Thermal, chemical, and enzymatic stability of the cyclotide kalata B1: the importance of the cyclic cystine knot

The cyclotides constitute a recently discovered family of plant-derived peptides that have the unusual features of a head-to-tail cyclized backbone and a cystine knot core. These features are thought to contribute to their exceptional stability, as qualitatively observed during experiments aimed at sequencing and characterizing early members of the family. However, to date there has been no quantitative study of the thermal, chemical, or enzymatic stability of the cyclotides. In this study, we demonstrate the stability of the prototypic cyclotide kalata B1 to the chaotropic agents 6 M guanidine hydrochloride (GdHCl) and 8 M urea, to temperatures approaching boiling, to acid, and following incubation with a range of proteases, conditions under which most proteins readily unfold. NMR spectroscopy was used to demonstrate the thermal stability, while fluorescence and circular dichroism were used to monitor the chemical stability. Several variants of kalata B1 were also examined, including kalata B2, which has five amino acid substitutions from B1, two acyclic permutants in which the backbone was broken but the cystine knot was retained, and a two-disulfide bond mutant. Together, these allowed determinations of the relative roles of the cystine knot and the circular backbone on the stability of the cyclotides. Addition of a denaturant to kalata B1 or an acyclic permutant did not cause unfolding, but the two-disulfide derivative was less stable, despite having a similar three-dimensional structure. It appears that the cystine knot is more important than the circular backbone in the chemical stability of the cyclotides. Furthermore, the cystine knot of the cyclotides is more stable than those in similar-sized molecules, judging by a comparison with the conotoxin PVIIA. There was no evidence for enzymatic digestion of native kalata B1 as monitored by LC-MS, but the reduced form was susceptible to proteolysis by trypsin, endoproteinase Glu-C, and thermolysin. Fluorescence spectra of kalata B1 in the presence of dithiothreitol, a reducing agent, showed a marked increase in intensity thought to be due to removal of the quenching effect on the Trp residue by the neighboring Cys5-Cys17 disulfide bond. In general, the reduced peptides were significantly more susceptible to chemical or enzymatic breakdown than the oxidized species.     

M42 aminopeptidase catalytic site: the structural and functional role of a strictly conserved aspartate residue

The M42 aminopeptidases are a family of dinuclear aminopeptidases widely distributed in Prokaryotes. They are potentially associated to the proteasome, achieving complete peptide destruction. Their most peculiar characteristic is their quaternary structure, a tetrahedron-shaped particle made of twelve subunits. The catalytic site of M42 aminopeptidases is defined by seven conserved residues. Five of them are involved in metal ion binding which is important to maintain both the activity and the oligomeric state. The sixth conserved residue, a glutamate, is the catalytic base deprotonating the water molecule during peptide bond hydrolysis. The seventh residue is an aspartate whose function remains poorly understood. This aspartate residue, however, must have a critical role as it is strictly conserved in all MH clan enzymes. It forms some kind of catalytic triad with the histidine residue and the metal ion of the M2 binding site. We assess its role in TmPep1050, an M42 aminopeptidase of Thermotoga maritima, through a mutational approach. Asp-62 was substituted with alanine, asparagine, or glutamate residue. The Asp-62 substitutions completely abolished TmPep1050 activity and impeded dodecamer formation. They also interfered with metal ion binding as only one cobalt ion is bound per subunit instead of two. The structure of Asp62Ala variant was solved at 1.5 Å showing how the substitution has an impact on the active site fold. We propose a structural role for Asp-62, helping to stabilize a crucial loop in the active site and to position correctly the catalytic base and a metal ion ligand of the M1 site.     

The structural basis of conserved residue variant effect on enzyme activity of UGT2B15

UDP-glucuronosyltransferase 2B15 (UGT2B15) is a crucial phase II drug-metabolizing enzyme, which glucuronidates various compounds, including clinical drugs and hormones. Mutants might affect glucuronidation, leading to a disruption of drug metabolism in vivo and decrease of therapeutic effect. Here, we mainly analyzed two representative mutants, H401P and L446S, on UGT2B15 activity using glucuronidation assays, molecular dynamic (MD) simulation and X-ray diffraction methods. The enzyme activity of L446S obviously increased six-fold than the wild type, although the enzyme activities of P191L, T374A, and H401P were lost apparently. Furthermore, we used MD simulations to calculate the energy change in the catalytic process of H401P and L446S, and the results indicated the free binding energies of H401P mutant to oxazepam and UDPGA were ?30.98 ± 1.00 kcal/mol and ?36.42 ± 1.04 kcal/mol, respectively, increased obviously compared to wild type, suggesting the mutation on position 401 had a crucial effect on the catalysis. Moreover, the three-dimensional structure of UGT2B15 C-terminal domain L446S was determined through protein crystallography and X-ray diffraction technology and the results suggested that one more hydrogen bonding between S446 and K410 was formed in the S446 crystal structure, compared to the wild type. Isothermal titration calorimetry assay further revealed the K_d values of C-terminal domain of UGT2B15 harbored L446S towards the cofactor UDPGA was similar to the value of wild type. Above all, our results pointed out that H401P and L446S affected the enzyme activity by different mechanism. Our work provided a helpful mechanism for variance explained in the UGTs catalyzation process.     

Decoding the membrane activity of the cyclotide kalata B1: the importance of phosphatidylethanolamine phospholipids and lipid organization on hemolytic and anti-HIV activities

Cyclotides, a large family of cyclic peptides from plants, have a broad range of biological activities, including insecticidal, cytotoxic, and anti-HIV activities. In all of these activities, cell membranes seem likely to be the primary target for cyclotides. However, the mechanistic role of lipid membranes in the activity of cyclotides remains unclear. To determine the role of lipid organization in the activity of the prototypic cyclotide, kalata B1 (kB1), and synthetic analogs, their bioactivities and affinities for model membranes were evaluated. We found that the bioactivity of kB1 is dependent on the lipid composition of target cell membranes. In particular, the activity of kB1 requires specific interactions with phospholipids containing phosphatidylethanolamine (PE) headgroups but is further modulated by nonspecific peptide-lipid hydrophobic interactions, which are favored in raft-like membranes. Negatively charged phospholipids do not favor high kB1 affinity. This lipid selectivity explains trends in antimicrobial and hemolytic activities of kB1; it does not target bacterial cell walls, which are negatively charged and lacking PE-phospholipids but can insert in the membranes of red blood cells, which have a low PE content and raft domains in their outer layer. We further show that the anti-HIV activity of kB1 is the result of its ability to target and disrupt the membranes of HIV particles, which are raft-like membranes very rich in PE-phospholipids.     

The critical structural role of a highly conserved histidine residue in group II amino acid decarboxylases

Glutamate decarboxylase is a pyridoxal 5'-phosphate (PLP)-dependent enzyme, belonging to the subset of PLP-dependent decarboxylases classified as group II. Site-directed mutagenesis of Escherichia coli glutamate decarboxylase, combined with analysis of the crystal structure, shows that a histidine residue buried in the protein core is critical for correct folding. This histidine is strictly conserved in the PF00282 PFAM family, which includes the group II decarboxylases. A similar role is proposed for residue Ser269, also highly conserved in this group of enzymes, as it provides one of the interactions stabilising His241.     

The fully conserved Asp residue in conserved sequence region I of the alpha-amylase family is crucial for the catalytic site architecture and activity

The alpha-amylase family is a large group of starch processing enzymes [Svensson, B. (1994) Plant Mol. Biol. 25, 141-157]. It is characterized by four short sequence motifs that contain the seven fully conserved amino acid residues in this family: two catalytic carboxylic acid residues and four substrate binding residues. The seventh conserved residue (Asp135) has no direct interactions with either substrates or products, but it is hydrogen-bonded to Arg227, which does bind the substrate in the catalytic site. Using cyclodextrin glycosyltransferase as an example, this paper provides for the first time definite biochemical and structural evidence that Asp135 is required for the proper conformation of several catalytic site residues and therefore for activity.     

The role of conserved extracellular cysteine residues in vasopressin V2 receptor function and properties of two naturally occurring mutant receptors with additional extracellular cysteine residues

The G protein-coupled vasopressin V2 receptor (V2 receptor) contains a pair of conserved cysteine residues (C112 and C192) which are thought to form a disulfide bond between the first and second extracellular loops. The conserved cysteine residues were found to be important for the correct formation of the ligand binding domain of some G protein-coupled receptors. Here we have assessed the properties of the V2 receptor after site-directed mutagenesis of its conserved cysteine residues in transiently transfected human embryonic kidney (HEK 293) cells. Mutant receptors (C112S, C112A and C192S, C192A) were non-functional and located mostly in the cell's interior. The conserved cysteine residues of the V2 receptor are thus not only important for the structure of the ligand binding domain but also for efficient intracellular receptor transport. In addition to the functional significance of the conserved cysteine residues, we have also analyzed the defects of two mutant V2 receptors which cause X-linked nephrogenic diabetes insipidus (NDI) by the introduction of additional cysteine residues into the second extracellular loop (mutants G185C, R202C). These mutations are assumed to impair normal disulfide bond formation. Mutant receptor G185C and R202C were efficiently transported to the plasma membrane but were defective in ligand binding. Only in the case of the mutant receptor R202C, the more sensitive adenylyl cyclase activity assay revealed vasopressin-stimulated cAMP formation with a 35-fold increased EC(50) value and with a reduced EC(max), indicating that ligand binding is not completely abolished. Taking the unaffected intracellular transport of both NDI-causing mutant receptors into account, our results indicate that the observed impairment of ligand binding by the additional cysteine residues is not due to the prevention of disulfide bond formation between the conserved cysteine residues.     

The biological and biochemical properties of L-canaline, a naturally occurring structural analogue of L-ornithine.

Many leguminous plants synthesize L-canavanine and sequester this nitrogen-rich, non-protein amino acid in the seed (1,2). Arginase-mediated hydrolytic cleavage of L-canavanine, in a manner analogous to L-ornithine and urea formation from L-arginine, produces urea and L-canaline (3,4). The resulting canaline is distinctive in being the only naturally occurring amino acid which possesses the aminooxy group (Fig. 1). Canaline decomposes in several organic solvents employed for its analysis by partition and ion-exchange chromatography (5,6) but the important question of the overall stability of this substituted hydroxylamine has not been investigated.     

Structural investigation of mutant Mycobacterium smegmatis arylamine N-acetyltransferase: a model for a naturally occurring functional polymorphism in Mycobacterium tuberculosis arylamine N-acetyltransferase

Arylamine N-acetyltransferase (NAT) acetylates the front-line anti-tuberculosis drug isoniazid (INH) and has been identified in Mycobacterium tuberculosis. A naturally occurring single nucleotide polymorphism (SNP) was recently found in the NAT gene in clinical isolates of M. tuberculosis. The nucleotide change from G-->A (619) produces an amino acid change Gly(207) Arg, which appears to reduce the activity of the NAT from M. tuberculosis (TBNAT). It has not been possible to generate sufficient soluble recombinant TBNAT for 3D structural studies. Therefore, Mycobacterium smegmatis NAT (SMNAT), which has 60% identity to TBNAT and has Gly at 207, was used as a model to investigate the possible structural effects of the G-->A 619 SNP. The mutant form of SMnat (SM207Rnat) was constructed by in vitro site-directed mutagenesis and was heterologously expressed with an N-terminal His tag in Escherichia coli, for comparison with the SMNAT. Both recombinant SMNATs were purified using Ni affinity chromatography and treated with thrombin to cleave the tag. Both proteins were produced with average yields of over 10 mg/L and were active. Substrate specificity and thermal stability of SM207RNAT were assessed and compared with the wild type SMNAT using kinetic assays and circular dichroism spectroscopy. SM207RNAT was crystallised and a data set of 2.00 A resolution was obtained. The SM207RNAT had different substrate specificities to the wild type protein and the 3D structures revealed that the Gly(207) Arg mutation caused slight changes in the orientation of His(203) in SMNAT.     

The role of an absolutely conserved tryptophan residue in octamer formation and stability in mitochondrial creatine kinases

In most organisms, mitochondrial creatine kinase (MtCK) is present as dimers and octamers with the latter predominating under physiological conditions. An absolutely conserved tryptophan residue (Trp-264 in chicken sarcomeric MtCK) appears to play a key role in octamer stability. Recently, it has been shown that the sponge Tethya aurantia, a member of the most ancient group of living multi-cellular animals, expresses an obligate, dimeric MtCK that lacks this absolutely conserved tryptophan residue, instead possessing a tyrosine in this position. In the present study we confirm that the absolutely conserved tryptophan residue is lacking in other sponge MtCKs where it is instead substituted by histidine or asparagine. Site directed mutations of the Trp-264 in expression constructs of chicken sarcomeric MtCK and the octameric MtCK from the marine worm Chaetopterus destabilized the octameric quaternary structure producing only dimers. A Tyr-->Trp mutation in an expression construct of the Tethya MtCK construct failed to produce octamerization; Tyr-->His and Tyr-->Asn mutations also yielded dimers. These results, in conjunction with analysis of homology models of Chaetopterus and Tethya MtCKs, strongly support the view that while the absolutely conserved tryptophan residue is important in octamer stability, octamer formation involves a complex suite of interactions between a variety of residues.     

Critical role of Glu175 on stability and folding of bacterial luciferase: stopped-flow fluorescence study

Bacterial luciferase is a heterodimeric enzyme, which catalyzes the light emission reaction, utilizing reduced FMN (FMNH2), a long chain aliphatic aldehyde and O(2), to produce green-blue light. This enzyme can be readily classed as slow or fast decay based on their rate of luminescence decay in a single turnover. Mutation of Glu175 in alpha subunit to Gly converted slow decay Xenorhabdus Luminescence luciferase to fast decay one. The following studies revealed that changing the luciferase flexibility and lake of Glu-flavin interactions are responsible for the unusual kinetic properties of mutant enzyme. Optical and thermodynamics studies have caused a decrease in free energy and anisotropy of mutant enzyme. Moreover, the role of Glu175 in transition state of folding pathway by use of stopped-flow fluorescence technique has been studied which suggesting that Glu175 is not involved in transition state of folding and appears as surface residue of the nucleus or as a member of one of a few alternative folding nuclei. These results suggest that mutation of Glu175 to Gly extended the structure of Xenorhabdus Luminescence luciferase, locally.     

The role of a conserved histidine residue, His324, in Trigonopsis variabilisd-amino acid oxidase

To investigate the functional role of an invariant histidine residue in Trigonopsis variabilis D-amino acid oxidase (DAAO), a set of mutant enzymes with replacement of the histidine residue at position 324 was constructed and their enzymatic properties were examined. Wild-type and mutant enzymes have been purified to homogeneity using the His-bound column and the molecular masses were determined to be 39.2 kDa. Western blot analysis revealed that the in vivo synthesized mutant enzymes are immuno-identical with that of the wild-type DAAO. The His324Asn and His324Gln mutants displayed comparable enzymatic activity to that of the wild-type enzyme, while the other mutant DAAOs showed markedly decreased or no detectable activity. The mutants, His324/Asn/Gln/Ala/Tyr/Glu, exhibited 38-181% increase in Km and a 2-10-fold reduction in kcat/Km. Based on the crystal structure of a homologous protein, pig kidney DAAO, it is suggested that His324 might play a structural role for proper catalytic function of T. variabilis DAAO.     

The conserved methionine residue of the metzincins: a site-directed mutagenesis study.

The metalloprotease clan of the metzincins derive their name from the presence of a conserved methionine residue that is located on the C-terminal side of the zinc-binding consensus sequence HEXXHXXGXXH. This methionine residue is located in a rather divergent part of the primary sequence but is structurally very well conserved. It is located under the pyramidal base of the three histidine residues that coordinate the catalytic zinc ion and is not involved in any direct contact with the metal nor the substrate. In order to clarify its role, this methionine residue (M226) of the protease C from Erwinia chrysanthemi has been mutated to various other amino acids. The mutants M226L, M226A, M226I were sufficiently stable to be isolated, while the mutants M226H, M226S and M226N could not be purified. The kinetic properties of these mutants were analysed. All mutants showed decreased activity, whereby increases in K(M) as well as decreases in k(cat) were observed. The M226L mutant and M226C-E189 K double mutant, which has the catalytic glutamic acid substituted as well, could be crystallised. The structure of the M226L mutant was determined to a resolution of 2.0 A and refined to R(free) of 0.20. The structure is isomorphous to the wild-type and does not show large differences, with the exception of a very small movement of the zinc-liganding histidine residues. The M226C-E189 K double mutant crystal structure has been refined to an R(free) of 0.20 at 2.1 A resolution. A small rearrangement of the zinc-liganding histidine residues can be detected, which leads to a slightly different zinc coordination and could explain the decrease in activity.     

Dissection of the structural and functional role of a conserved hydration site in RNase T1

The reoccurrence of water molecules in crystal structures of RNase T1 was investigated. Five waters were found to be invariant in RNase T1 as well as in six other related fungal RNases. The structural, dynamical, and functional characteristics of one of these conserved hydration sites (WAT1) were analyzed by protein engineering, X-ray crystallography, and (17)O and 2H nuclear magnetic relaxation dispersion (NMRD). The position of WAT1 and its surrounding hydrogen bond network are unaffected by deletions of two neighboring side chains. In the mutant Thr93Gln, the Gln93N epsilon2 nitrogen replaces WAT1 and participates in a similar hydrogen bond network involving Cys6, Asn9, Asp76, and Thr91. The ability of WAT1 to form four hydrogen bonds may explain why evolution has preserved a water molecule, rather than a side-chain atom, at the center of this intricate hydrogen bond network. Comparison of the (17)O NMRD profiles from wild-type and Thr93Gln RNase T1 yield a mean residence time of 7 ns at 27 degrees C and an orientational order parameter of 0.45. The effects of mutations around WAT1 on the kinetic parameters of RNase T1 are small but significant and probably relate to the dynamics of the active site.     

Structure and function of the catalytic site mutant Asp 99 Asn of phospholipase A2: absence of the conserved structural water.

To probe the role of the Asp-99 ... His-48 pair in phospholipase A2 (PLA2) catalysis, the X-ray structure and kinetic characterization of the mutant Asp-99-->Asn-99 (D99N) of bovine pancreatic PLA2 was undertaken. Crystals of D99N belong to the trigonal space group P3(1)21 and were isomorphous to the wild type (WT) (Noel JP et al., 1991, Biochemistry 30:11801-11811). The 1.9-A X-ray structure of the mutant showed that the carbonyl group of Asn-99 side chain is hydrogen bonded to His-48 in the same way as that of Asp-99 in the WT, thus retaining the tautomeric form of His-48 and the function of the enzyme. The NH2 group of Asn-99 points away from His-48. In contrast, in the D102N mutant of the protease enzyme trypsin, the NH2 group of Asn-102 is hydrogen bonded to His-57 resulting in the inactive tautomeric form and hence the loss of enzymatic activity. Although the geometry of the catalytic triad in the PLA2 mutant remains the same as in the WT, we were surprised that the conserved structural water, linking the catalytic site with the ammonium group of Ala-1 of the interfacial site, was ejected by the proximity of the NH2 group of Asn-99. The NH2 group now forms a direct hydrogen bond with the carbonyl group of Ala-1.     

Kinetic and structural studies on the catalytic role of the aspartic acid residue conserved in copper amine oxidase

Copper amine oxidase contains a post-translationally generated quinone cofactor, topa quinone (TPQ), which mediates electron transfer from the amine substrate to molecular oxygen. The overall catalytic reaction is divided into the former reductive and the latter oxidative half-reactions based on the redox state of TPQ. In the reductive half-reaction, substrate amine reacts with the C5 carbonyl group of the oxidized TPQ, forming the substrate Schiff base (TPQ(ssb)), which is then converted to the product Schiff base (TPQ(psb)). During this step, an invariant Asp residue with an elevated pKa is presumed to serve as a general base accepting the alpha proton of the substrate. When Asp298, the putative active-site base in the recombinant enzyme from Arthrobacter globiformis, was mutated into Ala, the catalytic efficiency dropped to a level of about 10(6) orders of magnitude smaller than the wild-type (WT) enzyme, consistent with the essentiality of Asp298. Global analysis of the slow UV/vis spectral changes observed during the reductive half-reaction of the D298A mutant with 2-phenylethylamine provided apparent rate constants for the formation and decay of TPQ(ssb) (k(obs) = 4.7 and 4.8 x 10(-4) s(-1), respectively), both of which are markedly smaller than those of the WT enzyme determined by rapid-scan stopped-flow analysis (k(obs) = 699 and 411 s(-1), respectively). Thus, Asp298 plays important roles not only in the alpha-proton abstraction from TPQ(ssb) but also in other steps in the reductive half-reaction. X-ray diffraction analyses of D298A crystals soaked with the substrate for 1 h and 1 week revealed the structures of TPQ(ssb) and TPQ(psb), respectively, as pre-assigned by single-crystal microspectrophotometry. Consistent with the stereospecificity of alpha-proton abstraction, the pro-S alpha-proton of TPQ(ssb) to be abstracted is positioned nearly perpendicularly to the plane formed by the Schiff-base imine double bond conjugating with the quinone ring of TPQ, so that the orbitals of sigma and pi electrons maximally overlap in the conjugate system. More intriguingly, the pro-S alpha proton of the substrate is released stereospecifically even in the reaction catalyzed by the base-lacking D298A mutant. On the basis of these results, we propose that the stereospecificity of alpha-proton abstraction is primarily determined by the conformation of TPQ(ssb), rather than the relative geometry of TPQ and the catalytic base.     

Mutagenesis study on the role of a lysine residue highly conserved in formate dehydrogenases and periplasmic nitrate reductases

Lysine 85 (K85) in the primary structure of the catalytic subunit of the periplasmic nitrate reductase (NAP-A) of Ralstonia eutropha H16 is highly conserved in periplasmic nitrate reductases and in the structurally related catalytic subunit of the formate dehydrogenases of various bacterial species. It is located between an [4Fe-4S] center and one of the molybdopterin-guanine dinucleotides mediating the through bonds electron flow to convert the specific substrate of the respective enzymes. To examine the role of K85, the structure of NAP-A of R. eutropha strain H16 was modeled on the basis of the crystal structure from the Desulfovibrio desulfuricans enzyme (Dias et al. Structure Fold Des. 7(1) (1999) 65) and K85 was replaced by site-directed mutagenesis, yielding K85R and K85M, respectively. The specific nitrate reductase activity was determined in periplasmic extracts. The mutant enzyme carrying K85R showed 23% of the wild-type activity, whereas the replacement by a polar, uncharged residue (K85M) resulted in complete loss of the catalytic activity. The reduced nitrate reductase activity of K85R was not due to different quantities of the expressed gene product, as controlled immunologically by NAP-specific antibodies. The results indicate that K85 is optimized for the electron transport flux to reduce nitrate to nitrite in NAP-A, and that the positive charge alone cannot meet further structural requirement for efficient electron flow.     

Involvement of residues Asp8, Asn13, Glu145, Asp168, and Thr173 in the chaperone activity of a recombinant DnaK from Bacillus licheniformis

Based on the sequence homology, we have modeled the three-dimensional structure of Bacillus licheniformis DnaK (BlDnaK), a counterpart of Hsp70, and identified five different amino acids that might be responsible for maintaining ADP-Mg(2+)-Pi in the correct configuration at the ATP-binding cleft of the protein. As compared with wild-type BlDnaK, site-directed mutant proteins D8A, N13D, E145A, D168A, and T173A had a dramatic reduction in their chaperone activities. Complementation test revealed that the mutant proteins lost completely the ability to rescue the temperature-sensitive growth defect of Escherichia colidnaK756-ts. Wild-type BlDnak assisted the refolding of denatured firefly luciferase, whereas a significant decrease in this ability was observed for the mutant proteins. Simultaneous addition of B. licheniformis DnaJ, BlGrpE, and NR-peptide, did not synergistically stimulate the ATPase activity of D8A, E145A, D168A and T173A. Circular dichroism spectra were nearly identical for wild-type and mutant proteins, and they, except D8A, also exhibited a similar sensitivity towards temperature-induced denaturation. These results suggest that the selected residues are critical for the proper function of BlDnaK.