Rigid Residues Research Articles

Structure and dynamics of human Nedd4-1 WW3 in complex with the αENaC PY motif

Nedd4-1 (neuronal precursor cell expressed developmentally downregulated gene 4-1) is an E3 ubiquitin ligase that interacts with and negatively regulates the epithelial Na+ channel (ENaC). The WW domains of Nedd4-1 bind to the ENaC subunits via recognition of PY motifs. Human Nedd4-1 (hNedd4-1) contains four WW domains with the third domain (WW3*) showing the strongest affinity to the PY motif. To understand the mechanism underlying this binding affinity, we have carried out NMR structural and dynamics analyses of the hNedd4-1 WW3* domain in complex with a peptide comprising the C-terminal tail of the human ENaC α-subunit. The structure reveals that the peptide interacts in a similar manner to other WW domain–ENaC peptide structures. Crucial interactions that likely provide binding affinity are the broad XP groove facilitating additional contacts between the WW3* domain and the peptide, compared to similar complexes, and the large surface area buried (83Å2) between R430 (WW3*) and L647′ (αENaC). This corroborates the model-free analysis of the 15N backbone relaxation data, which showed that R430 is the most rigid residue in the domain (S2=0.90±0.01). Carr–Purcell–Meiboom–Gill relaxation dispersion analysis identified two different conformational exchange processes on the μs–ms time-scale. One of these processes involves residues located at the peptide binding interface, suggesting conformational exchange may play a role in peptide recognition. Thus, both structural and dynamic features of the complex appear to define the high binding affinity. The results should aid interpretation of biochemical data and modeling interfaces between Nedd4-1 and other interacting proteins.

Identification of a Novel Binding Site Between HIV Type 1 Nef C-Terminal Flexible Loop and AP2 Required for Nef-Mediated CD4 Downregulation

HIV-1 Nef is an accessory protein necessary for HIV-1 virulence and rapid AIDS development. Nef promotes viral replication and infection by connecting CD4 and several other cell surface receptors to the clathrin adaptor protein AP2, resulting in the internalization and degradation of the receptors interacting with Nef. We investigated how Nef can mediate constitutive receptor endocytosis through the interaction of the dileucine motif in its C-terminal flexible loop (C-loop) with AP2, whereas AP2 binding of the transmembrane receptors usually results in an equilibrated (recycled) endocytosis. Our results indicated that in addition to the dileucine motif, there is a second motif in the Nef C-loop involved in the Nef-AP2 interaction. Nef-mediated CD4 downregulation was impaired when the residue in the hydrophobic region in the Nef C-loop (LL165HPMSLHGM173) was mutated to a basic residue K/R or an acidic residue E/D or to the rigid residue P, or when M168L170, L170H171, or G172M173 was mutated to AA. A pull-down assay indicated that AP2 was not coprecipitated with Nef mutants that did not downregulate CD4. Molecular modeling of the Nef C-terminal flexible loop in complex with AP2 suggests that M168L170 occupies a pocket in the AP2 σ2 subunit. Our data suggest a new model in the Nef-AP2 interaction in which the hydrophobic region in the Nef C-loop with the dileucine (L164L165) motif and M168L170 motif binds to AP2(σ2), while the acidic motif E174 and D175 binds to AP2(α), which explains how Nef through the flexible loop connects CD4 to AP2 for constitutive CD4 downregulation.

Neighborhood Properties Are Important Determinants of Temperature Sensitive Mutations

Temperature-sensitive (TS) mutants are powerful tools to study gene function in vivo. These mutants exhibit wild-type activity at permissive temperatures and reduced activity at restrictive temperatures. Although random mutagenesis can be used to generate TS mutants, the procedure is laborious and unfeasible in multicellular organisms. Further, the underlying molecular mechanisms of the TS phenotype are poorly understood. To elucidate TS mechanisms, we used a machine learning method–logistic regression–to investigate a large number of sequence and structure features. We developed and tested 133 features, describing properties of either the mutation site or the mutation site neighborhood. We defined three types of neighborhood using sequence distance, Euclidean distance, and topological distance. We discovered that neighborhood features outperformed mutation site features in predicting TS mutations. The most predictive features suggest that TS mutations tend to occur at buried and rigid residues, and are located at conserved protein domains. The environment of a buried residue often determines the overall structural stability of a protein, thus may lead to reversible activity change upon temperature switch. We developed TS prediction models based on logistic regression and the Lasso regularized procedure. Through a ten-fold cross-validation, we obtained the area under the curve of 0.91 for the model using both sequence and structure features. Testing on independent datasets suggested that the model predicted TS mutations with a 50% precision. In summary, our study elucidated the molecular basis of TS mutants and suggested the importance of neighborhood properties in determining TS mutations. We further developed models to predict TS mutations derived from single amino acid substitutions. In this way, TS mutants can be efficiently obtained through experimentally introducing the predicted mutations.

The balance of flexibility and rigidity in the active site residues of hen egg white lysozyme

The crystallographic temperature factors (B factor) of individual atoms contain important information about the thermal motion of the atoms in a macromolecule. Previously the theory of flexibility of active site has been established based on the observation that the enzyme activity is sensitive to low concentration denaturing agents. It has been found that the loss of enzyme activity occurs well before the disruption of the three-dimensional structural scaffold of the enzyme. To test the theory of conformational flexibility of enzyme active site, crystal structures were perturbed by soaking in low concentration guanidine hydrochloride solutions. It was found that many lysozyme crystals tested could still diffract until the concentration of guanidine hydrochloride reached 3 M. It was also found that the B factors averaged over individually collected data sets were more accurate. Thus it suggested that accurate measurement of crystal temperature factors could be achieved for medium-high or even medium resolution crystals by averaging over multiple data sets. Furthermore, we found that the correctly predicted active sites included not only the more flexible residues, but also some more rigid residues. Both the flexible and the rigid residues in the active site played an important role in forming the active site residue network, covering the majority of the substrate binding residues. Therefore, this experimental prediction method may be useful for characterizing the binding site and the function of a protein, such as drug targeting.

Interactions between oligopeptides and oxidised titanium surfaces detected by vibrational spectroscopy

AbstractFive alternating polar/hydrophobic oligopeptides derived from EAK 16 (AEAEAKAKAEAEAKAK) were examined in comparison with EAK 16 (peptide 1) both after solubilisation/lyophilisation and deposition on oxidised titanium surfaces. The peptides were synthesised for their possible use as biomimetic materials due to their self‐assembling properties and the presence, in one of them, of the arginine‐glycine‐aspartic (RGD) sequence, an active modulator of cell adhesion. Infrared (IR) and Raman spectroscopies were used to investigate the influence of the amino acid substitution on the self‐assembling properties of the peptides under both experimental conditions. In the lyophilised peptides, β‐sheet was the prevailing conformation (65–69%) as in EAK 16, irrespective of acid substitution (E→D, peptide 2), basic substitution (K→O, peptide 3), hydrophobic spacer substitution (A→Abu, peptide 4 and A→Y, peptide 5) and RGD insertion (peptide 6). After deposition on oxidised titanium, the main conformation remained β‐sheet. The side‐chain shortening of the acidic amino acid residue (peptide 2) or the insertion of a rigid and bulky residue such as Y (peptide 5) decreased the self‐assembling ability more than the side‐chain shortening of the basic amino acid residue (peptide 3) or the insertion of the RGD head (peptide 6). The interaction with the oxidised titanium surface was mainly due to carboxylate groups with a bidentate bridging coordination and C O peptidic groups. Copyright © 2010 John Wiley & Sons, Ltd.

Role of an isoform-specific substrate access channel residue in CO ligand accessibilities of neuronal and inducible nitric oxide synthase isoforms

The rates of the bimolecular CO rebinding to the oxygenase domains of inducible and neuronal NOS proteins (iNOSoxy and nNOSoxy, respectively) after photolytic dissociation have been determined by laser flash photolysis. The following mutants at the isoform-specific sites (murine iNOSoxy N115L and rat nNOSoxy L337N, L337F) have been constructed to investigate role of the residues in the CO ligand accessibilities of the NOS isoforms. These residues are in the NOS distal substrate access channel. The effect of the (6R)-5,6,7,8-tetrahydrobiopterin (H 4B) cofactor and l-arginine (Arg) substrate on the rates of CO rebinding have also been assessed. Addition of l-Arg to the iNOSoxy N115L mutant results in much faster CO rebinding rates, compared to the wild type. The results indicate that modifications to the iNOS channel in which the hydrophilic residue N115 is replaced by leucine (to resemble its nNOS cognate) open the channel somewhat, thereby improving access to the axial heme ligand binding position. On the other hand, introduction of a hydrophilic residue (L337N) or a bulky rigid aromatic residue (L337F) in the nNOS isoform does not significantly affect the kinetics profile, suggesting that the geometry of the substrate access pocket is not greatly altered. The bimolecular CO rebinding rate data indicate that the opening of the substrate access channel in the iNOS N115L mutant may be due to more widespread structural alterations induced by the mutation.

Optimum levels of exchangeable protons in perdeuterated proteins for proton detection in MAS solid-state NMR spectroscopy

We present a systematic study of the effect of the level of exchangeable protons on the observed amide proton linewidth obtained in perdeuterated proteins. Decreasing the amount of D(2)O employed in the crystallization buffer from 90 to 0%, we observe a fourfold increase in linewidth for both (1)H and (15)N resonances. At the same time, we find a gradual increase in the signal-to-noise ratio (SNR) for (1)H-(15)N correlations in dipolar coupling based experiments for H(2)O concentrations of up to 40%. Beyond 40%, a significant reduction in SNR is observed. Scalar-coupling based (1)H-(15)N correlation experiments yield a nearly constant SNR for samples prepared with < or =30% H(2)O. Samples in which more H(2)O is employed for crystallization show a significantly reduced NMR intensity. Calculation of the SNR by taking into account the reduction in (1)H T (1) in samples containing more protons (SNR per unit time), yields a maximum SNR for samples crystallized using 30 and 40% H(2)O for scalar and dipolar coupling based experiments, respectively. A sensitivity gain of 3.8 is obtained by increasing the H(2)O concentration from 10 to 40% in the CP based experiment, whereas the linewidth only becomes 1.5 times broader. In general, we find that CP is more favorable compared to INEPT based transfer when the number of possible (1)H,(1)H interactions increases. At low levels of deuteration (> or =60% H(2)O in the crystallization buffer), resonances from rigid residues are broadened beyond detection. All experiments are carried out at MAS frequency of 24 kHz employing perdeuterated samples of the chicken alpha-spectrin SH3 domain.

Evaluation of triproline and tri-α-methylproline chiral stationary phases: Retention and enantioseparation associated with hydrogen bonding

In this study, to demonstrate preparation strategy and improve understanding of chiral recognition mechanisms, triproline chiral stationary phases (CSPs) were evaluated with a series of analytes classified as having none, one, two or three H-bond donors. The average retention factors and mobile phase strength generally followed none < one < two < three hydrogen bond donors. The average solvent volume ratio (H r stands for average hexane volume ratio in the mobile phase, Hp r for heptane, ACN r for acetonitrile, or H 2O r for water) normalized chromatographic parameters calculated for di-, tri-, tetra-, penta-, hexa-, and decaproline CSPs facilitated the characterization of properties associated to the H-bond donor categorization. The H r of triproline CSP were 1.0, 0.96 and 0.88 for analyte of none, one and two hydrogen bond donors with hexane/2-propanol mobile phase, respectively. The number of hydrogen bond donors in an analyte was found to be a primary factor in influencing the retention and enantioseparation in the normal-phase and polar organic modes. Two H-bond acceptor solvents methyl tert-butyl ether and ethyl acetate increased chiral separation on oligoproline CSPs for some compounds. The role of carbon-donor hydrogen bonding at the H atom of proline asymmetric center was implied through testing a tri-α-methylproline stationary phase. On oligoproline CSPs, three factors including adjacent hydrogen bond acceptor and carbon-donor, and a rigid proline residue chain were recognized as important for contributing to the broad enantioselectivity. The α hydrogen atom on chiral center of stationary phase was found to play a crucial role in enantiomeric discrimination.

THERMAL DEGRADATION AND FLAME RETARDANCY OF CALCIUM ALGINATE FIBERS

Calcium alginate fibers were prepared by wet spinning of sodium alginate into a coagulating bath containing calcium chloride. The thermal degradation and flame retardancy of calcium alginate fibers were investigated with thermal gravimetry (TG), X-ray diffraction (XRD), limiting oxygen index (LOI) and cone calorimeter (CONE). The results show that calcium alginate fibers are inherently flame retardant with a LOI value of 34, and the heat release rate (HRR), total heat release (THR), CO and CO2 concentrations during combustion are much lower compared with those of viscose fibers. Calcium carbonate and calcium oxide were formed during thermal degradation of calcium alginate fibers at different temperatures. The shape of calcium alginate fibers is well kept after LOI test. The rigid combustion residue char acts as an effective barrier to the outward diffusion of flame and heat. The combustion process and flame retardant mechanism of calcium alginate fibers are also discussed.

Comparison of the Structure and Dynamics of the Antibiotic Peptide Polymyxin B and the Inactive Nonapeptide in Aqueous Trifluoroethanol by NMR Spectroscopy

The structure and dynamics of polymyxin B (PxB), an N-acylated cyclic decapeptide that displays antimicrobial activity against Gram-negative bacteria, is characterized by NMR and compared to results for the inactive nonapeptide, which is missing the N-terminal amino acid along with the attached acyl chain. Aqueous trifluoroethanol (TFE) was chosen as the solvent since the overall structure of PxB in TFE is similar to the structure when bound to vesicles. No differences were observed between the two peptides for (1)H H(alpha) chemical shifts or patterns of cross peaks in NOESY spectra, indicating that the overall structures are quite similar. The sign and intensity of NOESY spectra obtained at different temperatures were used to assess the relative mobility of the peptides. For both peptides, differential mobility is observed in different parts of the molecule, with greater mobility observed for the linear portion than the ring and faster motion seen for the side chains than the peptide backbone. However, all motion is faster in the nonapeptide, indicating that the presence of the N-terminal acyl chain restricts the mobility of PxB compared to the nonapeptide, which lacks this structural feature. For both peptides, differential mobility is also observed within the cyclic portion of the peptide. This supports a proposed model whereby the more rigid residues serve as pivot points, allowing the ring conformation to change in response to different binding partners. However, conformational flexibility within the cyclic ring is not sufficient for antimicrobial activity since both the active and inactive peptides exhibit the same flexibility. The N-terminal acyl chain on PxB, which is essential for activity, exhibits rapid, independent motion, and this flexibility may facilitate penetration of the outer membrane.

Sequence based residue depth prediction using evolutionary information and predicted secondary structure

BackgroundResidue depth allows determining how deeply a given residue is buried, in contrast to the solvent accessibility that differentiates between buried and solvent-exposed residues. When compared with the solvent accessibility, the depth allows studying deep-level structures and functional sites, and formation of the protein folding nucleus. Accurate prediction of residue depth would provide valuable information for fold recognition, prediction of functional sites, and protein design.ResultsA new method, RDPred, for the real-value depth prediction from protein sequence is proposed. RDPred combines information extracted from the sequence, PSI-BLAST scoring matrices, and secondary structure predicted with PSIPRED. Three-fold/ten-fold cross validation based tests performed on three independent, low-identity datasets show that the distance based depth (computed using MSMS) predicted by RDPred is characterized by 0.67/0.67, 0.66/0.67, and 0.64/0.65 correlation with the actual depth, by the mean absolute errors equal 0.56/0.56, 0.61/0.60, and 0.58/0.57, and by the mean relative errors equal 17.0%/16.9%, 18.2%/18.1%, and 17.7%/17.6%, respectively. The mean absolute and the mean relative errors are shown to be statistically significantly better when compared with a method recently proposed by Yuan and Wang [Proteins 2008; 70:509–516]. The results show that three-fold cross validation underestimates the variability of the prediction quality when compared with the results based on the ten-fold cross validation. We also show that the hydrophilic and flexible residues are predicted more accurately than hydrophobic and rigid residues. Similarly, the charged residues that include Lys, Glu, Asp, and Arg are the most accurately predicted. Our analysis reveals that evolutionary information encoded using PSSM is characterized by stronger correlation with the depth for hydrophilic amino acids (AAs) and aliphatic AAs when compared with hydrophobic AAs and aromatic AAs. Finally, we show that the secondary structure of coils and strands is useful in depth prediction, in contrast to helices that have relatively uniform distribution over the protein depth. Application of the predicted residue depth to prediction of buried/exposed residues shows consistent improvements in detection rates of both buried and exposed residues when compared with the competing method. Finally, we contrasted the prediction performance among distance based (MSMS and DPX) and volume based (SADIC) depth definitions. We found that the distance based indices are harder to predict due to the more complex nature of the corresponding depth profiles.ConclusionThe proposed method, RDPred, provides statistically significantly better predictions of residue depth when compared with the competing method. The predicted depth can be used to provide improved prediction of both buried and exposed residues. The prediction of exposed residues has implications in characterization/prediction of interactions with ligands and other proteins, while the prediction of buried residues could be used in the context of folding predictions and simulations.

Structural Basis for Recognition of CD20 by Therapeutic Antibody Rituximab

Rituximab is a widely used monoclonal antibody drug for treating certain lymphomas and autoimmune diseases. To understand the molecular mechanism of recognition of human CD20 by Rituximab, we determined the crystal structure of the Rituximab Fab in complex with a synthesized peptide comprising the CD20 epitope (residues 163-187) at 2.6-A resolution. The combining site of the Fab consists of four complementarity determining regions that form a large, deep pocket to accommodate the epitope peptide. The bound peptide assumes a unique cyclic conformation that is constrained by a disulfide bond and a rigid proline residue (Pro(172)). The (170)ANPS(173) motif of CD20 is deeply embedded into the pocket on the antibody surface and plays an essential role in the recognition and binding of Rituximab. The antigen-antibody interactions involve both hydrogen bonds and van der Waals contacts and display a high degree of structural and chemical complementarity. These results provide a molecular basis for the specific recognition of CD20 by Rituximab as well as valuable information for development of improved antibody drugs with better specificity and higher affinity.

Cyanoacrylate tissue sealant impairs tissue integration of macroporous mesh in experimental hernia repair

Tissue sealants have been proposed as an alternative to permanent fixation devices in hernia repair with the aim of reducing perforation-associated complications and chronic pain. Sealants can be divided into three main categories: synthetic glues (e.g., cyanoacrylate based), biologic products (e.g., fibrin sealant), and genetically engineered polymer protein glues. The beneficial effects of fibrin sealant have been reported in both experimental and clinical hernia repair. However, data on cyanoacrylate glues for mesh sealing are limited. In 20 Sprague-Dawley rats, two hernia defects (1.5 cm in diameter) per animal were created bilaterally in the midline of the abdominal wall. The peritoneum was spared. The lesions were left untreated for 10 days to achieve a chronic condition. Defects then were covered with TI-Mesh xl (2 x 2 cm), which was glued with Glubran-II. The time points of sacrifice were 17 days, 28 days, and 3 months. At autopsy, meshes were biomechanically tested, and histology was performed. Tissue integration of the meshes was impaired at all time points by impenetrable glue plaques. At application sites, the elasticity of the abdominal wall was significantly reduced because of nonresorbed, rigid glue residues. Mesh fixation by Glubran-II impairs tissue integration, elicits inflammation, and unfavorably alters the biomechanics of macroporous mesh and the abdominal wall.

PROFbval: predict flexible and rigid residues in proteins

The mobility of a residue on the protein surface is closely linked to its function. The identification of extremely rigid or flexible surface residues can therefore contribute information crucial for solving the complex problem of identifying functionally important residues in proteins. Mobility is commonly measured by B-value data from high-resolution three-dimensional X-ray structures. Few methods predict B-values from sequence. Here, we present PROFbval, the first web server to predict normalized B-values from amino acid sequence. The server handles amino acid sequences (or alignments) as input and outputs normalized B-value and two-state (flexible/rigid) predictions. The server also assigns a reliability index for each prediction. For example, PROFbval correctly identifies residues in active sites on the surface of enzymes as particularly rigid. http://www.rostlab.org/services/profbval profbval@rostlab.org Supplementary data are available at Bioinformatics online.

The Solution Structure of a Transient Photoreceptor Intermediate: Δ25 Photoactive Yellow Protein

The N-terminally truncated variant of photoactive yellow protein (Delta25-PYP) undergoes a very similar photocycle as the corresponding wild-type protein (WT-PYP), although the lifetime of its light-illuminated (pB) state is much longer. This has allowed determination of the structure of both its dark- (pG) as well as its pB-state in solution by nuclear magnetic resonance (NMR) spectroscopy. The pG structure shows a well-defined fold, similar to WT-PYP and the X-ray structure of the pG state of Delta25-PYP. In the long-lived photocycle intermediate pB, the central beta sheet is still intact, as well as a small part of one alpha helix. The remainder of pB is unfolded and highly flexible, as evidenced by results from proton-deuterium exchange and NMR relaxation studies. Thus, the partially unfolded nature of the presumed signaling state of PYP in solution, as suggested previously, has now been structurally demonstrated.

Kinetics of structural changes in the relay loop and SH3 domain of myosin

The intrinsic fluorescence of smooth muscle myosin signals conformational changes associated with different catalytic states of the ATPase cycle. To elucidate this relationship, we have examined the pre-steady-state kinetics of nucleotide binding, hydrolysis, and product release in motor domain-essential light chain mutants containing a single endogenous tryptophan, either residue 512 in the rigid relay loop or residue 29 adjacent to the SH3 domain. The intrinsic fluorescence of W512 is sensitive to both nucleotide binding and hydrolysis, and appears to report structural changes at the active site, presumably through a direct connection with switch II. The intrinsic fluorescence of W29 is sensitive to nucleotide binding but not hydrolysis, and does not appear to be tightly linked with structural changes occurring at the active site. We propose that the SH3 domain may be sensitive to conformational changes in the lever arm through contacts with the essential light chain.

Conformational flexibility of Corey lactone derivatives indicated by absorption and vibrational circular dichroism spectra.

Infrared absorption and vibrational circular dichroism (VCD) spectra of four Corey lactone derivatives (diol, benzoate, p-phenylbenzoate, and bisbenzoate) were measured and analyzed on the basis of ab initio computations. The analysis interpreted most of the spectral features as well as the differences among individual compounds. Despite the common rigid lactone residue, conformational behaviors and spectral features of the derivatives were found to be different, because of hydrogen bonding and solvent effects. Recognition of common molecular parts in the spectra of different molecules increases the potential of using VCD for monitoring the purity of intermediates in chiral syntheses. For the derivatives, a conserved spectral component corresponding to the lactone skeleton could be identified on the basis of theoretical analysis but was relatively weak in intensity.

Protein thermostability: structure-based difference of residual properties between thermophilic and mesophilic proteins

Structure-based differences of residual properties between 20 pairs of thermophilic and mesophilic proteins were statistically analyzed to elucidate the factors governing protein thermostability. This study analyzed the distributions of outer residues, inner residues, flexible residues, rigid residues, hydrogen bonds, salt bridges, cation–pi interactions, and disulfide bonds in each protein in terms of residual structural states, which were determined as five kinds of states under residual packing value. Their structural patterns found in thermophilic protein groups were compared with those of mesophilic protein groups for showing distinctive difference of residual properties. The results of statistical tests ( t-test) revealed that flexible residues in fully-exposed state and boundary state, salt bridges in exposed state, and hydrogen bonds in well-buried state could be critical factors related with protein thermostability. Such structure-based differences of residual properties would help to develop a strategy for enhancing protein thermostability.

Enhanced antitumour activity of 15-residue bovine lactoferricin derivatives containing bulky aromatic amino acids and lipophilic N-terminal modifications.

In a structure-antibacterial activity relationship study of a peptide fragment of bovine lactoferricin consisting of FKCRRWQWRMKKLGA (LFB 17-31), it was revealed that the two Trp residues were important for antibacterial activity. It has further been demonstrated that the size, shape and the aromatic character of the side chains were even more important than the Trp itself. In this study the antitumour effect of a series of LFB 17-31 derivatives are reported, in which the two Trp residues in position 6 and 8 were replaced with the larger non-coded aromatic amino acids Tbt, Tpc, Bip and Dip. The counterproductive Cys in position 3 was also substituted with these larger aromatic residues. In addition, the effect of introducing lipophilic groups of different size and shape in the N-terminal of the LFB 17-31 sequence was addressed. The resulting peptide derivatives were tested for activity against three human tumour cell lines and against normal human umbilical vein endothelial cells and fibroblasts. High antitumour activity by several of the peptides demonstrated that Trp successfully could be substituted by the bulky aromatic residues, and peptides containing the large and rigid Tbt residue in position 6 and/or 8 in LFB 17-31 were the most active candidates. The antitumour effect was even more increased by the Tbt-modified peptides when the three counterproductive amino acids Cys3, Gln7 and Gly14 were replaced by Ala. Enhanced antitumour activity was also obtained by modifying the N-terminal of LFB 17-31 with either long-chained fatty acids or bulky moieties. Thus, our results revealed that the size and shape of the lipophilic groups and their position in the peptide sequence were important for antitumour activity.

Crystal structure of D-Hydantoinase from Burkholderia pickettii at a resolution of 2.7 Angstroms: insights into the molecular basis of enzyme thermostability.

D-Hydantoinase (D-HYD) is an industrial enzyme that is widely used in the production of D-amino acids which are precursors for semisynthesis of antibiotics, peptides, and pesticides. This report describes the crystal structure of D-hydantoinase from Burkholderia pickettii (HYD(Bp)) at a 2.7-A resolution. The structure of HYD(Bp) consists of a core (alpha/beta)(8) triose phosphate isomerase barrel fold and a beta-sheet domain, and the catalytic active site consists of two metal ions and six highly conserved amino acid residues. Although HYD(Bp) shares only moderate sequence similarity with D-HYDs from Thermus sp. (HYD(Tsp)) and Bacillus stearothermophilus (HYD(Bst)), whose structures have recently been solved, the overall structure and the structure of the catalytic active site are strikingly similar. Nevertheless, the amino acids that compose the substrate-binding site are less conserved and have different properties, which might dictate the substrate specificity. Structural comparison has revealed insights into the molecular basis of the differential thermostability of D-HYDs. The more thermostable HYD(Tsp) contains more aromatic residues in the interior of the structure than HYD(Bp) and HYD(Bst). Changes of large aromatic residues in HYD(Tsp) to smaller residues in HYD(Bp) or HYD(Bst) decrease the hydrophobicity and create cavities inside the structure. HYD(Tsp) has more salt bridges and hydrogen-bonding interactions and less oxidation susceptible Met and Cys residues on the protein surface than HYD(Bp) and HYD(Bst). Besides, HYD(Tsp) also contains more rigid Pro residues. These factors are likely to make major contributions to the varying thermostability of these enzymes. This information could be exploited in helping to engineer more thermostable mesophilic enzymes.