NMR assignments of the N-terminal signaling domain of the TonB-dependent outer membrane transducer PupB

Outer membrane TonB-dependent transducers (TBDTs) actively transport ferric siderophore complexes from the extracellular environment into Gram-negative bacteria. They also participate in a cell-surface signaling regulatory pathway that results in upregulation of the transducer itself, in response to iron-deplete conditions. The TBDT PupB transports ferric pseudobactin, and signals through its N-terminal signaling domain (NTSD), while the TBDT homolog PupA is signaling-inactive. Here, we report the NMR chemical shift assignments of the PupB-NTSD. This information will provide the basis for structural characterization of the PupB-NTSD to further explore its signaling properties.     

The plug domain of a neisserial TonB-dependent transporter retains structural integrity in the absence of its transmembrane beta-barrel

Transferrin binding protein A (TbpA) is a TonB-dependent outer membrane protein expressed by pathogenic bacteria for iron acquisition from human transferrin. The N-terminal 160 residues (plug domain) of TbpA were overexpressed in both the periplasm and cytoplasm of Escherichia coli. We found this domain to be soluble and monodisperse in solution, exhibiting secondary structure elements found in plug domains of structurally characterized TonB-dependent transporters. Although the TbpA plug domain is apparently correctly folded, we were not able to observe an interaction with human transferrin by isothermal titration calorimetry or nitrocellulose binding assays. These experiments suggest that the plug domain may fold independently of the beta-barrel, but extracellular loops of the beta-barrel are required for ligand binding.     

The reversible heat denaturation of chymotrypsinogen

Within a restricted range of pH and protein concentration crystalline chymotrypsinogen undergoes thermal denaturation which is wholly reversed upon cooling. At a given temperature an equilibrium exists between native and reversibly denatured protein. Within the pH range 2 to 3 the amount of denatured protein is a function of the third power of the hydrogen ion activity. The presence of small amounts of electrolyte causes aggregation of the reversibly denatured protein. A specific anion effect has been observed at pH 2 but not at pH 3. Both the reversible denaturation reaction and the reversal reaction have been found to be first order reactions with respect to protein and the kinetic and thermodynamic constants for both reactions have been approximated at pH 2 and at pH 3. Renatured chymotrypsinogen has been found to be identical with native chymotrypsinogen with respect to crystallizability, solubility, activation to δ-chymotrypsin, sedimentation rate, and behavior upon being heated. Irreversible denaturation of chymotrypsinogen has been found to depend on pH, temperature, protein concentration, and time of heating. Irreversible denaturation results in an aggregation of the denatured protein.     

Chloride transporter KCC2-dependent neuroprotection depends on the N-terminal protein domain

Neurodegeneration is a serious issue of neurodegenerative diseases including epilepsy. Downregulation of the chloride transporter KCC2 in the epileptic tissue may not only affect regulation of the polarity of GABAergic synaptic transmission but also neuronal survival. Here, we addressed the mechanisms of KCC2-dependent neuroprotection by assessing truncated and mutated KCC2 variants in different neurotoxicity models. The results identify a threonine- and tyrosine-phosphorylation-resistant KCC2 variant with increased chloride transport activity, but they also identify the KCC2 N-terminal domain (NTD) as the relevant minimal KCC2 protein domain that is sufficient for neuroprotection. As ectopic expression of the KCC2-NTD works independently of full-length KCC2-dependent regulation of Cl⁻ transport or structural KCC2 C-terminus-dependent regulation of synaptogenesis, our study may pave the way for a selective neuroprotective therapeutic strategy that will be applicable to a wide range of neurodegenerative diseases.Neurodegeneration restricts neuron numbers during development but can become a serious issue in disease conditions such as temporal lobe epilepsy (TLE).¹ GABA-activated Cl⁻ channels contribute to activity-dependent refinement of neural networks by triggering the so-called giant depolarizing potentials providing developing neurons with a sense of activity essential for neuronal survival and co-regulation of excitatory glutamatergic and (inhibitory) GABAergic synapses.² By regulating transmembrane Cl⁻ gradients KCC2 plays a vital role in development and disease.³ In addition, KCC2 plays a protein structural role in spine formation through its C-terminal protein domain (CTD).^{4, 5} Hence, regulation of KCC2 expression and function is relevant for development and disease-specific plasticity of neural networks.^{6, 7, 8, 9}GlyR α3K RNA editing leads to proline-to-leucine substitution (P185L) in the ligand-binding domain and generates gain-of-function neurotransmitter receptors.^{10, 11, 12, 13} GlyR RNA editing is upregulated in the hippocampus of patients with TLE and leads to GlyR α3K^185L-dependent tonic inhibition of neuronal excitability associated with neurodegeneration.¹⁴ KCC2 expression promotes neuroprotection^{14, 15} but whether this involves regulation of transmembrane Cl⁻ gradient or protein structural role is a matter of debate.^{14, 15}Here, we assessed neuroprotection through several KCC2 variants in two different models of neurodegeneration including chronic neuronal silencing (α3K^185L model) and acute neuronal overexcitation (NMDA model).^{14, 15} The results identify a threonine- and tyrosine-phosphorylation-resistant KCC2 variant with increased Cl⁻ transport activity, but they also demonstrate that the N-terminal KCC2 protein domain (NTD) is sufficient for neuroprotection.     

PP3 forms stable tetrameric structures through hydrophobic interactions via the C-terminal amphipathic helix and undergoes reversible thermal dissociation and denaturation

The milk protein proteose peptone component 3 (PP3), also called lactophorin, is a small phosphoglycoprotein that is expressed exclusively in lactating mammary tissue. The C-terminal part of the protein contains an amphipathic helix, which, upon proteolytic liberation, shows antibacterial activity. Previous studies indicate that PP3 forms multimeric structures and inhibits lipolysis in milk. PP3 is the principal component of the proteose peptone fraction of milk. This fraction is obtained by heating and acidifying skimmed milk, and in the dairy industry milk products are also typically exposed to treatments such as pasteurization, which potentially could result in irreversible denaturation and inactivation of bioactive components. We show here, by the use of CD, that PP3 undergoes reversible thermal denaturation and that the α-helical structure of PP3 remains stable even at gastric pH levels. This suggests that the secondary structure survives treatment during the purification and possibly some of the industrial processing of milk. Finally, asymmetric flow field-flow fractionation and multi-angle light scattering reveal that PP3 forms a rather stable tetrameric complex, which dissociates and unfolds in guanidinium chloride. The cooperative unfolding of PP3 was completely removed by the surfactant n-dodecyl-β-d-maltoside and by oleic acid. We interpret this to mean that the PP3 monomers associate through hydrophobic interactions via the hydrophobic surface of the amphipathic helix. These observations suggest that PP3 tetramers act as reservoirs of PP3 molecules, which in the monomeric state may stabilize the milk fat globule.     

Nuclear magnetic resonance solution structure of the periplasmic signalling domain of the TonB-dependent outer membrane transporter FecA from Escherichia coli

Gram-negative bacteria possess outer membrane receptors that utilize energy provided by the TonB system to take up iron. Several of these receptors participate in extracytoplasmic factor (ECF) signalling through an N-terminal signalling domain that interacts with a periplasmic transmembrane anti-sigma factor protein and a cytoplasmic sigma factor protein. The structures of the intact TonB-dependent outer membrane receptor FecA from Escherichia coli and FpvA from Pseudomonas aeruginosa have recently been solved by protein crystallography; however, no electron density was detected for their periplasmic signalling domains, suggesting that it was either unfolded or flexible with respect to the remainder of the protein. Here we describe the well-defined solution structure of this domain solved by multidimensional nuclear magnetic resonance (NMR) spectroscopy. The monomeric protein construct contains the 79-residue N-terminal domain as well as the next 17 residues that are part of the receptor's plug domain. These form two clearly distinct regions: a highly structured domain at the N-terminal end followed by an extended flexible tail at the C-terminal end, which includes the 'TonB-box' region, and connects it to the plug domain of the receptor. The structured region consists of two alpha-helices that are positioned side by side and are sandwiched in between two small beta-sheets. This is a novel protein fold which appears to be preserved in all the periplasmic signalling domains of bacterial TonB-dependent outer membrane receptors that are involved in ECF signalling, because the hydrophobic residues that make up the core of the protein domain are highly conserved.     

Unusual cold denaturation of a small protein domain

A thermal unfolding study of the 45-residue α-helical domain UBA(2) using circular dichroism is presented. The protein is highly thermostable and exhibits a clear cold unfolding transition with the onset near 290 K without denaturant. Cold denaturation in proteins is rarely observed in general and is quite unique among small helical protein domains. The cold unfolding was further investigated in urea solutions, and a simple thermodynamic model was used to fit all thermal and urea unfolding data. The resulting thermodynamic parameters are compared to those of other small protein domains. Possible origins of the unusual cold unfolding of UBA(2) are discussed.     

Site-specific pegylation of G-CSF by reversible denaturation

A new strategy has been developed for extending the possibility of poly(ethylene glycol) (PEG) modification to accessible thiol groups of biologically active proteins. In particular, thiol-reactive PEGs have been coupled to the cysteine 17 of granulocyte colony stimulating factor (G-CSF), which is known to be partially buried in a hydrophobic protein pocket. The PEG linking was accomplished by partial protein denaturation with 3 M guanidine.HCl in the absence of any reducing agent in order to preserve the native protein's disulfide bridges. PEG coupling occurred also, but at a lower degree, by using a 3 M solution of urea as the denaturing agent. Following the PEGylation, which was carried out in the unfolded state, the conjugated protein was refolded using dialysis or gel filtration chromatography to eliminate the denaturant. Different thiol-reactive PEGs and polymer molecular weights (5, 10, or 20 kDa) were investigated for G-CSF conjugation under denaturation. The secondary structure of the protein in the G-CSF-PEG conjugates, evaluated using circular dichroism and biological activity assay in cell culture, was maintained with respect to the native protein. Unexpectedly, conjugation enhanced the G-CSF tendency to aggregate, a problem that was overcome by a proper formulation.     

The N-terminal domain of mammalian Lysyl-tRNA synthetase is a functional tRNA-binding domain.

Lysyl-tRNA synthetase from higher eukaryotes possesses a lysine-rich N-terminal polypeptide extension appended to a classical prokaryotic-like LysRS domain. Band shift analysis showed that this extra domain provides LysRS with nonspecific tRNA binding properties. A N-terminally truncated derivative of LysRS, LysRS-DeltaN, displayed a 100-fold lower apparent affinity for tRNA(3)Lys and a 3-fold increase in K(m) for tRNA(3)Lys in the aminoacylation reaction, as compared with the native enzyme. The isolated N-domain of LysRS also displayed weak affinity for tRNA, suggesting that the catalytic and N-domains of LysRS act synergistically to provide a high affinity binding site for tRNA. A more detailed analysis revealed that LysRS binds and specifically aminoacylates an RNA minihelix mimicking the amino acid acceptor stem-loop structure of tRNA(3)Lys, whereas LysRS-DeltaN did not. As a consequence, merging an additional RNA-binding domain into a bacterial-like LysRS increases the catalytic efficiency of the enzyme, especially at the low concentration of deacylated tRNA prevailing in vivo. Our results provide new insights into tRNA(Lys) channeling in eukaryotic cells and shed new light on the possible requirement of native LysRS for triggering tRNA(3)Lys packaging into human immunodeficiency virus, type 1 viral particles.     

1H, 13C and 15N resonance assignments of the periplasmic signalling domain of HasR, a TonB-dependent outer membrane heme transporter

TonB-dependent transporters (TBDTs) are bacterial outer membrane proteins that internalize nutrients such as vitamin B12, metal complexes, heme, some carbohydrates, etc. In addition to their transport activity, several TBDTs are also involved in a signalling cascade from the cell surface into the cytoplasm, via their periplasmic signalling domain. Here we report the backbone and side chain resonance assignments of the signalling domain of HasR, a TonB-dependent outer membrane heme transporter from Serratia marcescens as a first step towards its structural study.     

The two-step reversible denaturation of lactate dehydrogenase at low pH.

Upon exposure to conditions of low pH, beef B4 lactate dehydrogenase rapidly loses enzymatic activity, but this process can be completely reversed yielding 100% of the original activity if the enzyme is immediately returned to neutral conditions. As the time of exposure to low pH is increased, the fraction of activity recovered declines to a values of 50--60% and remains nearly constant over a period of many hours. Correlated with this behavior is a change in the kinetics of the recovery of activity. Recovery of activity has been shown to be a second-order process for enzyme exposed to low pH for brief periods of time (Anderson, S., and Weber, G. (1966), Arch. Biochem. Biophys. 116, 207). After several minutes at low pH recovery of activity is found to become first order and to occur at a considerably slower rate. Gel filtration chromatography at low pH separates the protein into two fractions. The lower molecular weight fraction is found to be primarily monomeric, as indicated by equilibrium ultracentrifugation, and is capable of recovering enzymatic activity. The higher molecular weight fraction is generated from the lower molecular weight fraction, and is incapable of recovering activity. These results are interpreted to indicate that the enzyme exists sequentially in three denatured forms at low pH, the first two capable of being restored to the native state, and the third irreversibly denatured.     

Theory of reversible denaturation of globular proteins.

A theoretical method is developed by which the character of the process of protein denaturation (e.g., whether or not it is of the all-or-none type) can be discussed in terms of conformation of native proteins and the forces stabilizing it. An important role is played by a quantity S(H): entropy of a protein molecule in solution in the conformational states with a given value of enthalpy H. It is demonstrated that the all-or-none type denaturation of proteins is a rather direct consequence of the globularity and specificity of the native conformations. Denaturations with significant intermediate states are discussed. Denaturations induced by added denaturants are also discussed.     

The kinetics and thermodynamics of reversible denaturation of crystalline soybean trypsin inhibitor

Crystalline soybean trypsin inhibitor protein undergoes denaturation on heating which is reversed on cooling. In the range of temperature of 35 to 50 degrees C. a solution of the protein consists of a mixture of native and denatured forms in equilibrium with each other. The equilibrium is only slowly established and its final value at any temperature is the same whether a heated, denatured solution of the protein is cooled to the given temperature or whether a fresh solution is raised to that temperature. The kinetics of reversible denaturation of the soybean protein as well as the reversal of denaturation is that of a reversible unimolecular reaction, each process consisting at a given temperature of the same two simultaneous reactions acting in opposite directions. The experimental data on the effect of temperature on the velocity and the equilibrium constants of the opposing reaction were utilized in evaluating the reaction energies and activation energies. The reaction energies for denaturation were found to be as follows:- Change in total heat of reaction DeltaH = 57,000 calories per mole Change in entropy of reaction DeltaS = 180 calories per degree per mole The heat of activation DeltaH(1) (double dagger) for denaturation = 55,000 The heat of activation DeltaH(2) (double dagger) for the reversal of denaturation = -1900 The entropy DeltaS(1) (double dagger) for denaturation = 95 The entropy DeltaS(2) (double dagger) for reversal of denaturation = -84     

The N-terminal domain of Homer/Vesl is a new class II EVH1 domain

Cellular activities controlled by signal transduction processes such as cell motility and cell growth depend on the tightly regulated assembly of multiprotein complexes. Adapter proteins that specifically interact with their target proteins are key components required for the formation of these assemblies. Ena/VASP-homology 1 (EVH1) domains are small constituents of large modular proteins involved in microfilament assembly that specifically recognize proline-rich regions. EVH1 domain-containing proteins are present in neuronal cells, like the Homer/Vesl protein family that is involved in memory-generating processes. Here, we describe the crystal structure of the murine EVH1 domain of Vesl 2 at 2.2 A resolution. The small globular protein consists of a seven-stranded antiparallel beta-barrel with a C-terminal alpha-helix packing alongside the barrel. A shallow groove running parallel with beta-strand VI forms an extended peptide-binding site. Using peptide library screenings, we present data that demonstrate the high affinity of the Vesl 2 EVH1 domain towards peptide sequences containing a proline-rich core sequence (PPSPF) that requires additional charged amino acid residues on either side for specific binding. Our functional data, substantiated by structural data, demonstrate that the ligand-binding of the Vesl EVH1 domain differs from the interaction characteristics of the previously examined EVH1 domains of the Evl/Mena proteins. Analogous to the Src homology 3 (SH3) domains that bind their cognate ligands in two distinct directions, we therefore propose the existence of two distinct classes of EVH1 domains.     

The N-terminal domain of the replication initiator protein RepE is a dimerization domain forming a stable dimer

The initiator protein RepE of the mini-F plasmid in Escherichia coli plays an essential role in DNA replication, which is regulated by the molecular chaperone-dependent oligomeric state (monomer or dimer). Crosslinking, ultracentrifugation, and gel filtration analyses showed that the solely expressed N-terminal domain (residues 1-144 or 1-152) exists in the dimeric state as in the wild-type RepE protein. This result indicates that the N-terminal domain functions as a dimerization domain of RepE and might be important for the interaction with the molecular chaperones. The N-terminal domain dimer has been crystallized in order to obtain structural insight into the regulation of the monomer/dimer conversion of RepE.     

The N-terminal zinc binding domain of ClpX is a dimerization domain that modulates the chaperone function

Clp ATPases are unique chaperones that promote protein unfolding and subsequent degradation by proteases. The mechanism by which this occurs is poorly understood. Here we demonstrate that the N-terminal domain of ClpX is a C4-type zinc binding domain (ZBD) involved in substrate recognition. ZBD forms a very stable dimer that is essential for promoting the degradation of some typical ClpXP substrates such as lambdaO and MuA but not GFP-SsrA. Furthermore, experiments indicate that ZBD contains a primary binding site for the lambdaO substrate and for the cofactor SspB. Removal of ZBD from the ClpX sequence renders the ATPase activity of ClpX largely insensitive to the presence of ClpP, substrates, or the SspB cofactor. All these results indicate that ZBD plays an important role in the ClpX mechanism of function and that ATP binding and/or hydrolysis drives a conformational change in ClpX involving ZBD.     

The N-terminal domain of Bcl-xL reversibly binds membranes in a pH-dependent manner

Bcl-xL regulates apoptosis by maintaining the integrity of the mitochondrial outer membrane by adopting both soluble and membrane-associated forms. The membrane-associated conformation does not require a conserved, C-terminal transmembrane domain and appears to be inserted into the bilayer of synthetic membranes as assessed by membrane permeabilization and critical surface pressure measurements. Membrane association is reversible and is regulated by the cooperative binding of approximately two protons to the protein. Two acidic residues, Glu153 and Asp156, that lie in a conserved hairpin of Bcl-xLDeltaTM appear to be important in this process on the basis of a 16% increase in the level of membrane association of the double mutant E153Q/D156N. Contrary to that for the wild type, membrane permeabilization for the mutant is not correlated with membrane association. Monolayer surface pressure measurements suggest that this effect is primarily due to less membrane penetration. These results suggest that E153 and D156 are important for the Bcl-xLDeltaTM conformational change and that membrane binding can be distinct from membrane permeabilization. Taken together, these studies support a model in which Bcl-xL activity is controlled by reversible insertion of its N-terminal domain into the mitochondrial outer membrane. Future studies with Bcl-xL mutants such as E153Q/D156N should allow determination of the relative contributions of membrane binding, insertion, and permeabilization to the regulation of apoptosis.     

The apical localization of SGLT1 glucose transporter is determined by the short amino acid sequence in its N-terminal domain

SGLT1, an isoform of Na+-dependent glucose cotransporters, is localized at the apical plasma membrane in the epithelial cells of the small intestine and the kidney, where it plays a pivotal role in the absorption and reabsorption of sugars, respectively. To search the domain responsible for the apical localization of SGLT1, we constructed an N-terminal deletion clone series of rat SGLT1 and analyzed the localization of the respective products in Madin-Darby canine kidney (MDCK) cells. The products of N-terminal deletion clones up to the 19th amino acid were localized at the apical plasma membrane, whereas the products of N-terminal 20- and 23-amino-acid deletion clones were localized along the entire plasma membrane. Since single-amino-acid mutations of either D28N or D28G in the N-terminal domain give rise to glucose/galactose malabsorption disease, we examined the localization of these mutants. The products of D28N and D28G clones were localized in the cytoplasm, showing that the aspartic acid-28 may be essential for the delivery of SGLT1 to the plasma membrane. These results suggest that a short amino acid sequence of the N-terminal domain of SGLT1 plays important roles in plasma membrane targeting and specific apical localization of the protein.