A native IgE in complex with profilin provides insights into allergen recognition and cross-reactivity.

Allergies have become a rising health problem, where plentiful substances can trigger IgE-mediated allergies in humans. While profilins are considered minor allergens, these ubiquitous proteins are primary molecules involved in cross-reactivity and pollen-food allergy syndrome. Here we report the first crystal structures of murine Fab/IgE, with its chains naturally paired, in complex with the allergen profilin from Hevea brasiliensis (Hev b 8). The crystallographic models revealed that the IgE's six complementarity-determining regions (CDRs) interact with the allergen, comprising a rigid paratope-epitope surface of 926 Å2, which includes an extensive network of interactions. Interestingly, we also observed previously unreported flexibility at Fab/IgE's elbow angle, which did not influence the shape of the paratope. The Fab/IgE exhibits a high affinity for Hev b 8, even when using 1 M NaCl in BLI experiments. Finally, based on the encouraging cross-reactivity assays using two mutants of the maize profilin (Zea m 12), this antibody could be a promising tool in IgE engineering for diagnosis and research applications.

Evolution of the Inhibitory and Non-Inhibitory ε, ζ, and IF1 Subunits of the F1FO-ATPase as Related to the Endosymbiotic Origin of Mitochondria.

The F1FO-ATP synthase nanomotor synthesizes >90% of the cellular ATP of almost all living beings by rotating in the "forward" direction, but it can also consume the same ATP pools by rotating in "reverse." To prevent futile F1FO-ATPase activity, several different inhibitory proteins or domains in bacteria (ε and ζ subunits), mitochondria (IF1), and chloroplasts (ε and γ disulfide) emerged to block the F1FO-ATPase activity selectively. In this study, we analyze how these F1FO-ATPase inhibitory proteins have evolved. The phylogeny of the α-proteobacterial ε showed that it diverged in its C-terminal side, thus losing both the inhibitory function and the ATP-binding/sensor motif that controls this inhibition. The losses of inhibitory function and the ATP-binding site correlate with an evolutionary divergence of non-inhibitory α-proteobacterial ε and mitochondrial δ subunits from inhibitory bacterial and chloroplastidic ε subunits. Here, we confirm the lack of inhibitory function of wild-type and C-terminal truncated ε subunits of P. denitrificans. Taken together, the data show that ζ evolved to replace ε as the primary inhibitor of the F1FO-ATPase of free-living α-proteobacteria. However, the ζ inhibitory function was also partially lost in some symbiotic α-proteobacteria and totally lost in some strictly parasitic α-proteobacteria such as the Rickettsiales order. Finally, we found that ζ and IF1 likely evolved independently via convergent evolution before and after the endosymbiotic origin mitochondria, respectively. This led us to propose the ε and ζ subunits as tracer genes of the pre-endosymbiont that evolved into the actual mitochondria.

New Insights into the Methylation of Mycobacterium tuberculosis Heparin Binding Hemagglutinin Adhesin Expressed in Rhodococcus erythropolis.

In recent years, knowledge of the role that protein methylation is playing on the physiopathogenesis of bacteria has grown. In Mycobacterium tuberculosis, methylation of the heparin binding hemagglutinin adhesin modulates the immune response, making this protein a subunit vaccine candidate. Through its C-terminal lysine-rich domain, this surface antigen interacts with heparan sulfate proteoglycans present in non-phagocytic cells, leading to extrapulmonary dissemination of the pathogen. In this study, the adhesin was expressed as a recombinant methylated protein in Rhodococcus erythropolis L88 and it was found associated to lipid droplets when bacteria were grown under nitrogen limitation. In order to delve into the role methylation could have in host-pathogen interactions, a comparative analysis was carried out between methylated and unmethylated protein produced in Escherichia coli. We found that methylation had an impact on lowering protein isoelectric point, but no differences between the proteins were found in their capacity to interact with heparin and A549 epithelial cells. An important finding was that HbhA is a Fatty Acid Binding Protein and differences in the conformational stability of the protein in complex with the fatty acid were observed between methylated and unmethylated protein. Together, these results suggest that the described role for this mycobacteria protein in lipid bodies formation could be related to its capacity to transport fatty acids. Obtained results also provide new clues about the role HbhA methylation could have in tuberculosis and point out the importance of having heterologous expression systems to obtain modified proteins.

Novel murine mAbs define specific and cross-reactive epitopes on the latex profilin panallergen Hev b 8.

The production of specific antibodies able to recognize allergens from different sources or block interactions between allergens and antibodies mediating allergic reactions is crucial for developing successful tools for diagnostics and therapeutics. Panallergens are highly conserved proteins present in widely different species, implicated in relevant cross-reactions. The panallergen latex profilin (Hev b 8) has been associated with the latex-food-pollen syndrome. We generated five monoclonal IgGs and one IgE from murine hybridomas against recombinant Hev b 8 and evaluated their interaction with this allergen using ELISA and biolayer interferometry (BLI). Affinity purified mAbs exhibited high binding affinities towards rHev b 8, with KD1 values ranging from 10-10 M to 10-11 M. Some of these antibodies also recognized the recombinant profilins from maize and tomato (Zea m 12 and Sola l 1), and the ash tree pollen (Fra e 2). Competition ELISA demonstrated that some mAb pairs could bind simultaneously to rHev b 8. Using BLI, we detected competitive, non-competitive, and partial-competition interactions between pairs of mAbs with rHev b 8, suggesting the existence of at least two non-overlapping epitopes on the surface of this allergen. Three-dimensional models of the Fv of 1B4 and 2D10 IgGs and docking simulations of these Fvs with rHev b 8 revealed these epitopes. Furthermore, these two mAbs inhibited the interaction of polyclonal IgE and IgG4 antibodies from profilin-allergic patients with rHev b 8, indicating that the mAbs and the antibodies present in sera from allergic patients bind to overlapping epitopes on the allergen. These mAbs can be useful tools for immune-localization studies, immunoassay development, or standardization of allergenic products.

The Contribution of the Ankyrin Repeat Domain of TRPV1 as a Thermal Module.

The TRPV1 cation nonselective ion channel plays an essential role in thermosensation and perception of other noxious stimuli. TRPV1 can be activated by low extracellular pH, high temperature, or naturally occurring pungent molecules such as allicin, capsaicin, or resiniferatoxin. Its noxious thermal sensitivity makes it an important participant as a thermal sensor in mammals. However, details of the mechanism of channel activation by increases in temperature remain unclear. Here, we used a combination of approaches to try to understand the role of the ankyrin repeat domain (ARD) in channel behavior. First, a computational modeling approach by coarse-grained molecular dynamics simulation of the whole TRPV1 embedded in a phosphatidylcholine and phosphatidylethanolamine membrane provides insight into the dynamics of this channel domain. Global analysis of the structural ensemble shows that the ARD is a region that sustains high fluctuations during dynamics at different temperatures. We then performed biochemical and thermal stability studies of the purified ARD by the means of circular dichroism and tryptophan fluorescence and demonstrate that this region undergoes structural changes at similar temperatures that lead to TRPV1 activation. Our data suggest that the ARD is a dynamic module and that it may participate in controlling the temperature sensitivity of TRPV1.

A screening platform to monitor RNA processing and protein-RNA interactions in ribonuclease P uncovers a small molecule inhibitor.

Ribonucleoprotein (RNP) complexes and RNA-processing enzymes are attractive targets for antibiotic development owing to their central roles in microbial physiology. For many of these complexes, comprehensive strategies to identify inhibitors are either lacking or suffer from substantial technical limitations. Here, we describe an activity-binding-structure platform for bacterial ribonuclease P (RNase P), an essential RNP ribozyme involved in 5' tRNA processing. A novel, real-time fluorescence-based assay was used to monitor RNase P activity and rapidly identify inhibitors using a mini-helix and a pre-tRNA-like bipartite substrate. Using the mini-helix substrate, we screened a library comprising 2560 compounds. Initial hits were then validated using pre-tRNA and the pre-tRNA-like substrate, which ultimately verified four compounds as inhibitors. Biolayer interferometry-based binding assays and molecular dynamics simulations were then used to characterize the interactions between each validated inhibitor and the P protein, P RNA and pre-tRNA. X-ray crystallographic studies subsequently elucidated the structure of the P protein bound to the most promising hit, purpurin, and revealed how this inhibitor adversely affects tRNA 5' leader binding. This integrated platform affords improved structure-function studies of RNA processing enzymes and facilitates the discovery of novel regulators or inhibitors.

The crystal structure of the chitinase ChiA74 of Bacillus thuringiensis has a multidomain assembly.

There is no structural information about any chitinase synthesized by Bacillus thuringiensis, the most successful microbial insect larvicide used worldwide. In this study, we solved the 3D structure of the chitinase ChiA74 at 2.26 Å. The crystal structure shows that ChiA74 is composed of a modular arrangement formed by (i) a catalytic region (CD), (ii) a chitinase insertion domain (CID), (iii) a fibronectin type III domain (FnIII), and (iv) a chitin binding domain (CBD). The location of the CBD with respect to the CD has no structural similarity to other chitinases with known structures. The activity of a ChiA74 lacking its secretion signal peptide (ChiA74Δsp) and a truncated version lacking its CBD/FnIII domains (ChiA74Δsp-50) did not have statistical differences in activity against colloidal chitin. However, ChiA74Δsp exhibits 4.5 and 2.0 higher activity than versions lacking the CBD (ChiA74Δsp-60) and CBD/FnIII domains (ChiA74Δsp-50), respectively, when crystalline chitin was used as substrate. Our data suggest that the CBD might plays a significant role in crystalline chitin hydrolysis. We also demonstrated the importance of the catalytic E211 in the CD, as mutants ChiA74ΔspE211N and ChiA74ΔspD207N, E211N were inactive against colloidal and crystalline chitins, chitosan and 4-MU-GlcNAc3. ChiA74 has a processive activity producing oligosaccharides with degree of polymerization (DP) of 1 (GlcNAc) and 2 (GlcNAc2).

Differential effects on enzyme stability and kinetic parameters of mutants related to human triosephosphate isomerase deficiency.

Human triosephosphate isomerase (TIM) deficiency is a very rare disease, but there are several mutations reported to be causing the illness. In this work, we produced nine recombinant human triosephosphate isomerases which have the mutations reported to produce TIM deficiency. These enzymes were characterized biophysically and biochemically to determine their kinetic and stability parameters, and also to substitute TIM activity in supporting the growth of an Escherichia coli strain lacking the tim gene. Our results allowed us to rate the deleteriousness of the human TIM mutants based on the type and severity of the alterations observed, to classify four "unknown severity mutants" with altered residues in positions 62, 72, 122 and 154 and to explain in structural terms the mutation V231M, the most affected mutant from the kinetic point of view and the only homozygous mutation reported besides E104D.

Redesign of LAOBP to bind novel l-amino acid ligands.

Computational protein design is still a challenge for advancing structure-function relationships. While recent advances in this field are promising, more information for genuine predictions is needed. Here, we discuss different approaches applied to install novel glutamine (Gln) binding into the Lysine/Arginine/Ornithine binding protein (LAOBP) from Salmonella typhimurium. We studied the ligand binding behavior of two mutants: a binding pocket grafting design based on a structural superposition of LAOBP to the Gln binding protein QBP from Escherichia coli and a design based on statistical coupled positions. The latter showed the ability to bind Gln even though the protein was not very stable. Comparison of both approaches highlighted a nonconservative shared point mutation between LAOBP_graft and LAOBP_sca. This context dependent L117K mutation in LAOBP turned out to be sufficient for introducing Gln binding, as confirmed by different experimental techniques. Moreover, the crystal structure of LAOBP_L117K in complex with its ligand is reported.

X-ray crystal structure of the passenger domain of plasmid encoded toxin(Pet), an autotransporter enterotoxin from enteroaggregative Escherichia coli (EAEC).

Autotransporters (ATs) represent a superfamily of proteins produced by a variety of pathogenic bacteria, which include the pathogenic groups of Escherichia coli (E. coli) associated with gastrointestinal and urinary tract infections. We present the first X-ray structure of the passenger domain from the Plasmid-encoded toxin (Pet) a 100 kDa protein at 2.3 Å resolution which is a cause of acute diarrhea in both developing and industrialized countries. Pet is a cytoskeleton-altering toxin that induces loss of actin stress fibers. While Pet (pdb code: 4OM9) shows only a sequence identity of 50% compared to the closest related protein sequence, extracellular serine protease plasmid (EspP) the structural features of both proteins are conserved. A closer structural look reveals that Pet contains a ß-pleaded sheet at the sequence region of residues 181-190, the corresponding structural domain in EspP consists of a coiled loop. Secondary, the Pet passenger domain features a more pronounced beta sheet between residues 135 and 143 compared to the structure of EspP.

Structural and functional perturbation of Giardia lamblia triosephosphate isomerase by modification of a non-catalytic, non-conserved region.

BACKGROUND: We have previously proposed triosephosphate isomerase of Giardia lamblia (GlTIM) as a target for rational drug design against giardiasis, one of the most common parasitic infections in humans. Since the enzyme exists in the parasite and the host, selective inhibition is a major challenge because essential regions that could be considered molecular targets are highly conserved. Previous biochemical evidence showed that chemical modification of the non-conserved non-catalytic cysteine 222 (C222) inactivates specifically GlTIM. The inactivation correlates with the physicochemical properties of the modifying agent: addition of a non-polar, small chemical group at C222 reduces the enzyme activity by one half, whereas negatively charged, large chemical groups cause full inactivation. RESULTS: In this work we used mutagenesis to extend our understanding of the functional and structural effects triggered by modification of C222. To this end, six GlTIM C222 mutants with side chains having diverse physicochemical characteristics were characterized. We found that the polarity, charge and volume of the side chain in the mutant amino acid differentially alter the activity, the affinity, the stability and the structure of the enzyme. The data show that mutagenesis of C222 mimics the effects of chemical modification. The crystallographic structure of C222D GlTIM shows the disruptive effects of introducing a negative charge at position 222: the mutation perturbs loop 7, a region of the enzyme whose interactions with the catalytic loop 6 are essential for TIM stability, ligand binding and catalysis. The amino acid sequence of TIM in phylogenetic diverse groups indicates that C222 and its surrounding residues are poorly conserved, supporting the proposal that this region is a good target for specific drug design. CONCLUSIONS: The results demonstrate that it is possible to inhibit species-specifically a ubiquitous, structurally highly conserved enzyme by modification of a non-conserved, non-catalytic residue through long-range perturbation of essential regions.

Ribonucleases P/MRP and the expanding ribonucleoprotein world.

One of the hallmarks of life is the widespread use of certain essential ribozymes. The ubiquitous ribonuclease P (RNase P) and eukaryotic RNase MRP are essential complexes where a structured, noncoding RNA acts in catalysis. Recent discoveries have elucidated the three-dimensional structure of the ancestral ribonucleoprotein complex, suggested the possibility of a protein-only composition in organelles, and even noted the absence of RNase P in a non-free-living organism. With respect to these last two findings, import mechanisms for RNases P/MRP into mitochondria have been demonstrated, and RNase P is present in organisms with some of the smallest known genomes. Together, these results have led to an ongoing debate regarding the precise definition of how "essential" these ribozymes truly are.

A ribosomal misincorporation of Lys for Arg in human triosephosphate isomerase expressed in Escherichia coli gives rise to two protein populations.

We previously observed that human homodimeric triosephosphate isomerase (HsTIM) expressed in Escherichia coli and purified to apparent homogeneity exhibits two significantly different thermal transitions. A detailed exploration of the phenomenon showed that the preparations contain two proteins; one has the expected theoretical mass, while the mass of the other is 28 Da lower. The two proteins were separated by size exclusion chromatography in 3 M urea. Both proteins correspond to HsTIM as shown by Tandem Mass Spectrometry (LC/ESI-MS/MS). The two proteins were present in nearly equimolar amounts under certain growth conditions. They were catalytically active, but differed in molecular mass, thermostability, susceptibility to urea and proteinase K. An analysis of the nucleotides in the human TIM gene revealed the presence of six codons that are not commonly used in E. coli. We examined if they were related to the formation of the two proteins. We found that expression of the enzyme in a strain that contains extra copies of genes that encode for tRNAs that frequently limit translation of heterologous proteins (Arg, Ile, Leu), as well as silent mutations of two consecutive rare Arg codons (positions 98 and 99), led to the exclusive production of the more stable protein. Further analysis by LC/ESI-MS/MS showed that the 28 Da mass difference is due to the substitution of a Lys for an Arg residue at position 99. Overall, our work shows that two proteins with different biochemical and biophysical properties that coexist in the same cell environment are translated from the same nucleotide sequence frame.

Identification of amino acids that account for long-range interactions in two triosephosphate isomerases from pathogenic trypanosomes.

For a better comprehension of the structure-function relationship in proteins it is necessary to identify the amino acids that are relevant for measurable protein functions. Because of the numerous contacts that amino acids establish within proteins and the cooperative nature of their interactions, it is difficult to achieve this goal. Thus, the study of protein-ligand interactions is usually focused on local environmental structural differences. Here, using a pair of triosephosphate isomerase enzymes with extremely high homology from two different organisms, we demonstrate that the control of a seventy-fold difference in reactivity of the interface cysteine is located in several amino acids from two structurally unrelated regions that do not contact the cysteine sensitive to the sulfhydryl reagent methylmethane sulfonate, nor the residues in its immediate vicinity. The change in reactivity is due to an increase in the apparent pKa of the interface cysteine produced by the mutated residues. Our work, which involved grafting systematically portions of one protein into the other protein, revealed unsuspected and multisite long-range interactions that modulate the properties of the interface cysteines and has general implications for future studies on protein structure-function relationships.

Different catalytic properties of two highly homologous triosephosphate isomerase monomers.

It is assumed that amino acid sequence differences in highly homologous enzymes would be found at the peripheral level, subtle changes that would not necessarily affect catalysis. Here, we demonstrate that, using the same set of mutations at the level of the interface loop 3, the activity of a triosephosphate isomerase monomeric enzyme is ten times higher than that of a homologous enzyme with 74% identity and 86% similarity, whereas the activity of the native, dimeric enzymes is essentially the same. This is an example of how the dimeric biological unit evolved to compensate for the intrinsic differences found at the monomeric species level. Biophysical techniques of size exclusion chromatography, dynamic light scattering, X-ray crystallography, fluorescence and circular dichroism, as well as denaturation/renaturation assays with guanidinium hydrochloride and ANS binding, allowed us to fully characterize the properties of the new monomer.

Expression, purification and preliminary X-ray diffraction studies of the transcriptional factor PyrR from Bacillus halodurans.

The PyrR transcriptional regulator is widely distributed in bacteria. This RNA-binding protein is involved in the control of genes involved in pyrimidine biosynthesis, in which uridyl and guanyl nucleotides function as effectors. Here, the crystallization and preliminary X-ray diffraction analysis of two crystal forms of Bacillus halodurans PyrR are reported. One of the forms belongs to the monoclinic space group P2(1) with unit-cell parameters a = 59.7, b = 87.4, c = 72.1 A, beta = 104.4 degrees , while the other form belongs to the orthorhombic space group P22(1)2(1) with unit-cell parameters a = 72.7, b = 95.9, c = 177.1 A. Preliminary X-ray diffraction data analysis and molecular-replacement solution revealed the presence of four and six monomers per asymmetric unit; a crystallographic tetramer is formed in both forms.

The pentatricopeptide repeats present in Pet309 are necessary for translation but not for stability of the mitochondrial COX1 mRNA in yeast.

Pet309 is a protein essential for respiratory growth. It is involved in translation of the yeast mitochondrial COX1 gene, which encodes subunit I of the cytochrome c oxidase. Pet309 is also involved in stabilization of the COX1 mRNA. Mutations in a similar human protein, Lrp130, are associated with Leigh syndrome, where cytochrome c oxidase activity is affected. The sequence of Pet309 reveals the presence of at least seven pentatricopeptide repeats (PPRs) located in tandem in the central portion of the protein. Proteins containing PPR motifs are present in mitochondria and chloroplasts and are in general involved in RNA metabolism. Despite the increasing number of proteins from this family found to play essential roles in mitochondria and chloroplasts, little is understood about the mechanism of action of the PPR domains present in these proteins. In a series of in vivo analyses we constructed a pet309 mutant lacking the PPR motifs. Although the stability of the COX1 mRNA was not affected, synthesis of Cox1 was abolished. The deletion of one PPR motif at a time showed that all the PPR motifs are required for COX1 mRNA translation and respiratory growth. Mutations of basic residues in PPR3 caused reduced respiratory growth. According to a molecular model, these residues are facing a central cavity that could be involved in mRNA-binding activity, forming a possible path for this molecule on Pet309. Our results show that the RNA metabolism function of Pet309 is found in at least two separate domains of the protein.