Phosphorylation-Competent Metastable State of Escherichia coli Toxin HipA.

Phosphorylation is a key post-translational modification that alters the functional state of many proteins. The Escherichia coli toxin HipA, which phosphorylates glutamyl-tRNA synthetase and triggers bacterial persistence under stress, becomes inactivated upon autophosphorylation of Ser150. Interestingly, Ser150 is phosphorylation-incompetent in the crystal structure of HipA since it is deeply buried ("in-state"), although in the phosphorylated state it is solvent exposed ("out-state"). To be phosphorylated, a minor population of HipA must exist in the phosphorylation-competent "out-state" (solvent-exposed Ser150), not detected in the crystal structure of unphosphorylated HipA. Here we report a molten-globule-like intermediate of HipA at low urea (∼4 kcal/mol unstable than natively folded HipA). The intermediate is aggregation-prone, consistent with a solvent exposed Ser150 and its two flanking hydrophobic neighbors (Val/Ile) in the "out-state". Molecular dynamics simulations showed the HipA "in-out" pathway to contain multiple free energy minima with an increasing degree of Ser150 solvent exposure with the free energy difference between the "in-state" and the metastable exposed state(s) to be ∼2-2.5 kcal/mol, with unique sets of hydrogen bonds and salt bridges associated with the metastable loop conformations. Together, the data clearly identify the existence of a phosphorylation-competent metastable state of HipA. Our results not only suggest a mechanism of HipA autophosphorylation but also add to a number of recent reports on unrelated protein systems where the common proposed mechanism for phosphorylation of buried residues is their transient exposure even without phosphorylation.

Signatures of tRNAGlx -specificity in proteobacterial glutamyl-tRNA synthetases.

The canonical function of glutamyl-tRNA synthetase (GluRS) is to glutamylate tRNAGlu . Yet not all bacterial GluRSs glutamylate tRNAGlu ; many glutamylate both tRNAGlu and tRNAGln , while some glutamylate only tRNAGln and not the cognate substrate tRNAGlu . Understanding the basis of the unique specificity of tRNAGlx is important. Mutational studies have hinted at hotspot residues, both on tRNAGlx and GluRS, which play crucial roles in tRNAGlx -specificity. However, its underlying structural basis remains unexplored. The majority of biochemical studies related to tRNAGlx -specificity have been performed on GluRS from Escherichia coli and other proteobacterial species. However, since the early crystal structures of GluRS and tRNAGlu -bound GluRS were from non-proteobacterial species (Thermus thermophilus), proteobacterial biochemical data have often been interpreted in the context of non-proteobacterial GluRS structures. Marked differences between proteobacterial and non-proteobacterial GluRSs have been demonstrated; therefore, it is important to understand tRNAGlx -specificity vis-a-vis proteobacterial GluRS structures. To this end, we solved the crystal structure of a double mutant GluRS from E. coli. Using the solved structure and several other currently available proteo- and non-proteobacterial GluRS crystal structures, we probed the structural basis of the tRNAGlx -specificity of bacterial GluRSs. Specifically, our analyses suggest a unique role played by the tRNAGlx D-helix contacting loop of GluRS in the modulation of tRNAGln -specificity. While earlier studies have identified functional hotspots on tRNAGlx that control the tRNAGlx -specificity of GluRS, this is the first report of complementary signatures of tRNAGlx -specificity in GluRS.

Sequence-Specific Dual DNA Binding Modes and Cytotoxicities of N-6-Functionalized Norcryptotackieine Alkaloids.

Norcryptotackieine (1a) belongs to the indoloquinoline class of alkaloids isolated from Cryptolepis sanguinolenta, a plant species that has been traditionally used as an antimalarial agent. Additional structural modifications of 1a can potentially enhance its therapeutic potency. Indoloquinolines such as cryptolepine, neocryptolepine, isocryptolepine, and neoisocryptolepine show restricted clinical applications owing to their cytotoxicity deriving from interactions with DNA. Here, we examined the effect of substitutions at the N-6 position of norcryptotackieine on the cytotoxicity, as well as structure-activity relationship studies pertaining to sequence specific DNA-binding affinities. The representative compound 6d binds DNA in a nonintercalative/pseudointercalative fashion, in addition to nonspecific stacking on DNA, in a sequence selective manner. The DNA-binding studies clearly establish the mechanism of DNA binding by N-6-substituted norcryptotackieines and neocryptolepine. The synthesized norcryptotackieines 6c,d and known indoloquinolines were screened on different cell lines (HEK293, OVCAR3, SKOV3, B16F10, and HeLa) to assess their cytotoxicity. Norcryptotackieine 6d (IC50 value of 3.1 µM) showed 2-fold less potency when compared to the natural indoloquinoline cryptolepine 1c (IC50 value of 1.64 µM) in OVCAR3 (ovarian adenocarcinoma) cell lines.

Multiple copies of a novel amphipathic α-helix forming segment in Physcomitrella patens dehydrin play a key role in abiotic stress mitigation.

Plants use a diverse set of proteins to mitigate various abiotic stresses. The intrinsically disordered protein dehydrin is an important member of this repertoire of proteins, characterized by a canonical amphipathic K-segment. It can also contain other stress-mitigating noncanonical segments-a likely reflection of the extremely diverse nature of abiotic stress encountered by plants. Among plants, the poikilohydric mosses have no inbuilt mechanism to prevent desiccation and therefore are likely to contain unique noncanonical stress-responsive motifs in their dehydrins. Here we report the recurring occurrence of a novel amphipathic helix-forming segment (D-segment: EGφφD(R/K)AKDAφ, where φ represents a hydrophobic residue) in Physcomitrella patens dehydrin (PpDHNA), a poikilohydric moss. NMR and CD spectroscopic experiments demonstrated the helix-forming tendency of the D-segment, with the shuffled D-segment as control. PpDHNA activity was shown to be size as well as D-segment dependent from in vitro, in vivo, and in planta studies using PpDHNA and various deletion mutants. Bimolecular fluorescence complementation studies showed that D-segment-mediated PpDHNA self-association is a requirement for stress abatement. The D-segment was also found to occur in two rehydrin proteins from Syntrichia ruralis, another poikilohydric plant like P. patens. Multiple occurrences of the D-segment in poikilohydric plant dehydrins/rehydrins, along with the experimental demonstration of the role of D-segment in stress abatement, implies that the D-segment mediates unique resurrection strategies, which may be employed by plant dehydrins that are capable of mitigating extreme stress.

DNA Minor Groove-Induced cis-trans Isomerization of a Near-Infrared Fluorescent Probe.

The discovery of small molecules that exhibit turn-on far-red or near-infrared (NIR) fluorescence upon DNA binding and understanding how they bind DNA are important for imaging and bioanalytical applications. Here we report the DNA-bound structure and the DNA binding mechanism of quinone cyanine dithiazole (QCy-DT), a recently reported AT-specific turn-on NIR fluorescent probe for double-stranded DNA. The nuclear magnetic resonance (NMR)-derived structure showed minor groove binding but no specific ligand-DNA interactions, consistent with an endothermic and entropy-driven binding mechanism deduced from isothermal titration calorimetry. Minor groove binding is typically fast because it minimally perturbs the DNA structure. However, QCy-DT exhibited unusually slow DNA binding. The cyanine-based probe is capable of cis-trans isomerization due to overlapping methine bridges, with 16 possible slowly interconverting cis/trans isomers. Using NMR, density functional theory, and free energy calculations, we show that the DNA-free and DNA-bound environments of QCy-DT prefer distinctly different isomers, indicating that the origin of the slow kinetics is a cis-trans isomerization and that the minor groove preferentially selects an otherwise unstable cis/trans isomer of QCy-DT. Flux analysis showed the conformational selection pathway to be the dominating DNA binding mechanism at low DNA concentrations, which switches to the induced fit pathway at high DNA concentrations. This report of cis/trans isomerization of a ligand, upon binding the DNA minor groove, expands the prevailing understanding of unique discriminatory powers of the minor groove and has an important bearing on using polymethine cyanine dyes to probe the kinetics of molecular interactions.

Insights on the disruption of the complex between human positive coactivator 4 and p53 by small molecules.

Interaction between human positive coactivator 4 (PC4), an abundant nuclear protein, and the tumor suppressor protein p53 plays a crucial role in initiating apoptosis. In certain neurodegenerative diseases PC4 assisted-p53-dependent apoptosis may play a central role. Thus, disruption of p53-PC4 interaction may be a good drug target for certain disease pathologies. A p53-derived short peptide (AcPep) that binds the C-terminal domain of PC4 (C-PC4) is known to disrupt PC4-p53 interaction. To fully characterize its binding mode and binding site on PC4, we co-crystallized C-PC4 with the peptide and determined its structure. The crystal, despite exhibiting mass spectrometric signature of the peptide, lacked peptide electron density and showed a novel crystal lattice, when compared to C-PC4 crystals without the peptide. Using peptide-docked models of crystal lattices, corresponding to our structure and the peptide-devoid structure we show the origin of the novel crystal lattice to be dynamically bound peptide at the previously identified putative binding site. The weak binding is proposed to be due to the lack of the N-terminal domain of PC4 (N-PC4), which we experimentally show to be disordered with no effect on PC4 stability. Taking cue from the structure, virtual screening of ∼18.6 million small molecules from the ZINC15 database was performed, followed by toxicity and binding free energy filtering. The novel crystal lattice of C-PC4 in presence of the peptide, the role of the disordered N-PC4 and the high throughput identification of potent small molecules will allow a better understanding and control of p53-PC4 interaction.

A Pyrimido-Quinoxaline Fused Heterocycle Lights Up Transfer RNA upon Binding at the Mg2+ Binding Site.

Transfer RNAs (tRNAs) are fundamental molecules in cellular translation. In this study we have highlighted a fluorescence-based perceptive approach for tRNAs by using a quinoxaline small molecule. We have synthesised a water-soluble fluorescent pyrimido-quinoxaline-fused heterocycle containing a mandatory piperazine tail (DS1) with a large Stokes shift (∼160 nm). The interaction between DS1 and tRNA results in significant fluorescence enhancement of the molecule with Kd ∼5 µM and multiple binding sites. Our work reveals that the DS1 binding site overlaps with the specific Mg2+ ion binding site in the D loop of tRNA. As a proof-of-concept, the molecule inhibited Pb2+ -induced cleavage of yeast tRNAPhe in the D loop. In competitive binding assays, the fluorescence of DS1-tRNA complex is quenched by a known tRNA-binder, tobramycin. This indicates the displacement of DS1 and, indeed, a substantiation of specific binding at the site of tertiary interaction in the central region of tRNA. The ability of compound DS1 to bind tRNA with a higher affinity compared to DNA and single-stranded RNA offers a promising approach to developing tRNA-based biomarker diagnostics in the future.

Intercalator-Induced DNA Superstructure Formation: Doxorubicin and a Synthetic Quinoxaline Derivative.

Small molecules that intercalate DNA have tremendous therapeutic potential. Typically, DNA intercalators do not alter the overall DNA double-helical structure, except locally at the intercalation sites. In a previous report, we showed that a quinoxaline-based intercalator with a mandatory benzyl substitution (1d) induced an unusually large circular dichroism signal upon DNA binding, suggesting the formation of intercalated DNA superstructures. However, no detailed structural studies have been reported. Using atomic force microscopy, we have probed the nature of the superstructure and report the formation of a plectonemically oversupercoiled structure of pBR322 plasmid DNA by 1d, where close association of distant DNA double-helical stretches is the predominant motif. Without the benzyl moiety (1a), no such DNA superstructure was observed. Similar superstructures were also observed with doxorubicin (dox), a therapeutically important DNA intercalator, suggesting that the superstructure is common to some intercalators. The superstructure formation, for both intercalators, was observed to be GC-specific. Interestingly, at higher concentrations (1d and dox), the DNA superstructure led to DNA condensation, a phenomenon typically associated with polyamines but not intercalators. The superstructure may have important biological relevance in connection to a recent study in which dox was shown to evict histone at micromolar concentrations.

Type VIa ß-turn-fused helix N-termini: A novel helix N-cap motif containing cis proline.

Helix N-capping motifs often form hydrogen bonds with terminal amide groups which otherwise would be free. Also, without an amide hydrogen, proline (trans) is over-represented at helix N-termini (N1 position) because this naturally removes the need to hydrogen bond one terminal amide. However, the preference of cisPro, vis-à-vis helix N-termini, is not known. We show that cisPro (αR or PPII ) often appears at the N-cap position (N0) of helices. The N-cap cisPro(αR ) is associated with a six-residue sequence motif - X(-2) -X(-1) -cisPro-X(1) -X(2) -X(3) - with preference for Glu/Gln at X(-1) , Phe/Tyr/Trp at X(1) and Ser/Thr at X(3) . The motif, formed by the fusion of a helix and a type VIa ß-turn, contains a hydrogen bond between the side chain of X(-1) and the side chain/backbone of X(3) , a α-helical hydrogen bond between X(-2) and X(2) and stacking interaction between cisPro and an aromatic residue at X(1) . NMR experiments on peptides containing the motif and its variants showed that local interactions associated with the motif, as found in folded proteins, were not enough to significantly tilt the cis/trans equilibrium towards cisPro. This suggests that some other evolutionary pressure must select the cisPro motif (over transPro) at helix N-termini. Database analysis showed that >C = O of the pre-cisPro(αR ) residue at the helix N-cap, directed opposite to the N→C helical axis, participates in long-range interactions. We hypothesize that the cisPro(αR ) motif is preferred at helix N-termini because it allows the helix to participate in long-range interactions that may be structurally and functionally important.

Effect of introducing aib in a designed helical inhibitor of hdm2-p53 interaction: A molecular dynamics study.

Although p53 is an intrinsically disordered protein, upon binding to Hdm2, a short stretch (residues 19-25) comprising the binding epitope assumes a helical backbone. Because the allowed conformational space of α-aminoisobutyric acid (Aib) is restricted to only the helical basin, Aib-containing helical mimics of p53 (binding epitope) are expected to inhibit interaction between p53 and Hdm2 with a much stronger affinity than the wild type p53 peptide (binding epitope), due to the entropic advantage associated with Aib. However, the IC50 values for the disruption of p53-Hdm2 interaction by Aib-p53 peptides and wild type p53 peptide were found to be comparable (J. Peptide Res. 2002, 60:88-94). To understand why incorporation of Aib didn't substantially increase Hdm2 affinity of Aib-p53 peptides, a series of molecular dynamics simulations were performed. It was found that despite stabilizing a helical backbone in the unbound state, the Aib residues in Aib-p53 peptide arrested two functionally important side-chains (F19 and W23) in non-productive conformations, resulting in relative side-chain orientations of the binding triad F19-W23-L26 incompatible with the bound conformation. Therefore, although a Aib-induced pre-formed helical peptide backbone in the unbound state is expected to favor binding, the locked side-chain orientations of the binding triad in non-productive modes would disfavor binding. This study shows that when using Aib to design functionally important helical peptides, care must be taken to consider potential interactions between side-chains of neighboring residues and Aib in the unbound state.

The Benzyl Moiety in a Quinoxaline-Based Scaffold Acts as a DNA Intercalation Switch.

Quinoxaline antibiotics intercalate dsDNA and exhibit antitumor properties. However, they are difficult to synthesize and their structural complexity impedes a clear mechanistic understanding of DNA binding. Therefore design and synthesis of minimal-intercalators, using only part of the antibiotic scaffold so as to retain the key DNA-binding property, is extremely important. Reported is a unique example of a monomeric quinoxaline derivative of a 6-nitroquinoxaline-2,3-diamine scaffold which binds dsDNA by two different modes. While benzyl derivatives bound DNA in a sequential fashion, with intercalation as the second event, nonbenzyl derivatives showed only the first binding event. The benzyl intercalation switch provides important insights about molecular architecture which control specific DNA binding modes and would be useful in designing functionally important monomeric quinoxaline DNA binders and benchmarking molecular simulations.

Protein-protein association rates captured in a single geometric parameter.

Understanding factors that drive protein-protein association is of fundamental importance. We show that a single geometric parameter in crystal structures of protein-protein complexes, the angle between the electric dipole of one subunit and the partner-generated electric field at the same subunit, linearly correlates with experimentally determined protein-protein association rates. Imprint of a dynamic kinetic process in a single static geometric parameter, associated with mutual electrostatic orientation of subunits in protein-protein complexes, is elegant and demonstrates the universality of electrostatic steering in attenuating protein-protein association rates. That the essence of a complex phenomenon could be captured by properties of the final crystal structure of the complex implies that the electrostatic orientations of protein subunits in crystal structures and the associated transition states are nearly identical. Further, the cosine of the angle, alone, is shown to be sufficient in predicting association rate constants, with accuracies comparable to currently available predictors that use more intricate methodologies. Our results offer mechanistic insights and could be useful in development of coarse-grained models.

Specific sequence of a Beta turn in human la protein may contribute to species specificity of hepatitis C virus.

UNLABELLED: Human La protein is known to be an essential host factor for translation and replication of hepatitis C virus (HCV) RNA. Previously, we have demonstrated that residues responsible for interaction of human La protein with the HCV internal ribosomal entry site (IRES) around the initiator AUG within stem-loop IV form a ß-turn in the RNA recognition motif (RRM) structure. In this study, sequence alignment and mutagenesis suggest that the HCV RNA-interacting ß-turn is conserved only in humans and chimpanzees, the species primarily known to be infected by HCV. A 7-mer peptide corresponding to the HCV RNA-interacting region of human La inhibits HCV translation, whereas another peptide corresponding to the mouse La sequence was unable to do so. Furthermore, IRES-mediated translation was found to be significantly high in the presence of recombinant human La protein in vitro in rabbit reticulocyte lysate. We observed enhanced replication with HCV subgenomic and full-length replicons upon overexpression of either human La protein or a chimeric mouse La protein harboring a human La ß-turn sequence in mouse cells. Taken together, our results raise the possibility of creating an immunocompetent HCV mouse model using human-specific cell entry factors and a humanized form of La protein. IMPORTANCE: Hepatitis C virus is known to infect only humans and chimpanzees under natural conditions. This has prevented the development of a small-animal model, which is important for development of new antiviral drugs. Although a number of human-specific proteins are responsible for this species selectivity and some of these proteins--mostly entry factors--have been identified, full multiplication of the virus in mouse cells is still not possible. In this study, we show that a turn in the human La protein that is responsible for the interaction with the viral RNA is highly specific for the human sequence. Replacement of the corresponding mouse sequence with the human sequence allows the mouse La to behave like its human counterpart and support viral growth in the mouse cell efficiently. This observation, in combination with previously identified cell entry factors, should open up the possibility of creating a mouse model of hepatitis C.

Evolutionary insights about bacterial GlxRS from whole genome analyses: is GluRS2 a chimera?

BACKGROUND: Evolutionary histories of glutamyl-tRNA synthetase (GluRS) and glutaminyl-tRNA synthetase (GlnRS) in bacteria are convoluted. After the divergence of eubacteria and eukarya, bacterial GluRS glutamylated both tRNAGln and tRNAGlu until GlnRS appeared by horizontal gene transfer (HGT) from eukaryotes or a duplicate copy of GluRS (GluRS2) that only glutamylates tRNAGln appeared. The current understanding is based on limited sequence data and not always compatible with available experimental results. In particular, the origin of GluRS2 is poorly understood. RESULTS: A large database of bacterial GluRS, GlnRS, tRNAGln and the trimeric aminoacyl-tRNA-dependent amidotransferase (gatCAB), constructed from whole genomes by functionally annotating and classifying these enzymes according to their mutual presence and absence in the genome, was analyzed. Phylogenetic analyses showed that the catalytic and the anticodon-binding domains of functional GluRS2 (as in Helicobacter pylori) were independently acquired from evolutionarily distant hosts by HGT. Non-functional GluRS2 (as in Thermotoga maritima), on the other hand, was found to contain an anticodon-binding domain appended to a gene-duplicated catalytic domain. Several genomes were found to possess both GluRS2 and GlnRS, even though they share the common function of aminoacylating tRNAGln. GlnRS was widely distributed among bacterial phyla and although phylogenetic analyses confirmed the origin of most bacterial GlnRS to be through a single HGT from eukarya, many GlnRS sequences also appeared with evolutionarily distant phyla in phylogenetic tree. A GlnRS pseudogene could be identified in Sorangium cellulosum. CONCLUSIONS: Our analysis broadens the current understanding of bacterial GlxRS evolution and highlights the idiosyncratic evolution of GluRS2. Specifically we show that: i) GluRS2 is a chimera of mismatching catalytic and anticodon-binding domains, ii) the appearance of GlnRS and GluRS2 in a single bacterial genome indicating that the evolutionary histories of the two enzymes are distinct, iii) GlnRS is more widespread in bacteria than is believed, iv) bacterial GlnRS appeared both by HGT from eukarya and intra-bacterial HGT, v) presence of GlnRS pseudogene shows that many bacteria could not retain the newly acquired eukaryal GlnRS. The functional annotation of GluRS, without recourse to experiments, performed in this work, demonstrates the inherent and unique advantages of using whole genome over isolated sequence databases.

Local control of cis-peptidyl-prolyl bonds mediated by CH···π interactions: the Xaa-Pro-Tyr motif.

Compared to generic peptide bonds, the peptidyl-prolyl bond shows a strong propensity for the cis conformer. The presence of a sequence-contiguous aromatic (Aro) residue can further stabilize the cis conformer, as observed for the Aro-Pro motif. The cis propensity of the reverse sequence motif, Pro-Aro, is not so well understood, especially the effect of N-capping the Pro-Aro motif with different amino acid residues. From a comparative nuclear magnetic resonance study of two peptide series with the general sequences Ac-Xaa-Pro-Tyr-NH2 and Ac-Xaa-Pro-Ala-NH2, we present a relative thermodynamic scale that reflects how the nature of the Xaa side chain influences the cis propensity of the Xaa-Pro-Tyr motif, with Gly, Pro, and Ala at position Xaa giving the greatest enhancement of the cis-peptidyl-prolyl population. We also show that CH···π interaction between Xaa and Tyr is responsible for the enhanced cis population. However, the mere presence of the CH···π interaction does not guarantee that the peptidyl-prolyl bond will have a higher cis content in Xaa-Pro-Tyr than in Xaa-Pro-Ala. Xaa-dependent intramolecular interactions present in Xaa-trans-Pro-Tyr can nullify favorable CH···π interactions in Xaa-cis-Pro-Tyr. The relative cis-peptidyl-prolyl stabilizing propensities of Xaa (Xaa-Pro-Tyr) in proteins and in our peptide series show strong linear correlation except when Xaa is aromatic. We also explore the Xaa-Pro-Gly-Tyr sequence motif and show that mediated by a Pro-Tyr CH···π interaction, the cis-peptidyl-prolyl bond in the motif is stabilized when Xaa is Pro.

Stable and potent analogues derived from the modification of the dicarbonyl moiety of curcumin.

Curcumin has shown promising therapeutic utilities for many diseases, including cancer; however, its clinical application is severely limited because of its poor stability under physiological conditions. Here we find that curcumin also loses its activity instantaneously in a reducing environment. Curcumin can exist in solution as a tautomeric mixture of keto and enol forms, and the enol form was found to be responsible for the rapid degradation of the compound. To increase the stability of curcumin, several analogues were synthesized in which the diketone moiety of curcumin was replaced by isoxazole (compound 2) and pyrazole (compound 3) groups. Isoxazole and pyrazole curcumins were found to be extremely stable at physiological pH, in addition to reducing atmosphere, and they can kill cancer cells under serum-depleted condition. Using molecular modeling, we found that both compounds 2 and 3 could dock to the same site of tubulin as the parent molecule, curcumin. Interestingly, compounds 2 and 3 also show better free radical scavenging activity than curcumin. Altogether, these results strongly suggest that compounds 2 and 3 could be good replacements for curcumin in future drug development.

Editors' Roundup: June 2023.

This commentary article represents the latest edition of the Biophysical Reviews 'Editors' Roundup' Series - a platform made available to the editorial board members of any journal with a genuine interest in promoting biophysical content. Each journal associated editor is able to submit a short description of up to five articles recently appearing in their journals with an explanation of why these articles are of interest. This edition (Vol. 15 Issue 3 June 2023) carries contributions from editorial members associated with Biophysics and Physicobiology (Biophysical Society of Japan), Biophysics (Russian Academy of Sciences), Cell Biochemistry and Biophysics (Springer), and Biophysical Reviews (IUPAB-International Union for Pure and Applied Biophysics).

Reprogramming of glucose metabolism via PFKFB4 is critical in FGF16-driven invasion of breast cancer cells.

Fibroblast growth factors (FGFs) are expressed in both developing and adult tissues and play important roles in embryogenesis, tissue homeostasis, angiogenesis, and neoplastic transformation. Here, we report the elevated expression of FGF16 in human breast tumor and investigate its potential involvement in breast cancer progression. The onset of epithelial-mesenchymal transition (EMT), a prerequisite for cancer metastasis, was observed in human mammary epithelial cell-line MCF10A by FGF16. Further study unveiled that FGF16 alters mRNA expression of a set of extracellular matrix genes to promote cellular invasion. Cancer cells undergoing EMT often show metabolic alteration to sustain their continuous proliferation and energy-intensive migration. Similarly, FGF16 induced a significant metabolic shift toward aerobic glycolysis. At the molecular level, FGF16 enhanced GLUT3 expression to facilitate glucose transport into cells, which through aerobic glycolysis generates lactate. The bi-functional protein, 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase 4 (PFKFB4) was found to be a mediator in FGF16-driven glycolysis and subsequent invasion. Furthermore, PFKFB4 was found to play a critical role in promoting lactate-induced cell invasion since silencing PFKFB4 decreased lactate level and rendered the cells less invasive. These findings support potential clinical intervention of any of the members of FGF16-GLUT3-PFKFB4 axis to control the invasion of breast cancer cells.

A functional loop spanning distant domains of glutaminyl-tRNA synthetase also stabilizes a molten globule state.

Molten globule and other disordered states of proteins are now known to play important roles in many cellular processes. From equilibrium unfolding studies of two paralogous proteins and their variants, glutaminyl-tRNA synthetase (GlnRS) and two of its variants [glutamyl-tRNA synthetase (GluRS) and its isolated domains, and a GluRS-GlnRS chimera], we demonstrate that only GlnRS forms a molten globule-like intermediate at low urea concentrations. We demonstrated that a loop in the GlnRS C-terminal anticodon binding domain that promotes communication with the N-terminal domain and indirectly modulates amino acid binding is also responsible for stabilization of the molten globule state. This loop was inserted into GluRS in the eukaryotic branch after the archaea-eukarya split, right around the time when GlnRS evolved. Because of the structural and functional importance of the loop, it is proposed that the insertion of the loop into a putative ancestral GluRS in eukaryotes produced a catalytically active molten globule state. Because of their enhanced dynamic nature, catalytically active molten globules are likely to possess broad substrate specificity. It is further proposed that the putative broader substrate specificity allowed the catalytically active molten globule to accept glutamine in addition to glutamic acid, leading to the evolution of GlnRS.

Glycine rescue of ß-sheets from cis-proline.

Proline is incompatible with ideal ß-sheet geometry, and the incompatibility gets magnified when Pro assumes the cis peptidyl-prolyl conformation. We show that Gly appears with high propensity at pre-cisPro positions in ß-sheets and rescues the ß-sheet from severe distortions by assuming a right-handed polyproline conformation (ß(PR)), effectively increasing the local ß-sheet register by one residue. The united residue, Gly(ß(PR))-cisPro, is evolutionarily conserved, functionally important, and dynamic in nature.