Structural studies of protein-nucleic acid complexes: A brief overview of the selected techniques.

Protein-nucleic acid complexes are involved in all vital processes, including replication, transcription, translation, regulation of gene expression and cell metabolism. Knowledge of the biological functions and molecular mechanisms beyond the activity of the macromolecular complexes can be determined from their tertiary structures. Undoubtably, performing structural studies of protein-nucleic acid complexes is challenging, mainly because these types of complexes are often unstable. In addition, their individual components may display extremely different surface charges, causing the complexes to precipitate at higher concentrations used in many structural studies. Due to the variety of protein-nucleic acid complexes and their different biophysical properties, no simple and universal guideline exists that helps scientists chose a method to successfully determine the structure of a specific protein-nucleic acid complex. In this review, we provide a summary of the following experimental methods, which can be applied to study the structures of protein-nucleic acid complexes: X-ray and neutron crystallography, nuclear magnetic resonance (NMR) spectroscopy, cryogenic electron microscopy (cryo-EM), atomic force microscopy (AFM), small angle scattering (SAS) methods, circular dichroism (CD) and infrared (IR) spectroscopy. Each method is discussed regarding its historical context, advancements over the past decades and recent years, and weaknesses and strengths. When a single method does not provide satisfactory data on the selected protein-nucleic acid complex, a combination of several methods should be considered as a hybrid approach; thus, specific structural problems can be solved when studying protein-nucleic acid complexes.

Structural Characteristics of the 5'-Terminal Region of Mouse p53 mRNA and Identification of Proteins That Bind to This mRNA Region.

A mouse model has often been used in studies of p53 gene expression. Detailed interpretation of functional studies is, however, hampered by insufficient knowledge of the impact of mouse p53 mRNA's structure and its interactions with proteins in the translation process. In particular, the 5'-terminal region of mouse p53 mRNA is an important region which takes part in the regulation of the synthesis of p53 protein and its N-truncated isoform Δ41p53. In this work, the spatial folding of the 5'-terminal region of mouse p53 mRNA and its selected sub-fragments was proposed based on the results of the SAXS method and the RNAComposer program. Subsequently, RNA-assisted affinity chromatography was used to identify proteins present in mouse fibroblast cell lysates that are able to bind the RNA oligomer, which corresponds to the 5'-terminal region of mouse p53 mRNA. Possible sites to which the selected, identified proteins can bind were proposed. Interestingly, most of these binding sites coincide with the sites determined as accessible to hybridization of complementary oligonucleotides. Finally, the high binding affinity of hnRNP K and PCBP2 to the 5'-terminal region of mouse p53 mRNA was confirmed and their possible binding sites were proposed.

Poly(C)-binding Protein 2 Regulates the p53 Expression via Interactions with the 5'-Terminal Region of p53 mRNA.

The p53 protein is one of the major transcriptional factors which guards cell homeostasis. Here, we showed that poly(C)-binding protein 2 (PCBP2) can bind directly to the 5' terminus of p53 mRNA by means of electrophoretic mobility shift assay. Binding sites of PCBP2 within this region of p53 mRNA were mapped using Pb2+-induced cleavage and SAXS methods. Strikingly, the downregulation of PCBP2 in HCT116 cells resulted in a lower level of p53 protein under normal and stress conditions. Quantitative analysis of p53 mRNA in PCBP2-downregulated cells revealed a lower level of p53 mRNA under normal conditions suggesting the involvement of PCBP2 in p53 mRNA stabilisation. However, no significant change in p53 mRNA level was observed upon PCBP2 depletion under genotoxic stress. Moreover, a higher level of p53 protein in the presence of rapamycin or doxorubicin and the combination of both antibiotics was noticed in PCBP2-overexpressed cells compared to control cells. These observations indicate the potential involvement of PCBP2 in cap-independent translation of p53 mRNA especially occurring under stress conditions. It has been postulated that the PCBP2 protein is engaged in the enhancement of p53 mRNA stability, probably via interacting with its 3' end. Our data show that under stress conditions PCBP2 also modulates p53 translation through binding to the 5' terminus of p53 mRNA. Thus PCBP2 emerges as a double-function factor in the p53 expression.

Structural studies of human muscle FBPase.

Muscle fructose-1,6-bisphosphatase (FBPase), which catalyzes the hydrolysis of fructose-1,6-bisphosphate (F1,6BP) to fructose-6-phosphate (F6P) and inorganic phosphate, regulates glucose homeostasis by controlling the glyconeogenic pathway. FBPase requires divalent cations, such as Mg2+, Mn2+, or Zn2+, for its catalytic activity; however, calcium ions inhibit the muscle isoform of FBPase by interrupting the movement of the catalytic loop. It has been shown that residue E69 in this loop plays a key role in the sensitivity of muscle FBPase towards calcium ions. The study presented here is based on five crystal structures of wild-type human muscle FBPase and its E69Q mutant in complexes with the substrate and product of the enzymatic reaction, namely F1,6BP and F6P. The ligands are bound in the active site of the studied proteins in the same manner and have excellent definition in the electron density maps. In all studied crystals, the homotetrameric enzyme assumes the same cruciform quaternary structure, with the κ angle, which describes the orientation of the upper dimer with respect to the lower dimer, of -85o. This unusual quaternary arrangement of the subunits, characteristic of the R-state of muscle FBPase, is also observed in solution by small-angle X-ray scattering (SAXS).

Crystal structures of plant inorganic pyrophosphatase, an enzyme with a moonlighting autoproteolytic activity.

Inorganic pyrophosphatases (PPases, EC 3.6.1.1), which hydrolyze inorganic pyrophosphate to phosphate in the presence of divalent metal cations, play a key role in maintaining phosphorus homeostasis in cells. DNA coding inorganic pyrophosphatases from Arabidopsis thaliana (AtPPA1) and Medicago truncatula (MtPPA1) were cloned into a bacterial expression vector and the proteins were produced in Escherichia coli cells and crystallized. In terms of their subunit fold, AtPPA1 and MtPPA1 are reminiscent of other members of Family I soluble pyrophosphatases from bacteria and yeast. Like their bacterial orthologs, both plant PPases form hexamers, as confirmed in solution by multi-angle light scattering and size-exclusion chromatography. This is in contrast with the fungal counterparts, which are dimeric. Unexpectedly, the crystallized AtPPA1 and MtPPA1 proteins lack ∼30 amino acid residues at their N-termini, as independently confirmed by chemical sequencing. In vitro, self-cleavage of the recombinant proteins is observed after prolonged storage or during crystallization. The cleaved fragment corresponds to a putative signal peptide of mitochondrial targeting, with a predicted cleavage site at Val31-Ala32. Site-directed mutagenesis shows that mutations of the key active site Asp residues dramatically reduce the cleavage rate, which suggests a moonlighting proteolytic activity. Moreover, the discovery of autoproteolytic cleavage of a mitochondrial targeting peptide would change our perception of this signaling process.

Borrelia outer surface protein C is capable of human fibrinogen binding.

Outer surface protein C (OspC) is one of the most abundant surface lipoproteins produced during early infection by the Borrelia spirochete, the causative agent of Lyme disease. The high sequence variability of the ospC gene results in the production of several and strongly divergent OspC types. One of the known roles of OspC is the recruitment of blood components, including complement regulators, to facilitate the bloodstream survival of Borrelia at an essential stage of host infection. Here, we identify and describe a new interaction between OspC and human fibrinogen. To test the ability of OspC to bind fibrinogen, we developed a microscale thermophoresis assay using four fluorescently labeled types of OspC. We show that OspC binds fibrinogen tightly, with nanomolar Kd , and that the binding depends on the OspC type. The binding assays combined with SAXS studies allowed us to map the OspC-binding site on the fibrinogen molecule. Spectrometric measurements of fibrinogen clotting in the presence of OspC indicate that OspC negatively influences the clot formation process. Taken together, our findings are consistent with the hypothesis that OspC interacts with blood protein partners to facilitate Borrelia spreading by the hematogenous route.

Unraveling the structural basis for the exceptional stability of RNA G-quadruplexes capped by a uridine tetrad at the 3' terminus.

Uridine tetrads (U-tetrads) are a structural element encountered in RNA G-quadruplexes, for example, in the structures formed by the biologically relevant human telomeric repeat RNA. For these molecules, an unexpectedly strong stabilizing influence of a U-tetrad forming at the 3' terminus of a quadruplex was reported. Here we present the high-resolution solution NMR structure of the r(UGGUGGU)4 quadruplex which, in our opinion, provides an explanation for this stabilization. Our structure features a distinctive, abrupt chain reversal just prior to the 3' uridine tetrad. Similar "reversed U-tetrads" were already observed in the crystalline phase. However, our NMR structure coupled with extensive explicit solvent molecular dynamics (MD) simulations identifies some key features of this motif that up to now remained overlooked. These include the presence of an exceptionally stable 2'OH to phosphate hydrogen bond, as well as the formation of an additional K+ binding pocket in the quadruplex groove.

Self-Assembly and Ordering of Peptide-Based Cavitands in Water and DMSO: The Power of Hydrophobic Effects Combined with Neutral Hydrogen Bonds.

Directional self-assembly of uncharged molecules in water is a major challenge in supramolecular chemistry. Herein, it is demonstrated that peptide-based cavitands wrap around a hydrophobic core (fullerene C60 ) by a combination of the hydrophobic effect and hydrogen-bonding interactions to form highly ordered three-component complexes in water that resemble the molten-globule stage of protein folding. The complexes were characterized by DOSY NMR spectroscopy, small-angle X-ray scattering, and circular dichroism, and their structures were confirmed by X-ray crystallography. Enhancement of the CD signals by nearly one order of magnitude and increased hydrolytic stability of hydrazone bonds of the complexes relative to the nonassembled species were observed. In contrast, DMSO and DMSO/water mixtures were found to be highly disintegrative for these complexes. Interestingly, some cavitands can only be synthesized in the presence of the hydrophobic template followed by disassembly of the complexes.

Biophysical analysis of BMV virions purified using a novel method.

Brome mosaic virus (BMV) has been successfully loaded with different types of nanoparticles. However, studies concerning its application as a nanoparticle carrier demand high-purity virions in large amounts. Existing BMV purification protocols rely on multiple differential ultracentrifugation runs of the initially purified viral preparation. Herein, we describe an alternative method for BMV purification based on ion-exchange chromatography and size-exclusion chromatography (SEC) yielding 0.2mg of virus from 1g of plant tissue. Our method is of similar efficiency to previously described protocols and can easily be scaled up. The method results in high-quality BMV preparations as confirmed by biophysical analyses, including cryogenic transmission electron microscopy (cryo-TEM), dynamic light scattering (DLS), static light scattering (SLS), and circular dichroism (CD) measurements and attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) spectroscopy. Our results revealed that purified BMV capsids are stable and monodisperse and can be used for further downstream applications. In this work, we also characterize secondary structure and size fluctuations of the BMV virion at different pH values.

Crystallographic and SAXS studies of S-adenosyl-l-homocysteine hydrolase from Bradyrhizobium elkanii.

S-Adenosyl-l-homocysteine hydrolase (SAHase) from the symbiotic bacterium Bradyrhizobium elkanii (BeSAHase) was crystallized in four ligand complexes with (i) mixed adenosine (Ado) and cordycepin (Cord; 3'-deoxyadenosine), (ii) adenine (Ade), (iii) Ado and (iv) mixed 2'-deoxyadenosine (2'-dAdo) and Ade. The crystal structures were solved at resolutions of 1.84, 1.95, 1.95 and 1.54 Å, respectively. Only the Ade complex crystallized with a dimer in the asymmetric unit, while all of the other complexes formed a crystallographically independent tetrameric assembly. In the Ado/Cord complex, adenosine is found in three subunits while the fourth subunit has cordycepin bound in the active site. In the Ade and Ado complexes only these ligand molecules are present in the active sites. The 2'-dAdo/Ade complex has Ade bound in two subunits and 2'-dAdo bound in the other two subunits. The BeSAHase fold adopted a closed conformation in the complexes with Ado, Ade and 2'-dAdo, and a semi-open conformation when cordycepin occupied the active site. An SAHase-specific molecular gate, consisting of residues His342 and Phe343, behaves differently in the different complexes, but there is no simple correlation with the ligand type. Additional small-angle X-ray scattering (SAXS) experiments confirm the tetrameric state of the protein in solution. The main conclusions from this work are (i) that the SAHase subunit does not simply oscillate between two discrete conformational open/closed states in correlation with the absence/presence of a ligand in the active site, but can also assume an intermediate form for some ligands; (ii) that the shut/open state of the molecular gate in the access channel to the active site is not correlated in a simple way with the open/closed subunit conformation or empty/occupied status of the active site, but that a variety of states are possible even for the same ligand; (iii) that a cation (typically sodium) coordinated in an intersubunit loop rigidifies a molecular hinge and thus stabilizes the closed conformation; (iv) that BeSAHase in solution is a tetramer, consistent with the model derived from crystallography.

The part of a long beta hairpin from the scrapie form of the human prion protein is reconstructed in the synthetic CC36 protein.

Mechanisms of beta sheet formation by the human prion protein are not clear yet. In this work, we clarified the role of the region containing C-half of the second helix and N-half of the third helix of that protein in the process of alpha helix to beta sheet transition. Solid phase automatic synthesis of the original peptide (CC36: Cys179-Cys214) failed because of the beta hairpin formation in the region 206-MERVVEQMC-214 with a high beta strand potential. Using Met206Arg and Val210Arg substitutions, we increased the probability of alpha helix formation by that sequence. After that modification, the complete CC36 peptide with disulfide bond has been synthesized. Modified peptide has been studied by circular dichroism (CD) and fluorescence spectrography. According to the CD spectra analysis, the CC36 peptide contains 37% of residues in beta sheet and just 15% in helix. Thermal analysis under the control of CD shows that the secondary structure content of the peptide is stable from 5°C to 80°C. Dissociation of oligomers of the CC36 peptide finishes at 37°C according to the fluorescence analysis. The CC36 peptide is able to bind Mn(2+) cations, which causes small temperature-associated structural shifts at concentrations of 2 - 10·10(-6) M. Predicted beta hairpin of the CC36 peptide (two beta strands are: 184-IKQHTVT-190 and 197-TETDVKM-205) should be the part of a longer beta hairpin from the scrapie form of the prion protein (PrPSc). Analogs of the CC36 peptide may be considered as antigens for the future development of a vaccine against PrPSc. Proteins 2016; 84:1462-1479. © 2016 Wiley Periodicals, Inc.

Tick receptor for outer surface protein A from Ixodes ricinus - the first intrinsically disordered protein involved in vector-microbe recognition.

The tick receptor for outer surface protein A (TROSPA) is the only identified factor involved in tick gut colonization by various Borrelia species. TROSPA is localized in the gut epithelium and can recognize and bind the outer surface bacterial protein OspA via an unknown mechanism. Based on earlier reports and our latest observations, we considered that TROSPA would be the first identified intrinsically disordered protein (IDP) involved in the interaction between a vector and a pathogenic microbe. To verify this hypothesis, we performed structural studies of a TROSPA mutant from Ixodes ricinus using both computational and experimental approaches. Irrespective of the method used, we observed that the secondary structure content of the TROSPA polypeptide chain is low. In addition, the collected SAXS data indicated that this protein is highly extended and exists in solution as a set of numerous conformers. These features are all commonly considered hallmarks of IDPs. Taking advantage of our SAXS data, we created structural models of TROSPA and proposed a putative mechanism for the TROSPA-OspA interaction. The disordered nature of TROSPA may explain the ability of a wide spectrum of Borrelia species to colonize the tick gut.

Structural characterization of a dimer of RNA duplexes composed of 8-bromoguanosine modified CGG trinucleotide repeats: a novel architecture of RNA quadruplexes.

Fragile X syndrome and fragile X-associated tremor/ataxia syndrome (FXTAS) are neurodegenerative disorders caused by the pathogenic expansion of CGG triplet repeats in the FMR1 gene. FXTAS is likely to be caused by a 'toxic' gain-of-function of the FMR1 mRNA. We provide evidence for the existence of a novel quadruplex architecture comprising CGG repeats. The 8-bromoguanosine ((Br)G)-modified molecule GC(Br)GGCGGC forms a duplex in solution and self-associates via the major groove to form a four-stranded, antiparallel (GC(Br)GGCGGC)4 RNA quadruplex with (Br)G3:G6:(Br)G3:G6 tetrads sandwiched between mixed G:C:G:C tetrads. Self-association of Watson-Crick duplexes to form a four-stranded structure has previously been predicted; however, no experimental evidence was provided. This novel four-stranded RNA structure was characterized using a variety of experimental methods, such as native gel electrophoresis, NMR spectroscopy, small-angle X-ray scattering and electrospray ionization mass spectrometry.

Crystallographic and CD probing of ligand-induced conformational changes in a plant PR-10 protein.

Plant pathogenesis-related class 10 (PR-10) proteins are a family of abundant proteins initially identified as elements of the plant defense system. The key structural feature suggesting PR-10 functionality is a huge hydrophobic cavity created in the protein interior by a scaffold composed of an extended ß-sheet wrapped around a long and flexible C-terminal α-helix. Several crystallographic and NMR studies have shown that the cavity can accommodate a variety of small molecule ligands, including phytohormones. The article describes ∼1.3 Å resolution crystal structures of a Lupinus luteus PR-10 isoform LlPR-10.1A, in its free form and in complex with trans-zeatin, a naturally occurring plant hormone belonging to the cytokinin group. Moreover we present the structure of the same protein where the saturation with zeatin is not complete. This set of three crystal structures allows us to track the structural adaptation of the protein upon trans-zeatin docking, as well as the sequence of the ligand-binding events, step-by-step. In addition, titration of LlPR-10.1A with trans-zeatin monitored in solution by CD spectra, confirmed the pattern of structural adaptations deduced from the crystallographic studies. The ligand-biding mode shows no similarity to other zeatin complexes of PR-10 proteins. The present work, which describes the first atomic models of the same PR-10 protein with and without a physiological ligand, reveals that the conformation of LlPR-10.1A undergoes a significant structural rearrangement upon trans-zeatin binding.

Structure of γ-conglutin: insight into the quaternary structure of 7S basic globulins from legumes.

γ-Conglutin from lupin seeds is an unusual 7S basic globulin protein. It is capable of reducing glycaemia in mammals, but the structural basis of this activity is not known. γ-Conglutin shares a high level of structural homology with glycoside hydrolase inhibitor proteins, although it lacks any kind of inhibitory activity against plant cell-wall degradation enzymes. In addition, γ-conglutin displays a less pronounced structural similarity to pepsin-like aspartic proteases, but it is proteolytically dysfunctional. Only one structural study of a legume 7S basic globulin, that isolated from soybean, has been reported to date. The quaternary assembly of soybean 7S basic globulin (Bg7S) is arranged as a cruciform-shaped tetramer comprised of two superposed dimers. Here, the crystal structure of γ-conglutin isolated from Lupinus angustifolius seeds (LangC) is presented. The polypeptide chain of LangC is post-translationally cleaved into α and ß subunits but retains its covalent integrity owing to a disulfide bridge. The protomers of LangC undergo an intricate quaternary assembly, resulting in a ring-like hexamer with noncrystallographic D3 symmetry. The twofold-related dimers are similar to those in Bg7S but their assembly is different as a consequence of mutations in a ß-strand that is involved in intermolecular ß-sheet formation in γ-conglutin. Structural elucidation of γ-conglutin will help to explain its physiological role, especially in the evolutionary context, and will guide further research into the hypoglycaemic activity of this protein in humans, with potential consequences for novel antidiabetic therapies.

Calponin-like Chd64 is partly disordered.

20-hydroxyecdysone (20E) and juvenile hormone (JH) signaling pathways interact to regulate insect development. Recently, two proteins, a calponin-like Chd64 and immunophilin FKBP39 have been found to play a pivotal role in the cross-talk between 20E and JH, although the molecular basis of interaction remains unknown. The aim of this work was to identify the structural features that would provide understanding of the role of Chd64 in multiple and dynamic complex that cross-links the signaling pathways. Here, we demonstrate the results of in silico and in vitro analyses of the structural organization of Chd64 from Drosophila melanogaster and its homologue from Tribolium castaneum. Computational analysis predicted the existence of disordered regions on the termini of both proteins, while the central region appeared to be globular, probably corresponding to the calponin homology (CH) domain. In vitro analyses of the hydrodynamic properties of the proteins from analytical size-exclusion chromatography and analytical ultracentrifugation revealed that DmChd64 and TcChd64 had an asymmetrical, elongated shape, which was further confirmed by small angle X-ray scattering (SAXS). The Kratky plot indicated disorderness in both Chd64 proteins, which could possibly be on the protein termini and which would give rise to specific hydrodynamic properties. Disordered tails are often involved in diverse interactions. Therefore, it is highly possible that there are intrinsically disordered regions (IDRs) on both termini of the Chd64 proteins that serve as platforms for multiple interaction with various partners and constitute the foundation for their regulatory function.

Interactions of a cationic surfactant--(benzyloxymethyl) dodecyldimethylammonium chloride with model biomembrane systems.

Phospholipids are the main components of biological membranes. The aim of the present study was to determine the influence of a cationic surfactant on phospholipid structure and dynamics. Fourier transform infrared (FTIR) and dielectric relaxation (DRS) spectroscopies as well as small-angle X-ray scattering (SAXS) with synchrotron radiation have been used to analyse the structure of fully hydrated 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) in the presence of a quaternary ammonium surfactant: (benzyloxymethyl) dodecyldimethylammoniumchloride (BzMDDAC). The presence of the surfactant caused changes in the temperature of the DMPC phase transition, as revealed using FTIR and DRS measurements. This change results from the disappearance of the multilamellar phase of DMPC and the formation of the unilamellar (most likely bicellar) phase, as indicated by the SAXS results.