Protein dynamics in a family of laboratory evolved thermophilic enzymes

Molecular dynamics simulations were employed to study how protein solution structure and dynamics are affected by adaptation to high temperature. Simulations were carried out on a para-nitrobenzyl esterase (484 residues) and two thermostable variants that were generated by laboratory evolution. Although these variants display much higher melting temperatures than wild-type (up to 18 degrees C higher) they are both >97% identical in sequence to the wild-type. In simulations at 300 K the thermostable variants remain closer to their crystal structures than wild-type. However, they also display increased fluctuations about their time-averaged structures. Additionally, both variants show a small but significant increase in radius of gyration relative to wild-type. The vibrational density of states was calculated for each of the esterases. While the density of states profiles are similar overall, both thermostable mutants show increased populations of the very lowest frequency modes (<10 cm(-1)), with the more stable mutant showing the larger increase. This indicates that the thermally stable variants experience increased concerted motions relative to wild-type. Taken together, these data suggest that adaptation for high temperature stability has resulted in a restriction of large deviations from the native state and a corresponding increase in smaller scale fluctuations about the native state. These fluctuations contribute to entropy and hence to the stability of the native state. The largest changes in localized dynamics occur in surface loops, while other regions, particularly the active site residues, remain essentially unchanged. Several mutations, most notably L313F and H322Y in variant 8G8, are in the region showing the largest increase in fluctuations, suggesting that these mutations confer more flexibility to the loops. As a validation of our simulations, the fluctuations of Trp102 were examined in detail, and compared with Trp102 phosphorescence lifetimes that were previously measured. Consistent with expectations from the theory of phosphorescence, an inverse correlation between out-of-plane fluctuations on the picosecond time scale and phosphorescence lifetime was observed.     

Surface-exposed tryptophan residues are essential for O-acetylserine sulfhydrylase structure,function, and stability

O-Acetylserine sulfhydrylase is a homodimeric enzyme catalyzing the last step of cysteine biosynthesis via a Bi Bi ping-pong mechanism. The subunit is composed of two domains, each containing one tryptophan residue, Trp50 in the N-terminal domain and Trp161 in the C-terminal domain. Only Trp161 is highly conserved in eucaryotes and bacteria. The coenzyme pyridoxal 5'-phosphate is bound in a cleft between the two domains. The enzyme undergoes an open to closed conformational transition upon substrate binding. The effect of single Trp to Tyr mutations on O-acetylserine sulfhydrylase structure, function, and stability was investigated with a variety of spectroscopic techniques. The mutations do not significantly alter the enzyme secondary structure but affect the catalysis, with a predominant influence on the second half reaction. The W50Y mutation strongly affects the unfolding pathway due to the destabilization of the intersubunit interface. The W161Y mutation, occurring in the C-terminal domain, produces a reduction of the accessibility of the active site to acrylamide and stabilizes thermodynamically the N-terminal domain, a result consistent with stronger interdomain interactions.     

Mutations on N-terminal region of Taiwan cobra phospholipase A(2) result in structurally distorted effects.

In the present study, three Taiwan cobra PLA(2) variants were prepared by adding an extra N-terminal Met, substituting Asn-1 by Met or deleting the N-terminal heptapeptide. Recombinant PLA(2) mutants were expressed in Escherichia coli (E. coli), and purified to homogeneity by reverse phase HPLC. Fluorescence measurement showed that the hydrophobic character of the catalytic site, the microenvironment of Trp residues and energy transfer from excited Trp to 8-anilinonaphthalene sulfonate (ANS) were affected by N-terminal mutations. An alteration in the structural flexibility of the active site was noted with the mutants lacking the N-terminal heptapeptide or with an extra N-terminal Met added as evidenced by the inability of the two variants to bind with Ba(2+). Moreover, modification of Lys residues and energy transfer within the protein-ANS complex revealed that the Ca(2+)-induced change in the global structure of PLA(2) was different from that in N-terminal variants. Together with the fact that an 'activation network' connects the N-terminus with the active site, our data suggest that mutagenesis on the N-terminal region affects directly the fine structure of the catalytic site, which subsequently transmits its influence in altering the structure outside the active site of PLA(2). Copyright (c) 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Physical studies of tyrosine and tryptophan residues in mammalian A1 heterogeneous nuclear ribonucleoprotein. Support for a segmented structure.

The mammalian heterogeneous ribonucleoprotein (hnRNP) A1 and its constituent N-terminal domain, termed UP1, have been studied by steady-state and dynamic fluorimetry, as well as phosphorescence and optically detected magnetic resonance (ODMR) spectroscopy at cryogenic temperatures. The results of these diverse techniques coincide in assigning the site of the single tryptophan residue of A1, located in the UP1 domain, to a partially solvent-exposed site distal to the protein's nucleic acid binding surface. In contrast, tyrosine fluorescence is significantly perturbed when either protein associates with single-stranded polynucleotides. Tyr to Trp energy transfer at the singlet level is found for both UP1 and A1 proteins. Single-stranded polynucleotide binding induces a quenching of their intrinsic fluorescence emission, which can be attributed to a significant reduction (greater than 50%) of the Tyr contribution, while Trp emission is only quenched by approximately 15%. Tyrosine quenching effects of similar magnitude are seen upon polynucleotide binding by either UP1 (1 Trp, 4 Tyr) or A1 (1 Trp, 12 Tyr), strongly suggesting that Tyr residues in both the N-terminal and C-terminal domain of A1 are involved in the binding process. Tyr phosphorescence emission was strongly quenched in the complexes of UP1 with various polynucleotides, and was attributed to triplet state energy transfer to nucleic acid bases located in the close vicinity of the fluorophore. These results are consistent with stacking of the tyrosine residues with the nucleic acid bases. While the UP1 Tyr phosphorescence lifetime is drastically shortened in the polynucleotide complex, no change of phosphorescence emission maximum, phosphorescence decay lifetime or ODMR transition frequencies were observed for the single Trp residue. The results of dynamic anisotropy measurements of the Trp fluorescence have been interpreted as indicative of significant internal flexibility in both UP1 and A1, suggesting a flexible linkage connecting the two sub-domains in UP1. Theoretical calculations based on amino acid sequence for chain flexibility and other secondary structural parameters are consistent with this observation, and suggest that flexible linkages between sub-domains may exist in other RNA binding proteins. While the dynamic anisotropy data are consistent with simultaneous binding of both the C-terminal and the N-terminal domains to the nucleic acid lattice, no evidence for simultaneous binding of both UP1 sub-domains was found.     

Insights into the catalytic roles of the polypeptide regions in the active site of thermolysin and generation of the thermolysin variants with high activity and stability

The active site of thermolysin is composed of one zinc ion and five polypeptide regions [N-terminal sheet (Asn112-Trp115), alpha-helix 1 (Val139-Thr149), C-terminal loop 1 (Asp150-Gly162), alpha-helix 2 (Ala163-Val176) and C-terminal loop 2 (Gln225-Ser234)]. To explore their catalytic roles, we introduced single amino-acid substitutions into these regions by site-directed mutagenesis and examined their effects on the activity and stability. Seventy variants, in which one of the twelve residues (Ala113, Phe114, Trp115, Asp150, Tyr157, Gly162, Ile168, Ser169, Asp170, Asn227, Val230 and Ser234) was replaced, were produced in Escherichia coli. The hydrolytic activities of thermolysin for N-[3-(2-furyl)acryloyl]-Gly-l-Leu amide (FAGLA) and casein revealed that the N-terminal sheet and alpha-helix 2 were critical in catalysis and the C-terminal loops 1 and 2 were in substrate recognition. Twelve variants were active for both substrates. In the hydrolysis of FAGLA and N-carbobenzoxy-L-Asp-L-Phe methyl ester, the k(cat)/K(m) values of the D150E (in which Asp150 is replaced with Glu) and I168A variants were 2-3 times higher than those of the wild-type (WT) enzyme. Thermal inactivation of thermolysin at 80 degrees C was greatly suppressed with the D150H, D150W, I168A, I168H, N227A, N227H and S234A. The evidence might provide the insights into the activation and stabilization of thermolysin.     

Effect of His-tag location on the catalytic activity of 3-hydroxybutyrate dehydrogenase

The effect of hexahistidine-tag (His-tag) location at either the C or N-terminus on the catalytic activity of 3-hydroxybutyrate dehydrogenase (3HBDH) from Alcaligenes faecalis was studied. The kinetic parameters of 3HBDHs with C and N-terminal His-tags were investigated, and the enzyme with an N-terminal His-tag was found to have approximately 1,200-fold higher catalytic efficiency than its C-terminal counterpart. Furthermore, the effect of His-tag location on the catalytic activity of 3 engineered variants of 3HBDH that were previously developed for the conversion of levulinic acid to 4-hydroxyvaleric acid was also investigated. All of the N-terminal variants exhibited higher catalytic efficiency for levulinic acid than did the C-terminal counterparts. The structural basis of the His-tag effect was studied by investigating the structure of 3HBDH obtained from in silico His-tag modification, and the results revealed that the modification of the C-terminal structure could deform the hinge region of the active site entry loop, disrupting the catalytic motion of the enzyme. In contrast, due to the location of the N-terminus far from the active site of the enzyme, the catalytic activity of the enzyme was not severely affected by the N-terminal His-tag.     

Fluorescence study of domain structure and lipid interaction of human apolipoproteins E3 and E4

Human apolipoprotein E (apoE) isoforms exhibit different conformational stabilities and lipid-binding properties that give rise to altered cholesterol metabolism among the isoforms. Using Trp-substituted mutations and site-directed fluorescence labeling, we made a comprehensive comparison of the conformational organization of the N- and C-terminal domains and lipid interactions between the apoE3 and apoE4 isoforms. Trp fluorescence measurements for selectively Trp-substituted variants of apoE isoforms demonstrated that apoE4 adopts less stable conformations in both the N- and C-terminal domains compared to apoE3. Consistent with this, the conformational reorganization of the N-terminal helix bundle occurs at lower guanidine hydrochloride concentration in apoE4 than in apoE3 as monitored by fluorescence resonance energy transfer (FRET) from Trp residues to acrylodan attached at the N-terminal helix. Upon binding of apoE3 and apoE4 variants to egg phosphatidylcholine small unilamellar vesicles, similar changes in Trp fluorescence or FRET efficiency were observed for the isoforms, indicating that the opening of the N-terminal helix bundle occurs similarly in apoE3 and apoE4. Introduction of mutations into the C-terminal domain of the apoE isoforms to prevent self-association and maintain the monomeric state resulted in great increase in the rate of binding of the C-terminal helices to a lipid surface. Overall, our results demonstrate that the different conformational organizations of the N- and C-terminal domains have a minor effect on the steady-state lipid-binding behavior of apoE3 and apoE4: rather, self-association property is a critical determinant in the kinetics of lipid binding through the C-terminal helices of apoE isoforms.     

Probing the role of oligomerization in the high thermal stability of Pyrococcus furiosus ornithine carbamoyltransferase by site-specific mutants.

The Pyrococcus furiosus ornithine carbamoyltransferase (OTCase) is extremely heat stable and maintains 50% of its catalytic activity after 60 min at 100 degrees C. The enzyme has an unusual quaternary structure when compared to anabolic OTCases from mesophilic organisms. It is built up of four trimers arranged in a tetrahedral manner, while other anabolic enzymes are single trimers. Residues Trp21, Glu25, Met29 and Trp33 are located in the main interfaces that occur between the catalytic trimers within the dodecamer. They participate in either hydrophobic clusters or ionic interactions. In order to elucidate the role played by the oligomerization in the enzyme stability at very high temperatures, we performed mutagenesis studies of these residues. All the variants show similar catalytic activities and kinetic properties when compared to the wild-type enzyme, allowing the interpretation of the mutations solely on heat stability and quaternary structure. The W21A variant has only a slight decrease in its stability, and is a dodecamer. The variants E25Q, M29A, W33A, W21A/W33A and E25Q/W33A show that altering more drastically the interfaces results in a proportional decrease in heat stability, correlated with a gradual dissociation of dodecamers into trimers. Finally, the E25Q/M29A/W33A variant shows a very large decrease in heat stability and is a trimer. These results suggest that extreme thermal stabilization of this OTCase is achieved in part through oligomerization.     

Biotin protein ligase from Saccharomyces cerevisiae. The N-terminal domain is required for complete activity.

Catalytically active biotin protein ligase from Saccharomyces cerevisiae (EC 6.3.4.15) was overexpressed in Escherichia coli and purified to near homogeneity in three steps. Kinetic analysis demonstrated that the substrates ATP, biotin, and the biotin-accepting protein bind in an ordered manner in the reaction mechanism. Treatment with any of three proteases of differing specificity in vitro revealed that the sequence between residues 240 and 260 was extremely sensitive to proteolysis, suggesting that it forms an exposed linker between an N-terminal 27-kDa domain and the C-terminal 50-kDa domain containing the active site. The protease susceptibility of this linker region was considerably reduced in the presence of ATP and biotin. A second protease-sensitive sequence, located in the presumptive catalytic site, was protected against digestion by the substrates. Expression of N-terminally truncated variants of the yeast enzyme failed to complement E. coli strains defective in biotin protein ligase activity. In vitro assays performed with purified N-terminally truncated enzyme revealed that removal of the N-terminal domain reduced BPL activity by greater than 3500-fold. Our data indicate that both the N-terminal domain and the C-terminal domain containing the active site are necessary for complete catalytic function.     

Conversion of feedback regulation in aspartate kinase by domain exchange

To elucidate the mechanism for the regulation of aspartate kinase (AK) via feedback inhibition, we constructed several chimeric enzymes between Bacillus subtilis AK II, a lysine-sensitive mesophilic enzyme, and Thermus flavus AK, a threonine-sensitive thermostable enzyme, each having the same alpha2beta2-type tetrameric structure. A chimeric AK, named BTT, composed of the chimeric alpha subunit that comprises of the N-terminal catalytic region from B. subtilis AK II and the C-terminal region from T. flavus, and the beta subunit from T. flavus, was inhibited only by threonine. Another chimeric enzyme, BT, which has a similar structure to that of BTT but lacks the beta subunit, having alpha2-type homo-dimeric structure, was also responsive only to threonine. However, the addition of threonine enhanced the activity of BT. These results indicate the regulatory function of C-terminal region and beta subunit in AK. BTT showed extremely high thermostability comparable to that of T. flavus, suggesting that the beta subunit also contributed to the stability of the AK.     

Molecular cloning of the gene encoding Vibrio metalloproteinase vimelysin and isolation of a mutant with high stability in organic solvents

Vimelysin is a unique metalloproteinase from Vibrio sp. T1800 exhibiting high activity at low temperature and high stability in organic solvents such as ethanol. A 1,821 bp open reading frame of the vimelysin gene encoded 607 amino acid residues consisting of an N-terminal pro-region, a mature enzyme, and a C-terminal pro-region. The mature enzyme region showed 80%, 57% and 35% sequence identity with the mature forms of vibriolysin from V. vulnificus, pseudolysin from Pseudomonas aeruginosa, and thermolysin from Bacillus thermoproteolyticus, respectively. The catalytic residues and zinc-binding motifs of metalloproteinases are well conserved in vimelysin. The vimelysin gene was expressed in E. coli JM109 cells and the recombinant enzyme was purified as a 38-kDa mature form from cell-free extracts. The purified recombinant enzyme is indistinguishable from the enzyme purified directly from Vibrio. To obtain mutants exhibiting higher stability in organic solvents, random mutations were introduced by error-prone PCR and 600 transformants were screened. The N123D mutant exhibits two times higher stability in organic solvents than the wild-type enzyme. A plausible mechanism for the stability of the N123D mutant in organic solvents was discussed based on homology models of vimelysin and the N123D mutant.     

Effect of mutations of N- and C-terminal charged residues on the activity of LCAT

On the basis of structural homology calculations, we previously showed that lecithin:cholesterol acyltransferase (LCAT), like lipases, belongs to the alpha/beta hydrolase fold family. As there is higher sequence conservation in the N-terminal region of LCAT, we investigated the contribution of the N- and C-terminal conserved basic residues to the catalytic activity of this enzyme. Most basic, and some acidic residues, conserved among LCAT proteins from different species, were mutated in the N-terminal (residues 1;-210) and C-terminal (residues 211;-416) regions of LCAT. Measurements of LCAT-specific activity on a monomeric substrate, on low density lipoprotein (LDL), and on reconstituted high density lipoprotein (rHDL) showed that mutations of N-terminal conserved basic residues affect LCAT activity more than those in the C-terminal region. This agrees with the highest conservation of the alpha/beta hydrolase fold and structural homology with pancreatic lipase observed for the N-terminal region, and with the location of most of the natural mutants reported for human LCAT. The structural homology between LCAT and pancreatic lipase further suggests that residues R80, R147, and D145 of LCAT might correspond to residues R37, K107, and D105 of pancreatic lipase, which form the salt bridges D105-K107 and D105-R37. Natural and engineered mutations at residues R80, D145, and R147 of LCAT are accompanied by a substantial decrease or loss of activity, suggesting that salt bridges between these residues might contribute to the structural stability of the enzyme.     

Roles of active site tryptophans in substrate binding and catalysis by alpha-1,3 galactosyltransferase

Aromatic amino acids are frequent components of the carbohydrate binding sites of lectins and enzymes. Previous structural studies have shown that in alpha-1,3 galactosyltransferase, the binding site for disaccharide acceptor substrates is encircled by four tryptophans, residues 249, 250, 314, and 356. To investigate their roles in enzyme specificity and catalysis, we expressed and characterized variants of the catalytic domain of alpha-1,3 galactosyltransferase with substitutions for each tryptophan. Substitution of glycine for tryptophan 249, whose indole ring interacts with the nonpolar B face of glucose or GlcNAc, greatly increases the K(m) for the acceptor substrate. In contrast, the substitution of tyrosine for tryptophan 314, which interacts with the beta-galactosyl moiety of the acceptor and UDP-galactose, decreases k(cat) for the galactosyltransferase reaction but does not affect the low UDP-galactose hydrolase activity. Thus, this highly conserved residue stabilizes the transition state for the galactose transfer to disaccharide but not to water. High-resolution crystallographic structures of the Trp(249)Gly mutant and the Trp(314)Tyr mutant indicate that the mutations do not affect the overall structure of the enzyme or its interactions with ligands. Substitutions for tryptophan 250 have only small effects on catalytic activity, but mutation of tryptophan 356 to threonine reduces catalytic activity for both transferase and hydrolase activities and reduces affinity for the acceptor substrate. This residue is adjacent to the flexible C-terminus that becomes ordered on binding UDP to assemble the acceptor binding site and influence catalysis. The results highlight the diverse roles of these tryptophans in enzyme action and the importance of k(cat) changes in modulating glycosyltransferase specificity.     

The role of tryptophan residues in the autoprocessing of prosubtilisin E

Subtilisin E, a serine protease from Bacillus subtilis, requires an N-terminal propeptide for its correct folding. The propeptide is autocleaved and digested by the subtilisin domain upon proper folding. Here we investigated the individual roles of the three Trp residues within the subtilisin domain (Trp106, Trp113 and Trp241) on propeptide processing, enzymatic activity and stability of subtilisin. When the propeptide processing was examined by SDS-PAGE after refolding by rapid dilution, the mutation at either position Trp106 or Trp113 was found to significantly delay the propeptide processing, while the mutation at Trp241 had no effect. Far-UV circular dichroism (CD) spectra of the mutants revealed that the mutations at the three positions did not affect appreciably the alpha-helix content of subtilisin. Secondary structure thermal unfolding monitored by CD spectroscopy revealed that none of the tryptophan residues had any significant effect on the stability of mature subtilisin. The enzymatic activity measurements showed that only Trp106 plays a major role in the enzymatic activity of subtilisin E. These results demonstrate that both Trp106 and Trp113 play a specific role in propeptide processing and enzymatic activity, while Trp241 plays no considerable role on any of these activities.     

Interdomain signaling in glutamine phosphoribosylpyrophosphate amidotransferase

The glutamine phosphoribosylpyrophosphate (PRPP) amidotransferase-catalyzed synthesis of phosphoribosylamine from PRPP and glutamine is the sum of two half-reactions at separated catalytic sites in different domains. Binding of PRPP to a C-terminal phosphoribosyltransferase domain is required to activate the reaction at the N-terminal glutaminase domain. Interdomain signaling was monitored by intrinsic tryptophan fluorescence and by measurements of glutamine binding and glutamine site catalysis. Enzymes were engineered to contain a single tryptophan fluorescence reporter in key positions in the glutaminase domain. Trp(83) in the glutamine loop (residues 73-84) and Trp(482) in the C-terminal helix (residues 471-492) reported fluorescence changes in the glutaminase domain upon binding of PRPP and glutamine. The fluorescence changes were perturbed by Ile(335) and Tyr(74) mutations that disrupt interdomain signaling. Fluoresence titrations of PRPP and glutamine binding indicated that signaling defects increased the K(d) for glutamine but had little or no effect on PRPP binding. It was concluded that the contact between Ile(335) in the phosphoribosyltransferase domain and Tyr(74) in the glutamine site is a primary molecular interaction for interdomain signaling. Analysis of enzymes with mutations in the glutaminase domain C-terminal helix and a 404-420 peptide point to additional signaling interactions that activate the glutamine site when PRPP binds.     

Dynamics-function correlation in Cu, Zn superoxide dismutase: a spectroscopic and molecular dynamics simulation study

A single mutation (Val29-->Gly) at the subunit interface of a Cu, Zn superoxide dismutase dimer leads to a twofold increase in the second order catalytic rate, when compared to the native enzyme, without causing any modification of the structure or the electric field distribution. To check the role of dynamic processes in this catalytic enhancement, the flexibility of the dimeric protein at the subunit interface region has been probed by the phosphorescence and fluorescence properties of the unique tryptophan residue. Multiple spectroscopic data indicate that Trp83 experiences a very similar, and relatively hydrophobic, environment in both wild-type and mutant protein, whereas its mobility is distinctly more restrained in the latter. Molecular dynamics simulation confirms this result, and provides, at the molecular level, details of the dynamic change felt by tryptophan. Moreover, the simulation shows that the loops surrounding the active site are more flexible in the mutant than in the native enzyme, making the copper more accessible to the incoming substrate, and being thus responsible for the catalytic rate enhancement. Evidence for increased, dynamic copper accessibility also comes from faster copper removal in the mutant by a metal chelator. These results indicate that differences in dynamic, rather than structural, features of the two enzymes are responsible for the observed functional change.     

Involvement of Val 315 located in the C-terminal region of thermolysin in its expression in Escherichia coli and its thermal stability

Thermolysin is a thermophilic and halophilic zinc metalloproteinase that consists of β-rich N-terminal (residues 1–157) and α-rich C-terminal (residues 158–316) domains. Expression of thermolysin variants truncated from the C-terminus was examined in E. coli culture. The C-terminal Lys316 residue was not significant in the expression, but Val315 was critical. Variants in which Val315 was substituted with fourteen amino acids were prepared. The variants substituted with hydrophobic amino acids such as Leu and Ile were almost the same as wild-type thermolysin (WT) in the expression amount, α-helix content, and stability. Variants with charged (Asp, Glu, Lys, and Arg), bulky (Trp), or small (Gly) amino acids were lower in these characteristics than WT. All variants exhibited considerably high activities (50–100% of WT) in hydrolyzing protein and peptide substrates. The expression amount, helix content, and stability of variants showed good correlation with hydropathy indexes of the amino acids substituted for Val315. Crystallographic study of thermolysin has indicated that V315 is a member of the C-terminal hydrophobic cluster. The results obtained in the present study indicate that stabilization of the cluster increases thermolysin stability and that the variants with higher stability are expressed more in the culture. Although thermolysin activity was not severely affected by the variation at position 315, the stability and specificity were modified significantly, suggesting the long-range interaction between the C-terminal region and active site.     

Structural studies of a two-domain chitinase from Streptomyces griseus HUT6037

Chitinase C (ChiC) from Streptomyces griseus HUT6037 was the first glycoside hydrolase family 19 chitinase that was found in an organism other than higher plants. An N-terminal chitin-binding domain and a C-terminal catalytic domain connected by a linker peptide constitute ChiC. We determined the crystal structure of full-length ChiC, which is the only representative of the two-domain chitinases in the family. The catalytic domain has an alpha-helix-rich fold with a deep cleft containing a catalytic site, and lacks three loops on the domain surface compared with the catalytic domain of plant chitinases. The chitin-binding domain is an all-beta protein with two tryptophan residues (Trp59 and Trp60) aligned on the surface. We suggest the binding mechanism of tri-N-acetylchitotriose onto the chitin-binding domain on the basis of molecular dynamics (MD) simulations. In this mechanism, the ligand molecule binds well on the surface-exposed binding site through two stacking interactions and two hydrogen bonds and only Trp59 and Trp60 are involved in the binding. Furthermore, the flexibility of the Trp60 side-chain, which may be involved in adjusting the binding surface to fit the surface of crystalline chitin by the rotation of chi2 angle, is shown.     

Mutational analysis of the role of tryptophan residues in an antimicrobial peptide

Antimicrobial peptides belonging to the pediocin-like family of bacteriocins (class IIa bacteriocins) produced by lactic acid bacteria contain several tryptophan residues that are highly conserved. Since tryptophan residues in membrane proteins are often positioned in the membrane-water interface, we hypothesized that Trp residues in bacteriocins could be important determinants of the structure of membrane-bound peptides and of anti-microbial activity. To test this hypothesis, the effects of mutating each of the 3 tryptophan residues (Trp18, Trp33, and Trp41) in the 43-residue pediocin-like bacteriocin sakacin P were studied. Trp18 and Trp33 are located at each end of an amphihilic alpha-helix, whereas Trp41 is near the end of an unstructured C-terminal tail. Replacement of Trp33 with the hydrophobic residues Leu and Phe had marginal effects on activity, whereas replacement with the more polar Tyr and Arg reduced activity 10-20 and 500-1000 times, respectively, indicating that Trp33 and the C-terminal part of the helix interact with the hydrophobic core of the membrane. Any mutation of Trp18 and Trp41 reduced activity, indicating that these two residues play unique roles. Substitutions with other aromatic residues were the least deleterious, indicating that both Trp18 and Trp41 interact with the membrane-water interface. The suggested locations of the three Trp residues are compatible with a structural model in which the helix and the C-terminal tail form a hairpin-like structure, bringing Trp18 and Trp41 close to each other in the interface, and placing Trp33 in the hydrophobic core of the membrane. Indeed, the deleterious effect of the W18L and W41L mutations could be overcome by stabilizing the hairpin-like structure by introduction of a disulfide bridge between residues 24 and 44. These results provide a basis for a refined structural model of pediocin-like bacteriocins and highlight the unique role that tryptophan residues can play in membrane-interacting peptides.     

Mutational analysis of Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKP)

Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKP) is a member of the serine/threonine protein phosphatases and shares 29% sequence identity with protein phosphatase 2Calpha (PP2Calpha) in its catalytic domain. To investigate the functional domains of CaMKP, mutational analysis was carried out using various recombinant CaMKPs expressed in Escherichia coli. Analysis of N-terminal deletion mutants showed that the N-terminal region of CaMKP played important roles in the formation of the catalytically active structure of the enzyme, and a critical role in polycation stimulation. A chimera mutant, a fusion of the N-terminal domain of CaMKP and the catalytic domain of PP2Calpha, exhibited similar substrate specificity to CaMKP but not to PP2Calpha, suggesting that the N-terminal region of CaMKP is crucial for its unique substrate specificity. Point mutations at Arg-162, Asp-194, His-196, and Asp-400, highly conserved amino acid residues in the catalytic domain of PP2C family, resulted in a significant loss of phosphatase activity, indicating that these amino acid residues may play important roles in the catalytic activity of CaMKP. Although CaMKP(1-412), a C-terminal truncation mutant, retained phosphatase activity, it was found to be much less stable upon incubation at 37 degrees C than wild type CaMKP, indicating that the C-terminal region of CaMKP is important for the maintenance of the catalytically active conformation. The results suggested that the N- and C-terminal sequences of CaMKP are essential for the regulation and stability of CaMKP.