Sequence-specific 1H, 15N and 13C resonance assignments of the 23.7-kDa homodimeric toxin CcdB from Vibrio fischeri

CcdB is the toxic component of a bacterial toxin–antitoxin system. It inhibits DNA gyrase (a type II topoisomerase), and its toxicity can be neutralized by binding of its antitoxin CcdA. Here we report the sequential backbone and sidechain ¹H, ¹⁵N and ¹³C resonance assignments of CcdB_Vfi from the marine bacterium Vibrio fischeri. The BMRB accession number is 16135.     

The thermodynamic stability of the proteins of the ccd plasmid addiction system

The two opponents, toxin (CcdB, LetB or LetD, protein G, LynB) and antidote (CcdA, LetA, protein H, LynA), in the plasmid addiction system ccd of the F plasmid were studied by different biophysical methods. The thermodynamic stability was measured at different temperatures combining denaturant and thermally induced unfolding. It was found that both proteins denature in a two-state equilibrium (native dimer versus unfolded monomer) and that CcdA has a significantly lower thermodynamic stability. Using a numerical model, which was developed earlier by us, and on the basis of the determined thermodynamic parameters the concentration dependence of the denaturation transition temperature was obtained for both proteins. This concentration dependence may be of physiological significance, as the concentration of both ccd addiction proteins cannot exceed a certain limit because their expression is controlled by autoregulation.The influence of DNA on the thermal stability of the two proteins was probed. It was found that cognate DNA increases the melting temperature of CcdA. In the presence of non-specific DNA the thermal stability was not changed. The melting temperature of CcdB was not influenced by the applied double-stranded oligonucleotides, neither cognate nor unspecific.     

Leukotriene BLT2 Receptor Monomers Activate the Gi2 GTP-binding Protein More Efficiently than Dimers

Accumulating evidence indicates that G protein-coupled receptors can assemble as dimers/oligomers but the role of this phenomenon in G protein coupling and signaling is not yet clear. We have used the purified leukotriene B₄ receptor BLT2 as a model to investigate the capacity of receptor monomers and dimers to activate the adenylyl cyclase inhibitory G_i2 protein. For this, we overexpressed the recombinant receptor as inclusion bodies in the Escherichia coli prokaryotic system, using a human α₅ integrin as a fusion partner. This strategy allowed the BLT2 as well as several other G protein-coupled receptors from different families to be produced and purified in large amounts. The BLT2 receptor was then successfully refolded to its native state, as measured by high-affinity LTB₄ binding in the presence of the purified G protein Gα_i2. The receptor dimer, in which the two protomers displayed a well defined parallel orientation as assessed by fluorescence resonance energy transfer, was then separated from the monomer. Using two methods of receptor-catalyzed guanosine 5′-3-O-(thio)triphosphate binding assay, we clearly demonstrated that monomeric BLT2 stimulates the purified Gα_i2β₁γ₂ protein more efficiently than the dimer. These data suggest that assembly of two BLT2 protomers into a dimer results in the reduced ability to signal.     

The 41 carboxy-terminal residues of the miniF plasmid CcdA protein are sufficient to antagonize the killer activity of the CcdB protein

Summary The ccd operon of plasmid F encodes two genes, ccdA and ccdB, which contribute to the high stability of the plasmid by post-segregational killing of plasmid-free bacteria. The CcdB protein is lethal to bacteria and the CcdA protein is an antagonist of this lethal action. A 520 by fragment containing the terminal part of the ccdA gene and the entire ccdB gene of plasmid F was cloned downstream of the tac promoter. Although the CcdB protein was expressed from this fragment, no killing of host bacteria was observed. We found that the absence of killing was due to the presence of a small polypeptide, CcdA41, composed of the 41 C-terminal residues of the CcdA protein. This polypeptide has retained the ability to regulate negatively the lethal activity of the CcdB protein.     

Lon-dependent proteolysis of CcdA is the key control for activation of CcdB in plasmid-free segregant bacteria

The ccd locus contributes to the stability of plasmid F by post-segregational killing of plasmid-free bacteria. The ccdB gene product is a potent cell-killing protein and its activity is negatively regulated by the CcdA protein, in this paper, we show that the CcdA protein is unstable and that the degradation of CcdA is dependent on the Lon protease. Differences in the stability of the killer CcdB protein and its antidote CcdA are the key to post-segregational killing. Because the half-life of active CcdA protein is shorter than that of active CcdB protein, persistence of the CcdB protein leads to the death of plasmid-free bacterial segregants.     

Proline Substitution of Dimer Interface β-strand Residues as a Strategy for the Design of Functional Monomeric Proteins

Proteins that exist in monomer-dimer equilibrium can be found in all organisms ranging from bacteria to humans; this facilitates fine-tuning of activities from signaling to catalysis. However, studying the structural basis of monomer function that naturally exists in monomer-dimer equilibrium is challenging, and most studies to date on designing monomers have focused on disrupting packing or electrostatic interactions that stabilize the dimer interface. In this study, we show that disrupting backbone H-bonding interactions by substituting dimer interface β-strand residues with proline (Pro) results in fully folded and functional monomers, by exploiting proline’s unique feature, the lack of a backbone amide proton. In interleukin-8, we substituted Pro for each of the three residues that form H-bonds across the dimer interface β-strands. We characterized the structures, dynamics, stability, dimerization state, and activity using NMR, molecular dynamics simulations, fluorescence, and functional assays. Our studies show that a single Pro substitution at the middle of the dimer interface β-strand is sufficient to generate a fully functional monomer. Interestingly, double Pro substitutions, compared to single Pro substitution, resulted in higher stability without compromising native monomer fold or function. We propose that Pro substitution of interface β-strand residues is a viable strategy for generating functional monomers of dimeric, and potentially tetrameric and higher-order oligomeric proteins.     

Proline Substitution of Dimer Interface β-strand Residues as a Strategy for?the Design of Functional Monomeric Proteins

Proteins that exist in monomer-dimer equilibrium can be found in all organisms ranging from bacteria to humans; this facilitates fine-tuning of activities from signaling to catalysis. However, studying the structural basis of monomer function that naturally exists in monomer-dimer equilibrium is challenging, and most studies to date on designing monomers have focused on disrupting packing or electrostatic interactions that stabilize the dimer interface. In this study, we show that disrupting backbone H-bonding interactions by substituting dimer interface β-strand residues with proline (Pro) results in fully folded and functional monomers, by exploiting proline’s unique feature, the lack of a backbone amide proton. In interleukin-8, we substituted Pro for each of the three residues that form H-bonds across the dimer interface β-strands. We characterized the structures, dynamics, stability, dimerization state, and activity using NMR, molecular dynamics simulations, fluorescence, and functional assays. Our studies show that a single Pro substitution at the middle of the dimer interface β-strand is sufficient to generate a fully functional monomer. Interestingly, double Pro substitutions, compared to single Pro substitution, resulted in higher stability without compromising native monomer fold or function. We propose that Pro substitution of interface β-strand residues is a viable strategy for generating functional monomers of dimeric, and potentially tetrameric and higher-order oligomeric proteins.     

Enhanced Formation of Disulfide-bridged Dimer (Fab-PE38)2 Utilizing Repeats of the Fab Binding Domain of Protein G

Fab-PE38 used in this study is B3(Fab)-ext-PE38, and it is an antibody toxin that is made by fusing the Pseudomonas exotoxin to the Fab domain of B3 antibody. This antibody toxin selectively binds to cancer cells and kills the target cancer cells. B3(Fab)-ext-PE38 has a cysteine residue on the ext sequence, and (B3(Fab)-ext-PE38)₂ is the disulfide-bridged dimer of the B3(Fab)-ext-PE38 monomer. (B3(Fab)-ext-PE38)₂ has been found to have 11-fold higher cytotoxicity on the CRL-1739 cell line than monomeric B3(scFv)-PE38. We made a recombinant tandem repeat of the domain III of Streptococcal protein G that has Fab binding property up to seven repeats. Multiple monomers were found to form non-covalent complexes with this tandem repeat. Complexes were purified by size-exclusion chromatography, and we could enhance the production of the disulfide-bridged dimer by reduction and oxidation of the complexes. The tandem repeat makes close intermolecular interactions between monomers possible, and the use of it greatly enhances the yield of the disulfide-bridged dimer.     

Structural and functional comparison between the stability systems ParD of plasmid R1 and Ccd of plasmid F

Summary The stability determined by the systems ParD of plasmid R1 and Ccd of plasmid F is due to the concerted action of two proteins, a cytotoxin and an antagonist of this function. In this paper we report that CcdA and Kis proteins, the antagonists of the Ccd and ParD systems respectively, share significant sequence homologies at both ends. In Kis, these regions seem to correspond to two different domains. Despite the structural similarities, Kis and CcdA are not interchangeable. In addition we have shown that the cytotoxins of these systems, the Kid and CcdB proteins, do not share structural homologies. In contrast to CcdB, the Kid protein of the ParD system induces RecA-dependent cleavage of the cl repressor of bacteriophage very inefficiently or not at all. The functional implications of these results are discussed.     

The Folding Unit of Phosphofructokinase-2 as Defined by the Biophysical Properties of a Monomeric Mutant

Escherichia coli phosphofructokinase-2 (Pfk-2) is an obligate homodimer that follows a highly cooperative three-state folding mechanism N₂ ↔ 2I ↔ 2U. The strong coupling between dissociation and unfolding is a consequence of the structural features of its interface: a bimolecular domain formed by intertwining of the small domain of each subunit into a flattened β-barrel. Although isolated monomers of E. coli Pfk-2 have been observed by modification of the environment (changes in temperature, addition of chaotropic agents), no isolated subunits in native conditions have been obtained. Based on in silico estimations of the change in free energy and the local energetic frustration upon binding, we engineered a single-point mutant to destabilize the interface of Pfk-2. This mutant, L93A, is an inactive monomer at protein concentrations below 30 μM, as determined by analytical ultracentrifugation, dynamic light scattering, size exclusion chromatography, small-angle x-ray scattering, and enzyme kinetics. Active dimer formation can be induced by increasing the protein concentration and by addition of its substrate fructose-6-phosphate. Chemical and thermal unfolding of the L93A monomer followed by circular dichroism and dynamic light scattering suggest that it unfolds noncooperatively and that the isolated subunit is partially unstructured and marginally stable. The detailed structural features of the L93A monomer and the F6P-induced dimer were ascertained by high-resolution hydrogen/deuterium exchange mass spectrometry. Our results show that the isolated subunit has overall higher solvent accessibility than the native dimer, with the exception of residues 240–309. These residues correspond to most of the β-meander module and show the same extent of deuterium uptake as the native dimer. Our results support the idea that the hydrophobic core of the isolated monomer of Pfk-2 is solvent-penetrated in native conditions and that the β-meander module is not affected by monomerizing mutations.     

Exploring the dihydrodipicolinate synthase tetramer: How resilient is the dimer-dimer interface?

Dihydrodipicolinate synthase (DHDPS, E.C. 4.2.1.52) is a tetrameric enzyme that catalyses the first committed step of the lysine biosynthetic pathway. Dimeric variants of DHDPS have impaired catalytic activity due to aberrant protein motions within the dimer unit. Thus, it is thought that the tetrameric structure functions to restrict these motions and optimise enzyme dynamics for catalysis. Despite the importance of dimer-dimer association, the interface between subunits of each dimer is small, accounting for only 4.3% of the total monomer surface area, and the structure of the interface is not conserved across species. We have probed the tolerance of dimer-dimer association to mutation by introducing amino acid substitutions within the interface. All point mutations resulted in destabilisation of the ‘dimer of dimers’ tetrameric structure. Both the position of the mutation in the interface and the physico-chemical nature of the substitution appeared to effect tetramerisation. Despite only weak destabilisation of the tetramer by some mutations, catalytic activity was reduced to ∼10-15% of the wild-type in all cases, suggesting that the dimer-dimer interface is finely tuned to optimise function.     

Engineering a Monomeric Fc Domain Modality by N-Glycosylation for the Half-life Extension of Biotherapeutics

Human IgG is a bivalent molecule that has two identical Fab domains connected by a dimeric Fc domain. For therapeutic purposes, however, the bivalency of IgG and Fc fusion proteins could cause undesired properties. We therefore engineered the conversion of the natural dimeric Fc domain to a highly soluble monomer by introducing two Asn-linked glycans onto the hydrophobic C_H3-C_H3 dimer interface. The monomeric Fc (monoFc) maintained the binding affinity for neonatal Fc receptor (FcRn) in a pH-dependent manner. We solved the crystal structure of monoFc, which explains how the carbohydrates can stabilize the protein surface and provides the rationale for molecular recognition between monoFc and FcRn. The monoFc prolonged the in vivo half-life of an antibody Fab domain, and a tandem repeat of the monoFc further prolonged the half-life. This monoFc modality can be used to improve the pharmacokinetics of monomeric therapeutic proteins with an option to modulate the degree of half-life extension.     

A Functional DnaK Dimer Is Essential for the Efficient Interaction with Hsp40 Heat Shock Protein

Highly conserved molecular chaperone Hsp70 heat shock proteins play a key role in maintaining protein homeostasis (proteostasis). DnaK, a major Hsp70 in Escherichia coli, has been widely used as a paradigm for studying Hsp70s. In the absence of ATP, purified DnaK forms low-ordered oligomer, whereas ATP binding shifts the equilibrium toward the monomer. Recently, we solved the crystal structure of DnaK in complex with ATP. There are two molecules of DnaK-ATP in the asymmetric unit. Interestingly, the interfaces between the two molecules of DnaK are large with good surface complementarity, suggesting functional importance of this crystallographic dimer. Biochemical analyses of DnaK protein supported the formation of dimer in solution. Furthermore, our cross-linking experiment based on the DnaK-ATP structure confirmed that DnaK forms specific dimer in an ATP-dependent manner. To understand the physiological function of the dimer, we mutated five residues on the dimer interface. Four mutations, R56A, T301A, N537A, and D540A, resulted in loss of chaperone activity and compromised the formation of dimer, indicating the functional importance of the dimer. Surprisingly, neither the intrinsic biochemical activities, the ATP-induced allosteric coupling, nor GrpE co-chaperone interaction is affected appreciably in all of the mutations except for R56A. Unexpectedly, the interaction with co-chaperone Hsp40 is significantly compromised. In summary, this study suggests that DnaK forms a transient dimer upon ATP binding, and this dimer is essential for the efficient interaction of DnaK with Hsp40.     

A Common Origin for the Bacterial Toxin-Antitoxin Systems parD and ccd,Suggested by Analyses of Toxin/Target and Toxin/Antitoxin Interactions

Bacterial toxin-antitoxin (TA) systems encode two proteins, a potent inhibitor of cell proliferation (toxin) and its specific antidote (antitoxin). Structural data has revealed striking similarities between the two model TA toxins CcdB, a DNA gyrase inhibitor encoded by the ccd system of plasmid F, and Kid, a site-specific endoribonuclease encoded by the parD system of plasmid R1. While a common structural fold seemed at odds with the two clearly different modes of action of these toxins, the possibility of functional crosstalk between the parD and ccd systems, which would further point to their common evolutionary origin, has not been documented. Here, we show that the cleavage of RNA and the inhibition of protein synthesis by the Kid toxin, two activities that are specifically counteracted by its cognate Kis antitoxin, are altered, but not inhibited, by the CcdA antitoxin. In addition, Kis was able to inhibit the stimulation of DNA gyrase-mediated cleavage of DNA by CcdB, albeit less efficiently than CcdA. We further show that physical interactions between the toxins and antitoxins of the different systems do occur and define the stoichiometry of the complexes formed. We found that CcdB did not degrade RNA nor did Kid have any reproducible effect on the tested DNA gyrase activities, suggesting that these toxins evolved to reach different, rather than common, cellular targets.     

Functional Roles of the Dimer-Interface Residues in Human Ornithine Decarboxylase

Ornithine decarboxylase (ODC) catalyzes the decarboxylation of ornithine to putrescine and is the rate-limiting enzyme in the polyamine biosynthesis pathway. ODC is a dimeric enzyme, and the active sites of this enzyme reside at the dimer interface. Once the enzyme dissociates, the enzyme activity is lost. In this paper, we investigated the roles of amino acid residues at the dimer interface regarding the dimerization, protein stability and/or enzyme activity of ODC. A multiple sequence alignment of ODC and its homologous protein antizyme inhibitor revealed that 5 of 9 residues (residues 165, 277, 331, 332 and 389) are divergent, whereas 4 (134, 169, 294 and 322) are conserved. Analytical ultracentrifugation analysis suggested that some dimer-interface amino acid residues contribute to formation of the dimer of ODC and that this dimerization results from the cooperativity of these interface residues. The quaternary structure of the sextuple mutant Y331S/Y389D/R277S/D332E/V322D/D134A was changed to a monomer rather than a dimer, and the K _d value of the mutant was 52.8 µM, which is over 500-fold greater than that of the wild-type ODC (ODC_WT). In addition, most interface mutants showed low but detectable or negligible enzyme activity. Therefore, the protein stability of these interface mutants was measured by differential scanning calorimetry. These results indicate that these dimer-interface residues are important for dimer formation and, as a consequence, are critical for enzyme catalysis.     

Differences in the Interactions between the Subunits of Photosystem II Dependent on D1 Protein Variants in the Thermophilic Cyanobacterium Thermosynechococcus elongatus

The main cofactors involved in the oxygen evolution activity of Photosystem II (PSII) are located in two proteins, D1 (PsbA) and D2 (PsbD). In Thermosynechococcus elongatus, a thermophilic cyanobacterium, the D1 protein is encoded by either the psbA₁ or the psbA₃ gene, the expression of which is dependent on environmental conditions. It has been shown that the energetic properties of the PsbA1-PSII and those of the PsbA3-PSII differ significantly (Sugiura, M., Kato, Y., Takahashi, R., Suzuki, H., Watanabe, T., Noguchi, T., Rappaport, F., and Boussac, A. (2010) Biochim. Biophys. Acta 1797, 1491–1499). In this work the structural stability of PSII upon a PsbA1/PsbA3 exchange was investigated. Two deletion mutants lacking another PSII subunit, PsbJ, were constructed in strains expressing either PsbA1 or PsbA3. The PsbJ subunit is a 4-kDa transmembrane polypeptide that is surrounded by D1 (i.e. PsbA1), PsbK, and cytochrome b₅₅₉ (Cyt b₅₅₉) in existing three-dimensional models. It is shown that the structural properties of the PsbA3/ΔPsbJ-PSII are not significantly affected. The polypeptide contents, the Cyt b₅₅₉ properties, and the proportion of PSII dimer were similar to those found for PsbA3-PSII. In contrast, in PsbA1/ΔPsbJ-PSII the stability of the dimer is greatly diminished, the EPR properties of the Cyt b₅₅₉ likely indicates a decrease in its redox potential, and many other PSII subunits are lacking. These results shows that the 21-amino acid substitutions between PsbA1 and PsbA3, which appear to be mainly conservative, must include side chains that are involved in a network of interactions between PsbA and the other PSII subunits.     

Assessing Structural Determinants of Zn2+ Binding to Human HV1 via Multiple MD Simulations

Voltage-gated proton channels (H_V1) are essential for various physiological tasks but are strongly inhibited by Zn²⁺ cations. Some determinants of Zn²⁺ binding have been elucidated experimentally and in computational studies. However, the results have always been interpreted under the assumption that Zn²⁺ binds to monomeric H_V1 despite evidence that H_V1 expresses as a dimer and that the dimer has a higher affinity for zinc than the monomer and experimental data that suggest coordination in the dimer interface. The results of former studies are also controversial, e.g., supporting either one single or two binding sites. Some structural determinants of the binding are still elusive. We performed a series of molecular dynamics simulations to address different structures of the human proton channel, the monomer and two plausible dimer conformations, to compare their respective potential to interact with and bind Zn²⁺ via the essential histidines. The series consisted of several copies of the system to generate independent trajectories and increase the significance compared to a single simulation. The amount of time simulated totals 29.9 μs for 126 simulations of systems comprising ∼59,000 to ∼187,000 atoms. Our approach confirms the existence of two binding sites in monomeric and dimeric human H_V1. The dimer interface is more efficient for attracting and binding Zn²⁺ via the essential histidines than the monomer or a dimer with the histidines in the periphery. The higher affinity is due to the residues in the dimer interface that create an attractive electrostatic potential funneling the zinc cations toward the binding sites.     

Effect of lysine to alanine mutations on the phosphate activation and BPTES inhibition of glutaminase

The GLS1 gene encodes a mitochondrial glutaminase that is highly expressed in brain, kidney, small intestine and many transformed cells. Recent studies have identified multiple lysine residues in glutaminase that are sites of N-acetylation. Interestingly, these sites are located within either a loop segment that regulates access of glutamine to the active site or the dimer:dimer interface that participates in the phosphate-dependent oligomerization and activation of the enzyme. These two segments also contain the binding sites for bis-2[5-phenylacetamido-1,2,4-thiadiazol-2-yl]ethylsulfide (BPTES), a highly specific and potent uncompetitive inhibitor of this glutaminase. BPTES is also the lead compound for development of novel cancer chemotherapeutic agents. To provide a preliminary assessment of the potential effects of N-acetylation, the corresponding lysine to alanine mutations were constructed in the hGAC_Δ1 plasmid. The wild type and mutated proteins were purified by Ni⁺-affinity chromatography and their phosphate activation and BPTES inhibition profiles were analyzed. Two of the alanine substitutions in the loop segment (K₃₁₁A and K₃₂₈A) and the one in the dimer:dimer interface (K₃₉₆A) form enzymes that require greater concentrations of phosphate to produce half-maximal activation and exhibit greater sensitivity to BPTES inhibition. By contrast, the K₃₂₀A mutation results in a glutaminase that exhibits near maximal activity in the absence of phosphate and is not inhibited by BPTES. Thus, lysine N-acetylation may contribute to the acute regulation of glutaminase activity in various tissues and alter the efficacy of BPTES-type inhibitors.