Unusually High-Affinity Mg(2+) Binding at the AU-Rich Sequence within the Antiterminator Hairpin of a Mg(2+) Riboswitch

Mg(2+) -Responsive riboswitches represent a fascinating example of bifunctional RNAs that sense Mg(2+) ions with high selectivity and autonomously regulate the expression of Mg(2+) -transporter proteins. The mechanism of the mgtA riboswitch is scarcely understood, and a detailed structural analysis is called for to study how this RNA can selectively recognize Mg(2+) and respond by switching between two alternative stem loop structures. In this work, we investigated the structure and Mg(2+) -binding properties of the lower part of the antiterminator loop C from the mgtA riboswitch of Yersinia enterocolitica by solution NMR and report a discrete Mg(2+) -binding site embedded in the AU-rich sequence. At the position of Mg(2+) binding, the helical axis exhibits a distinct kink accompanied by a widening of the major groove, which accommodates the Mg(2+) -binding pocket. An unusually large overlap between two adenine residues on the opposite strands suggests that the bending may be sequence-induced by strong stacking interactions, enabling Mg(2+) to bind at this so-far not described metal-ion binding site.     

Introduction of negative charge mimicking protein kinase C phosphorylation of cardiac troponin I. Effects on cardiac troponin C

Protein kinase C phosphorylation of cardiac troponin, the Ca(2+)-sensing switch in muscle contraction, is capable of modulating the response of cardiac muscle to a Ca(2+) ion concentration. The N-domain of cardiac troponin I contains two protein kinase C phosphorylation sites. Although the physiological consequences of phosphorylation at Ser(43)/Ser(45) are known, the molecular mechanisms responsible for these functional changes have yet to be established. In this work, NMR was used to identify conformational and dynamic changes in cardiac troponin C upon binding a phosphomimetic troponin I, having Ser(43)/Ser(45) mutated to Asp. Chemical shift perturbation mapping indicated that residues in helix G were most affected. Smaller chemical shift changes were observed in residues located in the Ca(2+)/Mg(2+)-binding loops. Amide hydrogen/deuterium exchange rates in the C-lobe of troponin C were compared in complexes containing either the wild-type or phosphomimetic N-domain of troponin I. In the presence of a phosphomimetic domain, exchange rates in helix G increased, whereas a decrease in exchange rates for residues mapping to Ca(2+)/Mg(2+)-binding loops III and IV was observed. Increased exchange rates are consistent with destabilization of the Thr(129)-Asp(132) helix capping box previously characterized in helix G. The perturbation of helix G and metal binding loops III and IV suggests that phosphorylation alters metal ion affinity and inter-subunit interactions. Our studies support a novel mechanism for protein kinase C signal transduction, emphasizing the importance of C-lobe Ca(2+)/Mg(2+)-dependent troponin interactions.     

Crystal structures of Streptococcus pneumoniae N-acetylglucosamine-1-phosphate uridyltransferase, GlmU, in apo form at 2.33 A resolution and in complex with UDP-N-acetylglucosamine and Mg(2+) at 1.96 A resolution

N-Acetylglucosamine-1-phosphate uridyltransferase (GlmU) is an essential bacterial enzyme with both an acetyltransferase and a uridyltransferase activity which have been mapped to the C-terminal and N-terminal domains, respectively. GlmU performs the last two steps in the synthesis of UDP-N-acetylglucosamine (UDP-GlcNAc), which is an essential precursor in both the peptidoglycan and the lipopolysaccharide metabolic pathways. GlmU is therefore an attractive target for potential antibiotics. Knowledge of its three-dimensional structure would provide a basis for rational drug design. We have determined the crystal structures of Streptococcus pneumoniae GlmU (SpGlmU) in apo form at 2.33 A resolution, and in complex with UDP-N-acetyl glucosamine and the essential co-factor Mg(2+) at 1.96 A resolution. The protein structure consists of an N-terminal domain with an alpha/beta-fold, containing the uridyltransferase active site, and a C-terminal domain with a long left-handed beta-sheet helix (LbetaH) domain. An insertion loop containing the highly conserved sequence motif Asn-Tyr-Asp-Gly protrudes from the left-handed beta-sheet helix domain. In the crystal, S. pneumoniae GlmU forms exact trimers, mainly through contacts between left-handed beta-sheet helix domains. UDP-N-acetylglucosamine and Mg(2+) are bound at the uridyltransferase active site, which is in a closed form. We propose a uridyltransferase mechanism in which the activation energy of the double negatively charged phosphorane transition state is lowered by charge compensation of Mg(2+) and the side-chain of Lys22.     

Solution structure of BmKK2, a new potassium channel blocker from the venom of chinese scorpion Buthus martensi Karsch

A natural K+ channel blocker, BmKK2 (a member of scorpion toxin subfamily alpha-KTx 14), which is composed of 31 amino acid residues and purified from the venom of the Chinese scorpion Buthus martensi Karsch, was characterized using whole-cell patch-clamp recording in rat hippocampal neurons. The three dimensional structure of BmKK2 was determined with two-dimensional NMR spectroscopy and molecular modelling techniques. In solution this toxin adopted a common alpha/beta-motif, but showed distinct local conformation in the loop between alpha-helix and beta-sheet in comparison with typical short-chain scorpion toxins (e.g., CTX and NTX). Also, the alpha helix is shorter and the beta-sheet element is smaller (each strand consisted only two residues). The unusual structural feature of BmKK2 was attributed to the shorter loop between the alpha-helix and beta-sheet and the presence of two consecutive Pro residues at position 21 and 22 in the loop. Moreover, two models of BmKK2/hKv1.3 channel and BmKK2/rSK2 channel complexes were simulated with docking calculations. The results demonstrated the existence of a alpha-mode binding between the toxin and the channels. The model of BmKK2/rSK2 channel complex exhibited favorable contacts both in electrostatic and hydrophobic, including a network of five hydrogen bonds and bigger interface containing seven pairs of inter-residue interactions. In contrast, the model of BmKK2/hKv1.3 channel complex, containing only three pairs of inter-residue interactions, exhibited poor contacts and smaller interface. The results well explained its lower activity towards Kv channel, and predicted that it may prefer a type of SK channel with a narrower entryway as its specific receptor.     

Solution structure and fluctuation of the Mg(2+)-bound form of calmodulin C-terminal domain

Calmodulin (CaM) is a Ca(2+)-binding protein that functions as a ubiquitous Ca(2+)-signaling molecule, through conformational changes from the "closed" apo conformation to the "open" Ca(2+)-bound conformation. Mg(2+) also binds to CaM and stabilizes its folded structure, but the NMR signals are broadened by slow conformational fluctuations. Using the E104D/E140D mutant, designed to decrease the signal broadening in the presence of Mg(2+) with minimal perturbations of the overall structure, the solution structure of the Mg(2+)-bound form of the CaM C-terminal domain was determined by multidimensional NMR spectroscopy. The Mg(2+)-induced conformational change mainly occurred in EF hand IV, while EF-hand III retained the apo structure. The helix G and helix H sides of the binding sequence undergo conformational changes needed for the Mg(2+) coordination, and thus the helices tilt slightly. The aromatic rings on helix H move to form a new cluster of aromatic rings in the hydrophobic core. Although helix G tilts slightly to the open orientation, the closed conformation is maintained. The fact that the Mg(2+)-induced conformational changes in EF-hand IV and the hydrophobic core are also seen upon Ca(2+) binding suggests that the Ca(2+)-induced conformational changes can be divided into two categories, those specific to Ca(2+) and those common to Ca(2+) and Mg(2+).     

The conformational landscape of the ribosomal protein S15 and its influence on the protein interaction with 16S RNA

The interaction between the ribosomal protein S15 and its binding sites in the 16S RNA was examined from two points of view. First, the isolated protein S15 was studied by comparing NMR conformer sets, available in the PDB and recalculated using the CNS-ARIA protocol. Molecular dynamics (MD) trajectories were then recorded starting from a conformer of each set. The recalculation of the S15 NMR structure, as well as the recording of MD trajectories, reveals that several orientations of the N-terminal alpha-helix alpha1 with respect to the structure core are populated. MD trajectories of the complex between the ribosomal protein S15 and RNA were also recorded in the presence and absence of Mg(2+) ions. The Mg(2+) ions are hexacoordinated by water and RNA oxygens. The coordination spheres mainly interact with the RNA phosphodiester backbone, reducing the RNA mobility and inducing electrostatic screening. When the Mg(2+) ions are removed, the internal mobility of the RNA and of the protein increases at the interaction interface close to the RNA G-U/G-C motif as a result of a gap between the phosphate groups in the UUCG capping tetraloop and of the disruption of S15-RNA hydrogen bonds in that region. On the other hand, several S15-RNA hydrogen bonds are reinforced, and water bridges appear between the three-way junction region and S15. The network of hydrogen bonds observed in the loop between alpha1 and alpha2 is consequently reorganized. In the absence of Mg(2+), this network has the same pattern as the network observed in the isolated protein, where the helix alpha1 is mobile with respect to the protein core. The presence of Mg(2+) ions may thus play a role in stabilizing the orientation of the helix alpha1 of S15.     

Recurrent alpha beta loop structures in TIM barrel motifs show a distinct pattern of conserved structural features.

A systematic survey of seven parallel alpha/beta barrel protein domains, based on exhaustive structural comparisons, reveals that a sizable proportion of the alpha beta loops in these proteins--20 out of a total of 49--belong to either one of two loop types previously described by Thornton and co-workers. Six loops are of the alpha beta 1 type, with one residue between the alpha-helix and beta-strand, and 13 are of the alpha beta 3 type, with three residues between the helix and the strand. Protein fragments embedding the identified loops, and termed alpha beta connections since they contain parts of the flanking helix and strand, have been analyzed in detail revealing that each type of connection has a distinct set of conserved structural features. The orientation of the beta-strand relative to the helix and loop portions is different owing to a very localized difference in backbone conformation. In alpha beta 1 connections, the chain enters the beta-strand via a residue adopting an extended conformation, while in alpha beta 3 it does so via a residue in a near alpha-helical conformation. Other conserved structural features include distinct patterns of side chain orientation relative to the beta-sheet surface and of main chain H-bonds in the loop and the beta-strand moieties. Significant differences also occur in packing interactions of conserved hydrophobic residues situated in the last turn of the helix. Yet the alpha-helix surface of both types of connections adopts similar orientations relative to the barrel sheet surface. Our results suggest furthermore that conserved hydrophobic residues along the sequence of the connections, may be correlated more with specific patterns of interactions made with neighboring helices and sheet strands than with helix/strand packing within the connection itself. A number of intriguing observations are also made on the distribution of the identified alpha beta 1 and alpha beta 3 loops within the alpha/beta-barrel motifs. They often occur adjacent to each other; alpha beta 3 loops invariably involve even numbered beta-strands, while alpha beta 1 loops involve preferentially odd beta-strands; all the analyzed proteins contain at least one alpha beta 3 loop in the first half of the eightfold alpha/beta barrel. Possible origins of all these observations, and their relevance to the stability and folding of parallel alpha/beta barrel motifs are discussed.     

Solution structure and dynamics of the human-Escherichia coli thioredoxin chimera: insights into thermodynamic stability

We have determined the high-resolution solution structure of the oxidized form of a chimeric human and Escherichia coli thioredoxin (TRX(HE)) by NMR. The overall structure is well-defined with a rms difference for the backbone atoms of 0.27 +/- 0.06 A. The topology of the protein is identical to those of the human and E. coli parent proteins, consisting of a central five-stranded beta-sheet surrounded by four alpha-helices. Analysis of the interfaces between the two domains derived from the human and E. coli sequences reveals that the general hydrophobic packing is unaltered and only subtle changes in the details of side chain interactions are observed. The packing of helix alpha(4) with helix alpha(2) across the hybrid interface is less optimal than in the parent molecules, and electrostatic interactions between polar side chains are missing. In particular, lysine-glutamate salt bridges between residues on helices alpha(2) and alpha(4), which were observed in both human and E. coli proteins, are not present in the chimeric protein. The origin of the known reduced thermodynamic stability of TRX(HE) was probed by mutagenesis on the basis of these structural findings. Two mutants of TRX(HE), S44D and S44E, were created, and their thermal and chemical stabilities were examined. Improved stability toward chaotropic agents was observed for both mutants, but no increase in the denaturation temperature was seen compared to that of TRX(HE). In addition to the structural analysis, the backbone dynamics of TRX(HE) were investigated by (15)N NMR relaxation measurements. Analysis using the model free approach reveals that the protein is fairly rigid with an average S(2) of 0.88. Increased mobility is primarily present in two external loop regions comprising residues 72-74 and 92-94 that contain glycine and proline residues.     

Calcium-induced folding of a fragment of calmodulin composed of EF-hands 2 and 3

Calmodulin (CaM) is an EF-hand protein composed of two calcium (Ca(2+))-binding EF-hand motifs in its N-domain (EF-1 and EF-2) and two in its C-domain (EF-3 and EF-4). In this study, we examined the structure, dynamics, and Ca(2+)-binding properties of a fragment of CaM containing only EF-2 and EF-3 and the intervening linker sequence (CaM2/3). Based on NMR spectroscopic analyses, Ca(2+)-free CaM2/3 is predominantly unfolded, but upon binding Ca(2+), adopts a monomeric structure composed of two EF-hand motifs bridged by a short antiparallel beta-sheet. Despite having an "even-odd" pairing of EF-hands, the tertiary structure of CaM2/3 is similar to both the "odd-even" paired N- and C-domains of Ca(2+)-ligated CaM, with the conformationally flexible linker sequence adopting the role of an inter-EF-hand loop. However, unlike either CaM domain, CaM2/3 exhibits stepwise Ca(2+) binding with a K (d1) = 30 +/- 5 microM to EF-3, and a K (d2) > 1000 microM to EF-2. Binding of the first equivalent of Ca(2+) induces the cooperative folding of CaM2/3. In the case of native CaM, stacking interactions between four conserved aromatic residues help to hold the first and fourth helices of each EF-hand domain together, while the loop between EF-hands covalently tethers the second and third helices. In contrast, these aromatic residues lie along the second and third helices of CaM2/3, and thus are positioned adjacent to the loop between its "even-odd" paired EF-hands. This nonnative hydrophobic core packing may contribute to the weak Ca(2+) affinity exhibited by EF-2 in the context of CaM2/3.     

Probing of metal-binding domains of RNA hairpin loops by laser-induced lanthanide(III) luminescence

Direct laser excitation of aqueous Eu(III) bound to specific RNA fragments was used to probe the metal-binding sites of the anticodon loop of tRNA(Phe) from E. coli and of a tetraloop containing a GNRA consensus sequence. Binding of Mg(II) or Eu(III) to either RNA fragment resulted in a higher melting transition, but no global change in structure was observed. Aqueous Eu(III) exhibits a single weak excitation peak at 17273 cm(-1), the intensity of which increased upon addition of the tRNA loop fragment. Analysis of incremental increases in the luminescence intensity upon complexation with the tRNA loop indicated a stoichiometry of one high-affinity Eu(III)-binding site per loop fragment, with a Kd of 1.3 +/- 0.2 microM. Competition experiments between Eu(III) and Mg(II) were consistent with the two metal ions binding to a common site and with an approximately 30-fold lesser affinity of the tRNA loop for Mg(II) than for Eu(III). The rate of luminescence decay following excitation of Eu(III) bound to the tRNA loop corresponded to displacement of up to 4-5 (of a possible 9) waters of hydration on binding to the tRNA loop. By comparison, Eu(III) binds to the DNA analogue of the tRNA loop with an 8-fold lesser affinity and one fewer direct coordination site than to the RNA sequence, suggesting that a 2'OH of RNA is one of the direct ligands. In contrast with the absence of a shift in the excitation peak of aqueous Eu(III) upon formation of the tRNA loop complex, direct excitation of Eu(III) bound to a GNRA tetraloop fragment resulted in a substantially blue-shifted excitation peak (17290 cm(-1)). The tetraloop fragment also has a single Eu(III)-binding site, with a Kd of 12 +/- 3 microM. The bound Eu(III) was competed by Mg(II), although the relative affinity for Mg(II) was approximately 150-450-fold less than that for Eu(III). The Eu(III)-binding site of the tetraloop site is highly dehydrated, with approximately 7 water molecules displaced upon binding by RNA ligands, suggesting that the blue-shift of the excitation peak is the result of Eu(III) specifically bound in a nonpolar site within the GNRA loop structure.     

The RTP site shared by the HIV-1 Tat protein and the 11S regulator subunit alpha is crucial for their effects on proteasome function including antigen processing

Nuclease A (NucA) from Anabaena sp. is a non-specific endonuclease able to degrade single and double-stranded DNA and RNA. The endonucleolytic activity is inhibited by the nuclease A inhibitor (NuiA), which binds to NucA with 1:1 stoichiometry and picomolar affinity. In order to better understand the mechanism of inhibition, the solution structure of NuiA was determined by NMR methods. The fold of NuiA is an alpha-beta-alpha sandwich but standard database searches by DALI and TOP revealed no structural homologies. A visual inspection of alpha-beta-alpha folds in the CATH database revealed similarities to the PR-1-like fold (SCOP nomenclature). The similarities include the ordering of secondary structural elements, a single helix on one face of the alpha-beta-alpha sandwich, and three helices on the other face. However, a major difference is in the IV helix, which in the PR-1 fold is short and perpendicular to the I and III helices, but in NuiA is long and parallel to the I and III helices. Additionally, a strand insertion in the beta-sheet makes the NuiA beta-sheet completely antiparallel in organization. The fast time-scale motions of NuiA, characterized by enhanced flexibility of the extended loop between helices III and IV, also show similarities to P14a, which is a PR-1 fold. We propose that the purpose of the PR-1 fold is to form a stable scaffold to present this extended structure for biological interactions with other proteins. This hypothesis is supported by data that show that when NuiA is bound to NucA significant changes in chemical shift occur in the extended loop between helices III and IV.     

Metal-ion affinity and specificity in EF-hand proteins: coordination geometry and domain plasticity in parvalbumin.

BACKGROUND: The EF-hand family is a large set of Ca(2+)-binding proteins that contain characteristic helix-loop-helix binding motifs that are highly conserved in sequence. Members of this family include parvalbumin and many prominent regulatory proteins such as calmodulin and troponin C. EF-hand proteins are involved in a variety of physiological processes including cell-cycle regulation, second messenger production, muscle contraction, microtubule organization and vision. RESULTS: We have determined the structures of parvalbumin mutants designed to explore the role of the last coordinating residue of the Ca(2+)-binding loop. An E101D substitution has been made in the parvalbumin EF site. The substitution decreases the Ca(2+)-binding affinity 100-fold and increases the Mg(2+)-binding affinity 10-fold. Both the Ca(2+)- and Mg(2+)-bound structures have been determined, and a structural basis has been proposed for the metal-ion-binding properties. CONCLUSIONS: The E101D mutation does not affect the Mg(2+) coordination geometry of the binding loop, but it does pull the F helix 1.1 A towards the loop. The E101D-Ca(2+) structure reveals that this mutant cannot obtain the sevenfold coordination preferred by Ca(2+), presumably because of strain limits imposed by tertiary structure. Analysis of these results relative to previously reported structural information supports a model wherein the characteristics of the last coordinating residue and the plasticity of the Ca(2+)-binding loop delimit the allowable geometries for the coordinating sphere.     

Structure of the Escherichia coli DNA polymerase III epsilon-HOT proofreading complex

The epsilon subunit of Escherichia coli DNA polymerase III possesses 3'-exonucleolytic proofreading activity. Within the Pol III core, epsilon is tightly bound between the alpha subunit (DNA polymerase) and subunit. Here, we present the crystal structure of epsilon in complex with HOT, the bacteriophage P1-encoded homolog of , at 2.1 A resolution. The epsilon-HOT interface is defined by two areas of contact: an interaction of the previously unstructured N terminus of HOT with an edge of the epsilon central beta-sheet as well as interactions between HOT and the catalytically important helix alpha1-loop-helix alpha2 motif of epsilon. This structure provides insight into how HOT and, by implication, may stabilize the epsilon subunit, thus promoting efficient proofreading during chromosomal replication.     

Structure of the Mg2+-loaded C-lobe of cardiac troponin C bound to the N-domain of cardiac troponin I: comparison with the Ca2+-loaded structure

Cardiac troponin C (cTnC) is the Ca(2+)-binding component of the troponin complex and, as such, is the Ca(2+)-dependent switch in muscle contraction. This protein consists of two globular lobes, each containing a pair of EF-hand metal-binding sites, connected by a linker. In the N lobe, Ca(2+)-binding site I is inactive and Ca(2+)-binding site II is primarily responsible for initiation of muscle contraction. The C lobe contains Ca(2+)/Mg(2+)-binding sites III and IV, which bind Mg(2+) with lower affinity and play a structural as well as a secondary role in modulating the Ca(2+) signal. To understand the structural consequences of Ca(2+)/Mg(2+) exchange in the C lobe, we have determined the NMR solution structure of the Mg(2+)-loaded C lobe, cTnC(81-161), in a complex with the N domain of cardiac troponin I, cTnI(33-80), and compared it with a refined Ca(2+)-loaded structure. The overall tertiary structure of the Mg(2+)-loaded C lobe is very similar to that of the refined Ca(2+)-loaded structure as evidenced by the root-mean-square deviation of 0.94 A for all backbone atoms. While metal-dependent conformational changes are minimal, substitution of Mg(2+) for Ca(2+) is characterized by condensation of the C-terminal portion of the metal-binding loops with monodentate Mg(2+) ligation by the conserved Glu at position 12 and partial closure of the cTnI hydrophobic binding cleft around site IV. Thus, conformational plasticity in the Ca(2+)/Mg(2+)-dependent binding loops may represent a mechanism to modulate C-lobe cTnC interactions with the N domain of cTnI.     

Specific recognition of the C-rich strand of human telomeric DNA and the RNA template of human telomerase by the first KH domain of human poly(C)-binding protein-2

Poly(C)-binding proteins (PCBPs) constitute a family of nucleic acid-binding proteins that play important roles in a wide spectrum of regulatory mechanisms. The diverse functions of PCBPs are dependent on the ability of the PCBPs to recognize poly(C) sequences with high affinity and specificity. PCBPs contain three copies of KH (hnRNP K homology) domains, which are responsible for binding nucleic acids. We have determined the NMR structure of the first KH domain (KH1) from PCBP2. The PCBP2 KH1 domain adopts a structure with three alpha-helices packed against one side of a three-stranded antiparallel beta-sheet. Specific binding of PCBP2 KH1 to a number of poly(C) RNA and DNA sequences, including the C-rich strand of the human telomeric DNA repeat, the RNA template region of human telomerase, and regulatory recognition motifs in the poliovirus-1 5'-untranslated region, was established by monitoring chemical shift changes in protein (15)N-HSQC spectra. The nucleic acid binding groove was further mapped by chemical shift perturbation upon binding to a six-nucleotide human telomeric DNA. The binding groove is an alpha/beta platform formed by the juxtaposition of two alpha-helices, one beta-strand, and two flanking loops. Whereas there is a groove in common with all of the DNA and RNA binders with a hydrophobic floor accommodating a three-residue stretch of C residues, nuances in recognizing flanking residues are provided by hydrogen bonding partners in the KH domain. Specific interactions of PCBP2 KH1 with telomeric DNA and telomerase RNA suggest that PCBPs may participate in mechanisms involved in the regulation of telomere/telomerase functions.     

Influence of ground-state structure and Mg2+ binding on folding kinetics of the guanine-sensing riboswitch aptamer domain

Riboswitch RNAs fold into complex tertiary structures upon binding to their cognate ligand. Ligand recognition is accomplished by key residues in the binding pocket. In addition, it often crucially depends on the stability of peripheral structural elements. The ligand-bound complex of the guanine-sensing riboswitch from Bacillus subtilis, for example, is stabilized by extensive interactions between apical loop regions of the aptamer domain. Previously, we have shown that destabilization of this tertiary loop-loop interaction abrogates ligand binding of the G37A/C61U-mutant aptamer domain (Gsw(loop)) in the absence of Mg(2+). However, if Mg(2+) is available, ligand-binding capability is restored by a population shift of the ground-state RNA ensemble toward RNA conformations with pre-formed loop-loop interactions. Here, we characterize the striking influence of long-range tertiary structure on RNA folding kinetics and on ligand-bound complex structure, both by X-ray crystallography and time-resolved NMR. The X-ray structure of the ligand-bound complex reveals that the global architecture is almost identical to the wild-type aptamer domain. The population of ligand-binding competent conformations in the ground-state ensemble of Gsw(loop) is tunable through variation of the Mg(2+) concentration. We quantitatively describe the influence of distinct Mg(2+) concentrations on ligand-induced folding trajectories both by equilibrium and time-resolved NMR spectroscopy at single-residue resolution.