Protein-nucleic Acid Complexes Research Articles

Automated NMR Resonance Assignment of Large Proteins for Protein−Ligand Interaction Studies

The detection and structural characterization of protein-ligand interactions by solution NMR is central to functional biology research as well as to drug discovery. Here we present a robust and highly automated procedure for obtaining the resonance assignments necessary for studies of such interactions. The procedure relies on a combination of three automated projection spectroscopy (APSY) experiments, including the new 4D APSY-HNCACB, and the use of fractionally deuterated protein samples. This labeling pattern increases the experimental sensitivity on the one hand, but it leads to peak multiplets on the other hand. The latter complications are however overcome by the geometric APSY analysis of the projection spectra. The three APSY experiments thus provide high precision chemical shift correlations of the backbone and side chain methyl groups, allowing a reliable and robust assignment of the protein by suitable algorithms. The present approach doubles the molecular size limit of APSY-based assignments to 25 kDa, thus providing the basis for efficient characterization of protein-ligand interactions at atomic resolution by NMR, such as structure-based drug design. We show the application to two human proteins with molecular weights of 15 and 22 kDa, respectively, at concentrations of 0.4 mM and discuss the general applicability to studies of protein-protein and protein-nucleic acid complexes.

13C/15N-19F intermolecular REDOR NMR study of the interaction of TAR RNA with Tat peptides.

The complex of the HIV TAR RNA with the viral regulatory protein Tat is of considerable interest, but the plasticity of this interaction has made it impossible so far to establish the structure of that complex. In order to explore a new approach to obtain structural information on protein-RNA complexes, we performed (13)C/(15)N-(19)F REDOR NMR experiments in the solid state on TAR bound to a peptide comprising the RNA-binding section of Tat. A critical arginine in the peptide was uniformly (13)C and (15)N labeled, and 5-fluorouridine was incorporated at the U23 position of TAR. REDOR irradiation resulted in dephasing of the (13)C and (15)N resonances, indicating the proximity of the U23(5F)-C and U23(5F)-N spin pairs. Best fits to the REDOR data show the U23(5F)-C distances and the U23(5F)-N distances are in good agreement with the distances obtained from solution NMR structures of partial complexes of Tat with TAR. These results demonstrate that it is possible to study protein-RNA complexes using solid-state REDOR NMR measurements, adding to a growing list of solid state techniques for studying protein-nucleic acid complexes.

Thermodynamics of Cooperative DNA Recognition at a Replication Origin and Transcription Regulatory Site

Binding cooperativity guides the formation of protein−nucleic acid complexes, in particular those that are highly regulated such as replication origins and transcription sites. Using the DNA binding domain of the origin binding and transcriptional regulator protein E2 from human papillomavirus type 16 as model, and through isothermal titration calorimetry analysis, we determined a positive, entropy-driven cooperativity upon binding of the protein to its cognate tandem double E2 site. This cooperativity is associated with a change in DNA structure, where the overall B conformation is maintained. Two homologous E2 domains, those of HPV18 and HPV11, showed that the enthalpic−entropic components of the reaction and DNA deformation can diverge. Because the DNA binding helix is almost identical in the three domains, the differences must lie dispersed throughout this unique dimeric β-barrel fold. This is in surprising agreement with previous results for this domain, which revealed a strong coupling between global dynamics and DNA recognition.

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Small-angle neutron scattering and contrast variation: a powerful combination for studying biological structures

The use of small-angle scattering (SAS) in the biological sciences continues to increase, driven as much by the need to study increasingly complex systems that are often resistant to crystallization or are too large for NMR as by the availability of user facilities and advancements in the modelling of biological structures from SAS data. SAS, whether with neutrons (SANS) or X-rays (SAXS), is a structural probe of length scales ranging from 10 to 10,000 Å. When applied to biological complexes in dilute solution, it provides size and shape information that can be used to produce structural models that can provide insight into function. SANS enables the use of contrast-variation methods through the unique interaction of neutrons with hydrogen and its isotope deuterium. SANS with contrast variation enables the visualization of components within multisubunit complexes, making it a powerful tool for probing protein-protein and protein-nucleic acid complexes, as well as the interaction of proteins with lipids and detergents.

Plant Proteins Involved in Agrobacterium-Mediated Genetic Transformation

Agrobacterium species genetically transform plants by transferring a region of plasmid DNA, T-DNA, into host plant cells. The bacteria also transfer several virulence effector proteins. T-DNA and virulence proteins presumably form T-complexes within the plant cell. Super-T-complexes likely also form by interaction of plant-encoded proteins with T-complexes. These protein-nucleic acid complexes traffic through the plant cytoplasm, enter the nucleus, and eventually deliver T-DNA to plant chromatin. Integration of T-DNA into the plant genome establishes a permanent transformation event, permitting stable expression of T-DNA-encoded transgenes. The transformation process is complex and requires participation of numerous plant proteins. This review discusses our current knowledge of plant proteins that contribute to Agrobacterium-mediated transformation, the roles these proteins play in the transformation process, and the modern technologies that have been employed to elucidate the cell biology of transformation.

A crystallization screen based on alternative polymeric precipitants

Most commercially available crystallization screens are sparse-matrix screens with a predominance of inorganic salts and polyethylene glycols (PEGs) as precipitants. It was noted that commercially available screens are largely unsatisfactory for the purpose of the crystallization of multimeric protein and protein-nucleic acid complexes. This was reasoned to be a consequence of the redundancy in screening crystallization parameter space by the predominance of PEG as a precipitant in standard screens and it was suggested that this limitation could be overcome by introducing a variety of other organic polymers. Here, a set of 288 crystallization conditions was devised based on alternative polymeric precipitants and tested against a set of 20 different proteins/complexes; finally, a screen comprising the 96 most promising conditions designed to complement PEG- and salt-based commercial screens was proposed.

Selenium Derivatization of Nucleic Acids for X‐Ray Crystal‐Structure and Function Studies

It is estimated that over two thirds of all new crystal structures of proteins are determined via the protein selenium derivatization (selenomethionine (Se-Met) strategy). This selenium derivatization strategy via MAD (multi-wavelength anomalous dispersion) phasing has revolutionized protein X-ray crystallography. Through our pioneer research, similarly, Se has also been successfully incorporated into nucleic acids to facilitate the X-ray crystal-structure and function studies of nucleic acids. Currently, Se has been stably introduced into nucleic acids by replacing nucleotide O-atom at the positions 2', 4', 5', and in nucleobases and non-bridging phosphates. The Se derivatization of nucleic acids can be achieved through solid-phase chemical synthesis and enzymatic methods, and the Se-derivatized nucleic acids (SeNA) can be easily purified by HPLC, FPLC, and gel electrophoresis to obtain high purity. It has also been demonstrated that the Se derivatization of nucleic acids facilitates the phase determination via MAD phasing without significant perturbation. A growing number of structures of DNAs, RNAs, and protein-nucleic acid complexes have been determined by the Se derivatization and MAD phasing. Furthermore, it was observed that the Se derivatization can facilitate crystallization, especially when it is introduced to the 2'-position. In addition, this novel derivatization strategy has many advantages over the conventional halogen derivatization, such as more choices of the modification sites via the atom-specific substitution of the nucleotide O-atom, better stability under X-ray radiation, and structure isomorphism. Therefore, our Se-derivatization strategy has great potentials to provide rational solutions for both phase determination and high-quality crystal growth in nucleic-acid crystallography. Moreover, the Se derivatization generates the nucleic acids with many new properties and creates a new paradigm of nucleic acids. This review summarizes the recent developments of the atomic site-specific Se derivatization of nucleic acids for structure determination and function study. Several applications of this Se-derivatization strategy in nucleic acid and protein research are also described in this review.

Heterogeneous nuclear ribonucleoproteinassociated nucleic acids trigger disease in a rat transfer model of arthritis

Toll-like receptor (TLR)-mediated autoimmunity to nucleic acid-protein complexes has been implicated in the pathogenesis of systemic lupus erythematosus, whereas information regarding the influence of nucleic acid sensing TLRs on the...

Statistics and Physical Origins of pK and Ionization State Changes upon Protein-Ligand Binding

This work investigates statistical prevalence and overall physical origins of changes in charge states of receptor proteins upon ligand binding. These changes are explored as a function of the ligand type (small molecule, protein, and nucleic acid), and distance from the binding region. Standard continuum solvent methodology is used to compute, on an equal footing, pK changes upon ligand binding for a total of 5899 ionizable residues in 20 protein-protein, 20 protein-small molecule, and 20 protein-nucleic acid high-resolution complexes. The size of the data set combined with an extensive error and sensitivity analysis allows us to make statistically justified and conservative conclusions: in 60% of all protein-small molecule, 90% of all protein-protein, and 85% of all protein-nucleic acid complexes there exists at least one ionizable residue that changes its charge state upon ligand binding at physiological conditions (pH = 6.5). Considering the most biologically relevant pH range of 4–8, the number of ionizable residues that experience substantial pK changes (ΔpK > 1.0) due to ligand binding is appreciable: on average, 6% of all ionizable residues in protein-small molecule complexes, 9% in protein-protein, and 12% in protein-nucleic acid complexes experience a substantial pK change upon ligand binding. These changes are safely above the statistical false-positive noise level. Most of the changes occur in the immediate binding interface region, where approximately one out of five ionizable residues experiences substantial pK change regardless of the ligand type. However, the physical origins of the change differ between the types: in protein-nucleic acid complexes, the pK values of interface residues are predominantly affected by electrostatic effects, whereas in protein-protein and protein-small molecule complexes, structural changes due to the induced-fit effect play an equally important role. In protein-protein and protein-nucleic acid complexes, there is a statistically significant number of substantial pK perturbations, mostly due to the induced-fit structural changes, in regions far from the binding interface.

Neutrons and Biology: Three Decades of Excitement and More to Come

When the first issue of Neutron News appeared, many years had passed since the first Brookhaven Symposium on Neutrons in Biology took place. The Symposium was dedicated to neutron results on systems as diverse as proteins in crystals, membranes, collagen, muscle and protein-nucleic acid complexes crucial for gene expression such as chromatin and the ribosome [1]. And if, as intense recent interest suggests [2], we considered confined water studies to be part of Neutrons in Biology, we would find experiments from Harwell on water dynamics in clays from earlier [3]. With wonderful foresight, the 1975 symposium also included sessions on the preparation of deuterated specimens, inelastic scattering, position-sensitive detectors and pulsed sources, highlighting the importance of instrumental innovation for the field! Two years later, a workshop on Small Angle Neutron Scattering (SANS) held in Villard-de-Lans, near Grenoble, gave rise to well remembered animated discussions involving competing groups using neutrons and X-rays.

Electron transfer from aromatic amino acids to guanine and adenine radical cations in π stacked and T-shaped complexes

Similar redox properties of the natural nucleobases and aromatic amino acids make it possible for electron transfer (ET) to occur between these sites in protein-nucleic acid complexes. Using DFT calculations, we estimate the ET rate from aromatic amino acid X (X = Phe, His, Tyr and Trp) to radical cations of guanine (G) and adenine (A) in dimers G-X and A-X with different arrangement of the subunits. We show that irrespective of the mutual orientation of the aromatic rings, the electronic interaction in the systems is strong enough to ensure effective ET from X to G(+) or A(+). Surprisingly, relatively high ET rates are found in T-shaped dimers. This suggests that pi stacking of nucleobases and aromatic amino acids is not required for feasible ET. In most complexes [G-X](+) and [A-X](+), we find the excess charge to be confined to a single site, either the nucleobase or amino acid X. Then, conformational changes may initiate migration of the radical cation state from the nucleobase to X and back. The ET process from Trp and Tyr to G(+) is found to be faster than deprotonation of G(+). Because the last reaction may lead to the formation of highly mutagenic species, the efficient repair of G(+) may play an important role in the protection of genomic DNA from oxidative damage.

Emerging roles of TLR7 and TLR9 in murine SLE

Systemic lupus erythematosus (SLE) is an autoimmune disorder characterized by B cell hyperactivity leading to the production of various autoantibodies and subsequent development of glomerulonephritis, i.e. lupus nephritis. Among the principal targets of autoantibodies produced in murine SLE are nucleic acid–protein complexes, such as chromatin and ribonucleoproteins, and the envelope glycoprotein gp70 of endogenous retroviruses. The preferential production of these autoantibodies is apparently promoted by the presence of genetic abnormalities leading to defects in the elimination of apoptotic cells and to an enhanced expression of endogenous retroviruses. Moreover, recent studies revealed that the innate receptors TLR7 and TLR9 are critically involved in the activation of dendritic cells and autoreactive B cells through the recognition of endogenous DNA- or RNA-containing antigens and subsequent development of autoimmune responses against nuclear autoantigens. Furthermore, the regulation of autoimmune responses against endogenous retroviral gp70 by TLR7 suggested the implication of endogenous retroviruses in this autoimmune response. Clearly, further elucidation of the precise molecular role of TLR7 and TLR9 in the development of autoimmune responses will help to develop novel therapeutic strategies and targets for SLE.

Counterion Atmosphere and Hydration Patterns near a Nucleosome Core Particle

The chromatin folding problem is an exciting and rich field for modern research. On the most basic level, chromatin fiber consists of a collection of protein-nucleic acid complexes, known as nucleosomes, joined together by segments of linker DNA. Understanding how the cell successfully compacts meters of highly charged DNA into a micrometer size nucleus while still enabling rapid access to the genetic code for transcriptional processes is a challenging goal. In this work we shed light on the way mobile ions condense around the nucleosome core particle, as revealed by an extensive all-atom molecular dynamics simulation. On a hundred nanosecond time scale, the nucleosome exhibited only small conformational fluctuations. We found that nucleosomal DNA is better neutralized by the combination of histone charges and mobile ions compared with free DNA. We provide a detailed physical explanation of this effect using ideas from electrostatics in continuous media. We also discovered that sodium condensation around the histone core is dominated by an experimentally characterized acidic patch, which is thought to play a significant role in chromatin compaction by binding with basic histone tails. Finally, we found that the nucleosome is extensively permeated by over a thousand water molecules, which in turn allows mobile ions to penetrate deeply into the complex. Overall, our work sheds light on the way ionic and hydration interactions within a nucleosome may affect internucleosomal interactions in higher order chromatin fibers.

Probing the limits of liquid droplet laser desorption mass spectrometry in the analysis of oligonucleotides and nucleic acids

In the present work we demonstrate the advantages of LILBID mass spectrometry (laser-induced liquid bead ion desorption) in the analysis of nucleic acids and large oligonucleotides. For established methods like matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI), the mass analysis of oligonucleotides or of noncovalent oligonucleotide-protein complexes, in particular of very large ones, still represents a considerable challenge either due to the lack of native solutions or nonspecific adduct formation or due to a reduced salt tolerance or a high charge state of the ions. With LILBID, oligonucleotides, solvated in micro-droplets of aqueous buffer at certain pH and ion strength, are brought into the gas phase by laser ablation. We show that our method is able to detect single- and double-stranded oligonucleotides with high softness, demonstrated by the buffer dependence of the melting of a duplex. The absolute sensitivity is in the attomole range concomitant with a total analyte consumption in the femtomole region. The upper mass limit of oligonucleotides still detected with good signal-to-noise ratio with LILBID is the 1.66 MDa plasmid pUC19. With DNA ladders from short duplexes with sticky ends, we show that LILBID correctly reflects the relative thermodynamic stabilities of the ladders. Moreover, as an example for a specific DNA-protein complex we show that a NF-kappaB p50 homodimer binds sequence specifically to its match DNA. In summary we demonstrate that LILBID, although presently performed only with low mass resolution, due to these advantages, is an alternative mass spectrometric method for the analysis of oligonucleotides in general and of specific noncovalent nucleic acid-protein complexes in particular.

Longitudinal-Relaxation-Enhanced NMR Experiments for the Study of Nucleic Acids in Solution

Atomic-resolution information on the structure and dynamics of nucleic acids is essential for a better understanding of the mechanistic basis of many cellular processes. NMR spectroscopy is a powerful method for studying the structure and dynamics of nucleic acids; however, solution NMR studies are currently limited to relatively small nucleic acids at high concentrations. Thus, technological and methodological improvements that increase the experimental sensitivity and spectral resolution of NMR spectroscopy are required for studies of larger nucleic acids or protein-nucleic acid complexes. Here we introduce a series of imino-proton-detected NMR experiments that yield an over 2-fold increase in sensitivity compared to conventional pulse schemes. These methods can be applied to the detection of base pair interactions, RNA-ligand titration experiments, measurement of residual dipolar (15)N-(1)H couplings, and direct measurements of conformational transitions. These NMR experiments employ longitudinal spin relaxation enhancement techniques that have proven useful in protein NMR spectroscopy. The performance of these new experiments is demonstrated for a 10 kDa TAR-TAR*(GA) RNA kissing complex and a 26 kDa tRNA.

Functional Characteristics of Small Proteins (70 Amino Acid Residues) Forming Protein-Nucleic Acid Complexes

The proteins composed of short polypeptides (about 70 amino acid residues) participating in large complexes (ribosome) and proteins interacting with DNA/RNA were taken for analysis and classified according to the hydrophobicity excess/deficiency distribution as a measure of structural and functional specificity and similarity. The characterization of this group of proteins is the introductory part to the analysis of the so called “Never Born Proteins” (NBP) in search for protein compounds exhibiting biological activity that may be valuable in pharmacological research. The entropy scale (classification between random and deterministic limits) organized in ranking list allows the comparative analysis of the proteins under consideration. The comparison of the hydropho- bicity deficiency appeared to be useful for similarity recognition, the examples of which are shown in the paper. The specificity of proteins participating in large protein-nucleic acid complexes generation is presented.

Detailed mechanism for transposition by TnpA transposase involves DNA shape rather than direct protein-DNA recognition to generate an active nucleoprotein complex

A series of single-crystal structures determined by Barabas and colleagues provides a detailed mechanism for how the TnpA transposase from Helicobacter pylori recognizes, cleaves, and integrates the IS200/IS605 class of transposable elements. An interesting aspect of the mechanism is that the transposase recognizes the transposon through the unique fold-back structure adopted by the sequences of the DNA components, rather than through direct protein-DNA interactions. This is an example of indirect readout that is reminiscent of how four-stranded junctions are recognized by recombination proteins, but is also analogous to ribonucleoproteins, in that the DNA facilitates formation of an active nucleic acid-protein complex.

Mitochondrial nucleoids from the yeast Candida parapsilosis: expansion of the repertoire of proteins associated with mitochondrial DNA

Molecules of mitochondrial DNA (mtDNA) are packed into nucleic acid-protein complexes termed mitochondrial nucleoids (mt-nucleoids). In this study, we analysed mt-nucleoids of the yeast Candida parapsilosis, which harbours a linear form of the mitochondrial genome. To identify conserved as well as specific features of mt-nucleoids in this species, we employed two strategies for analysis of their components. First, we investigated the protein composition of mt-nucleoids isolated from C. parapsilosis mitochondria, determined N-terminal amino acid sequences of 14 proteins associated with the mt-nucleoids and identified corresponding genes. Next, we complemented the list of mt-nucleoid components with additional candidates identified in the complete genome sequence of C. parapsilosis as homologues of Saccharomyces cerevisiae mt-nucleoid proteins. Our approach revealed several known mt-nucleoid proteins as well as additional components that expand the repertoire of proteins associated with these cytological structures. In particular, we identified and purified the protein Gcf1, which is abundant in the mt-nucleoids and exhibits structural features in common with the mtDNA packaging protein Abf2 from S. cerevisiae. We demonstrate that Gcf1p co-localizes with mtDNA, has DNA-binding activity in vitro, and is able to stabilize mtDNA in the S. cerevisiae Deltaabf2 mutant, all of which points to a role in the maintenance of the C. parapsilosis mitochondrial genome. Importantly, in contrast to Abf2p, in silico analysis of Gcf1p predicted the presence of a coiled-coil domain and a single high-mobility group (HMG) box, suggesting that it represents a novel type of mitochondrial HMG protein.

Application of FT-ICR MS for the Study of Protein Complexes

Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) represents a powerful technique for analyzing biological samples due to the ultrahigh resolving power, high mass accuracy, and multiple-stage tandem mass spectrometry (MSn). With the advent of electrospray ionization (ESI), determinations of binding stoichiometry and binding sites for protein complexes are available. This review summarizes the recent FT-ICR MS applications in characterization of protein complexes, such as protein-peptide complexes, protein-protein complexes, and protein-nucleic acid complexes. Especially, combined with ECD and SWIFT techniques, FT-ICR MS has unique ion manipulation capabilities and plays a critical role in the analysis of protein complexes.