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A transcriptional-switch model for Slr1738-controlled gene expression in the cyanobacterium Synechocystis
Background:
Protein-DNA interactions play a crucial role in the life of biological organisms in controlling transcription, regulation, as well as DNA recombination and repair. The deep understanding of these processes, which requires the atomic description of the interactions occurring between the proteins and their DNA partners is often limited by the absence of a 3D structure of such complexes.
Results:
In this study, using a method combining sequence homology, structural analogy modeling and biochemical data, we first build the 3D structure of the complex between the poorly-characterized PerR-like regulator Slr1738 and its target DNA, which controls the defences against metal and oxidative stresses in Synechocystis. In a second step, we propose an expanded version of the Slr1738-DNA structure, which accommodates the DNA binding of Slr1738 multimers, a feature likely operating in the complex Slr1738-mediated regulation of stress responses. Finally, in agreement with experimental data we present a 3D-structure of the Slr1738-DNA complex resulting from the binding of multimers of the FUR-like regulator onto its target DNA that possesses internal repeats.
Conclusion:
Using a combination of different types of data, we build and validate a relevant model of the tridimensional structure of a biologically important protein-DNA complex. Then, based on published observations, we propose more elaborated multimeric models that may be biologically important to understand molecular mechanisms.
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Structural adaptation of extreme halophilic proteins through decrease of conserved hydrophobic contact surface
Background:
Halophiles are extremophilic microorganisms growing optimally at high salt concentrations. There are two strategies used by halophiles to maintain proper osmotic pressure in their cytoplasm: accumulation of molar concentrations of potassium and chloride with extensive adaptation of the intracellular macromolecules ("salt-in" strategy) or biosynthesis and/or accumulation of organic osmotic solutes ("osmolyte" strategy). Our work was aimed at contributing to the understanding of the shared molecular mechanisms of protein haloadaptation through a detailed and systematic comparison of a sample of several three-dimensional structures of halophilic and non-halophilic proteins. Structural differences observed between the "salt-in" and the mesophilic homologous proteins were contrasted to those observed between the "osmolyte" and mesophilic pairs.
Results:
The results suggest that haloadaptation strategy in the presence of molar salt concentration, but not of osmolytes, necessitates a weakening of the hydrophobic interactions, in particular at the level of conserved hydrophobic contacts. Weakening of these interactions counterbalances their strengthening by the presence of salts in solution and confers the structure more flexibility that helps preventing aggregation and/or loss of function in hypersaline environments.
Conclusions:
Considering the significant increase of biotechnology applications of halophiles, the understanding of halophilicity can provide the theoretical basis for the engineering of proteins of great interest because stable at concentrations of salts that cause the denaturation or aggregation of the majority of macromolecules.
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The Host-Pathogen interaction of Human Cyclophilin A and HIV-1 Vpr requires specific N-terminal and novel C-terminal domains
Background:
Cyclophilin A (CypA) represents a potential key molecule in future antiretroviral therapy since inhibition of CypA suppresses human immunodeficiency virus type 1 (HIV-1) replication. CypA interacts with the virus proteins Capsid (CA) and Vpr, however, the mechanism through which CypA influences HIV-1 infectivity still remains unclear.
Results:
Here the interaction of full-length HIV-1 Vpr with the host cellular factor CypA has been characterized and quantified by surface plasmon resonance spectroscopy. A C-terminal region of Vpr, comprising the 16 residues 75GCRHSRIGVTRQRRAR90, with high binding affinity for CypA has been identified. This region of Vpr does not contain any proline residues but binds much more strongly to CypA than the previously characterized N-terminal binding domain of Vpr, and is thus the first protein binding domain to CypA described involving no proline residues. The fact that the mutant peptide Vpr75-90 R80A binds more weakly to CypA than the wild-type peptide confirms that Arg-80 is a key residue in the C-terminal binding domain. The N- and C-terminal binding regions of full-length Vpr bind cooperatively to CypA and have allowed a model of the complex to be created. The dissociation constant of full-length Vpr to CypA was determined to be approximately 320 nM, indicating that the binding may be stronger than that of the well characterized interaction of HIV-1 CA with CypA.
Conclusions:
For the first time the interaction of full-length Vpr and CypA has been characterized and quantified. A non-proline-containing 16-residue region of C-terminal Vpr which binds specifically to CypA with similar high affinity as full-length Vpr has been identified. The fact that this is the first non-proline containing binding motif of any protein found to bind to CypA, changes the view on how CypA is able to interact with other proteins. It is interesting to note that several previously reported key functions of HIV-1 Vpr are associated with the identified N- and C-terminal binding domains of the protein to CypA.
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Structural comparison of tRNA m1A58 methyltransferases revealed different molecular strategies to maintain their oligomeric architecture under extreme conditions.
Background:
tRNA m1A58 methyltransferases (TrmI) catalyze the transfer of a methyl group from S-adenosyl-L-methionine to nitrogen 1 of adenine 58 in the T-loop of tRNAs from all three domains of life. The m1A58 modification has been shown to be essential for cell growth in yeast and for adaptation to high temperatures in thermophilic organisms. These enzymes were shown to be active as tetramers. The crystal structures of five TrmIs from hyperthermophilic archaea and thermophilic or mesophilic bacteria have previously been determined, the optimal growth temperature of these organisms ranging from 37degreesC to 100degreesC. All TrmIs are assembled as tetramers formed by dimers of tightly assembled dimers.
Results:
In this study, we present a comparative structural analysis of these TrmIs, which highlights factors that allow them to function over a large range of temperature. The monomers of the five enzymes are structurally highly similar, but the inter-monomer contacts differ strongly. Our analysis shows that bacterial enzymes from thermophilic organisms display additional intermolecular ionic interactions across the dimer interfaces, whereas hyperthermophilic enzymes present additional hydrophobic contacts. Moreover, as an alternative to two bidentate ionic interactions that stabilize the tetrameric interface in all other TrmI proteins, the tetramer of the archaeal P. abyssi enzyme is strengthened by four intersubunit disulfide bridges.
Conclusions:
The availability of crystal structures of TrmI from mesophilic, thermophilic or hyperthermophilic organisms allows a detailed analysis of the architecture of this protein family. Our structural comparisons provide insight into the different molecular strategies used to achieve the tetrameric organization in order to maintain the enzyme activity under extreme conditions.
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Docking studies on Novel Analogues of 8 Methoxy Fluoroquinolones against GyrA Mutants of Mycobacterium tuberculosis
Background:
Fluoroquinolone resistance is a serious threat in the battle against the treatment of multi drug resistant tuberculosis (MDR-TB) and extensively drug resistant tuberculosis (XDR-TB). Fluoroquinolone resistant isolates from India had shown to have evolved several mutants in the quinolone resistance determining region (QRDR) of DNA gyrase A subunit (GyrA), the target of fluoroquinolone. In view of high prevalence of mutations in the 'hot spot' region, a study on combinatorial drug design was carried out to identify better analogues for the treatment of MDR-TB. The gyrA subunit 'hot spot' region of codons 90, 94 and 95 were modeled into their corresponding protein folds and used as receptors for the docking studies. Further, invitro tests were carried using the parent compounds, namely gatifloxacin and moxifloxacin and correlated with the obtained docking scores.
Results:
Molecular docking and in vitro studies correlated well in demonstrating the enhanced activity of moxifloxacin, when compared to gatifloxacin, on ofloxacin sensitive and resistant strains comprising of clinical isolates of MDR-TB. The evolved lead structures targeting against mutant QRDR receptors were guanosine and cholesteryl esters of moxifloxacin and gatifloxacin respectively. They showed consistently high binding affinity values of -10.3 and -10.1 kcal/mol respectively with the target receptors. Of these, the guanosine ester of moxifloxacin showed highest binding affinity score and its log P value lied within the Lipinski's range indicating that it could have better absorptivity when it is orally administered thereby having an enhanced activity against MTB.
Conclusions:
The docking results showed that the addition of the cholesteryl and guanosine esters to the 'DNA gyrase binding' region of gatifloxacin and moxifloxacin enhanced the binding affinity of these parent molecules with the mutant DNA gyrase receptors. Viewing the positive correlation for the docking and in vitro results with the parent compounds, these lead structures could be further evaluated for their in vitro and in vivo activity against MDR-TB.
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The redundancy of NMR restraints can be used to accelerate the unfolding behavior of an SH3 domain during molecular dynamics simulations
Background:
The simulation of protein unfolding usually requires recording long molecular dynamics trajectories. The present work aims to figure out whether NMR restraints data can be used to probe protein conformations in order to accelerate the unfolding simulation. The SH3 domain of nephrocystine (nph SH3) was shown by NMR to be destabilized by point mutations, and was thus chosen to illustrate the proposed method.
Results:
The NMR restraints observed on the WT nph SH3 domain were sorted from the least redundant to the most redundant ones. Protein NMR conformations were then calculated with: (i) the set full including all NMR restraints measured on nph SH3, (ii) the set reduced where the least redundant restraints with respect to the set full were removed,(iii) the sets random where randomly picked-up restraints were removed. From each set of conformations, we recorded series of 5-ns MD trajectories. The b barrel architecture of nph SH3 in the trajectories starting from sets (i) and (iii) appears to be stable. On the contrary, on trajectories based on the set (ii), a displacement of the hydrophobic core residues and a variation of the beta barrel inner cavity profile were observed. The overall nph SH3 destabilization agrees with previous experimental and simulation observations made on other SH3 domains. The destabilizing effect of mutations was also found to be enhanced by the removal of the least redundant restraints.
Conclusions:
We conclude that the NMR restraint redundancy is connected to the instability of the SH3 nph domain. This restraint redundancy generalizes the contact order parameter, which is calculated from the contact map of a folded protein and was shown in the literature to be correlated to the protein folding rate. The relationship between the NMR restraint redundancy and the protein folding is also reminiscent of the previous use of the Gaussian Network Model to predict protein folding parameters.
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Benchmarks for flexible and rigid transcription factor-DNA docking
Background:
Structural insight from transcription factor-DNA (TF-DNA) complexes is of paramount importance to our understanding of the affinity and specificity of TF-DNA interaction, and to the development of structure-based prediction of TF binding sites. Yet the majority of the TF-DNA complexes remain unsolved despite the considerable experimental efforts being made. Computational docking represents a promising alternative to bridge the gap. To facilitate the study of TF-DNA docking, carefully designed benchmarks are needed for performance evaluation and identification of the strengths and weaknesses of docking algorithms.
Results:
We constructed two benchmarks for flexible and rigid TF-DNA docking respectively using a unified non-redundant set of 38 test cases. The test cases encompass diverse fold families and are classified into easy and hard groups with respect to the degrees of difficulty in TF-DNA docking. The major parameters used to classify expected docking difficulty in flexible docking are the conformational differences between bound and unbound TFs and the interaction strength between TFs and DNA. For rigid docking in which the starting structure is a bound TF conformation, only interaction strength is considered.
Conclusions:
We believe these benchmarks are important for the development of better interaction potentials and TF-DNA docking algorithms, which bears important implications to structure-based prediction of transcription factor binding sites and drug design.
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Hydration studies on the archaeal protein Sso7d using NMR measurements and MD simulations.
Background:
How proteins approach surrounding molecules is fundamental to our understanding of the specific interactions that occur at the surface of proteins. The enhanced surface accessibility of small molecules such as organic solvents and paramagnetic probes to protein binding sites has been observed; however, the molecular basis of this finding has not been fully established. Recently, it has been suggested that hydration dynamics play a predominant role in controlling the distribution of hot spots on surface of proteins.
Results:
In the present study, the hydration of the archaeal multifunctional protein Sso7d from Solfolobus solfataricus was investigated using a combination of computational and experimental data derived from molecular dynamics simulations and ePHOGSY NMR spectroscopy.
Conclusions:
We obtained a convergent protein hydration landscape that indicated how the shape and stability of the Sso7d hydration shell could modulate the function of the protein. The DNA binding domain overlaps with the protein region involved in chaperon activity and this domain is hydrated only in a very small central region. This localized hydration seems to favor intermolecular approaches from a large variety of ligands. Conversely, high water density was found in surface regions of the protein where the ATP binding site is located, suggesting that surface water molecules play a role in protecting the protein from unspecific interactions.
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Computational analysis of a novel mutation in ETFDH gene highlights its long-range effects on the FAD-binding motif
Background:
Multiple acyl-coenzyme A dehydrogenase deficiency (MADD) is an autosomal recessive disease caused by the defects in the mitochondrial electron transfer system and the metabolism of fatty acids. Recently, mutations in electron transfer flavoprotein dehydrogenase (ETFDH) gene, encoding electron transfer flavoprotein:ubiquinone oxidoreductase (ETF:QO) have been reported to be the major causes of riboflavin-responsive MADD. To date, no studies have been performed to explore the functional impact of these mutations or their mechanism of disrupting enzyme activity.
Results:
High resolution melting (HRM) analysis and sequencing of the entire ETFDH gene revealed a novel mutation (p.Phe128Ser) and the hotspot mutation (p.Ala84Thr) from a patient with MADD. According to the predicted 3D structure of ETF:QO, the two mutations are located within the flavin adenine dinucleotide (FAD) binding domain; however, the two residues do not have direct interactions with the FAD ligand. Using molecular dynamics (MD) simulations and normal mode analysis (NMA), we found that the p.Ala84Thr and p.Phe128Ser mutations are most likely to alter the protein structure near the FAD binding site as well as disrupt the stability of the FAD binding required for the activation of ETF:QO. Intriguingly, NMA revealed that several reported disease-causing mutations in the ETF:QO protein show highly correlated motions with the FAD-binding site.
Conclusions:
Based on the present findings, we conclude that the changes made to the amino acids in ETF:QO are likely to influence the FAD-binding stability.
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Comparative Void-Volume Analysis of Psychrophilic and Mesophilic Enzymes: Structural bioinformatics of psychrophilic enzymes reveals sources of core flexibility
Background:
Psychrophiles, cold-adapted organisms, have adapted to live at low temperatures by using a variety of mechanisms. Their enzymes are active at cold temperatures by being structurally more flexible than mesophilic enzymes. Even though, there are some indications of the possible structural mechanisms by which psychrophilic enzymes are catalytic active at cold temperatures, there is not a generalized structural property common to all psychrophilic enzymes.
Results:
We examine twenty homologous enzyme pairs from psychrophiles and mesophiles to investigate flexibility as a key characteristic for cold adaptation. B-factors in protein X-ray structures are one way to measure flexibility. Comparing psychrophilic to mesophilic protein B-factors reveals that psychrophilic enzymes are more flexible in 5-turn and strand secondary structures. Enzyme cavities, identified using CASTp at various probe sizes, indicate that psychrophilic enzymes have larger average cavity sizes at probe radii of 1.4-1.5 Å, sufficient for water molecules. Furthermore, amino acid side chains lining these cavities show an increased frequency of acidic groups in psychrophilic enzymes.
Conclusions:
These findings suggest that embedded water molecules may play a significant role in cavity flexibility, and therefore, overall protein flexibility. Thus, our results point to the important role enzyme flexibility plays in adaptation to cold environments.
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