The trans labilization of cis-[PtCl2(13CH3NH2)2] by glutathione can be monitored at physiological pH by [1H,13C] HSQC NMR

In order to monitor the trans labilization of cisplatin at physiological pH we have prepared the complex cis-[PtCl₂(¹³CH₃NH₂)₂] and studied its interactions with excess glutathione in aqueous solution at neutral pH by two-dimensional [1H,13C] heteronuclear single-quantum correlation (HSQC) NMR spectroscopy. [1H,13C] HSQC spectroscopy is a good method for following the release of ¹³CH₃NH₂ but is not so good for characterizing the Pt species in solution. In the reaction of cisplatin with glutathione, Pt–S bonds are formed and Pt–NH₃ bonds are broken. The best technique for following the formation of Pt–S bonds of cisplatin is by UV spectroscopy. [1H,13C] HSQC spectroscopy is the best method for following the breaking of the Pt–N bonds. [1H,15N] HSQC spectroscopy is the best method for characterizing the different species in solution. However, the intensity of the peaks in the ¹⁵NH₃–Pt–S region, in [1H,15N] HSQC, reflects a balance between the formation of Pt–S bonds, which increases the signal intensity, and the trans labilization, which decreases the signal intensity. [1H,15N] HSQC spectroscopy and [1H,13C] HSQC spectroscopy are complementary techniques that should be used in conjunction in order to obtain the most accurate information on the interaction of platinum complexes with sulfur-containing ligands.     

NMR solution structure and characterization of substrate binding site of the PPIase domain of PrsA protein from Bacillus subtilis

PrsA is a peptidyl-prolyl isomerase (PPIase) from Bacillus subtilis belonging to the parvulin family of PPIases. It is a membrane bound lipoprotein at the membrane-wall interface, involved in folding of exported proteins. We present the NMR solution structure of the PPIase domain of PrsA, the first from a Gram-positive bacterium. In addition we mapped out the active site with NMR titration experiments. A high degree of conservation with other members of the parvulin family was revealed in the structure and binding site. Interactions with substrate peptides were also characterized by mutated domains revealing that H122 is indispensable for overall correct folding.     

The filamentous phages fd and IF1 use different mechanisms to infect Escherichia coli

The filamentous phage fd uses its gene 3 protein (G3P) to target Escherichia coli cells in a two-step process. First, the N2 domain of G3P attaches to an F pilus, and then the N1 domain binds to TolA-C. N1 and N2 are tightly associated, rendering the phage robust but noninfectious because the binding site for TolA-C is buried at the domain interface. Binding of N2 to the F pilus initiates partial unfolding, domain disassembly, and prolyl cis-to-trans isomerization in the hinge between N1 and N2. This activates the phage, and trans-Pro213 maintains this state long enough for N1 to reach TolA-C. Phage IF1 targets I pili, and its G3P contains also an N1 domain and an N2 domain. The pilus-binding N2 domains of the phages IF1 and fd are unrelated, and the N1 domains share a 31% sequence identity. We show that N2 of phage IF1 mediates binding to the I pilus, and that N1 targets TolA. Crystallographic and NMR analyses of the complex between N1 and TolA-C indicate that phage IF1 interacts with the same site on TolA-C as phage fd. In IF1-G3P, N1 and N2 are independently folding units, however, and the TolA binding site on N1 is permanently accessible. Activation by unfolding and prolyl isomerization, as in the case of phage fd, is not observed. In IF1-G3P, the absence of stabilizing domain interactions is compensated for by a strong increase in the stabilities of the individual domains. Apparently, these closely related filamentous phages evolved different mechanisms to reconcile robustness with high infectivity.     

NMR structural characterization of substrates bound to N-acetylglucosaminyltransferase V

N-Acetylglucosaminyltransferase V (GnT-V) is an enzyme involved in the biosynthesis of asparagine-linked oligosaccharides. It is responsible for the transfer of N-acetylglucosamine (GlcNAc) from the nucleotide sugar donor, uridine 5'-diphospho-N-acetylglucosamine (UDP-GlcNAc), to the 6 position of the alpha-1-6 linked Man residue in N-linked oligosaccharide core structures. GnT-V up-regulation has been linked to increased cancer invasiveness and metastasis and, appropriately, targeted for drug development. However, drug design is impeded by the lack of structural information on the protein and the way in which substrates are bound. Even though the catalytic domain of this type II membrane protein can be expressed in mammalian cell culture, obtaining structural information has proved challenging due to the size of the catalytic domain (95 kDa) and its required glycosylation. Here, we present an experimental approach to obtaining information on structural characteristics of the active site of GnT-V through the investigation of the bound conformation and relative placement of its ligands, UDP-GlcNAc and beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-(1-->6)-beta-D-GlcpOOctyl. Nuclear magnetic resonance (NMR) spectroscopy experiments, inducing transferred nuclear Overhauser effect (trNOE) and saturation transfer difference (STD) experiments, were used to characterize the ligand conformation and ligand-protein contact surfaces. In addition, a novel paramagnetic relaxation enhancement experiment using a spin-labeled ligand analogue, 5'-diphospho-4-O-2,2,6,6-tetramethylpiperidine 1-oxyl (UDP-TEMPO), was used to characterize the relative orientation of the two bound ligands. The structural information obtained for the substrates in the active site of GnT-V can be useful in the design of inhibitors for GnT-V.     

Global fold and backbone dynamics of the hepatitis C virus E2 glycoprotein transmembrane domain determined by NMR

E1 and E2 are two hepatitis C viral envelope glycoproteins that assemble into a heterodimer that is essential for membrane fusion and penetration into the target cell. Both extracellular and transmembrane (TM) glycoprotein domains contribute to this interaction, but study of TM–TM interactions has been limited because synthesis and structural characterization of these highly hydrophobic segments present significant challenges. In this NMR study, by successful expression and purification of the E2 transmembrane domain as a fusion construct we have determined the global fold and characterized backbone motions for this peptide incorporated in phospholipid micelles. Backbone resonance frequencies, relaxation rates and solvent exposure measurements concur in showing this domain to adopt a helical conformation, with two helical segments spanning residues 717–726 and 732–746 connected by an unstructured linker containing the charged residues D728 and R730 involved in E1 binding. Although this linker exhibits increased local motions on the ps timescale, the dominating contribution to its relaxation is the global tumbling motion with an estimated correlation time of 12.3 ns. The positioning of the helix–linker–helix architecture within the mixed micelle was established by paramagnetic NMR spectroscopy and phospholipid-peptide cross relaxation measurements. These indicate that while the helices traverse the hydrophobic interior of the micelle, the linker lies closer to the micelle perimeter to accommodate its charged residues. These results lay the groundwork for structure determination of the E1/E2 complex and a molecular understanding of glycoprotein heterodimerization.     

Probing the structure of the Ff bacteriophage major coat protein transmembrane helix dimer by solution NMR

The transmembrane (TM) segment of the major coat protein from Ff bacteriophage has been extensively studied as an example of dimerization in detergent and lipid bilayer systems. However, almost all the information regarding this interaction has been gained through mutagenesis studies, with little direct structural information being available. To this end solution NMR has the potential to provide new insights into structure of the dimer. In order to evaluate the utility of this approach we have studied a selectively ¹⁵N-labeled peptide containing the TM segment of MCP (MCP_TM) by solution NMR. This peptide was found to give rise to detergent concentration-dependent spectra that were assigned to monomeric and dimeric forms. The standard free energy of this interaction in SDS was estimated from these spectra and found to be consistent with weak but specific dimerization. In addition, similar spectra could be obtained in β-octyl glucoside with intermolecular paramagnetic relaxation experiments demonstrating a parallel arrangement of TM helices in the dimer. In both detergents backbone chemical shift differences between monomeric and dimeric forms of MCP_TM showed that the largest changes occur around its GXXXG motif. The resulting structural model is consistent with observations made for MCP mutants previously characterized in biological membranes, opening the door to detailed structural characterization of this form of MCP. These results also have general implications for the study of weakly interacting TM segments by solution NMR since the use of similar sample conditions should allow structural data to be accessed for oligomeric states from a wide range systems that undergo biologically relevant but weak associations in the membrane.     

The solution structure of the adhesion protein Bd37 from Babesia divergens reveals structural homology with eukaryotic proteins involved in membrane trafficking

Babesia divergens is the Apicomplexa agent of the bovine babesiosis in Europe: this infection leads to growth and lactation decrease, so that economical losses due to this parasite are sufficient to require the development of a vaccine. The major surface antigen of B. divergens has been described as a 37 kDa protein glycosyl phosphatidyl inositol (GPI)-anchored at the surface of the merozoite. The immuno-prophylactic potential of Bd37 has been demonstrated, and we present here the high-resolution solution structure of the 27 kDa structured core of Bd37 (Δ-Bd37) using NMR spectroscopy. A model for the whole protein has been obtained using additional small angle X-ray scattering (SAXS) data. The knowledge of the 3D structure of Bd37 allowed the precise epitope mapping of antibodies on its surface. Interestingly, the geometry of Δ-Bd37 reveals an intriguing similarity with the exocyst subunit Exo84p C-terminal region, an eukaryotic protein that has a direct implication in vesicle trafficking. This strongly suggests that Apicomplexa have developed in parallel molecular machines similar in structure and function to the ones used for endo- and exocytosis in eukaryotic cells.     

Lipid chain-length dependence for incorporation of alamethicin in membranes: electron paramagnetic resonance studies on TOAC-spin labeled analogs

Alamethicin is a 19-residue hydrophobic peptide, which is extended by a C-terminal phenylalaninol but lacks residues that might anchor the ends of the peptide at the lipid-water interface. Voltage-dependent ion channels formed by alamethicin depend strongly in their characteristics on chain length of the host lipid membranes. EPR spectroscopy is used to investigate the dependence on lipid chain length of the incorporation of spin-labeled alamethicin in phosphatidylcholine bilayer membranes. The spin-label amino acid TOAC is substituted at residue positions n = 1, 8, or 16 in the sequence of alamethicin F50/5 [TOAC(n), Glu(OMe)(7,18,19)]. Polarity-dependent isotropic hyperfine couplings of the three TOAC derivatives indicate that alamethicin assumes approximately the same location, relative to the membrane midplane, in fluid diC(N)PtdCho bilayers with chain lengths ranging from N = 10-18. Residue TOAC(8) is situated closest to the bilayer midplane, whereas TOAC(16) is located farther from the midplane in the hydrophobic core of the opposing lipid leaflet, and TOAC(1) remains in the lipid polar headgroup region. Orientational order parameters indicate that the tilt of alamethicin relative to the membrane normal is relatively small, even at high temperatures in the fluid phase, and increases rather slowly with decreasing chain length (from 13 degrees to 23 degrees for N = 18 and 10, respectively, at 75 degrees C). This is insufficient for alamethicin to achieve hydrophobic matching. Alamethicin differs in its mode of incorporation from other helical peptides for which transmembrane orientation has been determined as a function of lipid chain length.     

Molecular and structural analysis of C4-specific PEPC isoform from Pennisetum glaucum plays a role in stress adaptation

As an activator of adenylate cyclase, the neuropeptide Pituitary Adenylate Cyclase Activating Peptide (PACAP) impacts levels of cyclic AMP, a key second messenger available in brain cells. PACAP is involved in certain adult behaviors. To elucidate PACAP interactions, a compendium of microarrays representing mRNA expression in the adult mouse whole brain was pooled from the Phenogen database for analysis. A regulatory network was computed based on mutual information between gene pairs using gene expression data across the compendium. Clusters among genes directly linked to PACAP, and probable interactions between corresponding proteins were computed. Database “experts” affirmed some of the inferred relationships. The findings suggest ADCY7 is probably the adenylate cyclase isoform most relevant to PACAP's action. They also support intervening roles for kinases including GSK3B, PI 3-kinase, SGK3 and AMPK. Other high-confidence interactions are hypothesized for future testing. This new information has implications for certain behavioral and other disorders.     

NMR studies on fully hydrated membrane proteins, with emphasis on bacteriorhodopsin as a typical and prototype membrane protein

The 3D structures or dynamic feature of fully hydrated membrane proteins are very important at ambient temperature, in relation to understanding their biological activities, although their data, especially from the flexible portions such as surface regions, are unavailable from X-ray diffraction or cryoelectron microscope at low temperature. In contrast, high-resolution solid-state NMR spectroscopy has proved to be a very convenient alternative means to be able to reveal their dynamic structures. To clarify this problem, we describe here how we are able to reveal such structures and dynamic features, based on intrinsic probes from high-resolution solid-state NMR studies on bacteriorhodopsin (bR) as a typical membrane protein in 2D crystal, regenerated preparation in lipid bilayer and detergents. It turned out that their dynamic features are substantially altered upon their environments where bR is present. We further review NMR applications to study structure and dynamics of a variety of membrane proteins, including sensory rhodopsin, rhodopsin, photoreaction centers, diacylglycerol kinases, etc.     

Dissection of protein-protein interaction and CDK4 inhibition in the oncogenic versus tumor suppressing functions of gankyrin and P16

Protein-protein interactions usually involve a large number of residues; thus it is difficult to elucidate functional and structural roles of specific residues located in the interface. This problem is particularly challenging for ankyrin repeat proteins (ARs), which consist of linear arrays of small repeating units and play critical roles in almost every life process via protein-protein interactions, because the residues involved are discontinuously dispersed in both the ARs and their partners. Our previous studies showed that while both specific CDK4 inhibitor p16INK4A (P16) and gankyrin bind to cyclin-dependent kinase 4 (CDK4) in similar fashion, only P16 inhibits the kinase activity of CDK4. While this could explain why P16 is a tumor suppressor and gankyrin is oncogenic, the structural basis of these contrasting properties was unknown. Here we show that a double mutant of gankyrin, L62H/I79D, inhibits the kinase activity of CDK4, similar to P16, and such CDK4-inhibtory activity is associated with the I79D but not L62H mutation. In addition, mutations at I79 and L62 bring about a moderate decrease in the stability of gankyrin. Further structural and biophysical analyses suggest that the substitution of Ile79 with Asp leads to local conformational changes in loops I-III of gankyrin. Taken together, our results allow the dissection of the "protein-protein binding" and "CDK4 inhibition" functions of P16, show that the difference between tumor suppressing and oncogenic functions of P16 and gankyrin, respectively, mainly resides in a single residue, and provide structural insight to the contrasting biological functions of the two AR proteins.     

A hybrid structural model of the complete Brugia malayi cytoplasmic asparaginyl-tRNA synthetase

Aminoacyl-tRNA synthetases are validated molecular targets for anti-infective drug discovery because of their essentiality in protein synthesis. Thanks to genome sequencing, it is now possible to systematically study aminoacyl-tRNA synthetases from human eukaryotic parasites as putative targets for novel drug discovery. As part of a program targeting class IIb asparaginyl-tRNA synthetases (AsnRS) from the parasitic nematode Brugia malayi for anti-filarial drugs, we report the complete structure of a eukaryotic AsnRS. Metazoan and fungal AsnRS differ from their bacterial homologues by the addition of a conserved N-terminal extension of about 110 residues whose structure we have determined by solution NMR for the B. malayi enzyme. In addition, we solved by X-ray crystallography a series of structures of the catalytically active N-terminally truncated enzyme (residues 112-548), allowing the structural basis for the mechanism of asparagine activation to be elucidated. The N-terminal domain contains a structured region with a novel fold featuring a lysine-rich helix that is shown by NMR to interact with tRNA. This is connected by an unstructured tether to the remainder of the enzyme, which is highly similar to the known structure of bacterial AsnRS. These data enable a model of the complete AsnRS-tRNA complex to be constructed.     

Toward protein structure in situ: comparison of two bifunctional rhodamine adducts of troponin C

As part of a program to develop methods for determining protein structure in situ, sTnC was labeled with a bifunctional rhodamine (BR or BSR), cross-linking residues 56 and 63 of its C-helix. NMR spectroscopy of the N-terminal domain of BSR-labeled sTnC in complex with Ca(2+) and the troponin I switch peptide (residues 115-131) showed that BSR labeling does not significantly affect the secondary structure of the protein or its dynamics in solution. BR-labeling was previously shown to have no effect on the solution structure of this complex. Isometric force generation in isolated demembranated fibers from rabbit psoas muscle into which BR- or BSR-labeled sTnC had been exchanged showed reduced Ca(2+)-sensitivity, and this effect was larger with the BSR label. The orientation of rhodamine dipoles with respect to the fiber axis was determined by polarized fluorescence. The mean orientations of the BR and BSR dipoles were almost identical in relaxed muscle, suggesting that both probes accurately report the orientation of the C-helix to which they are attached. The BSR dipole had smaller orientational dispersion, consistent with less flexible linkers between the rhodamine dipole and cysteine-reactive groups.     

Ligand binding mode of GABAA receptor-associated protein

The γ-aminobutyric acid type A (GABA_A) receptor-associated protein is a versatile adaptor protein playing an important role in intracellular vesicle trafficking, particularly in neuronal cells. We present the X-ray structure of the soluble form of human GABA_A receptor-associated protein complexed with a high-affinity synthetic peptide at 1.3 Å resolution. The data shed light on the probable binding modes of key interaction partners, including the GABA_A receptor and the cysteine protease Atg4. The resulting models provide a structural background for further investigation of the unique biological properties of this protein.     

Identification of a novel ubiquitin binding site of STAM1 VHS domain by NMR spectroscopy

Interaction between the signal-transducing adapter molecule 1 (STAM1) Vps27/Hrs/Stam (VHS) domain and ubiquitin was investigated by nuclear magnetic resonance (NMR) spectroscopy. NMR evidence showed that the structure of STAM1 VHS domain resembles that of other VHS domains, especially the homologous domain of STAM2. We found that the VHS domain binds to ubiquitin via its hydrophobic patch consisting of N-terminus of helix 2 and C-terminus of helix 4 in which Trp26 on helix 2 plays a pivotal role in the binding. The binding between VHS and ubiquitin seems to be very similar to that between ubiquitin associated domain (UBA) and ubiquitin, however, the direction of α-helices involved in the ubiquitin binding is opposite. Here, we propose a novel ubiquitin binding site and the manner of ubiquitin recognition of the STAM1 VHS domain.

Structured summary

MINT-6804185:STAM1 (uniprotkb:Q92783) binds (MI:0407) to ubiquitin (uniprotkb:P62988) by nuclear magnetic resonance (MI:0077)     

NMR analysis of partially folded states and persistent structure in the alpha subunit of tryptophan synthase: implications for the equilibrium folding mechanism of a 29-kDa TIM barrel protein

Structural insights into the equilibrium folding mechanism of the alpha subunit of tryptophan synthase (αTS) from Escherichia coli, a (βα)₈ TIM barrel protein, were obtained with a pair of complementary nuclear magnetic resonance (NMR) spectroscopic techniques. The secondary structures of rare high-energy partially folded states were probed by native-state hydrogen-exchange NMR analysis of main-chain amide hydrogens. 2D heteronuclear single quantum coherence NMR analysis of several ¹⁵N-labeled nonpolar amino acids was used to probe the side chains involved in stabilizing a highly denatured intermediate that is devoid of secondary structure. The dynamic broadening of a subset of isoleucine and leucine side chains and the absence of protection against exchange showed that the highest energy folded state on the free-energy landscape is stabilized by a hydrophobic cluster lacking stable secondary structure. The core of this cluster, centered near the N-terminus of αTS, serves as a nucleus for the stabilization of what appears to be nonnative secondary structure in a marginally stable intermediate. The progressive decrease in protection against exchange from this nucleus toward both termini and from the N-termini to the C-termini of several β-strands is best described by an ensemble of weakly coupled conformers. Comparison with previous data strongly suggests that this ensemble corresponds to a marginally stable off-pathway intermediate that arises in the first few milliseconds of folding and persists under equilibrium conditions. A second, more stable intermediate, which has an intact β-barrel and a frayed α-helical shell, coexists with this marginally stable species. The conversion of the more stable intermediate to the native state of αTS entails the formation of a stable helical shell and completes the acquisition of the tertiary structure.     

Structural characterization of the pore forming protein TatAd of the twin-arginine translocase in membranes by solid-state N-NMR

The transmembrane protein TatA is the pore forming unit of the twin-arginine translocase (Tat), which has the unique ability of transporting folded proteins across the cell membrane. This ATP-independent protein export pathway is a recently discovered alternative to the general secretory (Sec) system of bacteria. To obtain insight in the translocation mechanism, the structure and alignment in the membrane of the well-folded segments 2-45 of TatA_d from Bacillus subtilis was studied here. Using solid-state NMR in bicelles containing anionic lipids, the topology and orientation of TatA_d was determined in an environment mimicking the bacterial membrane. A wheel-like pattern, characteristic for a tilted transmembrane helix, was observed in ¹⁵N chemical shift /¹⁵N-¹H dipolar coupling correlation NMR spectra. Analysis of this PISA wheel revealed a 14-16 residue long N-terminal membrane-spanning helix which is tilted by 17° with respect to the membrane normal. In addition, comparison of uniformly and selectively ¹⁵N-labeled TatA_2-45 samples allowed determination of the helix polarity angle.     

NADPH oxidase and eNOS control cardiomyogenesis in mouse embryonic stem cells on ascorbic acid treatment

Ascorbic acid (AA) increases cardiomyogenesis of embryonic stem (ES) cells. Herein we show that treatment of mouse ES cells with AA enhanced cardiac differentiation accompanied by an upregulation of the NADPH oxidase isoforms NOX2 and NOX4, phosphorylation of endothelial nitric oxide synthase (eNOS), and cyclic GMP (cGMP) formation, indicating that reactive oxygen species (ROS) as well as nitric oxide (NO) may be involved in cardiomyogenesis. In whole mount embryoid bodies as well as isolated Flk-1-positive (Flk-1⁺) cardiovascular progenitor cells ROS elevation by AA was observed in early stages of differentiation (Days 4-7), and absent at Day 10. In contrast NO generation following incubation with AA was absent at Day 4 and increased at Days 7 and 10. AA-mediated cardiomyogenesis was blunted by the NADPH oxidase inhibitors diphenylen iodonium (DPI) and apocynin, the free radical scavengers N-(2-mercaptopropionyl)-glycine (NMPG) and ebselen, and the NOS inhibitor L-NAME. Downregulation of NOX4 by short hairpin RNA (shRNA) resulted in significant inhibition of cardiomyogenesis and abolished the stimulation of MHC-ß and MLC2v gene expression observed on AA treatment. Our data demonstrate that AA stimulates cardiomyocyte differentiation from ES cells by signaling pathways that involve ROS generated at early stages and NO at late stages of cardiomyogenesis.     

Roles of reactive oxygen and nitrogen species in pain

Peroxynitrite (PN; ONOO⁻) and its reactive oxygen precursor superoxide (SO; O₂^•−) are critically important in the development of pain of several etiologies including pain associated with chronic use of opiates such as morphine (also known as opiate-induced hyperalgesia and antinociceptive tolerance). This is now an emerging field in which considerable progress has been made in terms of understanding the relative contributions of SO, PN, and nitroxidative stress in pain signaling at the molecular and biochemical levels. Aggressive research in this area is poised to provide the pharmacological basis for development of novel nonnarcotic analgesics that are based upon the unique ability to selectively eliminate SO and/or PN. As we have a better understanding of the roles of SO and PN in pathophysiological settings, targeting PN may be a better therapeutic strategy than targeting SO. This is because, unlike PN, which has no currently known beneficial role, SO may play a significant role in learning and memory [1]. Thus, the best approach may be to spare SO while directly targeting its downstream product, PN. Over the past 15 years, our team has spearheaded research concerning the roles of SO and PN in pain and these results are currently leading to the development of solid therapeutic strategies in this important area.