Plant organellar DNA polymerases repair double-stranded breaks by microhomology-mediated end-joining.

Double-stranded breaks (DSBs) in plant organelles are repaired via genomic rearrangements characterized by microhomologous repeats. These microhomologous signatures predict the existence of an unidentified enzymatic machinery capable of repairing of DSBs via microhomology-mediated end-joining (MMEJ) in plant organelles. Here, we show that organellar DNA polymerases from Arabidopsis thaliana (AtPolIA and AtPolIB) perform MMEJ using microhomologous sequences as short as six nucleotides. AtPolIs execute MMEJ by virtue of two specialized amino acid insertions located in their thumb subdomains. Single-stranded binding proteins (SSBs) unique to plants, AtWhirly2 and organellar single-stranded binding proteins (AtOSBs), hinder MMEJ, whereas canonical mitochondrial SSBs (AtmtSSB1 and AtmtSSB2) do not interfere with MMEJ. Our data predict that organellar DNA rearrangements by MMEJ are a consequence of a competition for the 3'-OH of a DSBs. If AtWhirlies or AtOSBs gain access to the single-stranded DNA (ssDNA) region of a DSB, the reaction will shift towards high-fidelity routes like homologous recombination. Conversely MMEJ would be favored if AtPolIs or AtmtSSBs interact with the DSB. AtPolIs are not phylogenetically related to metazoan mitochondrial DNA polymerases, and the ability of AtPolIs to execute MMEJ may explain the abundance of DNA rearrangements in plant organelles in comparison to animal mitochondria.

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 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.

Structural basis for the modulation of plant cytosolic triosephosphate isomerase activity by mimicry of redox-based modifications.

Reactive oxidative species (ROS) and S-glutathionylation modulate the activity of plant cytosolic triosephosphate isomerases (cTPI). Arabidopsis thaliana cTPI (AtcTPI) is subject of redox regulation at two reactive cysteines that function as thiol switches. Here we investigate the role of these residues, AtcTPI-Cys13 and At-Cys218, by substituting them with aspartic acid that mimics the irreversible oxidation of cysteine to sulfinic acid and with amino acids that mimic thiol conjugation. Crystallographic studies show that mimicking AtcTPI-Cys13 oxidation promotes the formation of inactive monomers by reposition residue Phe75 of the neighboring subunit, into a conformation that destabilizes the dimer interface. Mutations in residue AtcTPI-Cys218 to Asp, Lys, or Tyr generate TPI variants with a decreased enzymatic activity by creating structural modifications in two loops (loop 7 and loop 6) whose integrity is necessary to assemble the active site. In contrast with mutations in residue AtcTPI-Cys13, mutations in AtcTPI-Cys218 do not alter the dimeric nature of AtcTPI. Therefore, modifications of residues AtcTPI-Cys13 and AtcTPI-Cys218 modulate AtcTPI activity by inducing the formation of inactive monomers and by altering the active site of the dimeric enzyme, respectively. The identity of residue AtcTPI-Cys218 is conserved in the majority of plant cytosolic TPIs, this conservation and its solvent-exposed localization make it the most probable target for TPI regulation upon oxidative damage by reactive oxygen species. Our data reveal the structural mechanisms by which S-glutathionylation protects AtcTPI from irreversible chemical modifications and re-routes carbon metabolism to the pentose phosphate pathway to decrease oxidative stress.

Mimicking a p53-MDM2 interaction based on a stable immunoglobulin-like domain scaffold.

Antibodies recognize protein targets with great affinity and specificity. However, posttranslational modifications and the presence of intrinsic disulfide-bonds pose difficulties for their industrial use. The immunoglobulin fold is one of the most ubiquitous folds in nature and it is found in many proteins besides antibodies. An example of a protein family with an immunoglobulin-like fold is the Cysteine Protease Inhibitors (ICP) family I42 of the MEROPs database for protease and protease inhibitors. Members of this protein family are thermostable and do not present internal disulfide bonds. Crystal structures of several ICPs indicate that they resemble the Ig-like domain of the human T cell co-receptor CD8α As ICPs present 2 flexible recognition loops that vary accordingly to their targeted protease, we hypothesize that members of this protein family would be ideal to design peptide aptamers that mimic protein-protein interactions. Herein, we use an ICP variant from Entamoeba histolytica (EhICP1) to mimic the interaction between p53 and MDM2. We found that a 13 amino-acid peptide derived from p53 can be introduced in 2 variable loops (DE, FG) but not the third (BC). Chimeric EhICP1-p53 form a stable complex with MDM2 at a micromolar range. Crystal structure of the EhICP1-p53(FG)-loop variant in complex with MDM2 reveals a swapping subdomain between 2 chimeric molecules, however, the p53 peptide interacts with MDM2 as in previous crystal structures. The structural details of the EhICP1-p53(FG) interaction with MDM2 resemble the interaction between an antibody and MDM2.

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.

Structural Insights into the Polyphyletic Origins of Glycyl tRNA Synthetases.

Glycyl tRNA synthetase (GlyRS) provides a unique case among class II aminoacyl tRNA synthetases, with two clearly widespread types of enzymes: a dimeric (α2) species present in some bacteria, archaea, and eukaryotes; and a heterotetrameric form (α2ß2) present in most bacteria. Although the differences between both types of GlyRS at the anticodon binding domain level are evident, the extent and implications of the variations in the catalytic domain have not been described, and it is unclear whether the mechanism of amino acid recognition is also dissimilar. Here, we show that the α-subunit of the α2ß2 GlyRS from the bacterium Aquifex aeolicus is able to perform the first step of the aminoacylation reaction, which involves the activation of the amino acid with ATP. The crystal structure of the α-subunit in the complex with an analog of glycyl adenylate at 2.8 Å resolution presents a conformational arrangement that properly positions the cognate amino acid. This work shows that glycine is recognized by a subset of different residues in the two types of GlyRS. A structural and sequence analysis of class II catalytic domains shows that bacterial GlyRS is closely related to alanyl tRNA synthetase, which led us to define a new subclassification of these ancient enzymes and to propose an evolutionary path of α2ß2 GlyRS, convergent with α2 GlyRS and divergent from AlaRS, thus providing a possible explanation for the puzzling existence of two proteins sharing the same fold and function but not a common ancestor.

Structure of a bacterial ribonuclease P holoenzyme in complex with tRNA.

Ribonuclease (RNase) P is the universal ribozyme responsible for 5'-end tRNA processing. We report the crystal structure of the Thermotoga maritima RNase P holoenzyme in complex with tRNA(Phe). The 154 kDa complex consists of a large catalytic RNA (P RNA), a small protein cofactor and a mature tRNA. The structure shows that RNA-RNA recognition occurs through shape complementarity, specific intermolecular contacts and base-pairing interactions. Soaks with a pre-tRNA 5' leader sequence with and without metal help to identify the 5' substrate path and potential catalytic metal ions. The protein binds on top of a universally conserved structural module in P RNA and interacts with the leader, but not with the mature tRNA. The active site is composed of phosphate backbone moieties, a universally conserved uridine nucleobase, and at least two catalytically important metal ions. The active site structure and conserved RNase P-tRNA contacts suggest a universal mechanism of catalysis by RNase P.

The E104D mutation increases the susceptibility of human triosephosphate isomerase to proteolysis. Asymmetric cleavage of the two monomers of the homodimeric enzyme.

The deficiency of human triosephosphate isomerase (HsTIM) generates neurological alterations, cardiomyopathy and premature death. The mutation E104D is the most frequent cause of the disease. Although the wild type and mutant exhibit similar kinetic parameters, it has been shown that the E104D substitution induces perturbation of an interfacial water network that, in turn, reduces the association constant between subunits promoting enzyme inactivation. To gain further insight into the effects of the mutation on the structure, stability and function of the enzyme, we measured the sensitivity of recombinant E104D mutant and wild type HsTIM to limited proteolysis. The mutation increases the susceptibility to proteolysis as consequence of the loss of rigidity of its overall 3-D structure. Unexpectedly, it was observed that proteolysis of wild type HsTIM generated two different stable nicked dimers. One was formed in relatively short times of incubation with proteinase K; as shown by spectrometric and crystallographic data, it corresponded to a dimer containing a nicked monomer and an intact monomer. The formation of the other nicked species requires relatively long incubation times with proteinase K and corresponds to a dimer with two clipped subunits. The first species retains 50% of the original activity, whereas the second species is inactive. Collectively, we found that the E104D mutant is highly susceptible to proteolysis, which in all likelihood contributes to the pathogenesis of enzymopathy. In addition, the proteolysis data on wild type HsTIM illustrate an asymmetric conduct of the two monomers.

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.

Common evolutionary origins of the bacterial glycyl tRNA synthetase and alanyl tRNA synthetase.

Aminoacyl-tRNA synthetases (aaRSs) establish the genetic code. Each aaRS covalently links a given canonical amino acid to a cognate set of tRNA isoacceptors. Glycyl tRNA aminoacylation is unusual in that it is catalyzed by different aaRSs in different lineages of the Tree of Life. We have investigated the phylogenetic distribution and evolutionary history of bacterial glycyl tRNA synthetase (bacGlyRS). This enzyme is found in early diverging bacterial phyla such as Firmicutes, Acidobacteria, and Proteobacteria, but not in archaea or eukarya. We observe relationships between each of six domains of bacGlyRS and six domains of four different RNA-modifying proteins. Component domains of bacGlyRS show common ancestry with (i) the catalytic domain of class II tRNA synthetases; (ii) the HD domain of the bacterial RNase Y; (iii) the body and tail domains of the archaeal CCA-adding enzyme; (iv) the anti-codon binding domain of the arginyl tRNA synthetase; and (v) a previously unrecognized domain that we call ATL (Ancient tRNA latch). The ATL domain has been found thus far only in bacGlyRS and in the universal alanyl tRNA synthetase (uniAlaRS). Further, the catalytic domain of bacGlyRS is more closely related to the catalytic domain of uniAlaRS than to any other aminoacyl tRNA synthetase. The combined results suggest that the ATL and catalytic domains of these two enzymes are ancestral to bacGlyRS and uniAlaRS, which emerged from common protein ancestors by bricolage, stepwise accumulation of protein domains, before the last universal common ancestor of life.

Structural basis of neutralization of the major toxic component from the scorpion Centruroides noxius Hoffmann by a human-derived single-chain antibody fragment.

It has previously been reported that several single-chain antibody fragments of human origin (scFv) neutralize the effects of two different scorpion venoms through interactions with the primary toxins of Centruroides noxius Hoffmann (Cn2) and Centruroides suffusus suffusus (Css2). Here we present the crystal structure of the complex formed between one scFv (9004G) and the Cn2 toxin, determined in two crystal forms at 2.5 and 1.9 Å resolution. A 15-residue span of the toxin is recognized by the antibody through a cleft formed by residues from five of the complementarity-determining regions of the scFv. Analysis of the interface of the complex reveals three features. First, the epitope of toxin Cn2 overlaps with essential residues for the binding of ß-toxins to its Na(+) channel receptor site. Second, the putative recognition of Css2 involves mainly residues that are present in both Cn2 and Css2 toxins. Finally, the effect on the increase of affinity of previously reported key residues during the maturation process of different scFvs can be inferred from the structure. Taken together, these results provide the structural basis that explain the mechanism of the 9004G neutralizing activity and give insight into the process of directed evolution that gave rise to this family of neutralizing scFvs.

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.

Novel NADPH-cysteine covalent adduct found in the active site of an aldehyde dehydrogenase.

PaBADH (Pseudomonas aeruginosa betaine aldehyde dehydrogenase) catalyses the irreversible NAD(P)+-dependent oxidation of betaine aldehyde to its corresponding acid, the osmoprotector glycine betaine. This reaction is involved in the catabolism of choline and in the response of this important pathogen to the osmotic and oxidative stresses prevalent in infection sites. The crystal structure of PaBADH in complex with NADPH showed a novel covalent adduct between the C2N of the pyridine ring and the sulfur atom of the catalytic cysteine residue, Cys286. This kind of adduct has not been reported previously either for a cysteine residue or for a low-molecular-mass thiol. The Michael addition of the cysteine thiolate in the 'resting' conformation to the double bond of the α,ß-unsaturated nicotinamide is facilitated by the particular conformation of NADPH in the active site of PaBADH (also observed in the crystal structure of the Cys286Ala mutant) and by an ordered water molecule hydrogen bonded to the carboxamide group. Reversible formation of NAD(P)H-Cys286 adducts in solution causes reversible enzyme inactivation as well as the loss of Cys286 reactivity towards thiol-specific reagents. This novel covalent modification may provide a physiologically relevant regulatory mechanism of the irreversible PaBADH-catalysed reaction, preventing deleterious decreases in the intracellular NAD(P)+/NAD(P)H ratios.

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.

Crystal structure of the RNA component of bacterial ribonuclease P.

Transfer RNA (tRNA) is produced as a precursor molecule that needs to be processed at its 3' and 5' ends. Ribonuclease P is the sole endonuclease responsible for processing the 5' end of tRNA by cleaving the precursor and leading to tRNA maturation. It was one of the first catalytic RNA molecules identified and consists of a single RNA component in all organisms and only one protein component in bacteria. It is a true multi-turnover ribozyme and one of only two ribozymes (the other being the ribosome) that are conserved in all kingdoms of life. Here we show the crystal structure at 3.85 A resolution of the RNA component of Thermotoga maritima ribonuclease P. The entire RNA catalytic component is revealed, as well as the arrangement of the two structural domains. The structure shows the general architecture of the RNA molecule, the inter- and intra-domain interactions, the location of the universally conserved regions, the regions involved in pre-tRNA recognition and the location of the active site. A model with bound tRNA is in agreement with all existing data and suggests the general basis for RNA-RNA recognition by this ribozyme.

Genome-wide and structural analysis of the Myb-SHAQKYF family in Entamoeba histolytica.

Amoebiasis is the third leading cause of death among protozoon parasitic diseases in the lower-middle income countries. Understanding the molecular events that control gene expression such as transcription factors, their DNA binding mode and target sequences can help to develop new antiamoebic drugs against Entamoeba histolytica. In this paper we performed a genome and structural analysis of a specific transcription factor. The genome of E. histolytica codifies for 9 EhMybSHAQKYF proteins, which are a family within a large group of 34 Myb-DNA-binding domain (Myb-DBD) containing proteins. Here we compared Entamoeba Myb-SHAQKYF proteins with Myb-like proteins from the Reveille (RVE) family, important regulators of plant circadian networks. This comparison could lead to stablish their role in E. histolytica life cycle. We show that the ehmybshaqkyf genes are differentially expressed in trophozoites under basal cell culture conditions. An in-silico analysis predicts that members of this group harbor a highly conserved and structured Myb-DBD and a large portion of intrinsically disordered residues. As the Myb-DBD of these proteins harbors a distinctive Q[VI]R[ST]HAQK[YF]F sequence in its putative third α-helix, we consider relevant to determine the three-dimensional (3D) structure of one of them. An NMR structure of the Myb-DBD of EhMybS3 shows that this protein is composed of three α-helices stabilized by a hydrophobic core, similar to Myb proteins of different kingdoms. It is remarkable that despite not sharing similarities in their amino acid sequences, the structure of the Myb-DBD of the EhMybS3 is well conserved in this early branching eukaryote.

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.

Selection of a Single Domain Antibody, Specific for an HLA-Bound Epitope of the Mycobacterial Ag85B Antigen.

T cells recognizing epitopes on the surface of mycobacteria-infected macrophages can impart protection, but with associated risk for reactivation to lung pathology. We aimed to identify antibodies specific to such epitopes, which carry potentials for development toward novel therapeutic constructs. Since epitopes presented in the context of major histocompatibility complex alleles are rarely recognized by naturally produced antibodies, we used a phage display library for the identification of monoclonal human single domain antibody producing clones. The selected 2C clone displayed T cell receptor-like recognition of an HLA-A*0201 bound 199KLVANNTRL207 peptide from the Ag85B antigen, which is known to be an immunodominant epitope for human T cells. The specificity of the selected domain antibody was demonstrated by solid phase immunoassay and by immunofluorescent surface staining of peptide loaded cells of the T2 cell line. The antibody affinity binding was determined by biolayer interferometry. Our results validated the used technologies as suitable for the generation of antibodies against epitopes on the surface of Mycobacterium tuberculosis infected cells. The potential approaches forward the development of antibody in immunotherapy of tuberculosis have been outlined in the discussion.