Role of histidine-50, glutamic acid-96, and histidine-137 in the ribonucleolytic mechanism of the ribotoxin alpha-sarcin

alpha-Sarcin is a ribotoxin secreted by the mold Aspergillus giganteus that degrades the ribosomal RNA by acting as a cyclizing ribonuclease. Three residues potentially involved in the mechanism of catalysis--histidine-50, glutamic acid-96, and histidine-137--were changed to glutamine. Three different single mutation variants (H50Q, E96Q, H137Q) as well as a double variant (H50/137Q) and a triple variant (H50/137Q/E96Q) were prepared and isolated to homogeneity. These variants were spectroscopically (circular dichroism, fluorescence emission, and proton nuclear magnetic resonance) characterized. According to these results, the three-dimensional structure of these variants of alpha-sarcin was preserved; only very minor local changes were detected. All the variants were inactive when assayed against either intact ribosomes or poly(A). The effect of pH on the ribonucleolytic activity of alpha-sarcin was evaluated against the ApA dinucleotide. This assay revealed that only the H50Q variant still retained its ability to cleave a phosphodiester bond, but it did so to a lesser extent than did wild-type alpha-sarcin. The results obtained are interpreted in terms of His137 and Glu96 as essential residues for the catalytic activity of alpha-sarcin (His137 as the general acid and Glu96 as the general base) and His50 stabilizing the transition state of the reaction catalyzed by alpha-sarcin.     

Dissecting structural and electrostatic interactions of charged groups in alpha-sarcin. An NMR study of some mutants involving the catalytic residues

The cytotoxic ribonuclease alpha-sarcin is the best characterized member of the ribotoxin family. Ribotoxins share a common structural core, catalytic residues, and active site topology with members of the broader family of nontoxic microbial extracellular RNases. They are, however, much more specific in their biological action. To shed light on the highly specific alpha-sarcin activity, we have evaluated the structural and electrostatic interactions of its charged groups, by combining the structural and pK(a) characterization by NMR of several variants with theoretical calculations based on the Tanford-Kirkwood and Poisson-Boltzmann models. The NMR data reveal that the global conformation of wild-type alpha-sarcin is preserved in the H50Q, E96Q, H137Q, and H50/137Q variants, and that His137 is involved in an H-bond that is crucial in maintaining the active site structure and in reinforcing the stability of the enzyme. The loss of this H-bond in the H137Q and H50/137Q variants modifies the local structure of the active site. The pK(a) values of active site groups H50, E96, and H137 in the four variants have been determined by two-dimensional NMR. The catalytic dyad of E96 and H137 is not sensitive to charge replacements, since their pK(a) values vary less than +/-0.3 pH unit with respect to those of the wild type. On the contrary, the pK(a) of His50 undergoes drastic changes when compared to its value in the intact protein. These amount to an increase of 0.5 pH unit or a decrease of 1.1 pH units depending on whether a positive or negative charge is substituted at the active site. The main determinants of the pK(a) values of most of the charged groups in alpha-sarcin have been established by considering the NMR results in conjunction with those derived from theoretical pK(a) calculations. With regard to the active site residues, the H50 pK(a) is chiefly influenced by electrostatic interactions with E96 and H137, whereas the effect of the low dielectric constant and the interaction with R121 appear to be the main determinants of the altered pK(a) value of E96 and H137. Charge-charge interactions and an increased level of burial perturb the pK(a) values of the active site residues of alpha-sarcin, which can account for its reduced ribonucleolytic activity and its high specificity.     

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.     

Leucine 145 of the ribotoxin alpha-sarcin plays a key role for determining the specificity of the ribosome-inactivating activity of the protein

Secreted fungal RNases, represented by RNase T1, constitute a family of structurally related proteins that includes ribotoxins such as alpha-sarcin. The active site residues of RNase T1 are conserved in all fungal RNases, except for Phe 100 that is not present in the ribotoxins, in which Leu 145 occupies the equivalent position. The mutant Leu145Phe of alpha-sarcin has been recombinantly produced and characterized by spectroscopic methods (circular dichroism, fluorescence spectroscopy, and NMR). These analyses have revealed that the mutant protein retained the overall conformation of the wild-type alpha-sarcin. According to the analyses performed, Leu 145 was shown to be essential to preserve the electrostatic environment of the active site that is required to maintain the anomalous low pKa value reported for the catalytic His 137 of alpha-sarcin. Enzymatic characterization of the mutant protein has revealed that Leu 145 is crucial for the specific activity of alpha-sarcin on ribosomes.     

Glutamate 85 and glutamate 228 contribute to the pH-response of the soluble form of chloride intracellular channel 1

The chloride intracellular channel protein, CLIC1, is synthesised as a soluble monomer that can reversibly bind membranes. Soluble CLIC1 is proposed to respond to the low pH found at a membrane surface by partially unfolding and restructuring into a membrane-competent conformation. This transition is proposed to be controlled by strategically located “pH-sensor” residues that become protonated at acidic pH. In this study, we investigate the role of two conserved glutamate residues, Glu85 in the N-domain and Glu228 in the C-domain, as pH-sensors. E85L and E228L CLIC1 variants were created to reduce pH sensitivity by permanently breaking the bonds these residues form. The structure and stability of each variant was compared to the wild type at both pH 7.0 and pH 5.5. Neither substitution significantly altered the structure but both decreased the conformational stability. Furthermore, E85L CLIC1 formed a urea-induced unfolding intermediate state at both pH 7 and pH 5.5 compared to wild-type and E228L CLIC1 which only formed the intermediate at pH 5.5. We conclude that Glu85 and Glu228 are two of the five pH-sensor residues of CLIC1 and contribute to the pH-response in different ways. Glu228 lowers the stability of the native state at pH 5.5, while Glu85 contributes both to the stability of the native state and to the formation of the intermediate state. By putting these interactions into the context of the three previously described CLIC1 pH-sensor residues, we propose a mechanism for the conversion of CLIC1 from the soluble state to the pre-membrane form.     

Effect of active site residues in barnase on activity and stability.

We have mutated residues in the active site of the ribonuclease, barnase, in order to determine their effects on both enzyme activity and protein stability. Mutation of several of the positively charged residues that interact with the negatively charged RNA substrate (Lys27----Ala, Arg59----Ala and His102----Ala) causes large decreases in activity. This is accompanied, however, by an increase in stability. There is presumably electrostatic strain in the active site where positively charged side-chains are clustered. Mutation of several residues that make hydrogen bonds (Ser57----Ala, Asn58----Asp and Tyr103----Phe) causes smaller decreases in activity, but increases or has no effect on stability. Deletion of hydrogen bonding groups elsewhere in proteins has been found previously to decrease stability by 0.5 to 1.5 kcal mol-1. Conversely, we find that two mutations (Asp54----Asn and Gln104----Ala) decrease stability and increase activity. Another mutation (Glu73----Ala) decreases both activity and stability. It is clear that many residues in the active site do not contribute to stability and that for some, but not all, of the residues there is a compromise between activity and stability. This suggests that certain types of local instability may be necessary for substrate binding and catalysis by barnase. This has implications for the understanding of enzyme activity and the design of enzymes.     

Increasing protein stability by altering long-range coulombic interactions.

It is difficult to increase protein stability by adding hydrogen bonds or burying nonpolar surface. The results described here show that reversing the charge on a side chain on the surface of a protein is a useful way of increasing stability. Ribonuclease T1 is an acidic protein with a pI approximately 3.5 and a net charge of approximately -6 at pH 7. The side chain of Asp49 is hyperexposed, not hydrogen bonded, and 8 A from the nearest charged group. The stability of Asp49Ala is 0.5 kcal/mol greater than wild-type at pH 7 and 0.4 kcal/mol less at pH 2.5. The stability of Asp49His is 1.1 kcal/mol greater than wild-type at pH 6, where the histidine 49 side chain (pKa = 7.2) is positively charged. Similar results were obtained with ribonuclease Sa where Asp25Lys is 0.9 kcal/mol and Glu74Lys is 1.1 kcal/mol more stable than the wild-type enzyme. These results suggest that protein stability can be increased by improving the coulombic interactions among charged groups on the protein surface. In addition, the stability of RNase T1 decreases as more hydrophobic aromatic residues are substituted for Ala49, indicating a reverse hydrophobic effect.     

The refined 2.15 A X-ray crystal structure of human liver cathepsin B: the structural basis for its specificity.

From the lysosomal cysteine proteinase cathepsin B, isolated from human liver in its two-chain form, monoclinic crystals were obtained which contain two molecules per asymmetric unit. The molecular structure was solved by a combination of Patterson search and heavy atom replacement methods (simultaneously with rat cathepsin B) and refined to a crystallographic R value of 0.164 using X-ray data to 2.15 A resolution. The overall folding pattern of cathepsin B and the arrangement of the active site residues are similar to the related cysteine proteinases papain, actinidin and calotropin DI. 166 alpha-carbon atoms out of 248 defined cathepsin B residues are topologically equivalent (with an r.m.s. deviation of 1.04 A) with alpha-carbon atoms of papain. However, several large insertion loops are accommodated on the molecular surface and modify its properties. The disulphide connectivities recently determined for bovine cathepsin B by chemical means were shown to be correct. Some of the primed subsites are occluded by a novel insertion loop, which seems to favour binding of peptide substrates with two residues carboxy-terminal to the scissile peptide bond; two histidine residues (His110 and His111) in this "occluding loop' provide positively charged anchors for the C-terminal carboxylate group of such polypeptide substrates. These structural features explain the well-known dipeptidyl carboxypeptidase activity of cathepsin B. The other subsites adjacent to the reactive site Cys29 are relatively similar to papain; Glu245 in the S2 subsite favours basic P2-side chains. The above mentioned histidine residues, but also the buried Glu171 might represent the group with a pKa of approximately 5.5 near the active site, which governs endo- and exopeptidase activity. The "occluding loop' does not allow cystatin-like protein inhibitors to bind to cathepsin B as they do to papain, consistent with the reduced affinity of these protein inhibitors for cathepsin B compared with the related plant enzymes.     

Involvement of the amino-terminal beta-hairpin of the Aspergillus ribotoxins on the interaction with membranes and nonspecific ribonuclease activity

Ribotoxins are a family of potent cytotoxic proteins from Aspergillus whose members display a high sequence identity (85% for about 150 amino acid residues). The three-dimensional structures of two of these proteins, alpha-sarcin and restrictocin, are known. They interact with phospholipid bilayers, according to their ability to enter cells, and cleave a specific phosphodiester bond in the large subunit of ribosome thus inhibiting protein biosynthesis. Two nonconservative sequence changes between these proteins are located at the amino-terminal beta-hairpin of alpha-sarcin, a characteristic structure that is absent in other nontoxic structurally related microbial RNases. These two residues of alpha-sarcin, Lys 11 and Thr 20, have been substituted with the equivalent amino acids in restrictocin. The single mutants (K11L and T20D) and the corresponding K11L/T20D double mutant have been produced in Escherichia coli and purified to homogeneity. The spectroscopic characterization of the purified proteins reveals that the overall native structure is preserved. The ribonuclease and lipid-perturbing activities of the three mutants and restrictocin have been evaluated and compared with those of alpha-sarcin. These proteins exhibit the same ability to specifically inactivate ribosomes, although they show different activity against nonspecific substrate analogs such as poly(A). The mutant variant K11L and restrictocin display a lower phospholipid-interacting ability correlated with a decreased cytotoxicity. The results obtained are interpreted in terms of the involvement of the amino-terminal beta-hairpin in the interaction with both membranes and polyadenylic acid.     

Different positively charged amino acids have similar effects on the topology of a polytopic transmembrane protein in Escherichia coli.

Integral membrane proteins from a wide variety of sources conform to a "positive-inside rule," with many more positively charged amino acids in their cytoplasmic as compared to extracytoplasmic domains. A growing body of experimental work also points to positively charged residues in regions flanking the apolar transmembrane segments as being the main topological determinants. In this paper, we report a systematic comparison of the effects of positively (Arg, Lys, His) as well as negatively (Asp, Glu) charged residues on the membrane topology of a model Escherichia coli inner membrane protein. Our results show that positive charge is indeed the major factor determining the transmembrane topology, with Arg and Lys being of nearly equal efficiency. His, although normally a very weak topological determinant, can be potentiated by a lowering of the cytoplasmic pH. Asp and Glu affect the topology to similar extents and only when present in very high numbers.     

Characterization of the S1 binding site of the glutamic acid-specific protease from Streptomyces griseus.

The glutamic acid-specific protease from Streptomyces griseus (SGPE) is an 18.4-kDa serine protease with a distinct preference for Glu in the P1 position. Other enzymes characterized by a strong preference for negatively charged residues in the P1 position, e.g., interleukin-1 beta converting enzyme (ICE), use Arg or Lys residues as counterions within the S1 binding site. However, in SGPE, this function is contributed by a His residue (His 213) and two Ser residues (Ser 192 and S216). It is demonstrated that proSGPE is activated autocatalytically and dependent on the presence of a Glu residue in the -1 position. Based on this observation, the importance of the individual S1 residues is evaluated considering that enzymes unable to recognize a Glu in the P1 position will not be activated. Among the residues constituting the S1 binding site, it is demonstrated that His 213 and Ser 192 are essential for recognition of Glu in the P1 position, whereas Ser 216 is less important for catalysis out has an influence on stabilization of the ground state. From the three-dimensional structure, it appears that His 213 is linked to two other His residues (His 199 and His 228), forming a His triad extending from the S1 binding site to the back of the enzyme. This hypothesis has been tested by substitution of His 199 and His 228 with other amino acid residues. The catalytic parameters obtained with the mutant enzymes, as well as the pH dependence, do not support this theory; rather, it appears that His 199 is responsible for orienting His 213 and that His 228 has no function associated with the recognition of Glu in P1.     

Inverse electrostatic effect: electrostatic repulsion in the unfolded state stabilizes a leucine zipper

The pH-dependent stability of a protein is strongly affected by electrostatic interactions between ionizable residues in the folded as well as unfolded state. Here we characterize the individual contributions of charged Glu and His residues to stability and determine the NMR structure of the designed, heterodimeric leucine zipper AB consisting of an acidic A chain and a basic B chain. Thermodynamic parameters are compared with those of the homologous leucine zipper AB(SS) in which the A and B chains are disulfide-linked. NMR structures of AB based on (1)H NMR data collected at 600 MHz converge, and formation of the same six interchain salt bridges found previously in disulfide-linked AB(SS) [Marti, D. N., and Bosshard, H. R. (2003) J. Mol. Biol. 330, 621-637] is indicated. While the structures of AB and AB(SS) are very similar, their pH-dependent relative stabilities are strikingly different. The stability of AB peaks at pH approximately 4.5 and is higher at pH 8 than at pH 2. In contrast, AB(SS) is most stable at acidic pH where no interhelical salt bridges are formed. The different energetic contributions of charged Glu and His residues to stability of the two coiled coil structures were evaluated from pK(a) shifts induced by folding. The six charged Glu residues involved in salt bridges stabilize leucine zipper AB by 4.5 kJ/mol yet destabilize disulfide-linked AB(SS) by -1.1 kJ/mol. Two non-ion-paired Glu charges destabilize AB by only -1.8 kJ/mol but AB(SS) by -5.6 kJ/mol. The higher relative stability of AB at neutral pH is not caused by more favorable electrostatic interactions in the folded leucine zipper. It is due mainly to unfavorable electrostatic interactions in the unfolded A and B chains and may therefore be called an inverse electrostatic effect. This study illustrates the importance of residual interactions in the unfolded state and how the energetics of the unfolded state affect the stability of the folded protein.     

Rearrangement of charge-charge interactions in variant ubiquitins as detected by double-mutant cycles and NMR

Previous studies of ubiquitin disclosed numerous charge-charge interactions on the protein's surface. To investigate how neighboring residues influence the strength of these interactions, double-mutant cycles are combined with pK(a) determinations by 2D NMR. More specifically, the environment around the Asp21-Lys29 ion pair has been altered through mutations at position 25, which is an asparagine in mammalian ubiquitin and a positively-charged residue in many other ubiquitin-like proteins. The pK(a) value of Asp21 decreases by 0.4 to 0.7 pH unit when Asn25 is substituted with a positively charged residue, suggesting a new and favorable ion pair interaction between positions 21 and 25. However, analysis of double mutants reveals that the favorable interaction between Asp21 and Lys29 is weakened when position 25 is a positively charged residue. Interestingly, while the pK(a) value of His25 in the N25H variant agrees with model compound values, additional mutants reveal that this agreement is fortuitous, resulting from a balance of favorable and unfavorable interactions; similar results were observed previously for Glu34 in ubiquitin and His8 in staphylococcal nuclease. Ionizable groups may thus have pK(a) values similar to model compound values and yet still be involved in significant interactions with other protein groups. One surprising result of introducing positively charged residues at position 25 is a new interaction between Lys29 and Glu18, an interaction not present in wild-type ubiquitin. This unanticipated result illustrates a key advantage of using NMR to determine pK(a) values for many residues simultaneously in the variant proteins. Overall, the strength of an interaction between two residues at the surface of ubiquitin is sensitive to the identity of neighboring residues. The results also demonstrate that relatively conservative and common point mutations such as substitutions of polar with charged residues and vice versa can have effects on interactions beyond the site of mutation per se.     

Geometry of interaction of the histidine ring with other planar and basic residues

Among the aromatic residues in protein structures, histidine (His) is unique, as it can exist in the neutral or positively charged form at the physiological pH. As such, it can interact with other aromatic residues as well as form hydrogen bonds with polar and charged (both negative and positive) residues. We have analyzed the geometry of interaction of His residues with nine other planar side chains containing aromatic (residues Phe, Tyr, Trp, and His), carboxylate (Asp and Glu), carboxamide (Asn and Gln) and guanidinium (Arg) groups in 432 polypeptide chains. With the exception of the aspartic (Asp) and glutamic (Glu) acid side-chains, all other residues prefer to interact in a face-to-face or offset-face-stacked orientation with the His ring. Such a geometry is different from the edge-to-face relative orientation normally associated with the aromatic-aromatic interaction. His-His pair prefers to interact in a face-to-face orientation; however, when both the residues bind the same metal ion, the interplanar angle is close to 90 degrees. The occurrence of different interactions (including the nonconventional N-H...pi and C-H...pi hydrogen bonds) have been correlated with the relative orientations between the interacting residues. Several structural motifs, mostly involved in binding metal ions, have been identified by considering the cases where His residues are in contact with four other planar moieties. About 10% of His residues used here are also found in sequence patterns in PROSITE database. There are examples of the amino end of the Lys side chain interacting with His residues in such a way that it is located on an arc around a ring nitrogen atom.     

Contribution of active site residues to the activity and thermal stability of ribonuclease Sa

We have used site-specific mutagenesis to study the contribution of Glu 74 and the active site residues Gln 38, Glu 41, Glu 54, Arg 65, and His 85 to the catalytic activity and thermal stability of ribonuclease Sa. The activity of Gln38Ala is lowered by one order of magnitude, which confirms the involvement of this residue in substrate binding. In contrast, Glu41Lys had no effect on the ribonuclease Sa activity. This is surprising, because the hydrogen bond between the guanosine N1 atom and the side chain of Glu 41 is thought to be important for the guanine specificity in related ribonucleases. The activities of Glu54Gln and Arg65Ala are both lowered about 1000-fold, and His85Gln is totally inactive, confirming the importance of these residues to the catalytic function of ribonuclease Sa. In Glu74Lys, k(cat) is reduced sixfold despite the fact that Glu 74 is over 15 A from the active site. The pH dependence of k(cat)/K(M) is very similar for Glu74Lys and wild-type RNase Sa, suggesting that this is not due to a change in the pK values of the groups involved in catalysis. Compared to wild-type RNase Sa, the stabilities of Gln38Ala and Glu74Lys are increased, the stabilities of Glu41Lys, Glu54Gln, and Arg65Ala are decreased and the stability of His85Gln is unchanged. Thus, the active site residues in the ribonuclease Sa make different contributions to the stability.     

Cleavage specificities of aspartic proteinases toward oxidized insulin B chain at different pH values

The cleavage specificities of typical aspartic proteinases: pepsin A, gastricsin, cathepsin D and rhizopuspepsin, were examined at different pH values with oxidized insulin B chain as a substrate with special attention to the specificities near neutral pH. Significant differences in relative specificity for scissile bonds were observed between pH 2.0 and 5.5-6.5, which may be partly related with the changes in dissociation states of the His and Glu residues in the substrate and the ionizable residues in the active site of each enzyme.     

Contributions of all 20 amino acids at site 96 to the stability and structure of T4 lysozyme

To try to resolve the loss of stability in the temperature‐sensitive mutant of T4 lysozyme, Arg 96 → His, all of the remaining 18 naturally occurring amino acids were substituted at site 96. Also, in response to suggestions that the charged residues Lys85 and Asp89, which are 5–8 Å away, may have important effects, each of these amino acids was replaced with alanine. Crystal structures were determined for many of the variants. With the exception of the tryptophan and valine mutants R96W and R96V, the crystallographic analysis shows that the substituted side chain following the path of Arg96 in wildtype (WT). The melting temperatures of the variants decrease by up to ～16°C with WT being most stable. There are two site 96 replacements, with lysine or glutamine, that leave the stability close to that of WT. The only element that the side chains of these residues have in common with the WT arginine is the set of three carbon atoms at the C^α, C^β, and C^γ positions. Although each side chain is long and flexible with a polar group at the distal position, the details of the hydrogen bonding to the rest of the protein differ in each case. Also, the glutamine replacement lacks a positive charge. This shows that there is some adaptability in achieving full stabilization at this site. At the other extreme, to be maximally destabilizing a mutation at site 96 must not only eliminate favorable interactions but also introduce an unfavorable element such as steric strain or a hydrogen‐bonding group that remains unsatisfied. Overall, the study highlights the essential need for atomic resolution site‐specific structural information to understand and to predict the stability of mutant proteins. It can be very misleading to simply assume that conservative amino acid substitutions cause small changes in stability, whereas large stability changes are associated with nonconservative replacements.     

Binding interactions between the active center cleft of recombinant pokeweed antiviral protein and the alpha-sarcin/ricin stem loop of ribosomal RNA

Pokeweed antiviral protein (PAP) is a ribosome-inactivating protein that catalytically cleaves a specific adenine base from the highly conserved alpha-sarcin/ricin loop of the large ribosomal RNA, thereby inhibiting protein synthesis at the elongation step. Recently, we discovered that alanine substitutions of the active center cleft residues significantly impair the depurinating and ribosome inhibitory activity of PAP. Here we employed site-directed mutagenesis combined with standard filter binding assays, equilibrium binding assays with Scatchard analyses, and surface plasmon resonance technology to elucidate the putative role of the PAP active center cleft in the binding of PAP to the alpha-sarcin/ricin stem loop of rRNA. Our findings presented herein provide experimental evidence that besides the catalytic site, the active center cleft also participates in the binding of PAP to the target tetraloop structure of rRNA. These results extend our recent modeling studies, which predicted that the residues of the active center cleft could, via electrostatic interactions, contribute to both the correct orientation and stable binding of the substrate RNA molecules in PAP active site pocket. The insights gained from this study also explain why and how the conserved charged and polar side chains located at the active center cleft of PAP and certain catalytic site residues, that do not directly participate in the catalytic deadenylation of ribosomal RNA, play a critical role in the catalytic removal of the adenine base from target rRNA substrates by affecting the binding interactions between PAP and rRNA.     

Engineering of the pH-dependence of thermolysin activity as examined by site-directed mutagenesis of Asn112 located at the active site of thermolysin

Asn112 is located at the active site of thermolysin, 5-8 A from the catalytic Zn2+ and catalytic residues Glu143 and His231. When Asn112 was replaced with Ala, Asp, Glu, Lys, His, and Arg by site-directed mutagenesis, the mutant enzymes N112D and N112E, in which Asn112 is replaced with Asp and Glu, respectively, were secreted as an active form into Escherichia coli culture medium, while the other four were not. In the hydrolysis of a neutral substrate N-[3-(2-furyl)acryloyl]-Gly-L-Leu amide, the kcat/Km values of N112D and N112E exhibited bell-shaped pH-dependence, as did the wild-type thermolysin (WT). The acidic pKa of N112D was 5.7 +/- 0.1, higher by 0.4 +/- 0.2 units than that of WT, suggesting that the introduced negative charge suppressed the protonation of Glu143 or Zn2+-OH. In the hydrolysis of a negatively charged substrate, N-carbobenzoxy-l-Asp-l-Phe methyl ester (ZDFM), the pH-dependence of kcat/Km of the mutants decreased with increase in pH from 5.5 to 8.5, while that of WT was bell-shaped. This difference might be explained by the electrostatic repulsion between the introduced Asp/Glu and ZDFM, suggesting that introducing ionizing residues into the active site of thermolysin might be an effective means of modifying its pH-activity profile.     

Modulating the Stiffness of the Myosin VI Single α-Helical Domain

Highly charged, single α-helical (SAH) domains contain a high percentage of Arg, Lys, and Glu residues. Their dynamic salt bridge pairing creates the exceptional stiffness of these helical rods, with a persistence length of more than 200 Å for the myosin VI SAH domain. With the aim of modulating the stiffness of the helical structure, we investigated the effect, using NMR spectroscopy, of substituting key charged Arg, Lys, Glu, and Asp residues by Gly or His. Results indicate that such mutations result in the transient breaking of the helix at the site of mutation but with noticeable impact on amide hydrogen exchange rates extending as far as ±2 helical turns, pointing to a substantial degree of cooperativity in SAH stability. Whereas a single Gly substitution caused transient breaks ∼20% of the time, two consecutive Gly substitutions break the helix ∼65% of the time. NMR relaxation measurements indicate that the exchange rate between an intact and a broken helix is fast (>300,000 s⁻¹) and that for the wild-type sequence, the finite persistence length is dominated by thermal fluctuations of backbone torsion angles and H-bond lengths, not by transient helix breaking. The double mutation D27H/E28H causes a pH-dependent fraction of helix disruption, in which the helix breakage increases from 26% at pH 7.5 to 53% at pH 5.5. The ability to modulate helical integrity by pH may enable incorporation of externally tunable dynamic components in the design of molecular machines.