Ataxin-3, the disease protein in the neurodegenerative disorder Spinocerebellar Ataxia Type 3 or Machado Joseph disease, is a cysteine protease implicated in the ubiquitin proteasome pathway. It contains multiple ubiquitin binding sites through which it anchors polyubiquitin chains of different linkages that are then cleaved by the N-terminal catalytic (Josephin) domain. The properties of the ubiquitin interacting motifs (UIMs) in the C-terminus of ataxin-3 are well established. Very little is known, however, about how two recently identified ubiquitin-binding sites in the Josephin domain contribute to ubiquitin chain binding and cleavage. In the current study, we sought to define the specific contribution of the Josephin domain to the catalytic properties of ataxin-3 and assess how the topology and affinity of these binding sites modulate ataxin-3 activity. Using NMR we modeled the structure of diUb/Josephin complexes and showed that linkage preferences are imposed by the topology of the two binding sites. Enzymatic studies further helped us to determine a precise hierarchy between the sites. We establish that the structure of Josephin dictates specificity for K48-linked chains. Site 1, which is close to the active site, is indispensable for cleavage. Our studies open the way to understand better the cellular function of ataxin-3 and its link to pathology. Parsimony in nature reveals itself in many ways. A particularly elegant biological example is the capacity to regulate divergent cellular pathways through the versatile properties of a single modifier protein. An excellent example of a multiple-function adaptor is ubiquitin (Ub), a small protein implicated in diverse cellular processes and pathways including transcription, protein degradation, cell cycle progression, viral infection, and immune response Understanding how polyUb chains are recognized is an important, yet still poorly defined issue. It has been suggested that specificity is achieved by Ub chains interacting with multiple-domain proteins that contain different Ub binding motifs. One such protein is ataxin-3, a protein implicated in protein quality control pathways and transcriptional regulation Here, we have investigated the structural constraints imposed by these two Ub-binding sites and their preferences in polyUb binding and in selecting the exact branching of Ub chains for cleavage. We compare the properties of Josephin and full-length ataxin-3 to cleave different polyUb chains  and relate them to the binding and enzymatic properties of the domain. We observe a clear hierarchy between the two sites, which is dictated by their structural features and topological relationship. We also rationalize some of our results in terms of the structural constraints imposed by the Ub chains.To examine the importance of Ub-binding sites 1 and 2 to the ability of ataxin-3 to cleave poly-Ub chains independently from the C-terminal UIMs, we carried out enzymatic Ub cleavage studies using the isolated Josephin domain (amino acids 1–182). We followed established protocols Our results show that Ub-binding site 1 is necessary for the Josephin domain to cleave K63-linked hexa and K48-linked pentaUb chains, as well as mixed-linkage chains Fig. 1B,These data indicate that site 1 is critically involved in any DUB activity of the Josephin domain, and suggest an involvement of site 2 in specific linkage recognition or preference by the Josephin domain.We next compared our results for the isolated Josephin domain with those obtained for full-length, non-expanded (Q22) ataxin-3. Independent of the Jos_I77KQ78K and Jos_W87K mutants tested above, we generated I77AQ78A and W87A mutants in full-length ataxin-3. Replacing the bulky hydrophobic residues in sites 1 and 2 with a smaller hydrophobic residue rather than a charged residue represents a milder mutation. Even so, we obtained results similar to those observed with the isolated Josephin domain: site 1 proved to be essential for the ability of full-length ataxin-3 to cleave K63-linked or K48-linked chains Fig. 2. Together, these results confirm the crucial importance of site 1 in cleaving all Ub chains and a role of site 2 in conferring linkage preference to ataxin-3.To gather structural perspective into the role of sites 1 and 2 in Ub chain cleavage by the isolated Josephin domain, we first tested whether the Josephin enzymatic activity presents any preference towards polyUb chains of different lengths and/or linkage. We first incubated Josephin with either K48-linked or K63-linked diUb chains. We observed virtually no change in diUb levels or accumulation of monoUb in either case  to study the effects of titrating the isolated Josephin domain with K48- or K63-linked diUb chains and to collect distance restraints which could be translated into structural models. A C14A mutant was used to be sure that no enzymatic cleavage could occur. We observed similar chemical shift perturbation (CSP) effects for both diUb linkages . In contrast, the isolated Ubl domain of hHR23A does not affect cleavage by the isolated Josephin domain.The ability of ataxin-3 to cleave polyUb chains is not visibly affected by the presence of either full-length hHR23A or its isolated Ubl domain  as compared with the KFinally, we observed that, as for full-length ataxin-3 In summary, we have shown that the catalytic domain of the deubiquitinating enzyme and polyQ disease protein, ataxin-3, has a preference for interacting with K48-linked diUb chains compared to K63-linked diUb chains. Ub-binding site 1 on the Josephin domain is indispensible for the enzymatic activity of ataxin-3, while site 2 may be dedicated to the recognition of hHR23 proteins. Our data provide significant new insights into ataxin-3′s interactions with its substrates and the manner in which isopeptide bonds are positioned for cleavage.5′gccttctggaaatatggatgacagtggttttttctctgctgcggttataagcaatgccttg (forward) and 5′caaggcattgcttataaccgcagcagagaaaaaaccactgtcatccatatttccagaaggc (reverse) for the Iso77-Ala/Glu78-Ala mutant, 5′ggttataagcaatgccttgaaagttgcgggtttagaactaatcctg (forward) and 5′caggattagttctaaacccgcaactttcaaggcattgcttataacc (reverse) for the W98A ataxin-3 mutant. Mutations of the equivalent Josephin residues into lysines were obtained as previously described 5′actatccatggagtccatcttccacgagaaacaagaaggctcacttgctgctcaacattg (forward) and 5′atcttgcggccgcttacctaatcatctgcaggagttggtcagcttcgcaatctggcagatcacc (reverse) were used for a C14A Josephin mutant (cloned using NcoI and NotI restriction sites).The following primers were used to generate missense mutations of specific residues of ataxin-3 into alanine : 15NH4Cl as the sole source of nitrogen. The K48-linked diUb used for structural studies was produced as described elsewhere The proteins were expressed in E. coli BL21(DE3) and purified as previously described PolyUb chains  were incubated with full-length ataxin-3 or isolated Josephin domain species (100–400 nM) in DUB buffer  at 37°C. Fractions were collected in 2% SDS, 100 mM DTT sample buffer, boiled for 1 min and electrophoresed in 15% SDS-PAGE gels or in 4%–20% Ready Gels (BioRad). SDS-PAGE electrophoresis and western blotting were conducted as previously described Although we routinely observe monoUb generated during our enzymatic assays, monoUb is less readily detected by western blotting techniques than longer Ub chains. Thus, for diUb cleavage assays, to ascertain that diUb is indeed not a substrate for ataxin-3 we used a combination of western blotting and in-gel protein detection (as described above), as well as positive controls with the unrelated DUB USP28 with which the monoUb reaction product is easily detected. This approach, together with data from cleavage of polyUb chains by ataxin-3 (in which we detect monoUb) ensures us that the absence of monoUb from diUb cleavage assays is due to the actual absence of monoUb generated during the reaction rather than to a lack of detection on immunoblots.Ub4, IUb3, IUb2, IUb1)/Σ. Statistical analyses of data collected from independent experiments were conducted using two-tailed student t-tests. All graphical data are shown as means +/− standard deviation.For semi-quantification of western blots, images were collected below-saturation levels using VersaDoc 5000MP (BioRad). Quantification was conducted with Quantity One (BioRad). Background was subtracted equally among all lanes and the intensity of each band was integrated as the area under each intensity curve. The sum of intensities of all product bands was divided by the sum of intensities of all bands in that lane. For example, for cleavage of Ub5 chains, the equation was: Σ docking runs were performed to test the ability of K48 and K63 linked diUb to interact with Josephin: K48 or K63 linkages were imposed in two of them. In a third run, ambiguous linkage restraints were imposed to both K48 and K63 to see which one would be preferentially selected during docking. In all three runs, 5000 rigid-body docking solutions were generated and the best 200 were subjected to semi-flexible refinement in torsion angle space followed by refinement in explicit water. Random removal of restraints was turned off  and additional center-of-mass restraints were defined between the various molecules to ensure compact solutions. All other parameters were left to their HADDOCK default values. The C-terminal tail of each Ub was defined as fully flexible together with K48 and/or K63 depending on the linkage type.The isopeptide bond between the two Ubs was defined by imposing a loose distance restraint (10 Å) between the side chain ammonium group of either K48 or K63 and the carboxyl atom of G76. In a third docking run with an ambiguous linkage definition, an ambiguous distance restraint was defined including both K48 and K63. A final additional water refinement was performed in which the distance restraint was decreased to 1.5 Å in order to re-establish the iso-peptide bond.To check if positioning of Ub into the catalytic site could be consistent with the measured chemical shift data, the three runs were repeated with one additional distance restraint (10 Å) between the Josephin side chain sulphur of C14 and the C-terminal carbon of site 2. This distance was shortened to 5 Å for the additional water refinement step.The resulting models of each docking run were clustered with a 5 Å cut-off and the clusters ranked based on the average HADDOCK score calculated over the best four members of each cluster.15N labelled Josephin or C14A Josephin mutant  with diUB chains were performed with stepwise additions of concentrated stock solutions of K48-linked or K63-linked diUb chains  up to a 10 and 2.5 fold molar excess, respectively. Combined measurements of T1 and T2 relaxation times were performed to estimate the correlation times using a home modified Bruker sequence (hsqct2etf3gpsi3d). Overall correlation time values were estimated from the R2/R1 ratio using the program R2R1_TM available from the website of A.G. Palmer's group  NMR experiments were performed at 298 K on a Bruker Avance spectrometer operating at 600 MHz and equipped with cryoprobe. Titrations of


They are also candidate drug targets, in particular for the immunosuppressant cyclosporine A. In addition, cyclosporine is known to exhibit anti-parasitic effects on a wide range of organisms including several apicomplexa. In order to obtain new non-immunosuppressive drugs targeting apicomplexan cyclophilins, a profound knowledge of the cyclophilin repertoire of this phylum would be necessary.Cyclophilins (Cyps) are peptidyl Cryptosporidium hominis, Toxoplasma gondii, Plasmodium falciparum, Theileria annulata, Theileria parva, and Babesia bovis. In addition to good statistical support from the phylogenetic analysis, these subfamilies are also confirmed by comparison of cyclophilin domain architecture. Within an individual genome, the number of different Cyp genes that could be deduced varies between 7–9 for Cryptosporidia and 14 for T. gondii. Many of the putative apicomplexan cyclophilins are predicted to be nuclear proteins, most of them presumably involved in RNA processing.BLAST and maximum likelihood analyses identified 16 different cyclophilin subfamilies within the genomes of The genomes of apicomplexa harbor a cyclophilin repertoire that is at least as complex as that of most fungi. The identification of Cyp subfamilies that are specific for lower eukaryotes, apicomplexa, or even the genus Plasmodium is of particular interest since these subfamilies are not present in host cells and might therefore represent attractive drug targets. In contrast to PcaCyp52.5, neither OtCPR7 nor the archaebacterial FKBPs do contain TRP repeats separating the two PPIase domains . Finally, it should be mentioned that the OtCPR7 sequence might be COOH-terminally truncated since the Cyp domain itself is truncated. In contrast to all other FCBP proteins identified here, OtCPR7 contains an NH2-terminal mitochondrial localization signal as predicted with high significance by both PSORT II ) at its immediate NH2-terminus as identified by InterProScan suggesting that BhCFBP38 is exported by the bacterium. The domain architecture of all non-apicomplexan FCBPs and some representative CFBPs are shown in Figure S3.There are also several putative dual-family immunophilins with an NHi.e. in chlorophyta, oomycetes and alveolata. For OtCPR7 this conclusion is further supported by the fact that this FCBP does not contain any TRP repeats. Cyp domains of the putative archaebacterial FCBPs are not even closely related to this group and form a completely independent cluster. The Cyp domains of proteo-/flavobacterial CFBP proteins are monophyletic – in contrast to those of spirochaetes. However, for the latter group there are currently only members known from Treponema denticula and four Borrelia species. It is for instance possible that one of these two proteins is highly divergent from the average spirochaete CFBP due to secondary evolutionary changes. In particular, the presence of a lipoprotein anchor at the NH2-terminus of BhCFBP38 suggests an extracellular localization of the mature protein and therefore a significantly altered function.The discontinuous distribution pattern of FCBPs and CFBPs in phylogenetically unrelated clades raises the question whether these proteins evolved multiple times independently. Alternatively, a common evolutionary origin of proteins with this domain architecture might be assumed followed by either loss from most genomes or horizontal gene transfer. In order to address this question, BLAST analyses were used to identify those Cyps and FKBPs in archaebacteria, eubacteria, and eukaryotes that show the highest similarity to the diverse FCBPs and CFBPs. All proteins used for these analyses are listed in Tables S2 and S3 in Additional file Griffithsia japonica is proposed to be a member of the same cluster as revealed by maximum likelihood analysis. Since red algae are frequently supposed to be the evolutionary origin of the apicoplast, one explanation for this result may be that the Cyp domain of FKBPs in alveolata was derived from the genome of a rhodophyte-related secondary endosymbiont.The Cyp domains of FCBPs of ciliophora and apicomplexa are closely related, surprisingly, however, a non-FKBP Cyp from the rhodophyte OtCPR7 does not show any close relationship with FKBP domains from other eukaryotic FCBPs but appears to be closely related to bacterial FKBPs suggesting that it might have been acquired from a cyanobacterial endosymbiont. Though the FKBP domains of all alveolat FCBPs can be found in the same highly significant cluster (group with blue background), this group also contains non-FCBP FKBPs. Conspicuously, however, all FKBP proteins within this group also contain TRP repeats  and 12 introns (T. gondii) in the other species.The genomic organization differs largely between species with intronless genes in et al. ). Only two putative members of this subfamily can be found in the apicomplexan genomes analyzed here, i.e. TgCyp21 and PfCyp23.2  itself  itseP. falciparum) to six (B. bovis) exons  is present in the vicinity of the COOH-terminus  due to a longer COOH-terminus. Moreover, TgCyp72.9 contains a putative nuclear localization signal which is not present in its haemosporidian orthologs.Putative PPIL2-like Cyps form a very robust phylogenetic cluster Figure  and are s Figure . This suT. gondii, one intron in both Theileria species and no intron left in B. bovis.The genomic organization of Cyps with RING finger domain again shows signs of intron-loss during evolution with eight introns in CeCyp16-like domain (accession number: [cd01925]) does not contain any additional domain that could be identified by CD-BLAST or InterProScan . PfCyp51.8 is even predicted to contain two coiled-coil regions. Moreover, PfCyp51.8 has a large Lys-rich region encompassing both coiled-coil domains. A comparable but much smaller positively charged region consisting of a large number of Arg residues is present in TgCyp64.5.In contrast to most other moderate to large size Cyps, the subfamily containing a Cyp-n Figure . HoweverC. elegans ortholog CeCyp16 has been shown to be expressed predominantly in the intestine ) in its immediate NH2-terminus . In contrast to other PPIL4-like Cyps, the deduced sequence of ChCyp34.5 is missing a Ser/Arg-rich SR domain in its COOH-terminus. The predicted sequence of TgCyp66.3 is very unusual since it contains a Cyp domain that is interrupted by a large insertion which remains to be confirmed experimentally. However, in contrast to ChCyp34.5, TgCyp66.2 is more typical for PPIL2-like Cyps since it possesses a Ser-rich and highly positively charged domain in its COOH-terminus. Although it also contains multiple Lys in addition to Arg residues, it can be assumed that this domain fulfills a function similar to that of the SR domain of mammalian and fungal PPIL4-like Cyps. It is not unlikely that both ChCyp34.5 and TgCyp66.3 are not yet predicted accurately (e.g. a missing exon in ChCyp34.5 and a missing intron in TgCyp66.3 would explain the current results) and it will finally turn out that both possess normal Cyp domains and an SR domain.Putative PPIL4-like Cyps are only identifiable in the genomes of  Figures . Althougof fungi , this su2-terminus and a Cyp_ABH domain in their COOH-terminus.PPIL4-like Cyps should not be confused with the PPIE-like Cyps, a subfamily that is missing in all apicomplexan genomes. PPIE-like Cyps contain an RRM motif in the NHChCyp34.5 contains a nuclear localization signal within its Cyp domain and PSORTII predicts a nuclear localization. Due to its high content of positively charged amino acid residues, the putative TgCyp66.3 is predicted to have a multitude of overlapping nuclear localization signals in its COOH terminus in addition to one signal about 100 amino acids away from its NH2-terminus. Indeed, the orthologous AtCyp59 protein from A. thaliana has been described to be localized in the nucleus but outside of those nuclear speckles rich in SR domain proteins [AtCyp59 predominantly participates in transcriptional processes and that it is only marginally involved in splicing [AtCyp59 or other PPIL2-like Cyps since the SR domain responsible for all known AtCyp59 interactions is missing in ChCyp34.5 and the Cyp domain of TgCyp66.3 might well be non-functional. In this context it is also noteworthy that the Cyp domain of this protein is less conserved than that of other PPIL4-like Cyps. Whereas this domain in SpCyp6 and HsPPIL4 is recognized as Cyp_RRM domain (accession no.: [cd01921]), CD-Blast only recognizes a domain belonging to the Cyp superfamily (accession no.: [cd00197]). It is also possible that the truncated or disrupted PPIL4-like Cyps in Cryptosporidia and Toxoplasma containing a slightly degenerated Cyp domain represent transition states that ultimately led to complete loss of this gene in other apicomplexa.proteins . Althougsplicing . It is sLoss of introns during evolution of organisms exhibiting a parasitic live mode such as Giardia , TrypanoC. hominis, 4.5 for T. gondii, 2.0 for P. falciparum, 3.1 for T. parva, 3.5 for T. annulata, and 2.8 for B. bovis is very similar to that observed on the genome scale. Despite this overall similarity, however, there are some remarkable exeptions from the general pattern: First, the putative ChCyp22.9 with SP in C. hominis shows a conserved position of introns in comparison to its ortholog in T. gondii. Secondly, some Cyp subfamilies have a high number of introns in both haemosporidians and T. gondii although only minimal conservation of introns can be observed. This group includes the small apicomplexa-specific cyclophilins, the Cyps with WD40 repeats (with exception of PfCyp87), and the PPIL3-like Cyps. Finally, there is one Cyp gene subfamily, the CeCyp16-like Cyps, showing a completely reversed trend with more introns in the haemosporidian genomes than in T. gondii. Since several of the introns in haemosporidia appear to be of ancient origin, it is most likely that TgCyp65.5 has lost its introns since divergence from the last common anchestor with the haemosporidia.As expectable from the differences in the general genomic organization, the exon/intron structure of Cyp genes varies widely between species. The number of introns per Cyp gene, i.e. 0.5 for ChCyp22.9 (e.g. on regulation of gene expression) that might explain counterselection against their deletion during evolution.In summary, though the trend of moderate and nearly complete loss of introns observed on a genome-wide scale for haemo- and cryptosporidia, respectively, could also be observed for Cyp genes in general, there are exceptions to this rule in certain subfamilies that might be exploited in the future to decipher the selection forces that contribute to conservation of certain introns despite high overall frequency of intron loss. For instance, it would be highly intriguing to look for any functional roles for the three introns in Cryptosporidium and Toxoplasma (PPIL4-like Cyps) or even only in Toxoplasma (PPIL6-like Cyps), respectively. Six out of these 16 subfamilies  have been described to be a part of the extremely complex transcription and/or splicing machinery in mammalian or yeast cells indicating that regulation of protein conformation in these very large protein or ribonucleoprotein complexes catalyzing RNA processing is a highly conserved major function of eukaryotic Cyps.The present study was able to identify 16 different Cyp subfamilies in apicomplexa. While some of these subfamilies can be found in the genomes of all species analyzed, there are also two small subfamilies, that can only be found in the genus Theileria species are predicted to have an NH2-terminal apicoplast localization signal. Surprisingly, these are the only Cyps that are predicted to be transported to the apicoplast. Apicomplexa might be more easily able to cope with loss of cytosolic PPIA-like proteins than other eukaryota since members of the apicomplexa-specific group of relatively small Cyps with Cyp_ABH domain might be able to functionally replace PPIA-like cytosolic Cyps. Moreover, at least one member of the Cyp subfamily with signal peptides has been reported not to be confined to the secretory pathway but to be present in the cytosol as well [While most apicomplexa are predicted to posses typical cytoplasmic PPIA-like Cyps, these putative proteins in both  as well . This CyPlasmodium-specific Cyps, and Cyps with SYF2 domain, already points out obvious drug target candidates.Since the Cyp antagonist CsA has been shown to have anti-parasitc activity against a wide variety of apicomplexa ,17,19,20® protein and nucleic acid databases as well as in PlasmoDB, ToxoDB, CryptoDB, and in the Theileria parva genome database of TIGR. S. pombe Cyp1 and Cyp2 were used as query sequences. These Cyps were chosen because they are not closely related. If a Cyp subfamily member was not identified in one of the apicomplexan organisms, a Cyp of the same subfamily from a closely related apicomplexan parasite was used as query to search in protein, cDNA, EST and genome databases. This method ensures that no Cyps are missed in any of the taxa. In order to prevent that no complete subfamilies was overseen, BLAST analyses were also performed using the complete T. gondii Cyp repertoire as a query. However, no additional Cyp sequences could be identified.Initially, putative apicomplexan Cyps were identified using BLASTp and tBLASTn algorithms to search in GenBankS. pombe Cyps, names according to the entries in the ENSEMBL database were used.In contrast to conventional nomenclature for many Cyps, molecular mass suffixes in the names were given with one position after the decimal point since otherwise identical names would have resulted in a few cases. It was decided not to use suffix letters to avoid a possible confusion with mammalian Cyps. For instance, a Cyp19A might have been confused with a human CypA/PPIA. In addition it should be mentioned that all molecular mass suffixes used have been derived from the predicted sequence of unprocessed proteins. Although this can currently be only a provisional nomenclature, consecutive naming with numbers or letters would result in different names for orthologues Cyps and identical names for unrelated Cyps of different apicomplexa. A more function based nomenclature of apicomplexan Cyps should be introduced later, when at least for one apicomplexan genome all Cyps have been verified experimentally. For human and Homologous putative protein sequences were aligned using ClustalW2 . MaximumFor identification of protein domains, CD-BLAST ,32 and Icis/trans isomerase; snRNP: small nuclear ribonucleoprotein particle; SP: signal peptide; SR domain: Ser- and Arg-rich domain.ABC: ATP-binding casette; AP: apicoplast transit signal; CFBP: cyclosporin A- and FK506-binding proteins; CsA: cyclosporin A; Cyp: cyclophilin; ER: endoplasmic reticulum; FCBP: FK506- and cyclosporin A-binding proteins; FK506: tacrolimus; FKBP: FK506-binding proteins; ORF: open reading frames; PPIase: peptidyl-prolyl The authors declare that they have no competing interests.JK, GG, and GvSH designed the study and wrote the final manuscript. JK performed bioinformatic analyses and drafted the manuscript. All authors approved the final version.H. sapiens and S. pombeTable S1: Cyclophilins from . Listing of the human and fission yeast Cyp repertoire used for comparison with apicomplexan Cyps. Accession-no., protein size and domain architecture are summarized.Click here for filePlasmodium-specific CypsFigure S1 – . Domain architecture and genomic organization of Plasmodium-specific Cyps.Click here for fileTgCyp36.7Figure S2 – PPIL6-like Cyp . Domain architecture and genomic organization of TgCyp36.7.Click here for fileTables S2 and S3: Protein sequences used to analyse evolution of Cyp and FKBP domains in dual class immunophilins. Listing of the genes used for comparison with apicomplaxan FKBPs in Figures Click here for fileFigure S3 – FCBP and CFBP proteins in non-apicomplexa. Domain architecture of FCBPs and CFBPs from non-apicomplexan organisms.Click here for fileFigure S4 – PPIL1-like Cyps. Domain architecture and genomic organization of PPIL1-like Cyps.Click here for fileFigure S5 – PPIL4-like Cyps. Domain architecture and genomic organization of ChCyp34.5, the only apicomplexan PPIL4-like Cyp which contains an RNA recognition motif.Click here for file


Eps15 homology (EH) domains are protein interaction modules binding to peptides containing Asn-Pro-Phe (NPF) motifs and mediating critical events during endocytosis and signal transduction. The EH domain of POB1 associates with Eps15, a protein characterized by a striking string of DPF triplets, 15 in human and 13 in mouse Eps15, at the C-terminus and lacking the typical EH-binding NPF motif.By screening a multivalent nonapeptide phage display library we have demonstrated that the EH domain of POB1 has a different recognition specificity since it binds to both NPF and DPF motifs. The region of mouse Eps15 responsible for the interaction with the EH domain of POB1 maps within a 18 amino acid peptide (residues 623–640) that includes three DPF repeats. Finally, mutational analysis in the EH domain of POB1, revealed that several solvent exposed residues, while distal to the binding pocket, mediate specific recognition of binding partners through both hydrophobic and electrostatic contacts.In the present study we have analysed the binding specificity of the POB1 EH domain. We show that it differs from other EH domains since it interacts with both NPF- and DPF-containing sequences. These unusual binding properties could be attributed to a different conformation of the binding pocket that allows to accommodate negative charges; moreover, we identified a cluster of solvent exposed Lys residues, which are only found in the EH domain of POB1, and influence binding to both NPF and DPF motifs. The characterization of structures of the DPF ligands described in this study and the POB1 EH domain will clearly determine the involvement of the positive patch and the rationalization of our findings. The Eps homology (EH) domain is an evolutionary conserved protein interaction module originally identified as a tandem repeat of approximately 100 amino acids at the N-terminus of the proteins Eps15 and Eps15R , in actiScreening with a multivalent nonapeptide phage display library has identified peptides containing an NPF (Asn-Pro-Phe) motif as the preferred ligands of EH domains . While t155, Leu165 and Trp169 in the second EH domain of Eps15. Mutations in Leu165 and Trp169 were shown to abolish the binding to NPF-containing peptides . Ag. Ag18]. In order to confirm the EH targets inferred by the pull down experiment, we synthesized the full-length human Eps15 protein as 15 amino acid long peptides, overlapping by 12 amino acids, using the SPOT synthesis method . The mem623 to D633 are sufficient to promote binding to POB1_EH. Finally, the results suggest that some of the determinants involved in this interaction might promote a conformation necessary for binding without being involved in the establishment of contacts or, as an alternative, they could act as recognition sites for other binding partners which promote the association.By combining the results from pull down and the PepSpot experiments we conclude that many DPF peptides have the potential of binding the EH domain of POB but most of the Eps15 binding determinants are included in the fragment encompassing amino acids 623–670 and containing five DPF motifs. Furthermore, the two N-terminal DPF peptides included in the region from DCupers and collaborators proposed that Eps15 forms a head to head dimer and that two dimers further associate head to tail to form a tetramer . The tetTo test this hypothesis, we expressed as GST-fusions the N-terminal region of mouse Eps15 and we compared it with the EH domain of POB1 in a pull-down experiment: 50 μg of purified molecules bound to glutathione-sepharose beads were incubated with 3 mg of cell extract from Hek293. Bound proteins were resolved by SDS-PAGE and analyzed by western-blotting using an anti-Eps15 antibody. The result shown in figure In order to provide further evidence for the interaction of Eps15 with the N-terminal EH domain, we performed a pull-down assay using recombinant deletion constructs of Eps15, spanning the DPF region. The GST fusions were incubated with a Hek293 cell extract. Affinity purified proteins were resolved by SDS-PAGE and analyzed by western-blotting using an anti-Eps15 antibody to identify endogenous Eps15. The result Fig.  shows anNMR studies of the EH2 domain of Eps15, combined with mutational analysis, have identified a patch of hydrophobic residues  accommodating the proline in the NPF ligand Fig. . ResidueThe EH1 domain of Eps15 contains a similar hydrophobic binding pocket as the EH2 domain  delimitaThe corresponding residues in the POB1 EH domain are arranged differently ,22, thusThese observations could indicate that the topology of the POB1_EH complex bound to a DPF-containing peptide differs somewhat from the topology of a corresponding complex hosting a NPF peptide. Starting from this hypothesis, we investigated the contribution to Eps15 binding of the molecular surface of the POB1_EH domain as defined by the side chains of residues Ile347 and Phe344 and by the positive patch formed by Lys213 and Lys351 in the interaction with Eps15. To this end, we designed four POB1-EH mutant domains containing an Ala side chain at position K312, K351, and F344 respectively. A mutant in the conserved tryptophan (W324A) was also added as a control since this residue was already proven to be involved in NPF recognition. When tested in a pull down assay, mutants K312A, K351A and the double mutant K312A-K351A, similarly to W324A, have lost their ability to bind Eps15 Fig. . By contTaken together these results suggest that the EH domain of POB1 associates with peptides containing NPF and DPF motifs and that residues that are distal to the previously defined binding pocket may be involved in this recognition.Finally, the structural analysis  motifs are consistently found in EH-domain ligands . StructuAlthough EH domains have never been proven to bind to DPF target peptides, the observation that the association between the EH domain of POB1 and Eps15 is mediated by a C-terminal region containing several DPF has leadIn the present study we confirm that the POB1 EH domain can bind specific DPF motifs in the C-terminal region of Eps15. This interaction was firstly demonstrated by panning a phage displayed peptide library. With the SPOT synthesis method we have been able to identify many DPF peptides in the C-terminal region of Eps15 with the potential of binding the EH domain of POB, most of them included in the fragment encompassing amino acids 623–670 and containing five DPF motifs. Clearly, a potentially EH binding peptide identified in Eps15 with the SPOT experiment could be buried inside the folded protein and therefore it could be inaccessible to the interaction partner. On the other hand, the previous findings are consistent with the pull-down results as the same sequences also associate to POB1_EH domain. Therefore, the overlap between the results obtained by different approaches confirms that the POB1_EH domain can bind DPF peptides and, more in general, point out the effectiveness of combining the classical pull-down method with the SPOT technology to study protein interactions.Furthermore, we suggest that a similar association between the first EH domain of Eps15 and its C-terminus could participate in the stabilization of Eps15 tetramers.The DPF region of Eps15 is also the binding site for the AP-2 complex, the adaptor which connects the clathrin lattice to the plasma membrane and interacts with tyrosine-based signals of several integral membrane proteins . The binStructural and mutational analysis of several EH domains clearly demonstrate that the conserved tryptophan at the base of the NPF binding pocket is essential for domain functionality, while residues flanking the binding groove can influence specificity. For example, the EH3 domain of Eps15 binds to a peptide mimicking the FW-internalization motif of MPR; moreover, NMR and mutational analysis unequivocally shows that FW and NPF peptides bind in the same pocket and involve the same residues . NonetheThe mutational analysis reported in our manuscript confirms that the conserved tryptophan in the binding pocket of POB1_EH domain is essential for peptide recognition, since the mutation W324A affects the interaction with both NPF- and DPF peptides. In addition we provide evidence that the lysine residues K312 and K351, which are distal to the binding pocket, are involved in this recognition. On the contrary, mutating Phe344 to Ala doesn't affect binding.In the present study we have shown that the EH domains of POB1 binds the c-terminus of Eps15 thanks to its affinity for DPF containing peptides. In addition we have identified a cluster of solvent exposed Lys residues, which are only found in the EH domain of POB1, and influence binding to both NPF and DPF motifs. None of the mutants that we have characterized so far show a preferential binding for either NPF or DPF motifs suggesting that the two motifs have a similar binding mode and that both NPF and DPF binding require the hydrophobic pocket characterized in structural studies and the positively charged surface patch characterized in this study. The involvement of the positive patch, either by direct DPF binding or indirectly by modification of the tryptophan binding pocket, cannot be reconciled with the information provided by the structure of the peptide EH complexes described so far and the rationalization of these findings must await the characterization of structures of the DPF ligands described in this study and the POB1 EH domain.The plasmid vectors encoding mouse GST-Eps15 C-terminal constructs were a generous gift from G. Iannolo . The fraThe following primers were used to insert the mutations in the EH domain of POB1. CCAAGAACTTCTTCACCGCATCAAAGCTTTCCATTCC for the mutation K312A, AGAACTCTCCTATATAGCGGAGCTTAGTGATGCTG for the mutation W324A, TGAGTTCTGTGCTGCGGCTCATCTCATTGTGGCTC for the mutation F344A, CAATGGGTAGCCGTTCGCCCGAGCCACAATGA for the mutation K351A. Mutations were inserted using the Stratagene's QuikChange Mutagenesis Kit.The following primary antibodies were used in the experiments: rabbit anti-Eps15 (Santa Cruz), goat anti-GST , rabbit anti-Myc (Invitrogen) and rabbit anti-Epsin1 (a gift from G. Cestra). Secondary antibodies used in this work were secondary anti-goat monoclonal alkaline phosphatase-conjugated antibody (Sigma), anti-goat IgG peroxidase conjugated (SIGMA), anti-rabbit peroxidase-conjugated IgG and anti-mouse peroxidase-conjugated IgG (Jackson ImmunoResearch).10 infectious particles from a nonapeptide library. After washing 10times with PBS, 0.5% Tween 20, the bound phages were eluted with 100mM glycine HCl, pH 2.2. After three selection cycles, the binding of isolated clones was confirmed by ELISA.Library construction and panning were performed as described ,29. Brie9 particles and incubated with 0.2 μg of GST fusion protein. The wells were then washed 10times with PBS, 0.1Tween 20, and bound protein was detected with anti-GST goat primary antibody (Amersham Pharmacia Biotech) and a secondary anti-goat monoclonal alkaline phosphatase-conjugated antibody (Sigma). Clones with binding activity were selected for further analysis. The sequence of the peptides displayed by positive clones were determined by automatic (ABI PRISM 310Perkin-Elmer) sequencing of phage single-stranded DNA using universal M13-40 primer. For the ELISA with synthetic peptides, biotinylated peptides were bound to microtiter wells coated with 1 μg of streptavidin. Each well was incubated with 0,25 μg of GST-EH domain hybrid protein and the bound domain revealed with an anti GST antibody.Microtiter wells were coated with 102 incubator, in DMEM (Gibco/Invitrogen) supplemented with 10% fetal bovine serum (FBS) (SIGMA-Aldrich) and penicillin and streptomycin (Gibco/Invitrogen). Cells were transiently transfected with Lipofectamine 2000 reagent (Invitrogen) according to manufacture's instructions; alternatively, cells were transiently transfected with the calcium-phosphate method.293 cells were grown at 37°C in a 5% CO2O with 61 μl of 2 M CaCl2 to a final volume of 500 μl into a 2 ml tube. Drops of this DNA and CaCl2 mixture were added into a 15 ml tube containing 500 μl of HBS 2x, pH 7,05  while blowing bubbles into the tube with a 2 ml pipet. The mixture was maintained at room temperature for 20 minutes, then the DNA-CaPO4 co-precipitated drops were added to the surface of the media containing the cells and the plate was gently swirled to mix. After 8–16 hours the CaPO4 containing medium was removed and replaced with normal medium. Transient assays for gene expression in transfected cells were performed 24–72 hours post transfection.One day prior to transfection, 293 cells were plated on 10 cm dish to achieve 60–70% of confluence at the time of the transfection. Briefly, 10 μg of each DNA were diluted in ddHE.coli BL21(DE3) strain. Cultures were grown overnight in LB medium supplemented with ampicillin and used to inoculate fresh medium (v/v 1:100). The culture were subsequently grown to OD600 = 0,6 and induced for 4 hours at 30°C with 0,5 mM IPTG. Cells were collected by centrifugation and resuspended in 1/50 of the original volume with STE buffer  supplemented with protease inhibitor cocktail (SIGMA-Aldrich). For the purification of wild-type and mutated EH domains, cells were resuspended in EH buffer . Cells were disrupted by sonication and the lysates were clarified by centrifugation. To purify GST-fusion proteins from the extract, the lysates were incubated with glutathione-Sepharose 4B beads (Amersham Bioscience Inc.) for 1 hour at 4°C, then washed 5 times with ice-cold PBS. Were necessary, proteins were eluted from the beads by incubating them with 15 mM glutathione and eluted proteins were subjected to dialysis in cold PBS.All constructs were transformed into GST-fusion proteins and GST alone were expressed in bacteria, adsorbed to glutathione-Sepharose beads  and incubated for 2 hours at 4°C with 1 mg of 293 cellular extract in EH buffer. Beads were then washed 3 times with the lysis buffer and bound proteins were electrophoresed on a 10% polyacrylamide gel, transferred onto nitrocellulose membrane and probed with specific antibodies. Detection was performed with the enhanced chemoluminescence Supersignal West Pico Stable Peroxidase Solution (Pierce).The peptide arrays (15 amino acid long) were synthesized on cellulose-(3-amino-2-hydroxy-propyl)-ether (CAPE) membranes, because of a better signal-to-noise ratio in the incubation experiments.2.Preparation of CAPE membranes: a 18 × 28 cm Whatman 50 paper  was immersed in a stainless steel dish containing a solution of 400 mg of p-toluenesulfonic acid in methanol (50 ml) and shaken for 3 min. The membrane was removed from the tray and air-dried. Meanwhile a solution of 7.8 g of N--phathalimid in dioxane (60 ml) was heated to 80°C in a covered stainless steel dish placed on a shaking platform. Then, a solution of 400 mg of p-toluenesulfonic acid in 5 ml of dioxane was added. The membrane was placed in this solution and shaken at 80°C for 3–5 hours. Afterwards, the membrane was washed three times with 50 ml of dioxane and with ethanol  and subsequently incubated with a 10% (v/v) solution (50 ml) of hydrazine hydrate (80%) in ethanol for approximately 6 hours. Finally, the membrane was washed twice with ethanol, three times with dimethyl-acetamide, and once again with ethanol , and dried. The peptide loading of this type of amino-functionalized cellulose membrane is about 120–200 nmol/cmPeptides were synthesized according to standard SPOT synthesis protocols (Frank 1992) using an automatic spot synthesizer  as described in detail (Kramer and Schneider-Mergener 1998).Incubation with the domain was performed with 10 μg/ml of the GST-fused domain in blocking buffer overnight at 4°C. After washing three times for 10 min with TBS, the anti-GST monoclonal antibody  was added at a concentration of 1 μg/ml in blocking buffer for 2 hours at room temperature. After three washings with TBS (10 minutes each) a POD-labeled anti-mouse mAb (1 μg/ml in blocking buffer) was added and incubated for 1.5 hours at room temperature, followed by washing three times with TBS.Quantization of peptide-bound domain was carried out using a chemoluminescence substrate and the Lumi-ImagerTM instrument . Signal intensities were measured in BLUs (Boheringer Light Units).ES carried out the affinity selection, the ELISA and pull-down experiments, mutants analysis and drafted the manuscript. SP performed the Pep Spot analysis. MF helped in the structural analysis. LC participated in the design and coordination of the study. GC participated in the design and coordination of the study and drafts the manuscript. All authors read and approved the final manuscript.


NE1406, the first structural representative of PF09410, reveals a lipocalin-like fold with features that suggest involvement in lipid metabolism. In addition, NE1406 provides potential structural templates for two other protein families (PF07143 and PF08622). The first structural representative of the domain of unknown function DUF2006 family, also known as Pfam family PF09410, comprises a lipocalin-like fold with domain duplication. The finding of the calycin signature in the N-terminal domain, combined with remote sequence similarity to two other protein families (PF07143 and PF08622) implicated in isoprenoid metabolism and the oxidative stress response, support an involvement in lipid metabolism. Clusters of conserved residues that interact with ligand mimetics suggest that the binding and regulation sites map to the N-terminal domain and to the interdomain interface, respectively. Here, we report the crystal structure of NE1406, the first structural representation of this family, which was determined using the semiautomated high-throughput pipeline of the Joint Center for Structural Genomics  and I-PIPE (Insert) primers  that included sequences for the predicted 5′ and 3′ ends. The expression vector pSpeedET, which encodes an amino-terminal tobacco etch virus (TEV) protease-cleavable expression and purification tag (MGSDKIHHHHHHENLYFQ/G), was PCR-amplified with V-PIPE (Vector) primers. V-PIPE and I-PIPE PCR products were mixed to anneal the amplified DNA fragments together. Escherichia coli GeneHogs (Invitrogen) competent cells were transformed with the V-PIPE/I-PIPE mixture and dispensed onto selective LB-agar plates. The cloning junctions were confirmed by DNA sequencing. Using the PIPE method, the part of the gene encoding residues Met1–Pro22 was deleted. Expression was performed in a selenomethionine-containing medium with suppression of normal methionine synthesis. At the end of fermentation, lysozyme was added to the culture to a final concentration of 250 µg ml−1 and the cells were harvested and frozen. After one freeze–thaw cycle, the cells were sonicated in lysis buffer  and the lysate was clarified by centrifugation at 32 500g for 30 min. The soluble fraction was passed over nickel-chelating resin  pre-equilibrated with lysis buffer, the resin was washed with wash buffer  and the protein was eluted with elution buffer . The eluate was buffer-exchanged with TEV buffer  using a PD-10 column  and incubated with 1 mg TEV protease per 15 mg of eluted protein. The protease-treated eluate was run over nickel-chelating resin  pre-equilibrated with HEPES crystallization buffer  and the resin was washed with the same buffer. The flowthrough and wash fractions were combined and concentrated by centrifugal ultrafiltration (Millipore) to 19.4 mg ml−1 for crystallization trials. NE1406 was crystallized using the nanodroplet vapor-diffusion method ethanesulfonic acid] pH 9.0. Glycerol was added to the crystal to a final concentration of 10%(v/v) as a cryoprotectant. Initial screening for diffraction was carried out using the Stanford Automated Mounting system  cloning method (Klock 1 Table 1. The oli2.2.1), high-energy remote (λ2) and peak (λ3) points of the Se K absorption spectrum. The data sets were collected at 100 K using a MAR Mosaic300 CCD detector (Rayonix). The MAD data were integrated and reduced using MOSFLM  and TLS refinement with one group per chain. NCS restraints were applied as two sets: to the N-terminal residues 24–74 and the C-terminal residues 83–351. Data-collection and refinement statistics are summarized in Table 1Multiple-wavelength anomalous diffraction (MAD) data were collected at the APS on beamline 23-ID-D at wavelengths corresponding to the inflection  was adapted from an analysis using PDBsum . Atomic coordinates and experimental structure factors for NE1406 at 2.0 Å resolution have been deposited in the PDB with code 2ich.Analysis of the stereochemical quality of the model was accomplished using 3.3.1.a) was determined to 2.0 Å resolution using the MAD phasing technique. Data-collection, model and refinement statistics are summarized in Table 1A and B), two CHES molecules, three glycerol molecules, one sulfate ion and 394 water molecules in the asymmetric unit. No electron density was observed for Gly0 (from the purification tag), Val23 (the first residue after Gly0), Thr75–Pro82 and Arg352–SeMet356 in chain A or for Thr75–Asp80 and Pro353–SeMet356 in chain B. The side-chain atoms of Leu24, Arg144, Glu169, Gln200, Asp222 from chain A and Leu24, Gln89 and Arg352 from chain B were omitted owing to poor electron density. The two chains are nearly identical, with an r.m.s.d. of 0.30 Å over 320 Cα atoms . The Matthews coefficient . Lipocalins (PF00061) are an increasingly diverse family of predominantly small, single-domain, secreted proteins exhibiting high affinity and selectivity for hydrophobic molecules. Structurally, lipocalins form a subset of the calycin superfamily, which additionally includes avidins and fatty-acid binding proteins (FABPs) . The C-terminal domain (residues 221–352) is arranged perpendicular to the long axis of the N-terminal barrel and comprises ten β-strands. It can be superimposed on the N-terminal domain with a Cα r.m.s.d. of 2.4 Å over 105 residues , suggesting gene duplication, although the sequence identity of only 9% is nonsignificant . Strands β5–β6 are absent from the C-terminal domain, while β11 is replaced by another 310-helix . The 310-helix cap of the N-terminal barrel is replaced by two longer strands, β18–β19 , that extend over one end of the barrel  comprises 13 β-strands arranged in the form of a flattened barrel with a 33.2.FATCAT , avidin-related and streptavidin-related proteins , fatty-acid binding proteins (PDB codes 1g5w and 2q9s), nitrophorin (PDB codes 1d2u and 1u17) and a retinoic acid-binding protein (PDB code 1blr). The best score was for the outer membrane protein PagL from Pseudomonas aeruginosa (PDB code 2erv), which gave a Cα r.m.s.d. of 3.4 Å over 198 residues with a sequence identity of only 3%.A search with b) is conserved in the DUF2006 family. In the N-terminal domain of NE1406, the Arg214 side chain from β13 interacts with main-chain residues in both β1 and the N-terminal 310-helix, whereas hydrogen bonding of the Trp50 indole to the 310-helix is mediated via a glycerol molecule . Although the calycin signature is absent from the NE1406 C-terminal domain . The β-sheets forming the NE1406 β-barrel are both longer and flatter than those in lipocalins, resulting in a narrower opening at the bottom of the barrel where the lipocalin-binding site would reside. The difference is even more pronounced when NE1406 is compared with avidins , which have barrels that are more circular and compact than in lipocalins. In this respect, NE1406 resembles FABPs, which also exhibit a barrel that is flatter and more elliptical than in lipocalins. However, NE1406 lacks two additional helices at the top of the barrel that are a characteristic of FABPs. Secondary-structure elements, such as the long C-terminal α-helix characteristic of most lipocalin-like calycins, e.g. nitrophorin , which may also be bound inside the β-barrel and are thought to be involved in cell-envelope LPL transport , ApoD and lazarillo, are known to be peripherally anchored to biological membranes, where they are thought to play a role in membrane biogenesis and repair . However, calculations with the program PPM  of NE1406 shows a predicted functional association with the lipoprotein-releasing system ATP-binding protein LolD (lolD) and co-occurrence with an ATP-binding protein ABC transporter (NE1404). A high degree of confidence is predicted for the functional association of many DUF2006 homologs with ATP-dependent ABC transporters, as well as with other transmembrane proteins including Na+/H+ antiporters, sensor histidine kinases and lipoproteins (e.g. LprI precursor in Mycobacterium tuberculosis). The systematic presence of ATP-dependent cassettes and lipoproteins is compatible with a role for the DUF2006 family in lipid transport, while the presence of numerous signal transduction genes might indicate expression under specific conditions, such as environmental stress. Further experiments will be required in order to functionally characterize NE1406 and to determine whether it associates with lipids in vitro or in vivo and whether its transcription is subject to environmental regulation.The genome context (Vibrio, spirochaetes, firmicutes and archaea. Given the wide phylogenetic presence of the DUF2006 family, if an experimental connection to lipocalins is determined, this finding would present the first evidence of a lipocalin-related protein in the Archaea domain and would settle the question of whether or not this protein family may have arisen via horizontal transfer to eukaryotic cells from the endosymbiotic α-proteobacterial ancestor of the mitochondrion (Bishop, 2000The DUF2006 protein family contains over 400 homologs distributed among trypanosomata, fungi, mycobacteria, bacteroidetes, rhizobia, http://www1.jcsg.org/cgi-bin/models/get_mor.pl?key=2ichA.The availability of more DUF2006 sequences and structures might shed light on the evolutionary history of this intriguing protein family. The information presented here, in combination with further biochemical and biophysical studies, should yield valuable insights into the functional role of NE1406. Models of NE1406 homologs can be accessed at TOPSAN (Krishna et al., 2010http://www.topsan.org/explore?PDBid=2ich.Additional information about the protein described in this study is available from 4.NE1406 adopts a lipocalin-like fold with domain duplication. Analysis based on the calycin-superfamily signature present in the N-terminal domain reveals a potential binding site, while remote sequence homology and the genome context suggest involvement in isoprenoid metabolism and survival under oxidative stress.N. europaea, 2ich, r2ichsfNE1406 from             PDB reference: 10.1107/S1744309109037749/wd5116sup1.pdf            Supplementary material file. DOI:


The protein-remodeling machine Hsp104 dissolves amorphous aggregates as well as ordered amyloid assemblies such as yeast prions. Force generation originates from a tandem AAA+ (ATPases associated with various cellular activities) cassette, but the mechanism and allostery of this action remain to be established. Our cryoelectron microscopy maps of Hsp104 hexamers reveal substantial domain movements upon ATP binding and hydrolysis in the first nucleotide-binding domain (NBD1). Fitting atomic models of Hsp104 domains to the EM density maps plus supporting biochemical measurements show how the domain movements displace sites bearing the substrate-binding tyrosine loops. This provides the structural basis for N- to C-terminal substrate threading through the central cavity, enabling a clockwise handover of substrate in the NBD1 ring and coordinated substrate binding between NBD1 and NBD2. Asymmetric reconstructions of Hsp104 in the presence of ATPγS or ATP support sequential rather than concerted ATP hydrolysis in the NBD1 ring. Members of the AAA+ (ATPases associated with various cellular activities) super family of protein-remodeling factors employ the energy of ATP binding and hydrolysis to dissolve amorphous or amyloid aggregates. In contrast to structurally well characterized, ATP-fueled machines such as GroEL, kinesin, and myosin, the mechanisms by which the wide variety of AAA+ enzymes convert chemical energy into mechanical movement are poorly understood (reviewed in The protein-remodeling factor Hsp104 (heat shock protein 104) contains two nucleotide-binding domains (NBDs) per subunit. Kinetic studies to unravel the contribution of each ATPase domain to Hsp104's catalytic activity revealed cooperativity between AAA+ domains within rings and subunits . Owing tN728A mutation allows binding, but not hydrolysis, of ATP in NBD2 and enables us to control the hydrolysis state in one domain, stalling at least one AAA+ ring in a defined nucleotide state.We have shown that Hsp104 hexamers bound to ATPγS adopt an AAA+ domain arrangement that is incompatible with the classical hexameric packing . The ClpN728A hexamers in the presence of ADP and ATP and compared them to the structure in the presence of ATPγS. Second, we generated asymmetric reconstructions of the ATPγS and ATP Hsp104 hexamers and analyzed the domain orientations of individual subunits.In order to understand the conformational changes upon ATP binding and hydrolysis within Hsp104 hexamers, we pursued two cryoelectron microscopy (cryo-EM) strategies. First, we determined the 3D structures of Hsp104N728A in the presence of ATP and ADP and generated independent 3D reconstructions by angular reconstitution with 6-fold symmetry. Refinement yielded maps with good agreement between input class averages and corresponding reprojections  at surface thresholds enclosing a molecular mass of 612 kDa for each hexamer, but they differ considerably in outer diameter (78–95 Å) and width (15–28 Å) of the central pore. For the NBD1 ring, the outer diameter is identical in all three reconstructions, but the enclosed central cavity ranges from 28 Å in the ATP hexamer to 78 Å in the ATPγS complex. The hexamer with the largest pore in the N-terminal ring shows the smallest pore in the NBD1 ring (ATP), and, conversely, the hexamer with the smallest pore in the N-terminal ring shows the largest pore in the NBD1 ring (ATPγS). The NBD2 densities of the ATP and ADP hexamers are less well defined, hardly connected, and form an open ring structure in contrast to the ones in the ATPγS hexamer. They also lack the central density at the base of the cavity, which has been attributed to the C-terminal region of Hsp104 in the ATPγS state. Despite a similar overall height of the reconstructions, the double tier of the hexamer in the presence of ATP is about 10 Å shorter than the others.N728A mutation, NBD2 should be either ATP bound or empty in the presence of ATP or ATPγS. Because ATP binding to NBD2 is necessary for oligomerization of Hsp104, we assume that most NBD2 domains are nucleotide bound. NBD1, on the other hand, is able to hydrolyze ATP and can, therefore, also be captured in the ADP-bound state when ATP is present. Considering that the N728A mutant maintains 25% of the wild-type ATP turnover rate at NBD1 , the contacts between adjacent AAA+ domains are weak in both layers, whereas the N-terminal domains remain closely joined. The N-terminal domains, which are pulled slightly into the cavity, have a parallel alignment with an ∼17 Å central channel opening B. JudgedThe nature of the domain movements in Hsp104 is best seen in movies that show the maps obtained for the different nucleotide-bound states in alternation . In ordeWhen we interpret the domain rotations on the basis of the fits, the most obvious movements occur upon ATP hydrolysis in NBD1 (comparison between T/T and D/T states). Though both NBDs experience a 90° rotation in the plane of the ring NBD1: D and 3E,256YKG259; NBD2-Tyr: G661YVG664) are the only loops so far known to be involved in substrate binding and translocation , we prepared dot blots with monoclonal antibodies (mAbs), which recognize the N-terminal domain, the coiled coil, and the C-terminal domain of Hsp104 . StrikinN728A. They resemble the data obtained by Conformational changes in NBD2 were measured by nucleotide-dependent changes in fluorescence with the single tryptophan mutants Hsp104 Y662W and Hsp104 Y662W:N728A B. In comThe processivity of the disaggregation reaction of Hsp104 is unknown, but studies on single AAA+ hexamers such as ClpX and MCM helicase suggest a random rather than concerted or strictly sequential ATP hydrolysis model . To inveStrikingly, the T/T reconstruction without imposed symmetry shows a marked asymmetry in the hexamer, giving the complex a contracted appearance on one side A. On theIn contrast to the asymmetric Hsp104 T/T reconstruction, Hsp104 D/T deviates less from the corresponding symmetrized map F and 5G The reconstructions presented here of Hsp104 hexamers assembled in the presence of ATP and ADP reinforce the notion that the AAA+ domains in Hsp104 form expanded rings enclosing a large central cavity, unlike the classical packing observed in hexameric crystal structures of related proteins . ShieldiIt should be noted that a different hexamer structure of the closely related ClpB has been published, with a tighter packing of the AAA+ domains and the coiled coil extending radially outward . CompariATP hydrolysis in Hsp104 NBD1 generates a power stroke that displaces the substrate-binding motifs of NBD1 from the N-terminal end of the cavity wall to the center of the cavity. On the other hand, ATP binding to NBD1 moves the tyrosine motifs of NBD2 by ∼20 Å from the center of the cavity toward the C-terminal rim of the cavity. It has been shown that NBD1 activity alone is sufficient to reactivate some aggregated substrates . TherefoOur maps indicate that ATP binding to one AAA+ domain promotes homotypic domain contacts in the other domain. Accordingly, the NBD2 ring is unconnected unless NBD1 is occupied by ATP. ATP binding to NBD2 is necessary for hexamerization of Hsp104  but doesA wealth of biochemical studies have provided evidence for bidirectional communication between the two AAA+ domains in Hsp104  , but litN728A in the presence of ATP (D/T) (Starting with the hexameric state of Hsp104TP (D/T) A, we hypTP (D/T) . When ATTP (D/T) B, it enaN728A T/T map places NBD2-Tyr in the interface with the neighboring domain  and ATP (D/T) maps, with one subunit in the ATP-bound state in NBD1 and the other five in the ADP state , from 1.5 to 3.5 μm (ATP), and from 1.5–5.2 μm (ADP) (Hsp104μm (ADP) .N728A ATPγS), 11,128 (Hsp104N728A ATP), and 2379 particles (Hsp104 N728A ADP) were manually selected from 95, 43, and 36 micrographs, respectively, with the MRC program Ximdisp  at 1.4 Å per pixel at the specimen level. A total of 23,632 . All figures were produced with the UCSF Chimera package  was dialyzed into 20 mM HEPES-KOH (pH 7.5), 200 mM KCl, 10 mM MgClof ATPγS . Betweenof ATPγS . MembranY662W:N728A mutant was generated by Quikchange mutagenesis (Stratagene). Hsp104Y662W or Hsp104Y662W:N728A were buffer exchanged using Bio-Gel P6 columns into 20 mM HEPES-KOH (pH 7.5), 20 mM NaCl, 10 mM MgCl2, and 1 mM DTT. Under these conditions, Hsp104 is hexameric even in the absence of nucleotide (Y662W or Hsp104Y662W:N728A (2 μM) were then incubated in the absence or presence of ATPγS, ATP, or ADP (1 mM) for 10 min at 25°C. Tryptophan fluorescence at 346 nm (10 nm bandwidth) was measured after excitation at 295 nm (5 nm bandwidth) with a Safire2 plate reader (Tecan). The change in fluorescence (ΔF) was determined by comparing the fluorescence after 10 min (F) to the starting fluorescence , such that ΔF = (F/F0)−1.The Hsp104cleotide . Any par


DEAD-box RNA helicases play various, often critical, roles in all processes where RNAs are involved. Members of this family of proteins are linked to human disease, including cancer and viral infections. DEAD-box proteins contain two conserved domains that both contribute to RNA and ATP binding. Despite recent advances the molecular details of how these enzymes convert chemical energy into RNA remodeling is unknown. We present crystal structures of the isolated DEAD-domains of human DDX2A/eIF4A1, DDX2B/eIF4A2, DDX5, DDX10/DBP4, DDX18/myc-regulated DEAD-box protein, DDX20, DDX47, DDX52/ROK1, and DDX53/CAGE, and of the helicase domains of DDX25 and DDX41. Together with prior knowledge this enables a family-wide comparative structural analysis. We propose a general mechanism for opening of the RNA binding site. This analysis also provides insights into the diversity of DExD/H- proteins, with implications for understanding the functions of individual family members. DExD/H-box RNA helicases, from virus and bacteria to eukaryotes, play important roles in processes including ribosome biogenesis, RNA processing and folding, ribonucleoprotein (RNP) remodeling, RNA nuclear export, the regulation of RNA translation and transcription, and nonsense-mediated RNA decay. DExD/H-box RNA helicases have multiple functions in these processes: They can act as RNA chaperones, ATP-dependent RNA helicases and unwindases, as RNPases by mediating RNA-protein association and dissociation DExD/H-box proteins often contain accessory regulatory domains and localization modules, but their cores consist of two RecA-like domains joined by a short flexible linker. The N-terminal domain is commonly referred to as conserved domain-1, or DEAD-domain, and the C-terminal domain as conserved domain-2, or helicase domain We have solved crystal structures of single domains from eleven human DExD/H-box helicases of the DEAD-motif subfamily. A comparative analysis of these structures uncovered not only isoform specific features, but also nucleotide specific positioning of flexible elements that are common to several proteins. We suggest a structural mechanism for the linkage between binding of ATP and activation of the RNA binding site.We used X-ray crystallography to determine the structures of the DEAD-domains of DDX2A, DDX2B, DDX5, DDX10, DDX18, DDX20, DDX47, DDX52, and DDX53, as well as the helicase domains of DDX25 and DDX41. While the physiological roles of these proteins are diverse  all struSuperpostition of the different crystal structures illustrates the location of flexible regions . In gene2+-binding site. The negative charges originate from the side chains of the two helices, including the DEAD-motif on α-helix 8. As expected, the RNA binding cleft is positively charged in all DEAD domains, but the charged patches differ in size. The remainder of the DEAD-domain surfaces differs in electrostatic surface properties among the family members.We compared the surface charge distributions of the DEAD-domain structures . All DEAConserved motifs I (the P-loop), Ia, II, and the Q-motif participate in nucleotide binding Two of the structures show unique P-loop conformations. The DDX2B structure features an α-helix 4 that is longer than in other helicases, and leads into an unusually closed P-loop conformation . As a coThe AMPPNP-bound DDX20 structure contains no metal ion . Lack ofth residue upstream of the conserved glutamine is an aromatic residue in most DEAD-box helicases. Its side chain stacks with the nucleotide base, stabilizing it in its position. Interestingly this residue is not conserved: While phenylalanine is most common, DDX10 has a tyrosine and DDX47 has a tryptophan in the corresponding position. Moreover, an aromatic residue in this position is not obligatory: DDX53 features an isoleucine, with weaker van-der-Waals interactions with the adenosine ring than the base stacking interactions with the aromatic side chains , the BESSY , the Diamond , the ESRF , and the MaxLab  synchrotron radiation facilities. Data were indexed and integrated using XDS All proteins were expressed in Detailed The coordinates have been deposited in the Proteins Data Bank with accession codes 2G9N, 3BOR, 3FE2, 2PL3, 3LY5, 3B7G, 2RB4, 2P6N, 3BER, 3DKP, and 3IUY.Table S1(0.15 MB PDF)Click here for additional data file.