On the pathway of the formation of secondary structures in proteins.

Protein structures are typically made up of well-defined modules, called secondary structures. A hierarchical model of protein folding may start with the formation of five-membered non-covalently-linked ring motifs involving O⋅⋅⋅C=O and N-H···N interactions connecting two consecutive peptide groups. Some of these interactions lead to polyproline II structure, which are known to occur in the unfolded state of proteins. These interactions constitute different types of γ-turns, providing the sharpest reversal of the chain direction. Occurring transiently in the unfolded state, and in tandem, they can lead to ß-turns. One of the ß-turns (type I) is predisposed (from a consideration of residue usage) to form the N-terminal of an α-helix, which then propagates toward its C-terminal direction. O⋅⋅⋅C=O interactions encompass four distinct types of conformational features, and one of them has very similar backbone torsion angles as the polyproline II (PPII) conformation and can thus contribute to the formation of PPII helix. An adjustment from these angles can also drive the formation of ß-strand. N-H···N interactions can also constitute capping interaction at helix termini and can link a PPII helix to an α-helix. Thus, the polypeptide backbone is endowed with all the features that can initiate the formation of secondary structural elements, and the γ-turn motifs (resulting from O⋅⋅⋅C=O and N-H···N interactions) are the basic units the protein structures are made up of.

Adenosine dialdehyde, a methyltransferase inhibitor, induces colorectal cancer cells apoptosis by regulating PIMT:p53 interaction.

Post-translational modification (PTM) is important in controlling many biological processes by changing the structure and function of a protein. Protein methylation is an important PTM, and the role of methyltransferases has been implicated in numerous cellular functions. Protein L-isoaspartyl methyltransferase (PIMT) is ubiquitously expressed in almost all organisms and govern important cellular processes including apoptosis. Among other functions, PIMT has also been identified as a potent oncogene because it destabilizes the structure of the tumor suppressor p53 via methylation at the transactivation domain. In the present study we identified that out of the three methyltransferase inhibitors tested, namely, S-adenosyl-l-homocysteine (AdoHcy), adenosine and adenosine dialdehyde (AdOx), only AdOx augments p53 expression by destabilizing PIMT structure, as evident from far-UV CD. The effect of the inhibitors, AdOx in particular, to the structure of PIMT, and the binding of PIMT to the p53 transactivation domain have been investigated by docking and molecular dynamics simulations. AdOx significantly increases p53 accumulation and nuclear translocation in colon cancer cells, triggering the p53-mediated apoptotic pathway. To better understand the molecular mechanisms underlying p53 accumulation in colon cancer cells, we observed that the level of PIMT is considerably lower in AdOx-treated cells, reducing its association with p53, which stabilized p53. p53 then transactivated BAX, increasing the BAX: BCL-2 ratio and causing colon cancer cell death.

O‧‧‧C═O interaction, its occurrence and implications for protein structure and folding.

Various noncovalent interactions, long and short range, stabilize the native protein structure. We had observed a short-range interaction between two adjacent peptide groups in a nearly perpendicular orientation through the involvement of an NH‧‧‧N hydrogen bond. Here we show that the other half of the peptide group, namely the carbonyl moiety, can also be involved through the O‧‧‧C═O interaction. Considering the interacting residues, the second residue of the pair has distinct backbone conformational angles, occurring in four clusters, each engendering well-defined structural motifs. One of the motifs is the γ-turn, another being polyproline II helix. The interacting pair is found mostly in the irregular region in protein structures, and the propensities of residues and the identification of the nearest secondary structure show interesting patterns. The most conspicuous ß-turn conformation is built from two consecutive γ-turns, with embedded O‧‧‧C═O and NH‧‧‧N interactions, and there is considerable match of the residue usage at the central positions of the ß-turn and the γ-turn components. This clearly exemplifies the hierarchical growth of the protein secondary structures, which would be important in our understanding of protein folding. While the occurrence of the O‧‧‧C═O interaction in α-helices has been well documented, we find it to be equally important in making capping interactions at helix termini.

Inhibition of microtubule assembly and cytotoxic effect of graphene oxide on human colorectal carcinoma cell HCT116.

Nanomaterials, such as graphene oxide (GO), are increasingly being investigated for their suitability in biomedical applications. Tubulin is the key molecule for the formation of microtubules crucial for cellular function and proliferation, and as such an appealing target for developing anticancer drug. Here we employ biophysical techniques to study the effect of GO on tubulin structure and how the changes affect the tubulin/microtubule assembly. GO disrupts the structural integrity of the protein, with consequent retardation of tubulin polymerization. Investigating the anticancer potential of GO, we found that it is more toxic to human colon cancer cells (HCT116), as compared to human embryonic kidney epithelial cells (HEK293). Immunocytochemistry indicated the disruption of microtubule assembly in HCT116 cells. GO arrested cells in the S phase with increased accumulation in Sub-G1 population of cell cycle, inducing apoptosis by generating reactive oxygen species (ROS) in a dose- and time-dependent manner. GO inhibited microtubule formation by intervening into the polymerization of tubulin heterodimers both in vitro and ex vivo, resulting in growth arrest at the S phase and ROS induced apoptosis of HCT116 colorectal carcinoma cells. There was no significant harm to the HEK293 kidney epithelial cells used as control. Our report of pristine GO causing ROS-induced apoptosis of cancer cells and inhibition of tubulin-microtubule assembly can be of interest in cancer therapeutics and nanomedicine.

Delineation of a new structural motif involving NHN γ-turn.

Macromolecules are characterized by distinctive arrangement of hydrogen bonds. Different patterns of hydrogen bonds give rise to distinct and stable structural motifs. An analysis of 4114 non-redundant protein chains reveals the existence of a three-residue, (i - 1) to (i + 1), structural motif, having two hydrogen-bonded five-membered pseudo rings (the first, an NH···OC involving the first residue, and the second being NH∙∙∙N involving the last two residues), separated by a peptide bond. There could be an additional hydrogen bond between the side-chain at (i-1) and the main-chain NH of (i + 1). The average backbone torsion angles of -76(±21)° and - 12(±17)° at i creates a tight turn in the polypeptide chain, akin to a γ-turn. Indeed, a search of three-residue fragments with restriction on the terminal Cα ···Cα distance and the existence of the two pseudo rings on either side revealed the presence 14 846 cases of a variant, termed NHN γ-turn, distinct from the NHO γ-turn (2032 cases) that has traditionally been characterized by the presence of NHO hydrogen bond linking the terminal main-chain atoms. As in the latter, the newly identified γ-turns are also of two types-classical and inverse, occurring in the ratio of 1:6. The propensities of residues to occur in these turns and their secondary structural features have been enumerated. An understanding of these turns would be useful for structure prediction and loop modeling, and may serve as models to represent some of the unfolded state or disordered region in proteins.

Structural changes in DNA-binding proteins on complexation.

Characterization and prediction of the DNA-biding regions in proteins are essential for our understanding of how proteins recognize/bind DNA. We analyze the unbound (U) and the bound (B) forms of proteins from the protein-DNA docking benchmark that contains 66 binary protein-DNA complexes along with their unbound counterparts. Proteins binding DNA undergo greater structural changes on complexation (in particular, those in the enzyme category) than those involved in protein-protein interactions (PPI). While interface atoms involved in PPI exhibit an increase in their solvent-accessible surface area (ASA) in the bound form in the majority of the cases compared to the unbound interface, protein-DNA interactions indicate increase and decrease in equal measure. In 25% structures, the U form has missing residues which are located in the interface in the B form. The missing atoms contribute more toward the buried surface area compared to other interface atoms. Lys, Gly and Arg are prominent in disordered segments that get ordered in the interface on complexation. In going from U to B, there may be an increase in coil and helical content at the expense of turns and strands. Consideration of flexibility cannot distinguish the interface residues from the surface residues in the U form.

Self-Assembly of Ferritin: Structure, Biological Function and Potential Applications in Nanotechnology.

Protein cages are normally formed by the self-assembly of multiple protein subunits and ferritin is a typical example of a protein cage structure. Ferritin is a ubiquitous multi-subunit iron storage protein formed by 24 polypeptide chains that self-assemble into a hollow, roughly spherical protein cage. Ferritin has external and internal diameters of approximately 12 nm and 8 nm, respectively. Functionally, ferritin performs iron sequestration and is highly conserved in evolution. The interior cavity of ferritin provides a unique reaction vessel to carry out reactions separated from the exterior environment. In nature, the cavity is utilized for sequestration of iron and bio-mineralization as a mechanism to render iron inert and safe from the external environment. Material scientists have been inspired by this system and exploited a range of ferritin superfamily proteins as supramolecular templates to encapsulate different carrier molecules ranging from cancer drugs to therapeutic proteins, in addition to using ferritin proteins as well-defined building blocks for fabrication. Besides the interior cavity, the exterior surface and sub-unit interface of ferritin can be modified without affecting ferritin assembly.

Zinc oxide nanoparticles provide anti-cholera activity by disrupting the interaction of cholera toxin with the human GM1 receptor.

Vibrio cholerae causes cholera and is the leading cause of diarrhea in developing countries, highlighting the need for the development of new treatment strategies to combat this disease agent. While exploring the possibility of using zinc oxide (ZnO) nanoparticles (NPs) in cholera treatment, we previously found that ZnO NPs reduce fluid accumulation in mouse ileum induced by the cholera toxin (CT) protein. To uncover the mechanism of action of ZnO NPs on CT activity, here we used classical (O395) and El Tor (C6706) V. cholerae biotypes in growth and biochemical assays. We found that a ZnO NP concentration of 10 µg/ml did not affect the growth rates of these two strains, nor did we observe that ZnO NPs reduce the expression levels of CT mRNA and protein. It was observed that ZnO NPs form a complex with CT, appear to disrupt the CT secondary structure, and block its interaction with the GM1 ganglioside receptor in the outer leaflet of the plasma membrane in intestinal (HT-29) cells and thereby reduce CT uptake into the cells. In the range of 2.5-10 µg/ml, ZnO NPs exhibited no cytotoxicity on kidney (HEK293) and HT-29 cells. We conclude that ZnO NPs prevent the first step in the translocation of cholera toxin into intestinal epithelial cells without exerting measurable toxic effects on HEK293 and HT-29 cells.

Anoctamin 6 Contributes to Cl- Secretion in Accessory Cholera Enterotoxin (Ace)-stimulated Diarrhea: AN ESSENTIAL ROLE FOR PHOSPHATIDYLINOSITOL 4,5-BISPHOSPHATE (PIP2) SIGNALING IN CHOLERA.

Accessory cholera enterotoxin (Ace) of Vibrio cholerae has been shown to contribute to diarrhea. However, the signaling mechanism and specific type of Cl- channel activated by Ace are still unknown. We have shown here that the recombinant Ace protein induced ICl of apical plasma membrane, which was inhibited by classical CaCC blockers. Surprisingly, an Ace-elicited rise of current was neither affected by ANO1 (TMEM16A)-specific inhibitor T16A(inh)-AO1(TAO1) nor by the cystic fibrosis transmembrane conductance regulator (CFTR) blocker, CFTR inh-172. Ace stimulated whole-cell current in Caco-2 cells. However, the apical ICl was attenuated by knockdown of ANO6 (TMEM16F). This impaired phenotype was restored by re-expression of ANO6 in Caco-2 cells. Whole-cell patch clamp recordings of ANO currents in HEK293 cells transiently expressing mouse ANO1-mCherry or ANO6-GFP confirmed that Ace induced Cl- secretion. Application of Ace produced ANO6 but not the ANO1 currents. Ace was not able to induce a [Ca2+]i rise in Caco-2 cells, but cellular abundance of phosphatidylinositol 4,5-bisphosphate (PIP2) increased. Identification of the PIP2-binding motif at the N-terminal sequence among human and mouse ANO6 variants along with binding of PIP2 directly to ANO6 in HEK293 cells indicate likely PIP2 regulation of ANO6. The biophysical and pharmacological properties of Ace stimulated Cl- current along with intestinal fluid accumulation, and binding of PIP2 to the proximal KR motif of channel proteins, whose mutagenesis correlates with altered binding of PIP2, is comparable with ANO6 stimulation. We conclude that ANO6 is predominantly expressed in intestinal epithelia, where it contributes secretory diarrhea by Ace stimulation in a calcium-independent mechanism of RhoA-ROCK-PIP2 signaling.

Structure and function of Vibrio cholerae accessory cholera enterotoxin in presence of gold nanoparticles: Dependence on morphology.

BACKGROUND: Accessory cholera enterotoxin (Ace) is a classical enterotoxin produced by Vibrio cholerae, the causative agent for cholera. Considering the crucial role of Ace in pathogenesis of cholera, we explored the modulation of structure/function of Ace using gold nanoparticles (AuNPs) of different size and shape - spherical (AuNS10 and AuNS100, the number indicating the diameter in nm) and rod (AuNR10). METHODS: Biophysical techniques have been used to find out structural modulation of Ace by AuNPs. Effect of AuNP on Ace conformation was monitored by far-UV CD; urea-induced unfolding and binding of Ace to various AuNPs were studied by tryptophan fluorescence. In vivo experiments using mouse ileal loop and Ussing chamber were carried out to corroborate biophysical data. RESULTS: Biophysical data revealed degradation of Ace by AuNR10 and AuNS100, not by AuNS10. The feature of AuNR10 having high aspect ratio, but with the same transverse diameter as that of AuNS10 enabled us to explore the importance of morphology on modulation of protein structure/function. The equilibration time for adsorption shows dependence on the radius of curvature, being largest for AuNR10. In vivo experiments revealed the efficacy of AuNR10 and AuNS100 for reduced fluid accumulation, indicative of the loss of activity of Ace. CONCLUSIONS: We show how biophysical studies and in vivo experiments go hand-in-hand in establishing the efficacy and role of size/shape of AuNPs on a toxin structure. GENERAL SIGNIFICANCE: The effect of AuNP on toxin depends on its morphology. The targeted modulation of Ace could be of therapeutic benefit for gastrointestinal disorders.

Identification of the target DNA sequence and characterization of DNA binding features of HlyU, and suggestion of a redox switch for hlyA expression in the human pathogen Vibrio cholerae from in silico studies.

HlyU, a transcriptional regulator common in many Vibrio species, activates the hemolysin gene hlyA in Vibrio cholerae, the rtxA1 operon in Vibrio vulnificus and the genes of plp-vah1 and rtxACHBDE gene clusters in Vibrio anguillarum. The protein is also proposed to be a potential global virulence regulator for V. cholerae and V. vulnificus. Mechanisms of gene control by HlyU in V. vulnificus and V. anguillarum are reported. However, detailed elucidation of the interaction of HlyU in V. cholerae with its target DNA at the molecular level is not available. Here we report a 17-bp imperfect palindrome sequence, 5'-TAATTCAGACTAAATTA-3', 173 bp upstream of hlyA promoter, as the binding site of HlyU. This winged helix-turn-helix protein binds necessarily as a dimer with the recognition helices contacting the major grooves and the ß-sheet wings, the minor grooves. Such interactions enhance hlyA promoter activity in vivo. Mutations affecting dimerization as well as those in the DNA-protein interface hamper DNA binding and transcription regulation. Molecular dynamic simulations show hydrogen bonding patterns involving residues at the mutation sites and confirmed their importance in DNA binding. On binding to HlyU, DNA deviates by ∼68º from linearity. Dynamics also suggest a possible redox control in HlyU.

Antibacterial effect of silver nanoparticles and the modeling of bacterial growth kinetics using a modified Gompertz model.

BACKGROUND: An alternative to conventional antibiotics is needed to fight against emerging multiple drug resistant pathogenic bacteria. In this endeavor, the effect of silver nanoparticle (Ag-NP) has been studied quantitatively on two common pathogenic bacteria Escherichia coli and Staphylococcus aureus, and the growth curves were modeled. METHODS: The effect of Ag-NP on bacterial growth kinetics was studied by measuring the optical density, and was fitted by non-linear regression using the Logistic and modified Gompertz models. Scanning Electron Microscopy and fluorescence microscopy were used to study the morphological changes of the bacterial cells. Generation of reactive oxygen species for Ag-NP treated cells were measured by fluorescence emission spectra. RESULTS: The modified Gompertz model, incorporating cell death, fits the observed data better than the Logistic model. With increasing concentration of Ag-NP, the growth kinetics of both bacteria shows a decline in growth rate with simultaneous enhancement of death rate constants. The duration of the lag phase was found to increase with Ag-NP concentration. SEM showed morphological changes, while fluorescence microscopy using DAPI showed compaction of DNA for Ag-NP-treated bacterial cells. CONCLUSIONS: E. coli was found to be more susceptible to Ag-NP as compared to S. aureus. The modified Gompertz model, using a death term, was found to be useful in explaining the non-monotonic nature of the growth curve. GENERAL SIGNIFICANCE: The modified Gompertz model derived here is of general nature and can be used to study any microbial growth kinetics under the influence of antimicrobial agents.

Tumor-associated mesenchymal stem cells inhibit naïve T cell expansion by blocking cysteine export from dendritic cells.

Mesenchymal stem cells (MSCs) represent an important cellular constituent of the tumor microenvironment, which along with tumor cells themselves, serve to regulate protective immune responses in support of progressive disease. We report that tumor MSCs prevent the ability of dendritic cells (DC) to promote naïve CD4(+) and CD8(+) T cell expansion, interferon gamma secretion and cytotoxicity against tumor cells, which are critical to immune-mediated tumor eradication. Notably, tumor MSCs fail to prevent DC-mediated early T cell activation events or the ability of responder T cells to produce IL-2. The immunoregulatory activity of tumor MSCs is IL-10- and STAT3-dependent, with STAT3 repressing DC expression of cystathionase, a critical enzyme that converts methionine-to-cysteine. Under cysteine-deficient priming conditions, naïve T cells exhibit defective cellular metabolism and proliferation. Bioinformatics analyses as well as in vitro observations suggest that STAT3 may directly bind to a GAS-like motif within the cystathionase promoter (-269 to -261) leading to IL-10-STAT3 mediated repression of cystathionase gene transcription. Our collective results provide evidence for a novel mechanism of tumor MSC-mediated T cell inhibition within tumor microenvironment.

The antimicrobial activity of ZnO nanoparticles against Vibrio cholerae: Variation in response depends on biotype.

The potency of zinc oxide nanoparticles (NPs), with a core size of ~7-10nm, to inhibit cholera disease was investigated by demonstrating the effect on two biotypes (classical and El Tor) of O1 serogroup of Vibrio cholerae-El Tor was more susceptible both in planktonic and in biofilm forms. Interaction with ZnO NP results in deformed cellular architecture. Increased fluidity and depolarization of membrane, and protein leakage further confirmed the damages inflicted on Vibrio by NP. NP was shown to produce reactive oxygen species (ROS) and induce DNA damage. These results suggest that the antibacterial mechanism of ZnO action is most likely due to generation of ROS and disruption of bacterial membrane. The antimicrobial efficacy of NP has been validated in animal model. The synergistic action of NP and antibiotic suggests an alternative for the treatment of cholera.

Actin-curcumin interaction: insights into the mechanism of actin polymerization inhibition.

Curcumin, derived from rhizomes of the Curcuma longa plant, is known to possess a wide range of medicinal properties. We have examined the interaction of curcumin with actin and determined their binding and thermodynamic parameters using isothermal titration calorimetry. Curcumin is weakly fluorescent in aqueous solution, and binding to actin enhances fluorescence several fold with a large blue shift in the emission maximum. Curcumin inhibits microfilament formation, which is similar to its role in inhibiting microtubule formation. We synthesized a series of stable curcumin analogues to examine their affinity for actin and their ability to inhibit actin self-assembly. Results show that curcumin is a ligand with two symmetrical halves, each of which possesses no activity individually. Oxazole, pyrazole, and acetyl derivatives are less effective than curcumin at inhibiting actin self-assembly, whereas a benzylidiene derivative is more effective. Cell biology studies suggest that disorganization of the actin network leads to destabilization of filaments in the presence of curcumin. Molecular docking reveals that curcumin binds close to the cytochalasin binding site of actin. Further molecular dynamics studies reveal a possible allosteric effect in which curcumin binding at the "barbed end" of actin is transmitted to the "pointed end", where conformational changes disrupt interactions with the adjacent actin monomer to interrupt filament formation. Finally, the recognition and binding of actin by curcumin is yet another example of its unique ability to target multiple receptors.

Crystal structure of HlyU, the hemolysin gene transcription activator, from Vibrio cholerae N16961 and functional implications.

HlyU in Vibrio cholerae is known to be the transcriptional activator of the hemolysin gene, HlyA and possibly a regulator of other virulence factors influencing growth, colonization and pathogenicity of this infective agent. Here we report the crystal structure of HlyU from V. cholerae N16961 (HlyU_Vc) at 1.8Å. The protein, with five α-helices and three ß-strands in the topology of α1-α2-ß1-α3-α4-ß2-ß3-α5, forms a homodimer. Helices α3-α4 and a ß sheet form the winged helix-turn-helix (wHTH) DNA-binding motif common to the transcription regulators of the SmtB/ArsR family. In spite of an overall fold similar to SmtB/ArsR family, it lacks any metal binding site seen in SmtB. A comparison of the dimeric interfaces showed that the one in SmtB is much larger and have salt bridges that can be disrupted to accommodate metal ions. A model of HlyU-DNA complex suggests bending of the DNA. Cys38 in the structure was found to be modified as sulfenic acid; the oxidized form was not seen in another structure solved under reducing condition. Although devoid of any metal binding site, the presence of a Cys residue exhibiting oxidation-reduction suggests the possibility of the existence of a redox switch in transcription regulation. A structure-based phylogenetic analysis of wHTH proteins revealed the segregation of metal and non-metal binding proteins as well as those in the latter group that are under redox control.

Flexibility in the N-terminal actin-binding domain: clues from in silico mutations and molecular dynamics.

Dystrophin is a long, rod-shaped cytoskeleton protein implicated in muscular dystrophy (MDys). Utrophin is the closest autosomal homolog of dystrophin. Both proteins have N-terminal actin-binding domain (N-ABD), a central rod domain and C-terminal region. N-ABD, composed of two calponin homology (CH) subdomains joined by a helical linker, harbors a few disease causing missense mutations. Although the two proteins share considerable homology (>72%) in N-ABD, recent structural and biochemical studies have shown that there are significant differences (including stability, mode of actin-binding) and their functions are not completely interchangeable. In this investigation, we have used extensive molecular dynamics simulations to understand the differences and the similarities of these two proteins, along with another actin-binding protein, fimbrin. In silico mutations were performed to identify two key residues that might be responsible for the dynamical difference between the molecules. Simulation points to the inherent flexibility of the linker region, which adapts different conformations in the wild type dystrophin. Mutations T220V and G130D in dystrophin constrain the flexibility of the central helical region, while in the two known disease-causing mutants, K18N and L54R, the helicity of the region is compromised. Phylogenetic tree and sequence analysis revealed that dystrophin and utrophin genes have probably originated from the same ancestor. The investigation would provide insight into the functional diversity of two closely related proteins and fimbrin, and contribute to our understanding of the mechanism of MDys.

ω-Turn: a novel ß-turn mimic in globular proteins stabilized by main-chain to side-chain C−H···O interaction.

Mimicry of structural motifs is a common feature in proteins. The 10-membered hydrogen-bonded ring involving the main-chain C − O in a ß-turn can be formed using a side-chain carbonyl group leading to Asx-turn. We show that the N − H component of hydrogen bond can be replaced by a C(γ) -H group in the side chain, culminating in a nonconventional C − H···O interaction. Because of its shape this ß-turn mimic is designated as ω-turn, which is found to occur ∼ three times per 100 residues. Three residues (i to i + 2) constitute the turn with the C − H···O interaction occurring between the terminal residues, constraining the torsion angles ϕi + 1, ψi + 1, ϕi + 2 and χ'1(i + 2) (using the interacting C(γ) atom). Based on these angles there are two types of ω-turns, each of which can be further divided into two groups. C(ß) -branched side-chains, and Met and Gln have high propensities to occur at i + 2; for the last two residues the carbonyl oxygen may participate in an additional interaction involving the S and amino group, respectively. With Cys occupying the i + 1 position, such turns are found in the metal-binding sites. N-linked glycosylation occurs at the consensus pattern Asn-Xaa-Ser/Thr; with Thr at i + 2, the sequence can adopt the secondary structure of a ω-turn, which may be the recognition site for protein modification. Location between two ß-strands is the most common occurrence in protein tertiary structure, and being generally exposed ω-turn may constitute the antigenic determinant site. It is a stable scaffold and may be used in protein engineering and peptide design.

Crystal structure and activity of protein L-isoaspartyl-O-methyltransferase from Vibrio cholerae, and the effect of AdoHcy binding.

The repair enzyme Protein L-isoaspartyl-O-methyltransferase (PIMT) is widely distributed in various organisms. PIMT catalyzes S-adenosylmethionine (AdoMet) dependent methylation of abnormal L-isoaspartyl residues, formed by the deamidation of asparagines and isomerization of aspartates. We report the crystal structure of PIMT of Vibrio cholerae (VcPIMT), the aetiological agent for cholera, complexed with the demethylated cofactor S-adenosyl-L-homocysteine (AdoHcy) to 2.05 Å resolution. A stretch of residues (39-58), lining the substrate-binding site, is disordered. Urea-induced unfolding free energy for apo and VcPIMT-AdoHcy complex reveals greater stability for the cofactor-bound protein. The kinetic parameters for the methyltransferase activity of the recombinant VcPIMT was determined using a continuous spectrophotometric color-based assay using the peptide substrate [VYP(L-isoD)HA]. The enzyme exhibited activity higher than the Escherichia coli enzyme and closer to those from thermophilic bacteria and the mammalian source. The association constant for substrate binding is 2.29 × 10(6) M(-1), quite similar to that for AdoHcy. The crystal structure and the model of the peptide-bound structure indicate that the majority of the interactions used for cofactor/substrate binding are provided by the main-chain atoms. Evolutionary relationships derived based on a phylogenetic tree constructed using the PIMT sequences are in conformity with the crystal structures of nine AdoHcy-bound PIMTs.

Water and side-chain embedded π-turns.

Elucidating protein function from its structure is central to the understanding of cellular mechanisms. This involves deciphering the dependence of local structural motifs on sequence. These structural motifs may be stabilized by direct or water-mediated hydrogen bonding among the constituent residues. π-Turns, defined by interactions between (i) and (i + 5) positions, are large enough to contain a central space that can embed a water molecule (or a protein moiety) to form a stable structure. This work is an analysis of such embedded π-turns using a nonredundant dataset of protein structures. A total of 2965 embedded π-turns have been identified, as also 281 embedded Schellman motif, a type of π-turn which occurs at the C-termini of α-helices. Embedded π-turns and Schellman motifs have been classified on the basis of the protein atoms of the terminal turn residues that are linked by the embedded moiety, conformation, residue composition, and compared with the turns that have terminal residues connected by direct hydrogen bonds. Geometrically, the turns have been fitted to a circle and the position of the linker relative to its center analyzed. The hydroxyl group of Ser and Thr, located at (i + 3) position, is the most prominent linker for the side-chain mediated π-turns. Consideration of residue conservation among homologous sequences indicates the terminal and the linker positions to be the most conserved. The embedded π-turn as a binding site (for the linker) is discussed in the context of "nest," a concave depression that is formed in protein structures with adjacent residues having enantiomeric main-chain conformations.