Protoporphyrin IX Binds to Iron(II)-Loaded and to Zinc-Loaded Human Frataxin.

(1) Background: Human frataxin is an iron binding protein that participates in the biogenesis of iron sulfur clusters and enhances ferrochelatase activity. While frataxin association to other proteins has been extensively characterized up to the structural level, much less is known about the putative capacity of frataxin to interact with functionally related metabolites. In turn, current knowledge about frataxin's capacity to coordinate metal ions is limited to iron (II and III); (2) Methods: here, we used NMR spectroscopy, Molecular Dynamics, and Docking approaches to demonstrate new roles of frataxin; (3) Results: We demonstrate that frataxin also binds Zn2+ in a structurally similar way to Fe2+, but with lower affinity. In turn, both Fe2+-loaded and Zn2+-loaded frataxins specifically associate to protoporphyrin IX with micromolar affinity, while apo-frataxin does not bind to the porphyrin. Protoporphyrin IX association to metal-loaded frataxin shares the binding epitope with ferrochelatase; and (4) Conclusions: these findings expand the plethora of relevant molecular targets for frataxin and may help to elucidate the yet unknown different roles that this protein exerts in iron regulation and metabolism.

What is the Sugar Code?

A code is defined by the nature of the symbols, which are used to generate information-storing combinations (e. g. oligo- and polymers). Like nucleic acids and proteins, oligo- and polysaccharides are ubiquitous, and they are a biochemical platform for establishing molecular messages. Of note, the letters of the sugar code system (third alphabet of life) excel in coding capacity by making an unsurpassed versatility for isomer (code word) formation possible by variability in anomery and linkage position of the glycosidic bond, ring size and branching. The enzymatic machinery for glycan biosynthesis (writers) realizes this enormous potential for building a large vocabulary. It includes possibilities for dynamic editing/erasing as known from nucleic acids and proteins. Matching the glycome diversity, a large panel of sugar receptors (lectins) has developed based on more than a dozen folds. Lectins 'read' the glycan-encoded information. Hydrogen/coordination bonding and ionic pairing together with stacking and C-H/π-interactions as well as modes of spatial glycan presentation underlie the selectivity and specificity of glycan-lectin recognition. Modular design of lectins together with glycan display and the nature of the cognate glycoconjugate account for the large number of post-binding events. They give an entry to the glycan vocabulary its functional, often context-dependent meaning(s), hereby building the dictionary of the sugar code.

A conserved rRNA switch is central to decoding site maturation on the small ribosomal subunit.

While a structural description of the molecular mechanisms guiding ribosome assembly in eukaryotic systems is emerging, bacteria use an unrelated core set of assembly factors for which high-resolution structural information is still missing. To address this, we used single-particle cryo-electron microscopy to visualize the effects of bacterial ribosome assembly factors RimP, RbfA, RsmA, and RsgA on the conformational landscape of the 30S ribosomal subunit and obtained eight snapshots representing late steps in the folding of the decoding center. Analysis of these structures identifies a conserved secondary structure switch in the 16S ribosomal RNA central to decoding site maturation and suggests both a sequential order of action and molecular mechanisms for the assembly factors in coordinating and controlling this switch. Structural and mechanistic parallels between bacterial and eukaryotic systems indicate common folding features inherent to all ribosomes.

Characterizing ligand-induced conformational changes in clinically relevant galectin-1 by HN/H2O (D2O) exchange.

Glycans of cellular glycoconjugates serve as biochemical signals for a multitude of (patho)physiological processes via binding to their receptors (e.g. lectins). In the case of human adhesion/growth-regulatory galectin-1 (Gal-1), small angle neutron scattering and fluorescence correlation spectroscopy have revealed a significant decrease of its gyration radius and increase of its diffusion coefficient upon binding lactose, posing the pertinent question on the nature and region(s) involved in the underlying structural alterations. Requiring neither a neutron source nor labeling, diffusion measurements by 1H NMR spectroscopy are shown here to be sufficiently sensitive to detect this ligand-induced change. In order to figure out which region(s) of Gal-1 is (are) affected at the level of peptides, we first explored the use of H/D exchange mass spectrometry (HDX MS). Hereby, we found a reduction in proton exchange kinetics beyond the lactose-binding site. The measurement of fast HN/H2O exchange by phase-modulated NMR clean chemical exchange (CLEANEX) NMR on 15N-labeled Gal-1 then increased the spatial resolution to the level of individual amino acids. The mapped regions with increased protection from HN/H2O (D2O) exchange that include the reduction of solvent exposure around the interface can underlie the protein's compaction. These structural changes have potential to modulate this galectin's role in lattice formation on the cell surface and its interaction(s) with protein(s) at the F-face.

Backbone and sidechain NMR assignments for the ribosome maturation factor RbfA from Escherichia coli.

RbfA (ribosome binding factor A; 15.2 kDa) is a protein involved in ribosome biogenesis and has been shown to be important for growth at low temperatures and to act as a suppressor for a cold-sensitive mutation (C23U) in the ribosomal RNA of the small 30S ribosomal subunit. The 3D structure of isolated RbfA has been determined from several organisms showing that RbfA has type-II KH-domain fold topology similar to the KH domain of another assembly factor, Era, whose overexpression can compensate for the deletion of rbfA, suppressing both the cold sensitivity and abnormal accumulation of 17S rRNA in rbfA knockout stains. Interestingly, a RbfAΔ25 variant used in previous NMR studies, truncated at the C-terminal domain to remove 25 unstructured residues causing aggregation at room temperature, was biologically active in the sense that it could complement a knock-out of wildtype RbfA, although it did not act as a suppressor for a 16S cold-sensitive mutation (C23U), nor did it interact stably with the 30S subunit. To complement this work, we report the 1H, 13C, and 15 N backbone and sidechain NMR resonance assignments of full length RbfA from Escherichia coli measured under physiological conditions (pH 7.6). This construct contains seven additional C-terminal residues from the cloning (i.e. one alanine and six residues from the HRV 3C cleavage site) and no aggregation issues were observed over a 1-week period at 293 K. The assignment data has been deposited in the BMRB data bank under Accession No. 27857.

Backbone and sidechain NMR assignments for the ribosome maturation factor RimP from Escherichia coli.

Ribosome biogenesis is an energetically expensive and complex cellular process that involves the coordinated folding of the ribosomal RNA and dozens of ribosomal proteins. It proceeds along multiple parallel pathways and is guided by trans-acting factors called ribosome assembly factors. Although this process has been studied for decades, there are still many open questions regarding the role of the ribosome assembly factors in directing the folding of ribosome biogenesis intermediates. RimP is one of the early acting factors and guides the assembly of the small 30S ribosomal subunit by facilitating the binding of ribosomal proteins uS5 and uS12. Here we report the virtually complete 1H, 15N, and 13C chemical shift assignment of RimP from Escherichia coli. The NMR chemical shift data, deposited in the BMRB data bank under Accession No. 28014, indicates a widely folded protein composed of three alpha helices and eight beta strands.

Author Correction: Binding of the protein ICln to α-integrin contributes to the activation of IClswell current.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Binding of the protein ICln to α-integrin contributes to the activation of IClswell current.

IClswell is the chloride current induced by cell swelling, and plays a fundamental role in several biological processes, including the regulatory volume decrease (RVD). ICln is a highly conserved, ubiquitously expressed and multifunctional protein involved in the activation of IClswell. In platelets, ICln binds to the intracellular domain of the integrin αIIb chain, however, whether the ICln/integrin interaction plays a role in RVD is not known. Here we show that a direct molecular interaction between ICln and the integrin α-chain is not restricted to platelets and involves highly conserved amino acid motifs. Integrin α recruits ICln to the plasma membrane, thereby facilitating the activation of IClswell during hypotonicity. Perturbation of the ICln/integrin interaction prevents the transposition of ICln towards the cell surface and, in parallel, impedes the activation of IClswell. We suggest that the ICln/integrin interaction interface may represent a new molecular target enabling specific IClswell suppression in pathological conditions when this current is deregulated or plays a detrimental role.

RsgA couples the maturation state of the 30S ribosomal decoding center to activation of its GTPase pocket.

During 30S ribosomal subunit biogenesis, assembly factors are believed to prevent accumulation of misfolded intermediate states of low free energy that slowly convert into mature 30S subunits, namely, kinetically trapped particles. Among the assembly factors, the circularly permuted GTPase, RsgA, plays a crucial role in the maturation of the 30S decoding center. Here, directed hydroxyl radical probing and single particle cryo-EM are employed to elucidate RsgA΄s mechanism of action. Our results show that RsgA destabilizes the 30S structure, including late binding r-proteins, providing a structural basis for avoiding kinetically trapped assembly intermediates. Moreover, RsgA exploits its distinct GTPase pocket and specific interactions with the 30S to coordinate GTPase activation with the maturation state of the 30S subunit. This coordination validates the architecture of the decoding center and facilitates the timely release of RsgA to control the progression of 30S biogenesis.

The Novel Aminomethylcycline Omadacycline Has High Specificity for the Primary Tetracycline-Binding Site on the Bacterial Ribosome.

Omadacycline is an aminomethylcycline antibiotic with potent activity against many Gram-positive and Gram-negative pathogens, including strains carrying the major efflux and ribosome protection resistance determinants. This makes it a promising candidate for therapy of severe infectious diseases. Omadacycline inhibits bacterial protein biosynthesis and competes with tetracycline for binding to the ribosome. Its interactions with the 70S ribosome were, therefore, analyzed in great detail and compared with tigecycline and tetracycline. All three antibiotics are inhibited by mutations in the 16S rRNA that mediate resistance to tetracycline in Brachyspira hyodysenteriae, Helicobacter pylori, Mycoplasma hominis, and Propionibacterium acnes. Chemical probing with dimethyl sulfate and Fenton cleavage with iron(II)-complexes of the tetracycline derivatives revealed that each antibiotic interacts in an idiosyncratic manner with the ribosome. X-ray crystallography had previously revealed one primary binding site for tetracycline on the ribosome and up to five secondary sites. All tetracyclines analyzed here interact with the primary site and tetracycline also with two secondary sites. In addition, each derivative displays a unique set of non-specific interactions with the 16S rRNA.

Inhibition of translation initiation complex formation by GE81112 unravels a 16S rRNA structural switch involved in P-site decoding.

In prokaryotic systems, the initiation phase of protein synthesis is governed by the presence of initiation factors that guide the transition of the small ribosomal subunit (30S) from an unlocked preinitiation complex (30S preIC) to a locked initiation complex (30SIC) upon the formation of a correct codon-anticodon interaction in the peptidyl (P) site. Biochemical and structural characterization of GE81112, a translational inhibitor specific for the initiation phase, indicates that the main mechanism of action of this antibiotic is to prevent P-site decoding by stabilizing the anticodon stem loop of the initiator tRNA in a distorted conformation. This distortion stalls initiation in the unlocked 30S preIC state characterized by tighter IF3 binding and a reduced association rate for the 50S subunit. At the structural level we observe that in the presence of GE81112 the h44/h45/h24a interface, which is part of the IF3 binding site and forms ribosomal intersubunit bridges, preferentially adopts a disengaged conformation. Accordingly, the findings reveal that the dynamic equilibrium between the disengaged and engaged conformations of the h44/h45/h24a interface regulates the progression of protein synthesis, acting as a molecular switch that senses and couples the 30S P-site decoding step of translation initiation to the transition from an unlocked preIC to a locked 30SIC state.

Crystallographic characterization of the ribosomal binding site and molecular mechanism of action of Hygromycin A.

Hygromycin A (HygA) binds to the large ribosomal subunit and inhibits its peptidyl transferase (PT) activity. The presented structural and biochemical data indicate that HygA does not interfere with the initial binding of aminoacyl-tRNA to the A site, but prevents its subsequent adjustment such that it fails to act as a substrate in the PT reaction. Structurally we demonstrate that HygA binds within the peptidyl transferase center (PTC) and induces a unique conformation. Specifically in its ribosomal binding site HygA would overlap and clash with aminoacyl-A76 ribose moiety and, therefore, its primary mode of action involves sterically restricting access of the incoming aminoacyl-tRNA to the PTC.

Structural characterization of an alternative mode of tigecycline binding to the bacterial ribosome.

Although both tetracycline and tigecycline inhibit protein synthesis by sterically hindering the binding of tRNA to the ribosomal A site, tigecycline shows increased efficacy in both in vitro and in vivo activity assays and escapes the most common resistance mechanisms associated with the tetracycline class of antibiotics. These differences in activities are attributed to the tert-butyl-glycylamido side chain found in tigecycline. Our structural analysis by X-ray crystallography shows that tigecycline binds the bacterial 30S ribosomal subunit with its tail in an extended conformation and makes extensive interactions with the 16S rRNA nucleotide C1054. These interactions restrict the mobility of C1054 and contribute to the antimicrobial activity of tigecycline, including its resistance to the ribosomal protection proteins.

Solid-state NMR enhanced by dynamic nuclear polarization as a novel tool for ribosome structural biology.

The impact of Nuclear Magnetic Resonance (NMR) on studies of large macromolecular complexes hinges on improvements in sensitivity and resolution. Dynamic nuclear polarization (DNP) in the solid state can offer improved sensitivity, provided sample preparation is optimized to preserve spectral resolution. For a few nanomoles of intact ribosomes and an 800 kDa ribosomal complex we demonstrate that the combination of DNP and magic-angle spinning NMR (MAS-NMR) allows one to overcome current sensitivity limitations so that homo- and heteronuclear (13)C and (15)N NMR correlation spectra can be recorded. Ribosome particles, directly pelleted and frozen into an NMR rotor, yield DNP signal enhancements on the order of ~25-fold and spectra that exhibit narrow linewidths, suitable for obtaining site-specific information. We anticipate that the same approach is applicable to other high molecular weight complexes.

The C-terminus of ICln is natively disordered but displays local structural preformation.

ICln is a vital, ubiquitously expressed protein with roles in cell volume regulation, angiogenesis, cell morphology, activation of platelets and RNA processing. In previous work we have determined the 3D structure of the N-terminus of ICln (residues 1-159), which folds into a PH-like domain followed by an unstructured region (residues H134 - Q159) containing protein-protein interaction sites. Here we present sequence-specific resonance assignments of the C-terminus (residues Q159 - H235) of ICln by NMR, and show that this region of the protein is intrinsically unstructured. By applying (13)Cα- (13)Cß secondary chemical shifts to detect possible preferences for secondary structure elements we show that the C-terminus of ICln adopts a preferred α-helical organization between residues E170 and E187, and exists preferentially in extended conformations (ß-strands) between residues D161 to Y168 and E217 to T223.

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)ß(III) integrin and heparin. The conformational flexibility combined with the modular architecture of OPN may represent an important structural prerequisite for its functional diversity.

Autocorrelation analysis of NOESY data provides residue compactness for folded and unfolded proteins.

A novel spectral entropy interpretation for protein NOESY data is presented for the investigation of the spatial distribution of residues in protein structures without the requirement of NOE cross peak assignments. In this approach individual traces S(i)(omega) from a 3D (15)N NOESY-HSQC taken at frequency positions corresponding to different amide groups (residue position i) are subjected to a self-convolution procedure thus leading to the autocorrelation function C(i)(omega) of the NOESY-trace for a particular backbone residue position. The characteristic spatial surrounding of a particular residue position is reflected in the corresponding autocorrelation function and can be quantified by taking the (spectral) entropy S(nu) as an information measure. The feasibility of this novel approach is demonstrated with applications to the proteins Cyclophilin D and Osteopontin and the protein complex between the lipocalin Q83 and the bacterial siderophore Enterobactin. Typically, large entropy values were found for residues located in structurally loosely defined regions, whereas small entropy values were found for residues in hydrophobic core regions of the protein with tightly interacting side chains and distinct chemical shift patterns. The applications to the unfolded Osteopontin and the Q83/Enterobactin protein complex indicated that both local compaction of the polypeptide chain due to transiently formed structural elements and subtle changes in side-chain packing can be efficiently probed by this novel approach.

Direct methods and residue type specific isotope labeling in NMR structure determination and model-driven sequential assignment.

Direct methods in NMR based structure determination start from an unassigned ensemble of unconnected gaseous hydrogen atoms. Under favorable conditions they can produce low resolution structures of proteins. Usually a prohibitively large number of NOEs is required, to solve a protein structure ab-initio, but even with a much smaller set of distance restraints low resolution models can be obtained which resemble a protein fold. One problem is that at such low resolution and in the absence of a force field it is impossible to distinguish the correct protein fold from its mirror image. In a hybrid approach these ambiguous models have the potential to aid in the process of sequential backbone chemical shift assignment when (13)C(beta) and (13)C' shifts are not available for sensitivity reasons. Regardless of the overall fold they enhance the information content of the NOE spectra. These, combined with residue specific labeling and minimal triple-resonance data using (13)C(alpha) connectivity can provide almost complete sequential assignment. Strategies for residue type specific labeling with customized isotope labeling patterns are of great advantage in this context. Furthermore, this approach is to some extent error-tolerant with respect to data incompleteness, limited precision of the peak picking, and structural errors caused by misassignment of NOEs.

Backbone assignment of osteopontin, a cytokine and cell attachment protein implicated in tumorigenesis.

OPN is an RGD-containing protein overexpressed in cells transformed by v-myc and v-mil(raf) oncogenes. Here we report the resonance assignment of recombinant quail OPN and provide NMR evidence that quail OPN is an intrinsically unstructured protein in solution.