and 129 to 179 are flexibly disordered. The structure is analyzed in a search for possible correlations with the recently reported activity of nsp1 in the degradation of mRNA.     

Influence of key residues on the heterologous extracellular production of fungal ribonuclease U2 in the yeast Pichia pastoris

Ribonuclease U2, secreted by the smut fungus Ustilago sphaerogena, is a cyclizing ribonuclease that displays a rather unusual specificity within the group of microbial extracellular RNases, best represented by RNase T1. Superposition of the three-dimensional structures of RNases T1 and U2 suggests that the RNase U2 His 101 would be the residue equivalent to the RNase T1 catalytically essential His 92. RNase U2 contains three disulfide bridges but only two of them are conserved among the family of fungal extracellular RNases. The non-conserved disulfide bond is established between Cys residues 1 and 54. Mispairing of the disulfide network due to the presence of two consecutive Cys residues (54 and 55) has been invoked to explain the presence of wrongly folded RNase U2 species when produced in Pichia pastoris. In order to study both hypotheses, the RNase U2 H101Q and C1/54S variants have been produced, purified, and characterized. The results obtained support the major conclusion that His 101 is required for proper protein folding when secreted by the yeast P. pastoris. On the other hand, substitution of the first Cys residue for Ser results in a mutant version which is more efficiently processed in terms of a more complete removal of the yeast α-factor signal peptide. In addition, it has been shown that elimination of the Cys 1–Cys 54 disulfide bridge does not interfere with RNase U2 proper folding, generating a natively folded but much less stable protein.     

Crystal structure of a plant ribonuclease, RNase LE

Ribonuclease LE (RNase LE) from cultured tomato (Lycopersicon esculentum) cells is a member of the RNase T(2) family showing broad base specificity. The crystal structure of RNase LE has been determined at 1.65 A resolution. The structure consists of seven alpha-helices and seven beta-strands, belonging to an alpha+beta type structure. Comparison of the structure of RNase LE with that of RNase Rh, a microbial RNase belonging to the RNase T(2) family, reveals that while the overall folding topologies are similar to each other, major insertions and deletions are found at the N-terminal regions. The structural comparison, an amino acid sequence alignment of the RNase T(2) enzymes, and comparison of the disulfide-bonding pattern of these enzymes show that the structure of RNase LE shown here is the basic framework of the animal/plant subfamily of RNase T(2) enzymes (including a self-incompatibility protein called S-RNase), and the structure of RNase Rh is that of the fungal subfamily of RNase T(2) enzymes (including RNase T(2)). Subsequently, we superposed the active-site of the RNase LE with that of RNase Rh and found that (1) His39, Trp42, His92, Glu93, Lys96, and His97 of RNase LE coincided exactly with His46, Trp49, His104, Glu105, Lys108, and His109, respectively, of RNase Rh, and (2) two conserved water molecules were found at the putative P(1) sites of both enzymes. These facts suggest that plant RNase LE has a very similar hydrolysis mechanism to that of fungal RNase Rh, and almost all the RNase T(2) enzymes widely distributed in various species share a common catalytic mechanism. A cluster of hydrophobic residues was found on the active-site face of the RNase LE molecule and two large hydrophobic pockets exist. These hydrophobic pockets appear to be base binding sites mainly by hydrophobic interactions and are responsible for the base non-specificity of RNase LE.     

Crystal structure of an iron-dependent group III dehydrogenase that interconverts L-lactaldehyde and L-1,2-propanediol in Escherichia coli

The FucO protein, a member of the group III "iron-activated" dehydrogenases, catalyzes the interconversion between L-lactaldehyde and L-1,2-propanediol in Escherichia coli. The three-dimensional structure of FucO in a complex with NAD(+) was solved, and the presence of iron in the crystals was confirmed by X-ray fluorescence. The FucO structure presented here is the first structure for a member of the group III bacterial dehydrogenases shown experimentally to contain iron. FucO forms a dimer, in which each monomer folds into an alpha/beta dinucleotide-binding N-terminal domain and an all-alpha-helix C-terminal domain that are separated by a deep cleft. The dimer is formed by the swapping (between monomers) of the first chain of the beta-sheet. The binding site for Fe(2+) is located at the face of the cleft formed by the C-terminal domain, where the metal ion is tetrahedrally coordinated by three histidine residues (His200, His263, and His277) and an aspartate residue (Asp196). The glycine-rich turn formed by residues 96 to 98 and the following alpha-helix is part of the NAD(+) recognition locus common in dehydrogenases. Site-directed mutagenesis and enzyme kinetic assays were performed to assess the role of different residues in metal, cofactor, and substrate binding. In contrast to previous assumptions, the essential His267 residue does not interact with the metal ion. Asp39 appears to be the key residue for discriminating against NADP(+). Modeling L-1,2-propanediol in the active center resulted in a close approach of the C-1 hydroxyl of the substrate to C-4 of the nicotinamide ring, implying that there is a typical metal-dependent dehydrogenation catalytic mechanism.     

Structure-function studies of human granulocyte-macrophage colony-stimulating factor. Identification of residues required for activity

Human granulocyte-macrophage colony stimulating factor (hGM CSF), a protein containing 127 amino acids, was chemically synthesized by using automated stepwise solid-phase methods. The unpurified synthetic hGM-CSF had the same range of actions on hemopoietic cells as the purified recombinant protein. The structural requirements for the activities of synthetic hGM-CSF were examined by the design and synthesis of fragments and analogs. The synthetic fragment, hGM-CSF (54-127), containing all four of the cysteine residues found in the intact protein, lacked detectable activity. Assays of fragments shortened at the N terminus showed that the residues 1-13 were not required for activity, but that the integrity of residues 14-25, particularly residues 16, 17, and 18, was critical for biologic activity. The 14-25 region is predicted to form the first alpha-helix in hGM-CSF. Synthetic peptides within the N-terminal 53 residue region lacked detectable activity. The synthetic analog hGM-CSF (1-121), which lacks the C-terminal 6 residues, had similar activity to hGM-CSF (1-127) indicating that residues 122-127 are not required for activity. An analog, [Ala88] hGM-CSF (14-96), which lacks the hydrophobic C-terminal region and 2 cysteine residues, had low but readily detectable activity suggesting that residues 14-96 are sufficient for detectable synthetic hGM-CSF activity, although the presence of residues 97-121 are required for full activity. No dissociation of the multiple biological activities of hGM-CSF was detected.     

Chain termini cross-talk in the swapping process of bovine pancreatic ribonuclease

3D domain swapping is the process by which two or more protein molecules exchange part of their structure to form intertwined dimers or higher oligomers. Bovine pancreatic ribonuclease (RNase A) is able to swap the N-terminal α-helix (residues 1-13) and/or the C-terminal β-strand (residues 116-124), thus forming a variety of oligomers, including two different dimers. Cis-trans isomerization of the Asn113-Pro114 peptide group was observed when the protein formed the C-terminal swapped dimer. To study the effect of the substitution of Pro114 on the swapping process of RNase A, we have prepared and characterized the P114A monomeric and dimeric variants of the enzyme. In contrast with previous reports, the crystal structure and NMR data on the monomer reveals a mixed cis-trans conformation for the Asn113-Ala114 peptide group, whereas the X-ray structure of the C-terminal swapped dimer of the variant is very close to that of the corresponding dimer of RNase A. The mutation at the C-terminus affects the capability of the N-terminal α-helix to swap and the stability of both dimeric forms. The present results underscore the importance of the hydration shell in determining the cross-talk between the chain termini in the swapping process of RNase A.     

On a salmon (Oncorhynchus [corrected] keta) liver RNase, belonging to RNase T2 family: primary structure and some properties

A base-nonspecific and acid ribonuclease (RNase Ok2) was purified from the liver of a salmon (Oncorhnchus keta) to a homogeneous state by SDS-PAGE. The primary structure of RNase Ok2 was determined by protein chemistry and molecular cloning. The RNase Ok2 was a glycoprotein and consisted of 216 amino acid residues. Its molecular mass of protein moiety was 25,198, and its amino acid sequence showed that it belongs to the RNase T2 family of enzymes. The optimal pH of RNase Ok2 was around 5.5. The base preferences at the B1 and B2 sites were estimated from the rates of hydrolysis of 16 dinucleoside phosphates to be G>A>U, C, and G>A>U>C respectively. In this enzyme, one of the three histidine residues which have been thought to be important for catalysis of RNase Rh, a typical RNase of this family of enzymes, His104 was replaced by tyrosine residue. Based on the results, the role of H104, which has been proposed to be a phosphate binding site with a substrate, was reconsidered, and we proposed a revised role of this His residue in the hydrolysis mechanism of RNase T2 family enzymes.     

Global and local motions in ribonuclease A: a molecular dynamics study

The understanding of protein dynamics is one of the major goals of structural biology. A direct link between protein dynamics and function has been provided by x-ray studies performed on ribonuclease A (RNase A) (B. F. Rasmussen et al., Nature, 1992, Vol. 357, pp. 423-424; L. Vitagliano et al., Proteins: Structure, Function, and Genetics, 2002, Vol. 46, pp. 97-104). Here we report a 3 ns molecular dynamics simulation of RNase A in water aimed at characterizing the dynamical behavior of the enzyme. The analysis of local and global motions provides interesting insight on the dynamics/function relationship of RNase A. In agreement with previous crystallographic reports, the present study confirms that the RNase A active site is constituted by rigid (His12, Asn44, Thr45) and flexible (Lys41, Asp83, His119, Asp121) residues. The analysis of the global motions, performed using essential dynamics, shows that the two beta-sheet regions of RNase A move coherently in opposite directions, thus modifying solvent accessibility of the active site, and that the mixed alpha/3(10)-helix (residues 50-60) behaves as a mechanical hinge during the breathing motion of the protein. These data demonstrate that this motion, essential for RNase A substrate binding and release, is an intrinsic dynamical property of the ligand-free enzyme.     

Mutations in p97/VCP induce unfolding activity

A comparison of the protein sequences of various two-domain AAA+ ATPases revealed a striking difference in the residues lining the central pore of the D1 domain. The protein unfoldases of the bacterial Clp family and the archaeal VAT protein have at least one aromatic residue in the central D1 pore. In contrast, none of the members of the eukaryotic p97/VCP protein family has an aromatic residue in the D1 pore. The protein unfolding activity of VAT and other AAA+ ATPases is critically dependent on the presence of aromatic residues in this central pore. Unfoldase activity has not been demonstrated for the p97/VCP family in vitro. Thus, we exchanged the two aliphatic residues leucine and alanine of the D1 pore for aromatic tyrosine residues in full length p97 and in p97DeltaN, a truncated form of p97 lacking the N domain. We found that the mutant p97DeltaN variants with a single tyrosine or with two tyrosine residues in the central pore of D1 unfold the Clp family and VAT model substrate YFP-ssrA, whereas full length p97 with aromatic pore residues and wild-type p97 or p97DeltaN do not. Thus, p97 can exert unfoldase activity in vitro, provided that a single tyrosine residue is introduced into the D1 pore and that the N domain is deleted.