Cloning, expression, purification, and biochemical characterisation of the FIC motif containing protein of Mycobacterium tuberculosis

The role of FIC (Filamentation induced by cAMP)(2) domain containing proteins in the regulation of many vital pathways, mostly through the transfer of NMPs from NTPs to specific target proteins (NMPylation), in microorganisms, higher eukaryotes, and plants is emerging. The identity and function of FIC domain containing protein of the human pathogen, Mycobacterium tuberculosis, remains unknown. In this regard, M. tuberculosis fic gene (Mtfic) was cloned, overexpressed, and purified to homogeneity for its biochemical characterisation. It has the characteristic FIC motif, HPFREGNGRSTR (HPFxxGNGRxxR), spanning 144th to 155th residue. Neither the His-tagged nor the GST-tagged MtFic protein, overexpressed in Escherichia coil, nor expression of Mtfic in Mycobacterium smegmatis, yielded the protein in the soluble fraction. However, the maltose binding protein (MBP) tagged MtFic (MBP-MtFic) could be obtained partly in the soluble fraction. The cloned, overexpressed, and purified recombinant MBP-MtFic showed conversion of ATP, GTP, CTP, and UTP into AMP. GMP, CMP, and UMP, respectively. Sequence alignment with several FIC motif containing proteins, complemented with homology modeling on the FIC motif containing protein, VbhT of Bartonella schoenbuchensis as the template, showed conservation and interaction of residues constituting the FIC domain. Site-specific mutagenesis of the His144, or Glu148, or Asn150 of the FIC motif, or of Arg87 residue that constitutes the FIC domain, or complete deletion of the FIC motif, abolished the NTP to NMP conversion activity. The design of NMP formation assay using the recombinant, soluble MtFic would enable identification of its target substrate for NMPylation. (C) 2012 Elsevier Inc. All rights reserved.

Novel ATPase activity of the polyprotein intermediate, Viral Protein genome-linked-Nuclear Inclusion-a protease, of Pepper vein banding potyvirus

Potyviruses temporally regulate their protein function by polyprotein processing. Previous studies have shown that VPg (Viral Protein genome-linked) of Pepper vein banding virus interacts with the NIa-Pro (Nuclear Inclusion-a protease) domain, and modulates the kinetics of the protease. In the present study, we report for the first time that VPg harbors the Walker motifs A and B, and the presence of NIa-Pro, especially in cis (cleavage site (E191A) VPg-Pro mutant), is essential for manifestation of the ATPase activity. Mutation of Lys47 (Walker motif A) and Asp88:Glu89 (Walker motif B) to alanine in E191A VPg-Pro lead to reduced ATPase activity, confirming that this activity was inherent to VPg. We propose that potyviral VPg, established as an intrinsically disordered domain, undergoes plausible structural alterations upon interaction with globular NIa-Pro which induces the ATPase activity. (C) 2012 Elsevier Inc. All rights reserved.

Subdiffusion in hair bundle dynamics: The role of protein conformational fluctuations

The detection of sound signals in vertebrates involves a complex network of different mechano-sensory elements in the inner ear. An especially important element in this network is the hair bundle, an antenna-like array of stereocilia containing gated ion channels that operate under the control of one or more adaptation motors. Deflections of the hair bundle by sound vibrations or thermal fluctuations transiently open the ion channels, allowing the flow of ions through them, and producing an electrical signal in the process, eventually causing the sensation of hearing. Recent high frequency (0.1-10 kHz) measurements by Kozlov et al. Proc. Natl. Acad. Sci. U. S. A. 109, 2896 (2012)] of the power spectrum and the mean square displacement of the thermal fluctuations of the hair bundle suggest that in this regime the dynamics of the hair bundle are subdiffusive. This finding has been explained in terms of the simple Brownian motion of a filament connecting neighboring stereocilia (the tip link), which is modeled as a viscoelastic spring. In the present paper, the diffusive anomalies of the hair bundle are ascribed to tip link fluctuations that evolve by fractional Brownian motion, which originates in fractional Gaussian noise and is characterized by a power law memory. The predictions of this model for the power spectrum of the hair bundle and its mean square displacement are consistent with the experimental data and the known properties of the tip link. (C) 2012 American Institute of Physics. http://dx.doi.org/10.1063/1.4768902]

Single-Molecule DNA Analysis Reveals That Yeast Hop1 Protein Promotes DNA Folding and Synapsis: Implications for Condensation of Meiotic Chromosomes

During meiosis, long-range interaction between homologous chromosomes is thought to be crucial for homology recognition, exchange of DNA strands, and production of normal haploid gametes. However, little is known about the identity of the proteins involved and the actual molecular mechanism(s) by which chromosomes recognize and recombine with their appropriate homologous partners. Single-molecule analyses have the potential to provide insights into our understanding of this fascinating and long-standing question. Using atomic force microscopy and magnetic tweezers techniques, we discovered that Hop1 protein, a key structural component of Saccharomyces cerevisiae synaptonemal complex, exhibits the ability to bridge noncontiguous DNA segments into intramolecular stem-loop structures in which the DNA segments appear to be fully synapsed within the filamentous protein stems. Additional evidence suggests that Hop1 folds DNA into rigid protein DNA filaments and higher-order nucleoprotein structures. Importantly, Hop1 promotes robust intra- and intermolecular synapsis between double-stranded DNA molecules, suggesting that juxtaposition of DNA sequences may assist in strand exchange between homologues by recombination-associated proteins. Finally, the evidence from ensemble experiments is consistent with the notion that Hop1 causes rigidification of DNA molecules. These results provide the first direct evidence for long-range protein-mediated DNA DNA synapsis, independent of crossover recombination, which is presumed to occur during meiotic recombination.

Phospholipid Mediated Activation of Calcium Dependent Protein Kinase 1 (CaCDPK1) from Chickpea: A New Paradigm of Regulation

Phospholipids, the major structural components of membranes, can also have functions in regulating signaling pathways in plants under biotic and abiotic stress. The effects of adding phospholipids on the activity of stress-induced calcium dependent protein kinase (CaCDPK1) from chickpea are reported here. Both autophosphorylation as well as phosphorylation of the added substrate were enhanced specifically by phosphatidylcholine and to a lesser extent by phosphatidic acid, but not by phosphatidylethanolamine. Diacylgylerol, the neutral lipid known to activate mammalian PKC, stimulated CaCDPK1 but at higher concentrations. Increase in V-max of the enzyme activity by these phospholipids significantly decreased the K-m indicating that phospholipids enhance the affinity towards its substrate. In the absence of calcium, addition of phospholipids had no effect on the negligible activity of the enzyme. Intrinsic fluorescence intensity of the CaCDPK1 protein was quenched on adding PA and PC. Higher binding affinity was found with PC (K-1/2 = 114 nM) compared to PA (K-1/2 = 335 nM). We also found that the concentration of PA increased in chickpea plants under salt stress. The stimulation by PA and PC suggests regulation of CaCDPK1 by these phospholipids during stress response.

Inhibition of the Interaction Between NS3 Protease and HCV IRES With a Small Peptide: A Novel Therapeutic Strategy

Recently, we have demonstrated that the protease domain of NS3 alone can bind specifically to hepatitis C virus (HCV) internal ribosome entry site (IRES) near the initiator AUG, dislodges human La protein and inhibits translation in favor of viral RNA replication. Here, by using a computational approach, the contact points of the protease on the HCV IRES were putatively mapped. A 30-mer NS3 peptide was designed from the predicted RNA-binding region that retained RNA-binding ability and also inhibited IRES-mediated translation. This peptide was truncated to 15 mer and this also demonstrated ability to inhibit HCV RNA-directed translation as well as replication. More importantly, its activity was tested in an in vivo mouse model by encapsulating the peptide in Sendai virus virosomes followed by intravenous delivery. The study demonstrates for the first time that the HCV NS3-IRES RNA interaction can be selectively inhibited using a small peptide and reports a strategy to deliver the peptide into the liver.

Lacrimal Proline Rich 4 (LPRR4) Protein in the Tear Fluid Is a Potential Biomarker of Dry Eye Syndrome

Dry eye syndrome (DES) is a complex, multifactorial, immune-associated disorder of the tear and ocular surface. DES with a high prevalence world over needs identification of potential biomarkers so as to understand not only the disease mechanism but also to identify drug targets. In this study we looked for differentially expressed proteins in tear samples of DES to arrive at characteristic biomarkers. As part of a prospective case-control study, tear specimen were collected using Schirmer strips from 129 dry eye cases and 73 age matched controls. 2D electrophoresis (2DE) and Differential gel electrophoresis (DIGE) was done to identify differentially expressed proteins. One of the differentially expressed protein in DES is lacrimal proline rich 4 protein (LPRR4). LPRR4 protein expression was quantified by enzyme immune sorbent assay (ELISA). LPRR4 was down regulated significantly in all types of dry eye cases, correlating with the disease severity as measured by clinical investigations. Further characterization of the protein is required to assess its therapeutic potential in DES.

The origins of the evolutionary signal used to predict protein-protein interactions

Background: The correlation of genetic distances between pairs of protein sequence alignments has been used to infer protein-protein interactions. It has been suggested that these correlations are based on the signal of co-evolution between interacting proteins. However, although mutations in different proteins associated with maintaining an interaction clearly occur (particularly in binding interfaces and neighbourhoods), many other factors contribute to correlated rates of sequence evolution. Proteins in the same genome are usually linked by shared evolutionary history and so it would be expected that there would be topological similarities in their phylogenetic trees, whether they are interacting or not. For this reason the underlying species tree is often corrected for. Moreover processes such as expression level, are known to effect evolutionary rates. However, it has been argued that the correlated rates of evolution used to predict protein interaction explicitly includes shared evolutionary history; here we test this hypothesis. Results: In order to identify the evolutionary mechanisms giving rise to the correlations between interaction proteins, we use phylogenetic methods to distinguish similarities in tree topologies from similarities in genetic distances. We use a range of datasets of interacting and non-interacting proteins from Saccharomyces cerevisiae. We find that the signal of correlated evolution between interacting proteins is predominantly a result of shared evolutionary rates, rather than similarities in tree topology, independent of evolutionary divergence. Conclusions: Since interacting proteins do not have tree topologies that are more similar than the control group of non-interacting proteins, it is likely that coevolution does not contribute much to, if any, of the observed correlations.

Structural and molecular basis of interaction of HCV non-structural protein 5A with human casein kinase 1 alpha and PKR

Background: Interaction of non-structural protein 5A (NS5A) of Hepatitis C virus (HCV) with human kinases namely, casein kinase 1 alpha (ck1 alpha) and protein kinase R (PKR) have different functional implications such as regulation of viral replication and evasion of interferon induced immune response respectively. Understanding the structural and molecular basis of interactions of the viral protein with two different human kinases can be useful in developing strategies for treatment against HCV. Results: Serine 232 of NS5A is known to be phosphorylated by human ck1 alpha. A structural model of NS5A peptide containing phosphoacceptor residue Serine 232 bound to ck1 alpha has been generated using the known 3-D structures of kinase-peptide complexes. The substrate interacting residues in ck1 alpha has been identified from the model and these are found to be conserved well in the ck1 family. ck1 alpha - substrate peptide complex has also been used to understand the structural basis of association between ck1 alpha and its other viral stress induced substrate, tumour suppressor p53 transactivation domain which has a crystal structure available. Interaction of NS5A with another human kinase PKR is primarily genotype specific. NS5A from genotype 1b has been shown to interact and inhibit PKR whereas NS5A from genotype 2a/3a are unable to bind and inhibit PKR efficiently. This is one of the main reasons for the varied response to interferon therapy in HCV patients across different genotypes. Using PKR crystal structure, sequence alignment and evolutionary trace analysis some of the critical residues responsible for the interaction of NS5A 1b with PKR have been identified. Conclusions: The substrate interacting residues in ck1 alpha have been identified using the structural model of kinase substrate peptide. The PKR interacting NS5A 1b residues have also been predicted using PKR crystal structure, NS5A sequence analysis along with known experimental results. Functional significance and nature of interaction of interferon sensitivity determining region and variable region 3 of NS5A in different genotypes with PKR which was experimentally shown are also supported by the findings of evolutionary trace analysis. Designing inhibitors to prevent this interaction could enable the HCV genotype 1 infected patients respond well to interferon therapy.

Force and ATP hydrolysis dependent regulation of RecA nucleoprotein filament by single-stranded DNA binding protein

In Escherichia coli, the filament of RecA formed on single-stranded DNA (ssDNA) is essential for recombinational DNA repair. Although ssDNA-binding protein (SSB) plays a complicated role in RecA reactions in vivo, much of our understanding of the mechanism is based on RecA binding directly to ssDNA. Here we investigate the role of SSB in the regulation of RecA polymerization on ssDNA, based on the differential force responses of a single 576-nucleotide-long ssDNA associated with RecA and SSB. We find that SSB outcompetes higher concentrations of RecA, resulting in inhibition of RecA nucleation. In addition, we find that pre-formed RecA filaments de-polymerize at low force in an ATP hydrolysis- and SSB-dependent manner. At higher forces, re-polymerization takes place, which displaces SSB from ssDNA. These findings provide a physical picture of the competition between RecA and SSB under tension on the scale of the entire nucleoprotein SSB array, which have broad biological implications particularly with regard to competitive molecular binding.

Site-specific N-Linked Glycosylation of Receptor Guanylyl Cyclase C Regulates Ligand Binding, Ligand-mediated Activation and Interaction with Vesicular Integral Membrane Protein 36, VIP36

Guanylyl cyclase C (GC-C) is a multidomain, membrane-associated receptor guanylyl cyclase. GC-C is primarily expressed in the gastrointestinal tract, where it mediates fluid-ion homeostasis, intestinal inflammation, and cell proliferation in a cGMP-dependent manner, following activation by its ligands guanylin, uroguanylin, or the heat-stable enterotoxin peptide (ST). GC-C is also expressed in neurons, where it plays a role in satiation and attention deficiency/hyperactive behavior. GC-C is glycosylated in the extracellular domain, and differentially glycosylated forms that are resident in the endoplasmic reticulum (130 kDa) and the plasma membrane (145 kDa) bind the ST peptide with equal affinity. When glycosylation of human GC-C was prevented, either by pharmacological intervention or by mutation of all of the 10 predicted glycosylation sites, ST binding and surface localization was abolished. Systematic mutagenesis of each of the 10 sites of glycosylation in GC-C, either singly or in combination, identified two sites that were critical for ligand binding and two that regulated ST-mediated activation. We also show that GC-C is the first identified receptor client of the lectin chaperone vesicular integral membrane protein, VIP36. Interaction with VIP36 is dependent on glycosylation at the same sites that allow GC-C to fold and bind ligand. Because glycosylation of proteins is altered in many diseases and in a tissue-dependent manner, the activity and/or glycan-mediated interactions of GC-C may have a crucial role to play in its functions in different cell types.

Computational studies on histidine kinase protein BaeS to target multidrug-resistant Salmonella

Multidrug-resistant Salmonella serovars have been a recent concern in curing infectious diseases like typhoid. Salmonella BaeS and BaeR are the two-component system (TCS) that signal transduction proteins found to play an important role in its multidrug resistance. A canonical TCS comprises a histidine kinase (HK) and its cognate partner response regulator (RR). The general approaches for therapeutic targeting are either the catalytic ATP-binding domain or the dimerization domain HisKA (DHp) of the HK, and in some cases, the receiver or the regulatory domain of the RR proteins. Earlier efforts of identifying novel drugs targeting the signal transduction protein have not been quite successful, as it shares similar ATP-binding domain with the key house keeping gene products of the mammalian GHL family. However, targeting the dimerization domain of HisKA through which the signals are received from the RR can be a better approach. In this article, we show stepwise procedure to specifically identify the key interacting residues involved in the dimerization with the RR along with effective targeting by ligands screened from the public database. We have found a few inhibitors which target effectively the important residues for the dimerization activity. Our results suggest a plausible de novo design of better DHp domain inhibitors.

Development of a New Generation of Vectors for Gene Expression, Gene Replacement, and Protein-Protein Interaction Studies in Mycobacteria

Escherichia coli-mycobacterium shuttle vectors are important tools for gene expression and gene replacement in mycobacteria. However, most of the currently available vectors are limited in their use because of the lack of extended multiple cloning sites (MCSs) and convenience of appending an epitope tag(s) to the cloned open reading frames (ORFs). Here we report a new series of vectors that allow for the constitutive and regulatable expression of proteins, appended with peptide tag sequences at their N and C termini, respectively. The applicability of these vectors is demonstrated by the constitutive and induced expression of the Mycobacterium tuberculosis pknK gene, coding for protein kinase K, a serine-threonine protein kinase. Furthermore, a suicide plasmid with expanded MCS for creating gene replacements, a plasmid for chromosomal integrations at the commonly used L5 attB site, and a hypoxia-responsive vector, for expression of a gene(s) under hypoxic conditions that mimic latency, have also been created. Additionally, we have created a vector for the coexpression of two proteins controlled by two independent promoters, with each protein being in fusion with a different tag. The shuttle vectors developed in the present study are excellent tools for the analysis of gene function in mycobacteria and are a valuable addition to the existing repertoire of vectors for mycobacterial research.

Dramatic Structural Changes Resulting from the Loss of a Crucial Hydrogen Bond in the Hinge Region Involved in C-Terminal Helix Swapping in SurE: A Survival Protein from Salmonella typhimurium

Domain swapping is an interesting feature of some oligomeric proteins in which each protomer of the oligomer provides an identical surface for exclusive interaction with a segment or domain belonging to another protomer. Here we report results of mutagenesis experiments on the structure of C-terminal helix swapped dimer of a stationary phase survival protein from Salmonella typhimurium (StSurE). Wild type StSurE is a dimer in which a large helical segment at the C-terminus and a tetramerization loop comprising two beta strands are swapped between the protomers. Key residues in StSurE that might promote C-terminal helix swapping were identified by sequence and structural comparisons. Three mutants in which the helix swapping is likely to be avoided were constructed and expressed in E. coli. Three-dimensional X-ray crystal structures of the mutants H234A and D230A/H234A could be determined at 2.1 angstrom and 2.35 angstrom resolutions, respectively. Contrary to expectations, helix swapping was mostly retained in both the mutants. The loss of the crucial D230 OD2- H234 NE2 hydrogen bond (2.89 angstrom in the wild type structure) in the hinge region was compensated by new inter and intra-chain interactions. However, the two fold molecular symmetry was lost and there were large conformational changes throughout the polypeptide. In spite of these changes, the dimeric structure and an approximate tetrameric organization were retained, probably due to the interactions involving the tetramerization loop. Mutants were mostly functionally inactive, highlighting the importance of precise inter-subunit interactions for the symmetry and function of StSurE.

Annexin A2 and PSF proteins interact with p53 IRES and regulate translation of p53 mRNA

p53 mRNA has been shown to be translated into two isoforms, full-length p53 (FL-p53) and a truncated isoform Delta N-p53, which modulates the functions of FL-p53 and also has independent functions. Previously, we have shown that translation of p53 and Delta N-p53 can be initiated at Internal Ribosome Entry Sites (IRES). These two IRESs were shown to regulate the translation of p53 and Delta N-p53 in a distinct cell-cycle phase-dependent manner. Earlier observations from our laboratory also suggest that the structural integrity of the p53 RNA is critical for IRES function and is compromised by mutations that affect the structure as well as RNA protein interactions. In the current study, using RNA affinity approach we have identified Annexin A2 and PTB associated Splicing Factor (PSF/SFPQ) as novel ITAFs for p53 IRESs. We have showed that the purified Annexin A2 and PSF proteins specifically bind to p53 IRES elements. Interestingly, in the presence of calcium ions Annexin A2 showed increased binding with p53 IRES. Immunopulldown experiments suggest that these two proteins associate with p53 mRNA ex vivo as well. Partial knockdown of Annexin A2 and PSF showed decrease in p53 IRES activity and reduced levels of both the p53 isoforms. More importantly the interplay between Annexin A2, PSF and PTB proteins for binding to p53mRNA appears to play a crucial role in IRES function. Taken together, our observations suggest pivotal role of two new trans-acting factors in regulating the p53-IRES function, which in turn influences the synthesis of p53 isoforms.