AARS2 gene for mitochondrial alanyl-tRNA synthetase (mtAlaRS) have been reported both in patients with infantile-onset cardiomyopathy and in patients with childhood to adulthood-onset leukoencephalopathy. We present here an investigation of the effects of the described mutations on the structure of the synthetase, in an effort to understand the tissue-specific outcomes of the different mutations. The mtAlaRS differs from the other mtARSs because in addition to the aminoacylation domain, it has a conserved editing domain for deacylating tRNAs that have been mischarged with incorrect amino acids. We show that the cardiomyopathy phenotype results from a single allele, causing an amino acid change R592W in the editing domain of AARS2, whereas the leukodystrophy mutations are located in other domains of the synthetase. Nevertheless, our structural analysis predicts that all mutations reduce the aminoacylation activity of the synthetase, because all mtAlaRS domains contribute to tRNA binding for aminoacylation. According to our model, the cardiomyopathy mutations severely compromise aminoacylation whereas partial activity is retained by the mutation combinations found in the leukodystrophy patients. These predictions provide a hypothesis for the molecular basis of the distinct tissue-specific phenotypic outcomes.The accuracy of mitochondrial protein synthesis is dependent on the coordinated action of nuclear-encoded mitochondrial aminoacyl-tRNA synthetases (mtARSs) and the mitochondrial DNA-encoded tRNAs. The recent advances in whole-exome sequencing have revealed the importance of the mtARS proteins for mitochondrial pathophysiology since nearly every nuclear gene for mtARS (out of 19) is now recognized as a disease gene for mitochondrial disease. Typically, defects in each mtARS have been identified in one tissue-specific disease, most commonly affecting the brain, or in one syndrome. However, mutations in the  Mitochondrial protein synthesis produces key subunits of the oxidative phosphorylation (OXPHOS) complexes that generate the majority of the ATP for our cells. The protein synthesis in mitochondria requires careful synchrony between two genomes and cellular compartments, because the protein components of its machinery are encoded by the nuclear genome and imported into mitochondria, whereas the RNA components (2 rRNAs and 22 tRNAs) are encoded by mitochondrial DNA . Among tGARS and KARS encoding dual localized synthetases for cytoplasmic and mitochondrial translation), we can, however, conclude that the majority of the mtARS defects affect the central nervous system. Nevertheless, the range of CNS phenotypes is also remarkably wide, affecting specific neurons in many cases, and including patients with infantile-, early- and late-onset of disease.Defects of mitochondrial translation are a major cause of diseases that lead to mitochondrial OXPHOS dysfunction . Since 2DARS2; EARS2; MARS2; FARS2; RARS2; VARS2; TARS2; PARS2; NARS2; CARS2; HARS2) and leucyl-tRNA (LARS2) synthetases that cause sensorineural hearing loss and ovarian dysgenesis (YARS2) in a combination of myopathy and anemia  in a phenotype that differs from the other reported mtARS defects, being clinically a fatal early onset cardiomyopathy  is a common founder mutation and carried by all the identified patients with the severe infantile-onset phenotype.The mitochondrial alanyl-tRNA (mtAlaRS) synthetase encoded by ed tRNAs . The mised tRNAs . We havenslation . Based oAARS2 gene were selected for haplotype analysis, based on the haplotype blocks presented previously (Nine single-nucleotide polymorphisms (SNPs) flanking eviously . GenomicArchaeoglobus fulgidus as a template. This bacterial AlaRS is a homodimer with one subunit crystallized with bound tRNA substrate and an alanyl-adenylate analog in so-called “closed” conformation, while the second subunit is in “open,” substrate free conformation (A. fulgidus AlaRS structure (3WQY_A), and the “open” substrate free form was modeled using B chain of the same structure (3WQY_B). Sequence alignment of human mtAlaRS (985 aa) and template A. fulgidus AlaRS (906 aa) was done using similarity matrix BLOSUM62. Multiple sequence alignments of human mtAlaRS and template sequence with a number of prokaryote and eukaryote homologs were done using PROMALS3D server. Obtained alignment was then submitted for homology modeling to the SWISS-MODEL server. Modeling was performed using alignment mode using either 3WQY_A or 3WQY_B structure as template. Resulting “open” and “closed” structures were analyzed using Discovery Studio v4.1 (Accelrys) software. Overall architecture and arrangement of secondary structure elements in the obtained models was similar to those in the template structures except for two loops, encompassing residues 211–231 and 540–561, which were omitted in the analysis. Docking of the bacterial tRNAAla and alanyl-adenylate into the model was done after superimposition of template chain A with the modeled human mtAlaRS using Discovery Studio v4.1 (Accelrys) software.For human mtAlaRS homology modeling we used the recently solved structure of full-length alanyl-tRNA synthetase (AlaRS) from AARS2 mutations have been reported  mutation in one allele (Table 1).Out of these eleven patients six were homozygous for the c.1774C > T (R592W) mutation and five had compound heterozygous AARS2 cardiomyopathy patient originates from a common founder .Patients with British or German origin were recently suggested to share a common disease haplotype for R592W . We presTable 2), with the highest minor allele frequency of 0.0006 in Finland, where the first AARS2 cardiomyopathy patients were identified.Carriers of the c.1774C > T (R592W) mutation can be found in exome sequencing databases of different populations . The cardiomyopathy patients were either homozygous for the editing domain mutation R592W or had it in combination with a mutation located in the aminoacylation, editing or the C-terminal domain or with a truncating mutation. Mutations that cause premature termination or frameshift can be expected to be total loss-of-function alleles with no stable synthetase present. The mutation spectrum was very different for the leukodystrophy patients because these presented combinations of two missense mutations in the aminoacylation domain or an aminoacylation domain missense mutation with a truncating mutation, with one exception, which was a combination of missense mutations in the aminoacylation domain and the C-terminal domain. Thus none of the missense mutations in leukodystrophy patients were in the editing domain, which was in striking contrast to all of the cardiomyopathy patients .The R592W amino acid change locates to the editing domain of the mtAlaRS and we initially hypothesized that it causes mitochondrial mistranslation, which can be expected to harm OXPHOS assembly and function by amino acid misincorporation in the subunits that are synthesized by mitoribosomes . The newg domain . All of A. fulgidus AlaRS sequences shared overall 17.5% identity and 30.9% similarity . Domain-specific sequence identity and similarity was 17.5 and 31.2% for the aminoacylation domain, 25.0 and 39.8% for the editing domain and 14.7 and 26.5% for the C-terminal domain, respectively. Each domain can be further divided into two subdomains: the aminoacylation domain contains subdomains for aminoacylation and tRNA recognition, the editing domain consists of β-barrel and editing core, and the C-terminal or so-called C-Ala domain consists of helical and globular subdomains .The recent crystal structure of a full-length length AlaRS  made posA. fulgidus was resolved as a dimer, with one subunit in a substrate bound “closed” state and the other in a substrate free “open” state  for docking into our model and explore intermolecular interactions within the tertiary complex . The homology modeling suggests that human mtAlaRS shares with A. fulgidus AlaRS the unique structural features inherent for all AlaRSs. Firstly, it has a large tRNA-binding cavity whereas other ARSs adopt cognate tRNAs on their surface. Secondly, all three domains are involved in the binding of tRNA undergoing aminoacylation. Previous studies have shown that tRNA binding and the rate of aminoacylation are enhanced by the C-terminal domain as it has the highest tRNA binding affinity , and to the tRNA recognition subdomain and the surface of editing core . Close inspection of the A. fulgidus AlaRS structure as well as the modeled tertiary complex of mtAlaRS revealed that the “safety belt” folds along the surface of the β-barrel in the editing domain and establishes different interactions with its surface exposed residues in “open” and “closed” states . This suggests that the β-barrel may serve as a “buckle” for fastening the “safety belt” when the tRNA is bound in the aminoacylation site. In human mtAlaRS the interacting surfaces of the linker and β-barrel are comprised of different residues than in the template, but comparison of “open” and “closed” conformations shows rearrangements in their interactions upon tRNA binding . Interestingly, the linker has an amino acid sequence stretch, L494-Q505, which is highly conserved between mitochondrial homologs . Modeled structure shows that L502 from this stretch is involved in hydrophobic interactions predominantly with residues from β-hairpin (R592–A604) of the β-barrel. Since this motif is also very conserved between mitochondrial homologs and oriented toward the linker it is likely that these two fragments extend their interaction by establishing transient contacts upon sliding of the linker on the surface of the β-barrel during transition from “open” to “closed” conformations of the enzyme. Therefore, we propose that the editing domain of mtAlaRS, in addition to its proofreading activity of deacylating mischarged tRNAs, may also be involved in the aminoacylation activity of the enzyme by being involved in the binding of tRNAAla for aminoacylation and stabilizing position of its acceptor stem within the catalytic site.Since the template structure of AlaRS of n” state , it was affinity . The string core . We predFigure 7) to predict their effects on synthetase structure and function (Table 3). The missense mutations could be divided into three groups based on the effect on the aminoacylation activity: loss-of-function, severe, and moderate.We utilized our full-length mtAlaRS homology model to localize all amino acid changes found in mtARS in patients with cardiomyopathy  and leukodystrophy  are predicted to impair protein folding and stability resulting in loss of aminoacylation activity . Most of these residues are invariant between cytoplasmic, mitochondrial, and bacterial homologs of AlaRS and critical for maintaining the structure. Accordingly, the change L155R was recently modeled in yeast and had a deleterious effect on cell growth suggesting complete absence of aminoacylation activity  changes are not predicted to affect protein stability, as they are all surface exposed . However, each of these mutations is predicted to result in dramatic reduction of aminoacylation rate due to effects on substrate binding. In case of A77V  the binding and activation of alanine is likely to be affected. Y539C and R592W are predicted to impair tRNA binding and stabilization within the aminoacylation site by affecting interactions between the linker and β-barrel that we propose to be important for binding and securing acceptor stem of tRNAAla within the aminoacylation domain.The A77V, Y539C, and R592W  changes  affect structural residues and are predicted to cause local structural disturbances within the aminoacylation domain leading to partial reduction of aminoacylation activity. The F50C mutant modeled in yeast showed partial reduction of tRNAAla charging and protein instability, which became noticeable only at a higher temperature .In conclusion, our analysis of the homology model predicts that the missense mutations in the first two categories, affecting overall structure and substrate binding, severely compromise the aminoacylation by the synthetase, which is also true for the truncating mutations, whereas the missense mutations in the third category result in reduced function but with some retained aminoacylation activity. Combining the data of the compound heterozygous mutations in each patient clearly shows that all patients with leukodystrophy have combinations where one allele is in one of the two severe categories or is a truncating mutation and the other allele is in the third, moderate category, whereas all mutations of the cardiomyopathy patients are severe (AARS2, however, shows that mutations in the same mtARS can also give rise to highly different tissue-specific diseases.The tissue-specificity of mitochondrial diseases is puzzling. The pathogenic mechanisms behind the tissue-specific manifestations are likely to involve cell type-specific mitochondrial functions and metabolite requirements, in addition to defects of ATP production by oxidative phosphorylation . DiffereA. fulgidus, suggested instead that all domains of the synthetase, including the editing domain, have an important role in tRNA binding for aminoacylation. We speculate whether this unique way of cooperative tRNA binding for aminoacylation in AlaRSs might explain why the mtAlaRS has a preserved editing domain whereas most other mitochondrial ARSs do not. For example, editing activity was lost during the evolution of mtPheRS (Our previous hypothesis for the cardiomyopathy-related R592W was that it affected tRNA binding in the editing domain and thus interfered with the proofreading of mischarged tRNAs. The homology modeling of human mtAlaRS, based on the new resolved structure of AlaRS of  mtPheRS , and the mtPheRS . AlthougAARS2 mutations are simply caused by differential effects on aminoacylation, with cardiomyopathy resulting from highly severe reduction in aminoacylation activity, and leukodystrophy resulting from only partial reduction in activity. This conclusion could be reached only by careful analysis of the combined mutations found in the patients. The clinical feature of the cardiomyopathy patients was the heart defect, although OXPHOS defects could be seen also in muscle and brain. This suggests that the patients had a combined tissue-involvement that can be expected when mitochondrial protein synthesis is significantly compromised, and that other tissues would have also suffered, had the patients lived longer.We suggest that the two distinct phenotypes caused by AARS2 mutations manifested in the heart soon after birth – or even in late pregnancy – whereas the partial reduction of the synthetase function manifested from 2 to 40 years of age in the brain (We have previously proposed that severe defects of mitochondrial translation manifest in the heart immediately or soon after birth , most ofhe brain .in vivo studies on the partial retained activity of the leukodystrophy mutations – sufficient for heart function but limiting for brain function during later years – are warranted, to get further insight into the particular events taking place in the heart and in the brain, constituting ‘the narrow permissive window for disease manifestation’ (In the future, station’ . The rolThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


MaOFP1 was identified in banana (Musa acuminata L.AAA) fruit by a yeast two-hybrid (Y2H) method using the banana MADS-box gene MuMADS1 as bait and a 2 day postharvest (DPH) banana fruit cDNA library as prey. The interaction between MuMADS1 and MaOFP1 was further confirmed by Y2H and Bimolecular Fluorescence Complementation (BiFC) methods, which showed that the MuMADS1 K domain interacted with MaOFP1. Real-time quantitative PCR evaluation of MuMADS1 and MaOFP1 expression patterns in banana showed that they are highly expressed in 0 DPH fruit, but present in low levels in the stem, which suggests that simultaneous but different expression patterns exist for both MuMADS1 and MaOFP1 in different tissues and developing fruits. Meanwhile, MuMADS1 and MaOFP1 expression was highly stimulated and greatly suppressed, respectively, by exogenous ethylene. In contrast, MaOFP1 expression was highly stimulated while MuMADS1 was greatly suppressed by the ethylene competitor 1-methylcyclopropene (1-MCP). These results indicate that MuMADS1 and MaOFP1 are antagonistically regulated by ethylene and might play important roles in postharvest banana fruit ripening.The ovate family protein named  Transcription factors containing the MADS domain  are present in the majority of eukaryotic organisms –5. InitiArabidopsis and investigated the interaction map of more than one hundred Arabidopsis MADS-box transcription factors, which provided valuable information for identifying the functions of proteins involved in certain development processes [Arabidopsis, molecular and genetic evidence suggests that AG-SEP complexes interact with the homeodomain transcription factor BELL1 (BEL1) to regulate ovule development, in particular to repress activity of the homeobox protein WUSCHEL in the ovule chalaza to control outer integument development [MADS-box transcription factors meditate their prominent roles in plant development through a complex network of protein-DNA and homo- or heterodimeric protein-protein interactions . MADS-borocesses –24. Baserocesses –27. Theselopment . Moreoveelopment –31. DespArabidopsis, tomato and Petunia to investigate protein-protein interactions, this technique has rarely been reported in banana [MuMADS1 that was previously isolated from a banana suppression subtractive hybridization (SSH) library [MuMADS1 expression is closely related to ethylene biosynthesis and fruit ripening and is mainly expressed in the carpel and during different ovary developing stages, while its expression in fruit is strongly induced by exogenous ethylene [MaOFP1. The interaction mechanism of these two proteins in banana was further demonstrated by the Y2H method and Bimolecular Fluorescence Complementation (BiFC). The expression of MuMADS1 and MaOFP1 in different tissues and developing stages showed simultaneous but different patterns. Moreover, MuMADS1 and MaOFP1 expression in postharvest ripening stages following different treatments was antagonistically regulated at the transcriptional level by ethylene and 1-MCP. These results may increase the understanding of MADS-box gene mechanisms in regulating banana fruit development and ripening.Although yeast two-hybrid (Y2H) assays have been used in many plants such as n banana . In this library , was cloethylene . Using tMuMADS1 as bait and 2 day postharvest (DPH) banana fruit cDNA library as prey. After identification on different minimal synthetic dropout (SD) supplements media, plasmids from the blue clones were isolated. Through Y2H we obtained a full-length ovate family protein gene designated MaOFP1, which contained a 542 bp sequence with a 438 bp full-length open reading frame (ORF) that encodes a protein of 146 amino acids. Bioinformatics analysis showed that MaOFP1 has a well-conserved ovate domain in its C-terminus and shared 37.91% identity with four other ovate family protein genes from the banana A genome that are located on chromosomes 7, 5, 6 and 9 , respectively, under the control of the cauliflower mosaic virus 35S promoter (CaMV 35S). The two recombinant constructs were introduced into onion (Allium cepa) epidermal cells by the particle delivery method. Free YFP protein was distributed throughout the whole cell (nucleus and cytoplasm)  , whereas nucleus . As negacted Fig  and 2D.MaOFP1 were transformed into yeast strain AH109 and grown on auxotrophic selection media SD/-Ade/-His-Leu/-Trp+x-α-gal, blue clones appeared only for K+MaOFP1 and the positive control (pGBKT7-53+ pGADT7-T-antigen) as compared to the negative control. Other interactions of M + MaOFP1, I + MaOFP1 and C+ MaOFP1 yielded no blue clone growth. This result indicated that the K domain of MuMADS1 interacts with MaOFP1 in yeast . When the bait vectors containing the M, I, K and C domains and the prey vector containing east Fig  and 3B.MuMADS1 and MaOFP1 were mainly expressed in the ovary 4 (Ov4), Ov1 stages and postharvest 0 DPH fruits. In 0 DPH fruits, MuMADS1 and MaOFP1 reached relative expression levels of 31.13 and 59.72, respectively. Both genes were poorly expressed in the stems with relative expression levels of 0.62 and 0.36, respectively  and were shown to regulate plant growth and development [The OVATE gene is found exclusively in plants and encodes a protein with a putative nuclear localization signal and an approximately 70-aa C-terminal conserved domain, which was also known as the Domain of Unknown Function 623 (DUF623) . Subsequelopment –39.Arabidopsis KNOTTED1-like Arabidopsis thaliana (KNAT) members [Arabidopsis [Capsicum annuum), a relative of tomato in the Solanaceae family, an OVATE family member (CaOvate) was also shown to be involved in determining fruit shape by negatively affecting the expression of CaGA20ox1 [Arabidopsis and other plants remains relatively poor.AtOFPs were shown to have close functional interactions with three amino acid loop extension (TALE) homeodomain proteins, with AtOFP1 and AtOFP5 known to regulate the sub-cellular localization of TALE homeoproteins . AtOFP1  members –41, to r members . AtOFP5  members , and canbidopsis . Recentlbidopsis . In peppaGA20ox1 . HoweverMaOFP1 into bait and prey vectors, respectively, and subsequent transformation into yeast, the interaction of the K domain of MuMADS1 with MaOFP1 was again demonstrated by the appearance of blue clones upon cultivation on auxotrophic selection media SD/-Ade/-His-Leu/-Trp+ x-α-gal  roots, stems, leaves, flowers and developing ovaries were obtained from the Institute of Tropical Bioscience and Biotechnology banana plantation . Fruits were harvested at the mature green stage (100–110 d after flower shooting). Banana hands having a similar developmental stage were selected and five fingers from the hands were divided into three groups for different treatments. A control group was allowed to ripen naturally; the second and third groups were exposed for 12 h to 100 μL L-1 ethylene and 1 μL L-1 1-MCP , respectively [Banana (ectively . The trehttp://www.clontech.com/). A fragment of MuMADS1 was amplified by PCR with the primer pairs P1: 5′-CGGAATTCGATGGGAAGGGGTAAGAT-3′ and P2: 5′-CGGTCGA CTCTGATCCTTGTGATGC-3′. The PCR products were digested by EcoRI and SalI and cloned into the EcoRI- SalI site of the pGBKT7 bait vector and then immunodetected. In parallel, a 2 DPH cDNA expression library was constructed from 2 DPH banana fruit total RNA according to the manufacturer’s instructions . Subsequently, yeast cells containing the bait construct were transformed with the 2 DPH ‘‘prey” cDNA library according to the CLONTECH protocol. In total, 1.5×106 individual yeast transformants were generated.Y2H screening was performed using a MATCHMAKER GAL4 Two-Hybrid System 3 according to the manufacturer’s protocol . The thermal cycling conditions were 94°C for 1 min followed by 30 cycles of 94°C for 30 s, and 68°C for 3 min. The amplified product was then inserted into the PMD-18T (Takara) vector, sequenced and the sequence analyzed by BLAST (http://ncbi.nlm.nih.gov/blast). Putative conserved domains were detected at NCBI (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). Sequences from the banana A genome were aligned using DNAMAN software. The neighbor-joining method was used to construct the phylogenetic tree with MEGA software .Single clones obtained by Y2H were identified again on different minimal media with SD/-Leu/-Trp, SD/-His/-Leu/-Trp, SD/-Ade/-His/-Leu/-Trp and SD/-Ade/-His/-Leu/-Trp+x-α-gal. An ovate family protein gene was amplified using long distance (LD) PCR primers P1: MuMADS1 cDNA fragment was subcloned into the BamHI/SalI sites of pXY106  containing the N-terminal fragment of the yellow fluorescent protein , and a MaOFP1 cDNA fragment was subcloned into the KpnI/XbaI sites of pXY104  containing the C-terminal fragment of the YFP (YC). The resulting two plasmids, pXY106-MuMADS1 (MuMADS1-YN) and pXY104-MaOFP1 (MaOFP1-YC), were co-bombarded with gold particles (Bio-Rad) into onion (Allium cepa) epidermal cell layers as described [–1 4', 6-diamido-2-phenylindole (DAPI).A escribed . The CaMMuMADS1 were cloned into pGBKT7 to fuse with the bait domain (BD) using the primers . The PCR products were digested with EcoRI and SalI and cloned into the EcoRI- SalI site of the pGBKT7 bait vector. MaOFP1 was cloned into pGADT7 to fuse with the activation domain (AD) of GAL4. Self-activation was assayed on selective synthetic dropout medium plates . Then, combinations were simultaneously transformed into the yeast strain AH109 according to the protocol. The transformants containing plasmids pGADT7 and pGBKT7 were used as a negative control. The interactions were judged by growth on selective medium  according to the manufacturer’s protocol .Four domains: M (50 aa), I (40 aa), K (50 aa) and C (95 aa) of Total RNA was extracted from the roots, stems, flowers, leaves, developing ovaries and fruits using a modified cetyltrimethylammonium bromide (CTAB) method . First-s+mRNA (200 ng) was converted into cDNA using the SMART PCR cDNA Synthesis Kit (Clontech) in a final volume of 20 μL, which subsequently served as the template for real-time PCR. Primer sets used in real-time RT-PCR for MuMADS1 and MaOFP1 were: MADS1RTP1: 5′-GTGGAGCTTCAGAGTGACAACATG-3′; MADS1RTP2: 5′-TGGTCCTGGTG ATGCGAGTAGTG-3′; OFP1RTP1: 5′-GCCATTAACGGCCCACAGTTC-3′; OFP1 RTP2: 5′-TGGTGGAGAGGTGAATTCAAG-3′. RT-PCR of actin was used as an internal control. For amplification of actin, the following primer set was designed: forward, 5'-TGTAGCAATTCAGGCTGTTCTT-3' and reverse, 5'-TCAGAGATGG CTGGAAGAGAAC-3'. SYBR Premix Ex Taq (TaKaRa) was used in 25 μL reactions with 0.5 μL ROX reference dye. Primers were used at 100 nM each with the equivalent of 100 ng reverse-transcribed RNA template per reaction. In all experiments, appropriate negative controls containing no template RNA were subjected to the same procedure to exclude or detect any possible contamination. Before proceeding with the actual experiments, a series of template dilutions was performed to determine the optimal template concentration to be used in the experiments for maximal target amplification.Total RNA was isolated from the pulp of banana fruit at different ripening stages following different treatments. Poly (A)MuMADS1 and MaOFP1 products were compared. Differences in quantification cycle (Cq) values between the MuMADS1, MaOFP1 and MaActin transcripts were expressed as fold-change relative to actin.Each quantitative real-time PCR was performed using the Stratagene Mx3000P  device and SYBR chemistry. The thermal cycling conditions were 94°C for 3 min followed by 40 cycles of 94°C for 7 s, 55°C for 10 s, and 72°C for 15 s. Reactions were performed in triplicate and data analyzed using MxProTM QPCR software . Actin was used as the calibrator or control sample to which


The variable domains of antibodies and T-Cell receptors (TCRs) share similar structures. Both molecules act as sensors for the immune system but recognise their respective antigens in different ways. Antibodies bind to a diverse set of antigenic shapes whilst TCRs only recognise linear peptides presented by a major histocompatibility complex (MHC). The antigen specificity and affinity of both receptors is determined primarily by the sequence and structure of their complementarity determining regions (CDRs). In antibodies the binding site is also known to be affected by the relative orientation of the variable domains, VH and VL. Here, the corresponding property for TCRs, the Vβ-Vα orientation, is investigated and compared with that of antibodies. We find that TCR and antibody orientations are distinct. General antibody orientations are found to be incompatible with binding to the MHC in a canonical TCR-like mode. Finally, factors that cause the orientation of TCRs and antibodies to be different are investigated. Packing of the long Vα CDR3 in the domain-domain interface is found to be influential. In antibodies, a similar packing affect can be achieved using a bulky residue at IMGT position 50 on the VH domain. Along with IMGT VH 50, other positions are identified that may help to promote a TCR-like orientation in antibodies. These positions should provide useful considerations in the engineering of therapeutic TCR-like antibodies. The immune system needs to be able to sense molecules that might be harmful to the organism. Such harmful molecules are known as antigens. Two classes of receptor proteins that mediate antigen recognition are antibodies and T-Cell receptors (TCRs). Antibodies are able to bind a diverse range of antigen shapes whilst TCRs are specialised to recognise a cell-surface protein, the pMHC. Antibodies that bind the pMHC are rarely created naturally. However, such TCR-like antibodies are of therapeutic importance. The binding regions of the TCR and the antibody have very similar three dimensional structures. Both consist of two independent units, domains, which associate and form the antigen binding site between them. This work examines how the two domains orientate with respect to one another in TCRs and antibodies. Our results show that the conformations that exist in TCRs and antibodies are distinct. Consequently it is difficult for an antibody to bind to a pMHC in the same way a TCR would. However, a similar conformation can be achieved in antibodies as in TCRs by the presence of certain amino-acids in the domain interface. This knowledge should aid the development of therapeutic TCR-like antibodies. The immunoglobulin fold provides the scaffold for many different proteins with diverse a set of functions A key task of the immune system is to specifically recognise molecules that are potentially pathogenic or foreign to the organism. Examples of components that enable this are B-cell receptors or, in their soluble form, antibodies. These are able to bind to antigens without the aid of other cellular machinery. The portion of the molecule that mediates antigen binding, the variable fragment (Fv), consists of two immunoglobulin domains, VH and VL.In contrast, another component of the immune system, the T-cell receptor (TCR), binds only to peptide antigens and only when they are presented on the surface of a cell by the major histocompatibility complex (MHC). However, like the antibody, the TCR binds using its variable region that consists of two domains, Vα and Vβ, which are analogous to the antibody VL and VH domains.Each of the variable domain types have three CDRs that are generally characterised as loop structures Recently, the way in which the antibody variable domains are orientated has been recognised as a determinant of antigen binding We and others have studied the geometry of the variable domain orientation in antibodies Although the diversity in potential T-Cell epitopes is much smaller than for antibodies, variation is present in the MHC class, allele, and the bound peptide The relative similarity between T-Cell epitopes is also reflected in the structure of the complex formed between the TCR and MHC/peptide. Typically, the TCR is found to bind diagonally with respect to the MHC's peptide groove. This geometry of interaction is referred to as the canonical binding mode. Those cases where the TCR is positioned in a different orientation are known as non-canonical Naturally, T-Cell and B-Cell receptors perform different functions in the immune system. An example of a non-natural interface between the two receptor types are TCR-like antibodies. These antibodies are specific for T-cell epitopes. Given that they bind to a component of the organism, they are not normally found naturally in the body. However, in diseases where T-cell functionality is inhibited, engineered TCR-like antibodies provide a method for delivering cytotoxic drugs and induction of infected cell apoptosis Antibodies able to recognise T-cell epitopes must mimic a TCR and replicate their binding site properties to enable pMHC specificity. Producing TCR-like antibodies is a difficult procedure inhibited by the process of isolating the desired MHC/peptide epitope In this study we investigate the Vβ-Vα domain orientations in TCR variable regions and compare them to the analogous structural space in antibodies. Functional reasons for differences in the two receptor types are proposed by analysing the influence of domain orientation on the structural properties of a TCR-MHC complex. Given that the orientation space is different we investigate which sequence positions may be contributing factors and suggest residues on the domain interface of antibodies that may induce a more TCR-like orientation.All X-Ray crystal structures containing a paired TCR α and β chain were extracted from the protein data bank (PDB) A non-redundant antibody dataset was created using the structural antibody database (SAbDab) The structures in SAbDab were filtered to identify TCR-like antibodies bound to MHCs. A BLAST search To compare the variable domain orientations of antibodies and TCRs it is necessary to define which domains are equivalent in the two receptor types. Is VH equivalent to Vβ or Vα?Comparing the mean sequence identity between the domain types showed that Vκ and Vλ (collectively VL) domains were more similar to both Vα and Vβ than VH was to either. Therefore, sequence identity alone provides no clear indication.To compare the use of the VH-Vα/VL-Vβ or the VH-Vβ/VL-Vα equivalence we examined the residue conservation at individual IMGT positions in the sets of domains. To check the use of VL/Vα and VH/Vβ equivalence we identified IMGT positions where the VH and Vα domains have the same conserved residue whilst in VL domains a different conserved amino-acid is present. Positions such as these suggest that the alternative equivalence should be used. These positions were identified for both equivalences i.e. VH-Vβ/VL-Vα and VH-Vα/VL-Vβ. A position was deemed to be conserved if it had the same amino-acid in 50% or more sequences. Five positions support making VH-Vα/VL-Vβ equivalent whilst 10 positions support making VH-Vβ/VL-Vα equivalent. This equivalence is also supported by the similarity in the combination of genes that make up corresponding domains. Both Vβ and VH are generated from v, d and j genes whilst Vα and VL are built from just v and j genes. We therefore use the VH-Vβ/VL-Vα equivalence herein.The variable domain orientation root mean square deviation (RMSD) is a measure of the difference in orientation between two structures. As an example, the orientation RMSD between two TCR structures (Tx and Ty) is calculated as follows. Tx and Ty are structurally aligned using the Cα coordinates of their shared Vα domain framework positions. Tx's Vβ domain is then independently aligned to Ty's Vβ domain. The RMSD is calculated between Tx's Vβ domain in its native orientation and the transformed position. The same procedure is also performed using the Vβ domains for the initial alignment and calculating RMSD between Vα domains. The mean of these two values describes the difference in the relative orientation. All structural alignments were performed using the Biopython SVD superimposer The orientation RMSD was calculated for each pair of structures in the non-redundant antibody set, the non-redundant TCR set and between the sets. These measurements were used as distances between the structures allowing them to be clustered using a complete-linkage hierarchal clustering method how structures vary. The ABangle methodology was developed to characterise the orientation between the VH and VL domains in antibodies. It gives six absolute measures, five angles and a distance, to fully describe the orientation between the two domains. The same method can be applied to any set of structurally defined homologous domain pairs. The process is presented in detail in Dunbar et al 2013 Whilst relative measures of orientation differences such as the orientation RMSD give a magnitude to changes in pose they do not reveal coresets).Define the most structurally conserved positions for both domains  structures for both domains.Compute or choose the pivot axis about which to measure orientation.Define six measures about the resulting coordinate system to describe orientation.coreset positions are listed in the supplementary material. To allow for direct comparisons to be made with antibodies, the pivot axis, C, was chosen to be the same as in the antibody ABangle work. However, the pivot axis was also calculated for TCRs and has been used in a separate study This process was followed for the TCR structures in our dataset. The The resulting coordinate system allows for 6 absolute measures of orientation to be defined, five angles and a distance. The distance, dc, is the length of C. HL describes a torsion angle of one domain with respect to the other and is similar to the packing angle defined by Abhinandan et al TCRs are often observed to bind to the MHC in a diagonal mode with respect to the peptide binding groove. Qualitative observations have labelled this association geometry as the canonical binding mode. The binding geometry can also be described quantitatively using the MHC-TCR docking angle The effect that changing the Vβ-Vα orientation has on the structure of a TCR-MHC complex was investigated. A single TCR-MHC-peptide complex was chosen PDB 1mi5  as the nThe native complex was compared to structures made when the Vβ-Vα orientation was changed to assume the poses in the TCR and antibody decoy sets. The following protocol was used to change the Vβ-Vα orientation.Given a decoy structure, the VL domain (or Vα domain for TCR decoys) was aligned to the Vα domain of the native complex . This stAfter remodelling the residue side chains with Scwrl4 Antibody and TCR structures were clustered based on their orientation RMSD . The TCRWhilst the RMSD measure shows that the orientations of antibodies and TCRs have distinct conformations it does not show us how the structures differ. We therefore used the ABangle methodology to identify structural differences.−16). In fact, antibodies are almost never observed to reach the extreme HC2 orientation seen in TCRs. Two of the other orientation angles, LC1 and LC2 are also significantly different  whilst the two receptor types do not have significantly different HL torsion angles or HC1 bend angles. The distributions of the dc length are significantly different with a p-value of 3.46×10−8. The difference in orientation between antibodies and TCRs can be best described as a twisting-like change of the variable domains with respect to one another. ABangle's six orientation measures were calculated for each structure in the dataset. For such similar molecules in terms of their function and domain structure, it is somewhat surprising that TCRs and antibodies have such different orientations. As discussed previously, TCRs bind specifically to peptide antigens only when they are presented by MHCs on the surface of a cell. In comparison, antibodies are not restricted in the same way and bind to a far more varied set of antigenic shapes.To investigate whether the variable domain orientation is likely to affect function we calculated the influence that changing the Vβ-Vα orientation had on the structure of a single TCR-MHC complex PDB 1mi5). Two sets of orientation decoys were used, one from antibody structures and the other from other TCR structures . In comparison, medians of 5 and 8 clashes were induced for the equivalent domains using the antibody decoys (10 and 14 before side chain rearrangement). A Mann-Whitney U test found the increase in the total number of clashes to be statistically significant . The increased number of clashes between the proteins suggests that antibody orientations are incompatible with binding in a canonical TCR-like mode. For the native complex and the re-orientated complex sets, the mean Cβ-Cβ contact distances were calculated. The contact matrices for each set are shown in Supplementary Figure S5 in The difference between the antibody-orientated complexes and the TCR-orientated complexes can also be measured using the energetics of the structures. Supplementary Figure S1 in These results suggest that binding in a TCR-like mode requires the receptor to have a TCR-like variable domain orientation. Although the Vβ-Vα orientation is at least as variable as the VH-VL orientation, TCR conformations are generally compatible with binding to the MHC in a canonical mode. In contrast, the space of antibody orientations is such that the canonical mode becomes more difficult to obtain. Despite this observation, antibodies may be engineered such that they bind to MHC-peptide epitopes. These molecules are known as TCR-like antibodies.The orientations of each of the TCR-like antibodies found in SAbDab were calculated using the ABangle methodology. Supplementary Figure S2 in To determine whether these TCR-like antibodies behave similarly to TCRs we investigated the geometry of the complex they form with the MHC using the docking angle see . Most ofOnly one of the TCR-like antibodies, 3cvh, binds in a similar mode to the TCR structures and with the expected equivalence. As discussed above, 3cvh, is the only antibody that had an variable domain orientation that approached that seen in the TCR space. Although only a single example, this structure suggests that in order to be able bind in a TCR-like mode to the MHC, a TCR-like variable domain orientation should be promoted.Given that variable domain orientation may be related to the functions of antibodies and TCRs, we investigated which factors give rise to the structural differences between them. The type of antigen an antibody binds does not determine its absolute VH-VL orientation Supplementary Figures S3a and S3b in To test the effect of the VL CDR3 length on orientation in antibodies we identified all non-redundant structures in SAbDab with CDRL3 loops of 13 residues. Nine such antibodies exist  but with a different amino acid in each set. Ten positions were identified and are listed in Each of the antibody compatible mutations is located on the periphery of the VH-VL interface. Positions from a similar region of the domain interface were previously found to be influential for variation in the HC2 measure of VH-VL orientation In summary, the packing of the CDR3 of Vα and VL domains is likely to be an influential factor for domain orientation. However, having a long antibody CDRL3 does not necessarily replicate the TCR orientation. Instead, the VH-VL orientation can be made more TCR-like by adopting specific amino acids at certain interface positions: phe-H50, gln-H6 and val-H4.The orientation between the antibody variable domains, VH and VL, has previously been found to be influential in determining the geometry of the antigen binding site The functional implications of variable domain orientation were investigated by analysing its effect on the structure of a TCR-MHC complex. The Vβ-Vα orientation was changed to assume the orientation of a set of antibody structures and a set of other TCR structures. The contacts, clashes and energetics of the transformed complex structures were analysed. Antibody orientations are found to be incompatible with binding to the MHC in a canonical manner. In contrast, assuming orientations of other TCRs disrupted the complex structure far less. The TCR and MHC are thought to have co-evolved over millions of years allowing for their interaction to be optimised Despite the apparent steric restriction imposed by their variable domain orientations, it is possible to engineer antibodies that bind to the MHC. The small number of structures of TCR-like antibodies bound to an MHC were examined and their docking angle calculated. Only one of the three cases, 3cvh, binds in the canonical TCR binding mode. This antibody has a VH-VL orientation that is similar to the TCRs. The other TCR-like antibodies also bind in a diagonal mode but use their VH domains in the same way as a TCR uses its Vα domain. Reversing the equivalence of the domains allows them to overcome the steric restrictions imposed by the variable domain orientation. The current number of available structures is small so it is not possible to make statistically robust conclusions. As more structures become available how TCR-like antibodies bind the MHC might be better understood. To understand how an antibody may be engineered to bind specifically to the MHC in a canonical manner, we examined the factors that cause antibodies and TCRs to be distinct in the HC2 angle.TCRs tend to have longer Vα CDR3 loops than antibodies have VL CDR3 loops. The Vα CDR3 packs into the domain-domain interface and acts to open up the Vβ-Vα orientation. However, antibody structures with similar longer VL CDR3 loops are generally not observed to share the TCR orientation. The length of the loop alone is therefore not predictive of the variable domain orientation. However, a TCR-like orientation is found in antibodies with particular interface residues. The most influential position we identified was at IMGT position 50 on the VH domain. Here, antibodies with a phenylalanine instead of the germline leucine tend to have a TCR-like orientation. The TCR itself predominantly has a leucine at this position. Thus in TCRs the interface is twisted open due to packing a longer Vα CDR3 in the interface but in antibodies the same effect is achieved by the incorporation of a bulky residue, phenylalanine, in the interface. Along with IMGT 50 on the VH domain, additional candidate positions were also identified that could be mutated to increase the similarity of the VH-VL interface to the Vβ-Vα interface. Together, these positions provide promising targets for the rational engineering of antibodies specific to MHCs.Text S1All supplementary figures and tables are contained within this file.(DOCX)Click here for additional data file.


Plasmodium knowlesi, previously associated only with infection of macaques, is now known to infect humans as well and has become a significant public health problem in Southeast Asia. This species should therefore be targeted in vaccine and therapeutic strategies against human malaria. Apical Membrane Antigen 1 (AMA1), which plays a role in Plasmodium merozoite invasion of the erythrocyte, is currently being pursued in human vaccine trials against P. falciparum. Recent vaccine trials in macaques using the P. knowlesi orthologue PkAMA1 have shown that it protects against infection by this parasite species and thus should be developed for human vaccination as well. Here, we present the crystal structure of Domains 1 and 2 of the PkAMA1 ectodomain, and of its complex with the invasion-inhibitory monoclonal antibody R31C2. The Domain 2 (D2) loop, which is displaced upon binding the Rhoptry Neck Protein 2 (RON2) receptor, makes significant contacts with the antibody. R31C2 inhibits binding of the Rhoptry Neck Protein 2 (RON2) receptor by steric blocking of the hydrophobic groove and by preventing the displacement of the D2 loop which is essential for exposing the complete binding site on AMA1. R31C2 recognizes a non-polymorphic epitope and should thus be cross-strain reactive. PkAMA1 is much less polymorphic than the P. falciparum and P. vivax orthologues. Unlike these two latter species, there are no polymorphic sites close to the RON2-binding site of PkAMA1, suggesting that P. knowlesi has not developed a mechanism of immune escape from the host’s humoral response to AMA1.The malaria parasite  Plasmodium species: P. falciparum, P. vivax, P. malariae and P. ovale. It has now been confirmed, however, that natural human infection also occurs with P. knowlesi [P. knowlesi, previously confused with infection by the less virulent P. malariae, is quite widespread in Southeast Asia and can lead to mortality [P. knowlesi in therapeutic and vaccine strategies against human malaria.Human malaria was long thought to be restricted to infection by four knowlesi , a speciortality –4. AccorPlasmodium parasite, includes an ectodomain, a transmembrane region and a cytoplasmic domain. The ectodomain comprises three domains referred to as Domain 1, Domain 2 and Domain 3. AMA1 is produced in the microneme organelles and transferred to the parasite surface just prior to or during red blood cell (RBC) invasion [P. knowlesi [Plasmodium species, as well as in other members of the Apicomplexa phylum [Plasmodium species, antibodies raised against the ectoplasmic region of the protein have been shown to inhibit invasion, and immunization with AMA1 in animal models protects against infection [P. falciparum AMA1 ectodomain are currently being pursued in clinical trials [Apical Membrane Antigen 1 (AMA1), a type 1 transmembrane protein of the invasion . First dknowlesi , AMA1 waa phylum –9. AMA1 nfection –14. In sl trials , 16.Plasmodium species and other members of the Apicomplexa phylum  have re caninum , 22, sug caninum , 24. In receptor ,26. Furtreceptor , 28. Morreceptor . This moreceptor –33, showP. knowlesi merozoites, is specific for Plasmodium knowlesi AMA1 (PkAMA1) and inhibits in vitro multiplication of the parasite [P. knowlesi merozoites in the presence of R31C2 have shown that the parasite makes extensive contacts with the RBC surface, characteristic of the random attachment that occurs during the first stage of invasion [The monoclonal antibody (mAb) R31C2, raised in rats against the W1 variant of parasite . R31C2 wparasite . Electroinvasion . Howeverinvasion , 36–38.P. knowlesi that could be specifically used in Southeast Asia. Here we report the crystal structure of Domains 1 and 2 of the ectoplasmic region of PkAMA1 and of its complex with the Fab fragment of R31C2 which shows that the mAb binds to the hydrophobic groove. In addition, the D2 loop (residues Pro295 to Ser332), which is displaced upon binding the RON2 receptor, remains fixed in the same conformation as the unbound PkAMA1 even though the antibody makes a significant number of direct contacts with this region of the antigen. We also examine the polymorphism of PkAMA1 in the light of these structural data.We have recently tested PkAMA1 as a vaccine in monkeys . These eP. knowlesi H strain  and an additional 23 C-terminal residues that include the c-myc tag and a hexa-His tag. The synthetic gene was adapted to P. pastoris codon usage and four potential N-glycosylation sites were mutated as follows: Asn107Lys, Ser178Asn, Asn189Glu and Ser240Arg. . The recombinant gene was maintained in frame with the c-myc-hexahistidine tag by the addition of two bases to the restriction site (XbaI); the sequence at the end of the gene sequence thus codes for an additional glycine [The recombinant PkAMA1 construct comprises residues Pro43 to Pro387 from the PkAMA1 sequence of the  glycine .P. vivax AMA1 construction [The culture protocol was similar to that reported for the truction , except P. knowlesi strain [H) and light-chain variable domain (VL) PCR products using variable domain primers were cloned into the Invitrogen sequencing vector pCR2.1 and transformed into TOP10 cells. Positive clones were identified by colony PCR. The sequences have been deposited in GenBank with entry number KM225619 for VL and KM225620 for VH.R31C2, a monoclonal antibody raised in an A0 rat against the W1 i strain , belongsThe Fab fragment of R31C2 was obtained by papain digestion and purified on a DEAE-Sephacel support in batch, then on a mono Q column to resolve different crystallizable isoforms. The complex was obtained by 1 h incubation in a 1:1 molar ratio of the recombinant protein with the Fab fragment of R31C2. A volume of 1 μl of the complex was mixed with 1 μl of buffer containing 8% PEG 6000, 40 mM Hepes pH7 and 80 mM NaCl. The drop was set up over a reservoir of 500 μl of buffer and stored at 17°C. Crystals were flash-cooled in liquid nitrogen after brief soaking in a cryoprotectant consisting of 20% PEG 6000, 50 mM Hepes pH 7, 100 mM sodium chloride and 20% glycerol.H coordinates from PDB entry 2GCY and the VL coordinates from 1D5I, which showed the best sequence identity with the respective domains of R31C2, were used for the R31C2 variable dimer, and the CH and CL domains from PDB entry 1IGF were used as the search model for the C dimer. Two independent protein molecules were identified in the crystallographic asymmetric unit for PkAMA1 and a single complex for PkAMA1-Fab R31C2.X-ray diffraction data for PkAMA1 were collected on beamline BM14 at the ESRF , and for the PkAMA1-Fab R31C2 complex on beamline PROXIMA-1 at synchrotron SOLEIL . The diffraction images were integrated with the program XDS  and cryshttp://www.pymol.org) [The structures were refined with the program BUSTER  and manumol.org) . The strThe free PkAMA1 protein crystallized in space group C2 with two molecules (A and B) in the asymmetric unit and the structure was refined at 2.45 Å resolution , Table 22 of solvent-accessible surface is buried at the antibody-antigen interface . The heavy-chain variable domain (VH) contributes 838 Å2 to the buried surface while the light-chain variable domain (VL) contributes 342 Å2. All three Complementarity-Determining Regions (CDR) of both VH and VL make direct contacts with PkAMA1. The shape complementarity factor  is 0.74, which is significantly above values typical of antibody-antigen complexes [H is also reflected in the number and nature of the contacts across the interface; for VH there are 116 interatomic distances less than 3.8 Å, of which 22 are polar  while for VL there are only 25 contacts, of which two are polar , with 46 interatomic contacts < 3.8 Å.The PkAMA1-Fab R31C2 complex, which crystallized in the space group C2 with one molecule of complex in the asymmetric unit, was refined at 3.1 Å resolution , Table 2omplexes . The morre polar , Fig 4. Conformational changes that occur in PkAMA1 upon binding R31C2 are restricted mainly to flexible loops that come into contact with the antibody. The largest changes are induced in the PkAMA1 loop Phe169-Asn178, where differences exceed 7 Å at its extremity. A single turn of α-helix is formed from residues Ala172 to Gln175, placing Glu173 and Asp174 in position to form a salt bridge with R31C2 heavy chain residues Arg-H95 and Arg-H56, respectively. This region of PkAMA1 makes the most extensive interactions with R31C2, with residues from CDR-H1, CDR-H2, CDR-H3 and CDR-L1 participating in direct contacts with the antigen. The loop 128–136 becomes more ordered in comparison to the unbound PkAMA1 structure with differences of up to 6 Å between the unbound and bound states. Although the D2 loop makes extensive interactions with R31C2, the regions in direct contact show only small differences. The largest of these differences occur close to the N- and C-termini of the loop, which are very mobile in both PkAMA1 structures (as well as in PfAMA1 and PvAMA1) and do not contact the antibody. Most intramolecular contacts of the D2 loop are with Domain 1 of the PkAMA1 core; Glu288 is the only Domain 2 residue to contact the D2 loop but this is with Arg296 at its N-terminus.H and VL sequences were compared with germline sequences using the International Immunogenetics Information server [L domain derives from the Vκ germline gene IGKV14S1 (98.9% identity) and the J gene segment IGKJ5. Five somatic mutations in the amino acid sequence are present in the regions encoded by the Vκ and Jκ gene segments but none of these make direct contacts with PkAMA1. The VH domain derives from the germline gene IGHV5S36 (92.7% identity), the IGHD1-6 D minigene and the IGHJ2 J gene segment. Eighteen somatic amino acid mutations are present, five of which contact PkAMA1: Tyr50Thr, Gly53Ser, Gly55Ser and Ser56Arg in CDR-H2 and Asp100bGlu in CDR-H3. The higher selection pressure on VH probably reflects the significantly more important role of this domain in binding to PkAMA1. The somatic mutation Tyr50Thr leads to a hydrogen bond between Oγ and the glutamate group of Glu173, which would not be sterically possible for the germline residue tyrosine. The mutation Gly55Ser does not lead to additional polar interactions but probably improves the complementarity at the antibody-antigen interface. The mutation Ser56Arg appears to have the most significant impact in the affinity maturation of R31C2 as this antibody residue forms a salt bridge to Glu81 and Asp174 of PkAMA1. CDR-H3 residue Arg95, which forms a salt bridge to Glu173, probably results from nucleotide deletions and additions occurring at the junction between VH and D (used in the third reading frame).The Vn server  Fig 5)..H and VLPlasmodium merozoites and highlighting the potential of this parasite protein as a malaria vaccine candidate [P. knowlesi merozoites are able to attach to the RBC surface in the presence of R31C2 but no penetration of the host cell is observed, implying that AMA1 comes into play after initial contact by the parasite [Plasmodium and other Apicomplexa parasites have since confirmed this view. AMA1 has been shown to bind to RON2, a component of the parasite RON protein complex that is transferred to the RBC membrane during invasion [R31C2 was the first invasion-inhibitory anti-AMA1 mAb to be characterized, providing the first demonstration that AMA1 plays a crucial role in the infection of RBCs by andidate . P. knowparasite . The accinvasion , 27, 28.The RON2-binding site on AMA1 comprises the hydrophobic groove and an adjacent region that becomes exposed after displacement of the D2 loop by the receptor , 28. In Plasmodium homologues than is the sequence variability over the complete ectodomain [The D2 loop is functionally important as it must be displaced to fully expose the binding site for the RON receptor. Nonetheless, this region is more variable among the different todomain . InteresP. falciparum [P. knowlesi malaria.Polymorphism in AMA1 has been studied most extensively in lciparum , 52. In lciparum , 53–56. lciparum . A totallciparum . Moreovelciparum . It is u


Dear Editor,Protein domain is usually defined as distinct, compact and stable protein structural unit that folds independently of other such units  network, first we established a baseline human protein-protein interaction network composed of 19,139 individual PPIs by integrating several protein-protein interaction databases. We then mapped domains into the PPI network based on Pfam definitions  distribution of domains approximately follow power-law distribution Fig. S1), which shows that most of the domains are linked to only few other domains. In contrast, some domains such as the “SH2” domain on Grb2 protein are connected to many other domains (k = 122), which is consistent with its central role in dynamic regulation of tyrosine kinase signal, the key signal of eukaryotic cell growth . The average value of H is equal to 0.16 and whole distribution is shown in Fig. P < 10−4 by a Wilcoxon rank-sum test), while C domains’ are higher (P < 10−4 by a Wilcoxon rank-sum test). About 30% of C domains have degree k ≥ 10. Similarly, for clustering coefficient, which measures the density of network module, P domains are lower than average, while C domains are higher. Therefore, the highly interacted C domains are “hubs” in the network, which function to organize the local network modules. Interestingly, the betweenness of domains, which measures the number of shortest paths between any domain pair that involves a given domain . In contrast, no GO terms were found to be enriched among C domains, suggesting that each C domain may have unique, non-overlapping functions.We further analyzed the node degrees of P and C domains, as shown in Fig. P < 10−4, Wilcoxon rank-sum test) and this effect still exists even if the difference in the contact degree was taken into effect. So the result suggests that the evolution of P domains was constrained by the diversity of their interaction partners.Some previous studies . Considering that C domains and P domains are hubs and bottlenecks of the DDI network respectively, this observation suggests that oncogenic mutations tend to avoid the topologically important nodes of the biological networks, probably because such mutations in key domains would lead to immediate breakdown of the whole system so become highly deleterious for cancer cell survival.To identify possibly different roles of P and C domains in diseases, we investigated the distribution of oncogenic mutations in the DDI network. Previous reports  frequently than average as “frequent DDIs” (or “rare DDIs”). The most frequent and rare DDIs are listed in Table S1. Within the list, we noticed that the domains in frequent DDIs were functionally similar to each other; instead, domains in rare DDIs usually have different (or complementary) functions. This observation is expected as domains with similar functions tend to coordinate with each other to function together. Evolutionary rate calculation also showed that co-evolving domains  interact with each other more frequently . This is also consistent with our knowledge that protein binding of double-stranded viral RNA and processing it are two closely related biological processes. The top 20 frequent function combinations are listed in Fig. To understand the pattern how different biological functions are coordinated through combination of domains, we integrated the domain function information onto the DDI network. We found that there are some function combination appears more frequently than by chance. For example, in the network there are 73 domains annotated with “double-stranded RNA binding” function and 29 domains annotated with “RNA processing” function, and they form 11 function combinations with the frequency (=0.239) much higher than statistically expected Supplementary material 2 (TIF 131 kb)Supplementary material 3 (TIF 1676 kb)


Previous studies suggest that superoxide anions are possibly traveling along acupuncture meridians. The electrical potential difference (EPD) between acupoints may be related to the movement. To test the above hypothesis, we conducted a study investigating the effects of acupoint antioxidant interventions on the meridian EPD. Firstly, ST39 (L) and ST44 (L) were screened out for the EPD detection along the stomach meridian, and ST36 (L) was selected for interventions including acumassage with the control cream, as well as the TAT-SOD cream for 30 minutes, or injection with reduced glutathione sodium. The EPD between ST39 and ST44 was recorded for 80 minutes and measured again 48 h later. While the EPD increased during the acumassage, the acumassage with TAT-SOD cream and the glutathione injection generated waves of EPD increased, indicating the migration or removal from the visceral organ of a greater quantity of superoxide. Remarkably lower EPD readings 48 h later with both antioxidant acupoint interventions than the mere acumassage imply a more complete superoxide flushing out due to the restored superoxide pathway at the acupoint after interventions. The results confirm superoxide transportation along the meridians and demonstrate a possibility of acupoint EPD measurement as a tool to monitor changes in the meridians and acupoints. Reactive oxygen species (ROS), molecules or ions formed by the incomplete one-electron reduction of oxygen, have drawn considerable interest because of their regulations ranging from cell signaling to inflammatory response and cell death , 2. An iAcupoints lie along the meridians. What acupoints will be in relation to this “ROS network”? In the issue of Free Radical Biology and Medicine, a controlled study  has showWithin the acupuncture community, it is a commonly held opinion that acupuncture points have distinct electrical properties, for example, increased conductance , 9, reduSuperoxide is negatively charged, and its migration is driven by the voltage difference. Therefore, any antioxidant intervention at acupoints is possible to be monitored by the electrical potential difference between acupoints along the meridians. To test the hypothesis, the effects of antioxidant interventions at one acupoint on the EPD between another two acupoints along the stomach meridian are investigated in hope to confirm superoxide's involvement in meridians and establish a new and effective tool for the studies of meridians and superoxide. E. coli as follows: constructs preparation: the nucleic acid sequence encoding TAT-SOD fusion protein was constructed by DNA recombinant technology and inserted into expression vector pGEX-2 T; cell culture and transfections: E. coli  cells were transformed with the expression vector pGEX-2 T containing the inserted TAT-SOD; TAT-SOD fusion protein preparation: TAT-SOD was expressed in the E. coli by the induction of IPTG. After purification of affinity chromatography, electrophoretically pure TAT-SOD protein was obtained [μm diameter and 40 mm length stainless steel  were used for acupuncture.The vehicle cream for TAT-SOD cream was also used as control cream. The vehicle cream was baby lotion , which contains water, propylene glycol, myristyl myristate, glyceryl stearate, oleic acid, and stearic acid. 3000 U SOD/mL TAT-SOD cream was prepared by the homogenization of membrane permeable TAT-SOD with the vehicle cream. TAT-SOD was prepared by recombinant expression of a fusion protein of human Cu, Zn-SOD fused with TAT peptide inobtained . 0.12 g/A total of 30 healthy volunteers were recruited in this study after giving full informed consent. Participants should meet the following criteria: (1) 20 and 35 years of age; (2) no history or physical examination suggestive of renal, hepatic, or cardiovascular diseases or any other severe organic diseases; (3) having regular diet; minimal liquor, tobacco, tea, and coffee; normal sleeping patterns (before 12 a.m.); (4) no long-term medications; (5) no history of drug abuse; (6) having not undergone acupuncture or other acupoint interventions within 1 month before the test. This study was approved by the Medical Research Ethics Committee and Institutional Review Board of Fujian Institute of Traditional Chinese Medicine.μL, 0.12 g/mL), and (F) acupuncture. When the EPD between the two selected detection acupoints was relatively stable after a balance of about 20 min, the cream was applied on the intervention acupoint assisted by massage stick for 30 min, reduced glutathione sodium was injected into the intervention acupoint quickly, and acupuncture was conducted, respectively. Make a record of the EPD between the two detection acupoints for 60 min, since the start-time of acupoint intervention. Besides, the EPD between the same acupoints was monitored again 48 h after treatments.Before the trial, several acupoints along the ST meridian were selected for their convenience to operate. The subjects first underwent the measurement of EPD between acupoints. Based on the stability and individual difference of the EPD between acupoints, two acupoints were screened out for the EPD detection along the ST meridian. Meanwhile, one downstream acupoint along the ST meridian for interventions was selected. Subsequently, 30 subjects were divided into 6 groups. The treatments applied to different groups on acupoint were as follows: (A) no treatment, (B) acumassage without cream, (C) acumassage with control cream, (D) acumassage with TAT-SOD cream (3000 U/mL), (E) injection of reduced glutathione sodium injection  was used for acupoint massage. Acupoint for intervention was localized according to name and location of acupoints: Chinese National Standards GB/T12346 . B groupμm diameter and 40 mm length stainless steel  were used for acupuncture in this study. Acupoint for intervention was localized according to name and location of acupoints: Chinese National Standards GB/T12346 [Disposable sterilized acupuncture needles of 300 B/T12346 . Skin waB/T12346 . After mt-test, and paired t-test was used to analyze interclass variance. A one-factor ANOVA (SPSS version 21.0), followed by Duncan's test, was used to test for significant differences of the reduction of the EPD of each group 48 h after treatments.Data are reported as means (SEM). All statistical analyses were carried out using SPSS version 21.0 software. Results in left EPD and right EPD of the two same detection acupoints were compared using two-sample T1 test (paired t-test), the EPD of most acupoint combinations showed significant individual difference; only the EPD between ST39 and ST44 (L) showed no significances (P > 0.05). There are also generally existing differences between the left side and the right side of the same two acupoints, regarding T2 test (two-sample t-test).As shown in Therefore, ST39 and ST44 (L) were screened out for the EPD detection along the ST meridian for the stable and reliable voltage reading. In the meantime, the downstream acupoint ST36, admittedly an important and effective acupoint along the ST meridian in dredging meridian channels and relieving fatigue –18, was A diagrammatic sketch of how to monitor the variation of the EPD between ST39 and ST44 caused by different acupoint interventions on ST36 was shown in μL, 0.12 g/mL), and the EPD between ST39 and ST44 was recorded for 80 min and measured again 48 h later. The results of the variation trends of different interventions during the first 80 min were presented in ST36 was subject to acumassage alone, acumassage with the control cream, and the cream containing membrane permeable TAT-superoxide dismutase for 30 min or injected with reduced glutathione sodium . The fall of the EPD readings of D and F groups was similar without significance (P < 0.01). E group had the largest decline between all groups (P < 0.01).The reduction of EPD between ST39 and ST44 of each group 48 h after the intervention was demonstrated in TAT-SOD, a fusion protein of human Cu, Zn-SOD fused with TAT peptide, is permeable membrane and well capable of eliminating intracellular superoxide. As a negatively charged anion, superoxide migration is caused by the EPD. Therefore, the EPD between acupoints may reflect the migration of superoxide along the meridian. The phenomenon that the EPD readings of B and C groups gradually descended when interventions ended, while D and E groups still fluctuated at high levels in the next 30 min follow-up, indicates that ROS, or more specifically superoxide anion, may be traveling along meridians.All the interventions at ST36 in this study resulted in the immediate increase in EPD reading. The elevation of EPD reading stopped almost right after the interventions stopped with the acumassage alone and the acumassage with the control lotion, indicating that the intervention at ST36 could cause the increased flow of superoxide anion along the meridians. With both antioxidant oxidant intervention and acupuncture, it is clear that the increased superoxide flow could last for some time even after the intervention stopped. The continuation of the elevated flow of superoxide resulted in a lower EPD reading 48 h later, implying a lower level of superoxide migration as the result of the intervention. ST36 is like a gating point modulating the superoxide flow from its source organ. It turned on while acumassage intervention lasted but went off as soon as it stopped. Antioxidant interventions and acupuncture seemed to generate a lasting effect to turn on the connection so as to enable the flow of waves of superoxide even after the intervention stopped, so much as to clear up the storage of superoxide at its source, as indicated by the remarkable lower EPD level 48 h later.In addition, the EPD readings were much lower with both antioxidant acupoint interventions than the mere acumassage 48 h later, implying a more potent superoxide flushing out due to the restored superoxide pathway at the acupoint after the interventions. The effect of acumassage with TAT-SOD cream was similar to that of acupuncture intervention, as shown in Moreover, acupoint EPD measurement can quickly respond synchronously, when the status of intervention changes. For example, after acupuncture intervention at ST36, the needle was removed quickly; in the meantime, the EPD reading between ST39 and ST44 sharply ascended. There the same thing happened at the start-time of interventions. Therefore, acupoint EPD measurement hold great potential as a powerful tool to monitor changes in the meridian and acupoints.Different responses of superoxide flow along the meridian caused by different interventions may be attributed to the biological nature of acupoints. It is reported that acupoints are high in mast cells which will respond to pressures such as acumassage . MeridiaThe results confirm superoxide transportation along the meridian and demonstrate a great potential of acupoint EPD measurement as a powerful tool to monitor changes in the meridians and acupoints.


Filamins are multi-domain, actin cross-linking, and scaffolding proteins. In addition to the actin cross-linking function, filamins have a role in mechanosensor signaling. The mechanosensor function is mediated by domain-domain interaction in the C-terminal region of filamins. Recently, we have shown that there is a three-domain interaction module in the N-terminal region of filamins, where the neighboring domains stabilize the structure of the middle domain and thereby regulate its interaction with ligands. In this study, we have used small-angle X-ray scattering as a tool to screen for potential domain-domain interactions in the N-terminal region. We found evidence of four domain-domain interactions with varying flexibility. These results confirm our previous study showing that domains 3, 4, and 5 exist as a compact three domain module. In addition, we report interactions between domains 11–12 and 14–15, which are thus new candidate sites for mechanical regulation. FLNA) and filamin B (FLNB) are expressed ubiquitously whereas filamin C (FLNC) is muscle specific Filamins (FLNs) are a family of actin cross-linking proteins essential for the maintenance of cellular architecture and function In this study, we conduct a systematic small-angle X-ray scattering (SAXS) screen of all possible two-domain fragments of FLNc to search for domain-domain interactions in the Rod 1 region of FLNc. These results confirm the previously observed interactions between domains 3–4 and 4–5 and reveal two new interactions.g) (Supporting Information S2 in g (mean square distances from the center of mass weighted by electron densities) and Dmax (maximum dimension of the particle) for the fragments was 2.3 and 7.7 nm respectively. Based on smaller than average values of Dmax and Rg, four fragments were compact, domains 3–4, 4–5, 11–12 and 14–15. Further, the shape of the pairwise distance distribution plot (P(r) vs r) for domains 4–5 showed a compact bell-shaped profile, suggesting this fragment was globular max, the distance distribution plot showed a bimodal peak distribution, indicating some degree of flexibility between the domains  nsembles  of thesensembles .Hence, both dimensionless Kratky and EOM analyses suggested that the fragment of domains 4–5 was the least flexible, followed by domains 3–4 and 14–15. Domains 11–12 were at the borderline between partly and fully flexible two-domain fragments.Ab-initio envelopes  values  and the In conclusion, the inter-domain interactions in the Rod 2 region of FLNs have been shown to regulate ligand binding For expression of recombinant protein in Escherichia coli BL21 Gold strain (Agilent Technologies), two-domain fragments of human FLNc cDNA (GenBank AJ012737) were cloned in the GST-fusion protein vector pGTVL1  g) were calculated for each concentration with the program GUINIER The beamlines for SAXS data collection were BM29 at the European Synchrotron Research Facility (ESRF) Grenoble  Hamburg . The details of the beamline setups and detectors are given in the publications max for all the fragments. The molecular mass of the constructs were evaluated by comparing the forward scattering from the fragments with that of reference solution of bovine serum albumin (BSA) with molecular mass, 66 kDa using the equation, Mw sample  = I(0) sample × Mw ref/I(0)ref. Porod's law was used to find out the excluded volume of the hydrated particle (V), assuming the samples were monodispersed:Based on the analysis of concentration-dependent changes and the Guinier region (Supporting Information S2 and S3 in −4 decay in scattering intensity at higher angles 2 versus s× Rg) was used to probe for the flexibility of the proteins compared to BSA ab-initio envelopes using the scattering data. The chi values of each of the envelopes against the scattering data are tabulated in Supporting Information S7 in ab-initio envelope with the respective rigid body model was done with SUPCOMB The same law was used to check the SWe thank Ms. Arja Mansikkaviita for excellent technical assistance; Dr. Salla Ruskmao for help during data collection; and Jonne Seppälä and Dr. Ulla Pentikäinen for discussions. We are extremely thankful to Dr. Alexander V. Shkumatov for his valuable inputs and suggestions during the SAXS data analysis. We acknowledge European Molecular Biology Lab (EMBL) and European Synchrotron Radiation Facility (ESRF) for providing synchrotron access for the SAXS data collection. We express gratitude to Michal J. Gajda (EMBL Hamburg) and Matthew Bowler, Adam Round, and Petra Pernot  for their assistance during the data collection at the beamlines.File S1Supporting files. Supporting Information S1, Preparative size exclusion chromatography profiles of all the constructs studied. (A–N): The y-axis is Absorbance at 280 nm wavelength and the x-axis is the elution volume in ml. In each purification, the main peak eluting between 150–200 ml was collected for further concentration and analysis. Supporting Information S2, Check for concentration dependence of scattering. (A–N): Logarithmic plot of scattering (I) versus momentum transfer (s) is shown for all concentrations of each construct. The low angle region is highlighted with a dotted box and the inset is shown on the upper right of each panel. Rg vs concentration plot for each construct is also shown on the bottom right of each panel. Supporting Information S3, Guinier region representation of all the constructs. (A–N): Experimental scattering in the low s range is shown with black dots. The fit is shown in red. The residuals points from the scattering curve that fall outside the Guinier fit are shown in green. Supporting Information S4,Low angle fit of the reciprocal space scattering for the P(r) function analysis is shown (A–N). Supporting Information S5, Ab-initio envelopes and rigid body models of the rest of the two-domain fragments not shown inAb-initio and rigid body models are superimposed and their fits are expressed in terms of NSD (A–H lower panel). Fit of the rigid body model with the experimental data and the goodness of the fit (expressed in terms of X2) are given for each fragment (A–H upper panel). Supporting Information S6, Superposition of the most typical (blue mesh) and the least typical (red mesh) ab-initio models with the averaged envelope (grey surface). Supporting Information S7, The chi values of each of the 10 GASBOR generated ab-initio models. Supporting Information S8, The average NSD of each of the 10 GASBOR generated ab-initio models.(DOCX)Click here for additional data file.


N-terminal putative α-helix of the large subunit plays a key role in the homodimeric assembly and the self-cyclization of the large subunit and is deeply involved in the archaeal PolD stability and activity. We also discuss the evolutional transformation from archaeal D-family to eukaryotic B-family polymerase on the basis of the structural information.Archaea-specific D-family DNA polymerase (PolD) forms a dimeric heterodimer consisting of two large polymerase subunits and two small exonuclease subunits. According to the protein-protein interactions identified among the domains of large and small subunits of PolD, a symmetrical model for the domain topology of the PolD holoenzyme is proposed. The experimental evidence supports various aspects of the model. The conserved amphipathic nature of the  Replicative DNA polymerases (DNA Pols) are divided into archaeal-eukaryotic and bacterial types that appear not to be homologous to each other ,2. All aPyrococcus abyssi (PabPolD) plays an important role in chromosomal replication, together with the B-family DNA polymerase, and is capable of RNA primer elongation  was reported to be essential for the folding of PolD and is probably the oligomerization domain 2, was analyzed using a circular dichroism (CD) spectrometer and a fluorometric method between 20 °C and 85 °C 2 was reported to be heat stable and a uniform molecule [2 forms a central core of the dimeric large subunit, (DP2)2, in which DP2(1-50) is an essential element for the refolding of DP2(792-1163) to maintain the complex in soluble form. On the basis of the results, here we propose a revised domain topology of the PolD holoenzyme, probably associated in a symmetric manner as shown in 2 by multiple protein-protein interactions shown with arrows in orange  and surface plasmon resonance (SPR) assays . Refoldimolecule . The resn orange b. It is N-terminal residues of DP2 form the dimeric assembly, we manually built a model of residues 1 to 47 in the crystal packing of DP2(48-291). The modeled α-helices were fitted well in the remaining space of the crystal packing for DP2(48-291) as shown in a stereo view of N-terminal residues after trapping in the crystal lattice. One DP2(48-291) molecule and the tentative model of DP2(1-47) are shown in red and blue, and symmetry-related molecules are shown in pink and cyan, respectively.According to secondary-structure prediction using the amino acid sequence and the PSIPRED program , the regN-terminal residues was likely to form an α-helix with complex formation according to secondary-structure prediction and molecular modeling, helical wheel analysis of DP2(8-33) was carried out with the DNASIS-Mac ver 2.0 software. As shown in C-terminus of DP2(792-1163) [P. abyssi were rapidly degraded, suggesting the (G)-PYF box inside the hydrophobic domain DP2(792-1163) has a major role in PolD stability and in polymerase activity [N-terminal residues play a key role in PolD folding and stability. The (G)-PYF box motif might be a counterpart to assemble with the putative α-helix, DP2(8-33), although further work would be necessary to confirm the interaction. Since PolDs are essential in DNA replication and repair [N-terminal residues is conserved completely in Euryarchaeota and Korarchaeota as shown in N-terminus of DP2 seems to be a major target for the DNA replication control.Since the disordered ~50 92-1163) . One or activity . We haveactivity . We alsod repair  and the Archaea-specific D-family DNA polymerase (PolD) forms a tetramer consisting of two large polymerase subunits (DP2) and two small exonuclease subunits (DP1). PolDs, originally discovered from Euryarchaeota, were also identified in the putative phyla Nanoarchaeota, Thaumarchaeota , and Korarchaeota, which may have diverged early from the major archaeal phyla Crenearchaeota and Euryarchaeota.C-terminal part of DP1, eukaryotic Mre11-like nuclease domain, shows low but significant homology to the non-catalytic second subunit of eukaryotic B-family DNA polymerases . We reported that the N-terminal domain of Pyrococcus horikoshii DP1 interacts with a synthetic peptide corresponding to the C-terminal zinc finger-like region of the DP2 [N-terminal domain of DP1 suggested that the interacting pair of DP1 and the C-terminal zinc finger-like region of the DP2 are conserved during the evolutionary transformation from archaeal D-family to eukaryotic B-family polymerases, probably in order to maintain the holoenzyme structure [Interestingly, the  the DP2 . The NMRtructure .N-terminal (1–300) domain structure determined by X-ray crystallography, although ~50 N-terminal residues were disordered [N-terminal domain of DP2 suggested that the disordered N-terminus (~50 residues) play a key role in self-cyclization and homodimeric assembly of DP2. According to the molecular structure of the N-terminal region of DP2 and the symmetrical topology model of the PolD holoenzyme, the amphipathic nature of the N-terminus interacting with the active center of DP2 might be a potent tool to control the archaeal PolD stability and polymerase activity.We reported the sordered . The det


A lack of inhibition of PI5P4Kγ ATPase activity suggested that NIH-12848 does not interact with the enzyme's ATP-binding site and direct exploration of binding using hydrogen–deuterium exchange (HDX)-MS (HDX-MS) revealed the putative PI5P-binding site of PI5P4Kγ to be the likely region of interaction. This was confirmed by a series of mutation experiments which led to the identification of a single PI5P4Kγ amino acid residue that can be mutated to its PI5P4Ks α and β homologue to render PI5P4Kγ resistant NIH-12848 inhibition. NIH-12848 (10 μM) was applied to cultured mouse principal kidney cortical collecting duct (mpkCCD) cells which, we show, express PI5P4Kγ that increases when the cells grow to confluence and polarize. NIH-12848 inhibited the translocation of Na+/K+-ATPase to the plasma membrane that occurs when mpkCCD cells grow to confluence and also prevented reversibly their forming of ‘domes’ on the culture dish. Both these NIH-12848-induced effects were mimicked by specific RNAi knockdown of PI5P4Kγ, but not that of PI5P4Ks α or β. Overall, the data reveal a probable contribution of PI5P4Kγ to the development and maintenance of epithelial cell functional polarity and show that NIH-12848 is a potentially powerful tool for exploring the cell physiology of PI5P4Ks.NIH-12848 (NCGC00012848-02), a putative phosphatidylinositol 5-phosphate 4-kinase γ (PI5P4Kγ) inhibitor, was explored as a tool for investigating this enigmatic, low activity, lipid kinase. PI5P4K assays  We have characterised a specific inhibitor of the enzyme Phosphatidylinositol 5-phosphate 4-kinase γ, including establishing where on the enzyme the inhibitor binds, and then applied this inhibitor to a kidney cell line to elucidate the intracellular functions of the enzyme. Lipids were extracted using an acidic phase-separation  at 37°C in a 5% CO2, 95% air atmosphere. Medium was changed every 2 days and all studies were performed on cells that had been grown on plastic Petridishes or glass slides.Immortalized mouse cortical collecting duct (mpkCCD) cells ,23 were plus SMART pool). Control siRNAs were sequences that would not be recognized by any part of the mouse genome. mpkCCD cells were plated at a density of 6 × 104 per well (24-well/plate) 1 day before transfection, then were transfected with each siRNA at a final concentration of 25 pmol/ml using DharmaFECT 1 Transfection Reagent (Dharmacon). At 72-h post-transfection, a new round of transfection was performed and the cells were cultured for another 48 h.Depletion of specific PI5P4K isoforms was achieved by transfecting cells with siRNAs specific to each PI5P4K isoform. The siRNAs were obtained from Thermo Scientific Dharmacon  and anti-zona occludens-1 (anti-ZO-1) polyclonal antibodies (Millipore) and were examined under a Leica confocal microscope.Confluent cells grown on plastic Petridishes were examined under an inverted microscope (Zeiss) equipped with phase-contrast optics. Indirect immunofluorescence studies were performed on confluent cells grown on glass slides fixed with ice-cold methanol or 4% formaldehyde. Cells were processed for immunofluorescence using anti-Nag for 5 min. The supernatant was run on 10% polyacrylamide gels, transferred to nitrocellulose membranes, blocked in TBS/0.05% Tween 20/5% non-fat dry milk and probed with antibodies diluted in the same solution. The following primary antibodies were used: polyclonal anti-PI5P4Kγ 1:2500 .Direct competition experiments  into PI5P4Kγ and PI5P4Kγ+ and both these mutant enzymes were completely resistant to NIH-12848 inhibition .Examining this region closely in the structure F suggestPI5P4Kγ+ . We ther164 and/or Asn165 are the major contributors to the sensitivity of PI5P4Kγ to NIH-12848. So we mutated only those two residues in PI5P4Kγ to their PI5P4Ks α/β homologues individually or together and found that whereas S164N PI5P4Kγ is as sensitive to NIH-12848 inhibition as WT PI5P4Kγ , N165I PI5P4Kγ is completely insensitive, with 50 μM NIH-12848 having no effect on this mutant  summarizes the activities and sensitivities to NIH12848 of the mutants discussed in the present study.These data support the HDX–MS data suggesting that NIH-12848 is binding to this area of the PI5P4Kγ protein and they also imply that Sers mutant . Note th165 is the residue in PI5P4Kγ that is almost entirely responsible for its sensitivity to NIH-12848. There was no detectable change in the amide exchange rate at Asn165 when NIH-12848 binds to PI5P4Kγ or PI5P4Kγ+  it is highly expressed in the pro-nephric duct throughout development (ZFIN Database: ZDB-IMAGE-030829-506).PI5P4Kγ is widely expressed in mammalian tissues, but is particularly high and is indeed the principal PI5P4K isoform in kidney . It is econtacts ,23. We fcontacts  and in z+/K+-ATPase from the cytoplasm to the basolateral plasma membrane that occurs when the cells reach confluence [One easily monitored parameter is the redistribution of Nanfluence . We explConsistent with this possibility, the formation of ‘domes’ (see above) was also markedly inhibited B by NIH-+/K+-ATPase re-localization (Our extensive molecular exploration of the interaction of NIH-12848 with PI5P4Kγ (above) emphasizes its remarkable specificity, but nevertheless it is possible that the effects we see are due to an off-target action. To gather some independent evidence that inhibition of PI5P4Kγ underlies the effects of NIH-12848 and thus to begin a validation of this inhibitor as a tool for exploring PI5P4Kγ function, we knocked down PI5P4Kγ in the mpkCCD cells by RNAi. lization B and domlization C. WithinControl experiments knocking down PI5P4Kα and β did not show the same effect on dome formation. PI5P4Kβ knockdown had no detectable effect , whereas knockdown of PI5P4Kα had a pronounced opposite (stimulatory) effect . We have no immediate explanation for this latter action, which we believe is likely to be independent of PI5P4Kγ activity because knockdown of PI5P4Kγ and PI5P4Kα together showed that PI5P4Kγ causes a similar and internally consistent inhibitory effect on the higher level of dome formation resulting from PI5P4Kα knockdown . Further studies, including using PI5P4Kα inhibitors (e.g. ) will beOverall, these experiments yield the first functional insight into PI5P4Kγ's physiological role in epithelial cells. Much remains to be explored to clarify exactly what the enzyme is doing in this context, but the very similar effects induced in mpkCCD cells by inhibition with NIH-12848 and knockdown of PI5P4Kγ are a crucial first stage in a proof of principle of the potential usefulness of NIH-12848 in such an exploration. Our detailed mapping and manipulation of the molecular interaction of NIH-12848 with PI5P4Kγ also suggests ways to a wider use of this inhibitor in the exploration of functions of the PI5P4K family.