Robert Konrat

RNA chaperones, RNA annealers and RNA helicases.

RNA molecules face difficulties when folding into their native structures. In the cell, proteins can assist RNAs in reaching their functionally active states by binding and stabilizing a specific structure or, in a quite opposite way, by interacting in a non-specific manner. These proteins can either facilitate RNA-RNA interactions in a reaction termed RNA annealing, or they can resolve non-functional inhibitory structures. The latter is defined as “RNA chaperone activity” and is the main topic of this review. Here we define RNA chaperone activity in a stringent way and we review those proteins for which RNA chaperone activity has been clearly demonstrated. These proteins belong to quite diverse families such as hnRNPs, histone-like proteins, ribosomal proteins, cold shock domain proteins and viral nucleocapsid proteins. DExD/H-box containing RNA helicases are discussed as a special family of enzymes that restructure RNA or RNPs in an ATP-dependent manner. We further address the different mechanisms RNA chaperones might use to promote folding including the recently proposed theory of protein disorder as a key element in triggering RNA-protein interactions. Finally, we present a new website for proteins with RNA chaperone activity which compiles all the information on these proteins with the perspective to promote the understanding of their activity.

Direct observation of the dynamic process underlying allosteric signal transmission.

Allosteric regulation is an effective mechanism of control in biological processes. In allosteric proteins a signal originating at one site in the molecule is communicated through the protein structure to trigger a specific response at a remote site. Using NMR relaxation dispersion techniques we directly observe the dynamic process through which the KIX domain of CREB binding protein communicates allosteric information between binding sites. KIX mediates cooperativity between pairs of transcription factors through binding to two distinct interaction surfaces in an allosteric manner. We show that binding the activation domain of the mixed lineage leukemia (MLL) transcription factor to KIX induces a redistribution of the relative populations of KIX conformations toward a high-energy state in which the allosterically activated second binding site is already preformed, consistent with the Monod-Wyman-Changeux (WMC) model of allostery. The structural rearrangement process that links the two conformers and by which allosteric information is communicated occurs with a time constant of 3 ms at 27 degrees C. Our dynamic NMR data reveal that an evolutionarily conserved network of hydrophobic amino acids constitutes the pathway through which information is transmitted.

BEST-TROSY experiments for time-efficient sequential resonance assignment of large disordered proteins

The characterization of the conformational properties of intrinsically disordered proteins (IDPs), and their interaction modes with physiological partners has recently become a major research topic for understanding biological function on the molecular level. Although multidimensional NMR spectroscopy is the technique of choice for the study of IDPs at atomic resolution, the intrinsically low resolution, and the large peak intensity variations often observed in NMR spectra of IDPs call for resolution- and sensitivity-optimized pulse schemes. We present here a set of amide proton-detected 3D BEST-TROSY correlation experiments that yield the required sensitivity and spectral resolution for time-efficient sequential resonance assignment of large IDPs. In addition, we introduce two proline-edited 2D experiments that allow unambiguous identification of residues adjacent to proline that is one of the most abundant amino acids in IDPs. The performance of these experiments, and the advantages of BEST-TROSY pulse schemes are discussed and illustrated for two IDPs of similar length (~270 residues) but with different conformational sampling properties.

High-Affinity Binding of the Staphylococcal HarA Protein to Haptoglobin and Hemoglobin Involves a Domain with an Antiparallel Eight-Stranded β-Barrel Fold

Iron scavenging from the host is essential for the growth of pathogenic bacteria. In this study, we further characterized two staphylococcal cell wall proteins previously shown to bind hemoproteins. HarA and IsdB harbor homologous ligand binding domains, the so called NEAT domain (for “near transporter”) present in several surface proteins of gram-positive pathogens. Surface plasmon resonance measurements using glutathione S-transferase (GST)-tagged HarAD1, one of the ligand binding domains of HarA, and GST-tagged full-length IsdB proteins confirmed high-affinity binding to hemoglobin and haptoglobin-hemoglobin complexes with equilibrium dissociation constants (KD) of 5 to 50 nM. Haptoglobin binding could be detected only with HarA and was in the low micromolar range. In order to determine the fold of this evolutionarily conserved ligand binding domain, the untagged HarAD1 protein was subjected to nuclear magnetic resonance spectroscopy, which revealed an eight-stranded, purely antiparallel β-barrel with the strand order (-β1↓-β2↑-β3↓-β6↑-β5↓-β4↑-β7↓-β8↑), forming two Greek key motifs. Based on structural-homology searches, the topology of the HarAD1 domain resembles that of the immunoglobulin (Ig) fold family, whose members are involved in protein-protein interactions, but with distinct structural features. Therefore, we consider that the HarAD1/NEAT domain fold is a novel variant of the Ig fold that has not yet been observed in other proteins.

NMR contributions to structural dynamics studies of intrinsically disordered proteins.

Intrinsically disordered proteins (IDPs) are characterized by substantial conformational plasticity. Given their inherent structural flexibility X-ray crystallography is not applicable to study these proteins. In contrast, NMR spectroscopy offers unique opportunities for structural and dynamic studies of IDPs. The past two decades have witnessed significant development of NMR spectroscopy that couples advances in spin physics and chemistry with a broad range of applications. This article will summarize key advances in basic physical-chemistry and NMR methodology, outline their limitations and envision future R&D directions.

Synthesis of a 13C-methyl-group-labeled methionine precursor as a useful tool for simplifying protein structural analysis by NMR spectroscopy.

In the past decade NMR spectroscopy has emerged as one of the most powerful tools for the determination of protein structure and dynamics. The introduction of uniform N,C labeling, coupled with the development of triple-resonance experiments has permitted structural and dynamic studies of systems that are up to 20 kDa in molecular mass. The characterization of larger macromolecules requires techniques that maximize both spectral resolution and sensitivity due to chemical-shift overlap and increasing linewidth in high molecular-weight systems. Consequently, research has focused on correlation spectroscopy of side-chain methyl groups. New labeling techniques have been established by using a-ketoacids as biosynthetic precursors for the production of isotope-labeled proteins. Attention has therefore been turned towards the synthesis of a-ketobutyric and a-ketovaleric acid, biosynthetic precursors for the aliphatic amino acids isoleucine, as well as leucine and valine, respectively. NMR investigations of the trimeric E. coli enzyme IIA (34 kDa), the monomeric 82 kDa enzyme malate synthase G from E. coli, or studies of the protein p21-KID within a 45 kDa binary complex (p21-KID/Cdk2) are applications of these ACHTUNGTRENNUNGdevelopments. Furthermore, new methods for quantifying the amplitudes of motion of methyl-containing side chains have been developed, which are essential for a better understanding of dynamics and its relationship to function. Here we report a novel synthetic route towards a precursor of the S-C-methyl-bearing amino acid, methionine. The synthesis sequence is outlined in Scheme 1. Starting from easily accessible tert-butyl a-bromomethacrylate (1), homoallylic alcohol 2 was obtained through an indium mediated aqueous Barbier-type reaction between 1 and formaldehyde. The highest yields were obtained by using 4.5 mmol allyl ester (1), 9 mmol indium powder, and 36 mmol depolymerized paraformaldehyde in a mixture of THF and water (5:1) after 16 h of sonication. Primary alcohol 2 was converted to the corresponding tosylate 3 by using standard procedures. Treatment of 3 with potassium thioacetate in acetone gave thioacetyl-compound 4 after 6 h of sonication in excellent yield. Subsequent ozonolysis of the double bond at 78 8C in dichloromethane afforded 2-oxo-derivate 5. The a-keto functionality of 5 was further transformed into its N,N’-dimethylhydrazone 6. Although the thioester of 5 could have been cleaved by N,N’-dimethylhydrazine, the obviously greater reactivity of the a-keto functionality allowed for selective protection in high yield. All attempts to generate a thiol moiety starting from ester 6 by using a ZemplBn saponification protocol with sodium methanolate in dry methanol failed. Compound 7 was finally obtained by treatment of thioester 6 with ethylamine and silver triflate at 20 8C, followed by methylation of the thiolate with CH3I in THF. To prevent disulfide formation, silver triflate was applied in equimolar amounts; this pushed the reaction towards product formation due to silver iodide precipitation. Subsequently, the hydrazone of 7 was cleaved by using an aqueous HCl solution (1m) and THF to obtain a-ketoester 8. Hydrolysis of the tert-butyl ester 8 was performed in a mixture of an aqueous HCl solution (0.025m) and acetonitrile to yield the target precursor compound 9 in 32% overall yield (starting from 1). [a] M. Fischer, Prof. J. H usler, Prof. W. Schmid Institute of Organic Chemistry, University of Vienna W hringer Strasse 38, 1090 Vienna (Austria) Fax: (+43)1-4277-9 521 E-mail : walther.schmid@univie.ac.at

Structural Analysis of the Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) Intracellular Domain Reveals a Conserved Interaction Epitope

Background: PfEMP1 localization and connection to the red blood cell cytoskeleton is necessary for cytoadherence. Results: The PfEMP1 intracellular domain (ATS) is structurally conserved and interacts directly with a novel parasite protein through a flexible epitope. Conclusion: The ATS epitope mediate interactions that may be critical for cytoadherence. Significance: This is the first demonstration of ATS interacting with PHIST domains. Plasmodium falciparum-infected red blood cells adhere to endothelial cells, thereby obstructing the microvasculature. Erythrocyte adherence is directly associated with severe malaria and increased disease lethality, and it is mediated by the PfEMP1 family. PfEMP1 clustering in knob-like protrusions on the erythrocyte membrane is critical for cytoadherence, however the molecular mechanisms behind this system remain elusive. Here, we show that the intracellular domains of the PfEMP1 family (ATS) share a unique molecular architecture, which comprises a minimal folded core and extensive flexible elements. A conserved flexible segment at the ATS center is minimally restrained by the folded core. Yeast-two-hybrid data and a novel sequence analysis method suggest that this central segment contains a conserved protein interaction epitope. Interestingly, ATS in solution fails to bind the parasite knob-associated histidine-rich protein (KAHRP), an essential cytoadherence component. Instead, we demonstrate that ATS associates with PFI1780w, a member of the Plasmodium helical interspersed sub-telomeric (PHIST) family. PHIST domains are widespread in exported parasite proteins, however this is the first specific molecular function assigned to any variant of this family. We propose that PHIST domains facilitate protein interactions, and that the conserved ATS epitope may be targeted to disrupt the parasite cytoadherence system.

5-Fluoro pyrimidines: labels to probe DNA and RNA secondary structures by 1D 19F NMR spectroscopy

19F NMR spectroscopy has proved to be a valuable tool to monitor functionally important conformational transitions of nucleic acids. Here, we present a systematic investigation on the application of 5-fluoro pyrimidines to probe DNA and RNA secondary structures. Oligonucleotides with the propensity to adapt secondary structure equilibria were chosen as model systems and analyzed by 1D 19F and 1H NMR spectroscopy. A comparison with the unmodified analogs revealed that the equilibrium characteristics of the bistable DNA and RNA oligonucleotides were hardly affected upon fluorine substitution at C5 of pyrimidines. This observation was in accordance with UV spectroscopic melting experiments which demonstrated that single 5-fluoro substitutions in double helices lead to comparable thermodynamic stabilities. Thus, 5-fluoro pyrimidine labeling of DNA and RNA can be reliably applied for NMR based nucleic acid secondary structure evaluation. Furthermore, we developed a facile synthetic route towards 5-fluoro cytidine phosphoramidites that enables their convenient site-specific incorporation into oligonucleotides by solid-phase synthesis.

The metastasis-associated extracellular matrix protein osteopontin forms transient structure in ligand interaction sites.

Osteopontin (OPN) is an acidic hydrophilic glycophosphoprotein that was first identified as a major sialoprotein in bones. It functions as a cell attachment protein displaying a RGD cell adhesion sequence and as a cytokine that signals through integrin and CD44 cell adhesion molecules. OPN is also implicated in human tumor progression and cell invasion. OPN has intrinsic transforming activity, and elevated OPN levels promote metastasis. OPN gene expression is also strongly activated in avian fibroblasts simultaneously transformed by the v-myc and v-mil(raf) oncogenes. Here we have investigated the solution structure of a 220-amino acid recombinant OPN protein by an integrated structural biology approach employing bioinformatic sequence analysis, multidimensional nuclear magnetic resonance spectroscopy, synchrotron radiation circular dichroism spectroscopy, and small-angle X-ray scattering. These studies suggest that OPN is an intrinsically unstructured protein in solution. Although OPN does not fold into a single defined structure, its conformational flexibility significantly deviates from random coil-like behavior. OPN comprises distinct local secondary structure elements with reduced conformational flexibility and substantially populates a compact subspace displaying distinct tertiary contacts. These compacted regions of OPN encompass the binding sites for α(V)β(III) integrin and heparin. The conformational flexibility combined with the modular architecture of OPN may represent an important structural prerequisite for its functional diversity.

Cooperative Unfolding of Compact Conformations of the Intrinsically Disordered Protein Osteopontin

Intrinsically disordered proteins (IDPs) constitute a class of biologically active proteins that lack defined tertiary and often secondary structure. The IDP Osteopontin (OPN), a cytokine involved in metastasis of several types of cancer, is shown to simultaneously sample extended, random coil-like conformations and stable, cooperatively folded conformations. By a combination of two magnetic resonance methods, electron paramagnetic resonance and nuclear magnetic resonance spectroscopy, we demonstrate that the OPN ensemble exhibits not only characteristics of an extended and flexible polypeptide, as expected for an IDP, but also simultaneously those of globular proteins, in particular sigmoidal structural denaturation profiles. Both types of states, extended and cooperatively folded, are populated simultaneously by OPN in its apo state. The heterogeneity of the structural properties of IDPs is thus shown to even involve cooperative folding and unfolding events.