On the abundance and importance of AXXXA sequence motifs in globular proteins and their involvement in CßCß interaction.

The AXXXA and GXXXG motifs are frequently observed in helices, especially in membrane proteins. The motif GXXXG is known to stabilize helix-helix association in membrane proteins via CαHO bonding. AXXXA sequence motif additionally stabilizes the folded state of proteins. We found 27,000 and 18,000 occurrences of AXXXA and GXXXG motifs in a non-redundant set of 6000 obligate homodimeric (OD) complexes. Interestingly, this is less pronounced in transient homodimers (TD) and heterodimers (HetD). On average each obligate homodimer contains four AXXXA motifs, it is 2 and 3.5 for HetD and TD, respectively. Focusing on the binding surface it is seen that 27 % of the ODs contain at least one AXXXA motif at the interface, whereas it is 17 % and 15 % for HetD and TD respectively. AXXXA predominantly stabilizes the OD quaternary structure via the side chain CßCß interactions. This interaction is energetically favorable and is found to be a major driving force for OD quaternary structure stability. Cß-Cß interactions are observed ∼6 times higher than the known CαHO interaction for helix-helix stabilization. Two additional new interactions of CßO and OO are observed at the AXXXA containing interface regions. The occurrence of the motif gets drastically reduced if any of the terminal Ala residues are replaced by Gly. Our findings show the importance of AXXXA in providing stability to the quaternary structure through specific hydrophobic interactions and the specificity of the Ala residue at motif termini. The knowledge gained can be used for designing synthetic proteins of improved stability and for designing peptide-based therapeutics.

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.