Domains are the building blocks of proteins. During evolution, they have been duplicated, fused and recombined, to produce proteins with novel structures and functions. Structural and genome-scale studies have shown that pairs or groups of domains observed together in a protein are almost always found in only one N to C terminal order and are the result of a single recombination event that has been propagated by duplication of the multi-domain unit.Previous studies of domain organisation have used graph theory to represent the co-occurrence of domains within proteins. We build on this approach by adding directionality to the graphs and connecting nodes based on their relative order in the protein. Most of the time, the linear order of domains is conserved. However, using the directed graph representation we have identified non-linear features of domain organization that are over-represented in genomes. Recognising these patterns and unravelling how they have arisen may allow us to understand the functional relationships between domains and understand how the protein repertoire has evolved.We identify groups of domains that are not linearly conserved, but instead have been shuffled during evolution so that they occur in multiple different orders. We consider 192 genomes across all three kingdoms of life and use domain and protein annotation to understand their functional significance.To identify these features and assess their statistical significance, we represent the linear order of domains in proteins as a directed graph and apply graph theoretical methods. We describe two higher-order patterns of domain organisation: clusters and bi-directionally associated domain pairs and explore their functional importance and phylogenetic conservation.Taking into account the order of domains, we have derived a novel picture of global protein organization. We found that all genomes have a higher than expected degree of clustering and more domain pairs in forward  and reverse orientation in different proteins relative to random graphs with identical degree distributions. While these features were statistically over-represented, they are still fairly rare. Looking in detail at the proteins involved, we found strong functional relationships within each cluster. In addition, the domains tended to be involved in protein-protein interaction and are able to function as independent structural units. A particularly striking example was the human Jak-STAT signalling pathway which makes use of a set of domains in a range of orders and orientations to provide nuanced signaling functionality. This illustrated the importance of functional and structural constraints (or lack thereof) on domain organisation. One of the driving forces behind protein evolution is the duplication and shuffling of domains -3. ThereSome domains occur in multiple of different domain architectures. They are relatively rare, typically involved in protein-protein interactions  and mostPrevious global studies of domain organisation have used undirected graphs to represent the co-occurrence of domains within proteins -23. ThesWe describe a directed domain graph that takes into account the sequential order of domains. To begin, we evaluate the global properties of the directed networks and compare them with random and previously studied examples. We then assess the domain graphs for individual genomes and phylogenetic groups. Aside from global properties, our analysis also identified cases of non-linear domain organisation. For example, domains form clusters that are highly inter-linked, in particular with links between pairs of neighbours [see Additional file We consider proteins in terms of their domains based on the SCOP database  superfamThis section presents the global properties of the directed domain graph, establishes its topology and compares it with the previously studied "bag-of-domains" model ,17. The The degree distribution [see Additional file In order to assess the significance of the domain graph's global network properties, we compared the observed values with those expected at random. The randomly expected values were determined by calculating the network properties for 1000 random graphs. We were specifically interested in the network properties that are impacted by domain order: the clustering relationships of domains, the density and connectedness of the network.In designing our randomisation strategy it was important to consider both the known properties of the graph and the parameters of interest. We knew that the domain graph was scale free. A simple randomisation approach that preserves only the number of nodes and edges generates a truly random graph, with a degree distribution that follow a Poisson distribution . By defiTo overcome this problem, we generated random graphs with precisely the same degree distribution as the observed graph ,27 expl. This isdifferent domain superfamilies. There are only a small number of cases where one pair of domains has links in both directions, these are discussed below.New to our directed model of domain organisation is the notion of inward and outward edges; as a result, we have two degree distributions (incoming and outgoing). Both the in- and out-degree obey a power-law distribution. Highly connected nodes (i.e. those with more than 50 edges) have in- and out-degrees that are correlated so that any particular node has approximately equal numbers  of inward and outward links Figure . This shThe domain graph above included domain architectures from all proteins in our set of 192 genomes, providing a broad picture of protein organization. By including proteins from different genomes in a single graph, the domains represented may not all interact physically or during evolution. In order focus on evolutionary and functional features of domain organisation, we considered the global network parameters for the graphs of domains from individual genomes and phylogenetic groups of genomes. We calculated the graph parameters expected at random separately for each genome or group through 1000 randomisation of each graph.Restricting the domain set to single or groups of genomes does not affect the global graph topology compared to random; all are scale-free and small-world. However, there is considerable variation in the network parameters  than expected; while around 20 bacterial species have values slightly higher than expected. The average path lengths present a more complicated picture. For the all-genome graph, the observed path length is longer than expected. However, for all the archaea, a large proportion of eukaryotes and around half of the bacteria, the average path length is slightly lower than expected. For the most part the single-genome values fall within two standard deviations of the mean and may not be significant. This suggests that average path lengths are no different from the values expected at random and therefore not influenced or controlled by functional or evolutionary constraints.Section 1 investigates this variability between genomes further by looking on a case-by-case basis at the functional significance of these clusters and their phylogenetic distribution. and 9 deFrom the genome level domain graphs, we identified all bi-directional paths  includeThe clusters we describe are an extreme form of re-use, because not only have individual domains recombined with multiple partners, but their partners have recombined with each-other. Aside from signal-transduction domains, the two central human clusters  can lead to exceptional arrangements of domains. For instance, multi-domain structures that are essentially beads-on-a-string with no fixed interface between domains are more likely to be functional in multiple orientations than their tightly  inter-linked counterparts. Many of the domains we observe within our clusters can function independently of their neighbours.Homo sapiens are almost exclusively eukaryote-specific  and the characteristic path length . This table lists, for each cluster with a clustering coefficient greater than 0 , the common GO category to which they belong and the number of nodes that are included in the cluster.Click here for file


The first structures from the FmdE Pfam family (PF02663) reveal that some members of this family form tightly intertwined dimers consisting of two domains , whereas others contain only the core domain. The presence of the zinc-finger domain suggests that some members of this family may perform functions associated with transcriptional regulation, protein–protein interaction, RNA binding or metal-ion sensing. fmdE gene from the methanogenic archaeon Methanobacterium thermoautotrophicum, indicates that 13 of them are co-transcribed with genes encoding subunits of molybdenum formylmethanofuran dehydrogenase (EC 1.2.99.5), an enzyme that is involved in microbial methane production. Here, the first crystal structures from PF02663 are described, representing two bacterial and one archaeal species: B8FYU2_DESHY from the anaerobic dehalogenating bacterium Desulfitobacterium hafniense DCB-2, Q2LQ23_SYNAS from the syntrophic bacterium Syntrophus aciditrophicus SB and Q9HJ63_THEAC from the thermoacidophilic archaeon Thermoplasma acidophilum. Two of these proteins, Q9HJ63_THEAC and Q2LQ23_SYNAS, contain two domains: an N-terminal thioredoxin-like α+β core domain (NTD) consisting of a five-stranded, mixed β-sheet flanked by several α-helices and a C-terminal zinc-finger domain (CTD). B8FYU2_DESHY, on the other hand, is composed solely of the NTD. The CTD of Q9HJ63_THEAC and Q2LQ23_SYNAS is best characterized as a treble-clef zinc finger. Two significant structural differences between Q9HJ63_THEAC and Q2LQ23_SYNAS involve their metal binding. First, zinc is bound to the putative active site on the NTD of Q9HJ63_THEAC, but is absent from the NTD of Q2LQ23_SYNAS. Second, whereas the structure of the CTD of Q2LQ23_SYNAS shows four Cys side chains within coordination distance of the Zn atom, the structure of Q9HJ63_THEAC is atypical for a treble-cleft zinc finger in that three Cys side chains and an Asp side chain are within coordination distance of the zinc.Examination of the genomic context for members of the FmdE Pfam family (PF02663), such as the protein encoded by the  In thermophilic methanogenic archaea, co-transcription of the fmdE gene with downstream genes encoding catalytic subunits of formylmethanofuran dehydrogenase (EC 1.2.99.5) has been reported , affecting the levels of atmospheric methane as a greenhouse gas , an anaerobic dehalogenating bacterium; Ta1109 from Thermoplasma acidophilum (UniProt Q9HJ63_THEAC), a thermoacidophilic archaeon; and SYN_00638 from Syntrophus aciditrophicus SB (UniProt Q2LQ23_SYNAS), a syntrophic bacterium, encode proteins with molecular weights of 17.4, 23.1 and 21.5 kDa with calculated isoelectric points of 5.95, 6.13 and 6.21, respectively. Their structures, which are the first reported for the PF02663 Pfam family, were determined using the semi-automated high-throughput pipeline of the Joint Center for Structural Genomics  cloning method  and I-PIPE 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. Expression was performed in a selenomethionine-containing medium at 310 K. Cells were induced after 1.5 h using 0.11%(w/v) arabinose and were allowed to grow for an additional 3 h before harvesting. Selenomethionine was incorporated via inhibition of methionine biosynthesis phosphine–HCl (TCEP)] 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 to 15 mg ml−1 by centrifugal ultrafiltration (Millipore) for crystallization trials. B8FYU2_DESHY was crystallized at 277 K using the nanodroplet vapor-diffusion method . Initial screening for diffraction was carried out using the Stanford Automated Mounting system  sodium azide.At the end of fermentation, lysozyme was added to the culture to a final concentration of 250 µg mlTa1109 gene  was amplified from T. acidophilum DSM1728 genomic DNA. Cloning , expression and purification were performed as described for B8FYU2_DESHY. Purified Q9HJ63_THEAC was concentrated to 14 mg ml−1 for crystallization trials and was crystallized at 277 K using the nanodroplet vapor-diffusion method . Initial screening for diffraction and oligomeric state determination were performed as described for B8FYU2_DESHY.The SYN_00638 gene  was amplified from S. aciditrophicus SB genomic DNA. Cloning , expression and purification were performed as described for B8FYU2_DESHY. Purified Q2LQ23_SYNAS was concentrated to 20 mg ml−1 for crystallization trials and was crystallized at 277 K using the nanodroplet vapor-diffusion method . Initial screening for diffraction and oligomeric state determination were carried out as described for B8FYU2_DESHY. The crystal was indexed in the tetragonal space group P41212.The 2.2.1), inflection (λ2) and peak (λ3) wavelengths of a three-wavelength selenium multi-wavelength anomalous diffraction (Se-MAD) experiment for the P212121 crystal form of B8FYU2_DESHY and the C2 crystal form of Q9HJ63_THEAC. Three-wavelength Se-MAD data were collected on beamline 11-1 at SSRL for Q2LQ23_SYNAS. Additional diffraction data for Q9HJ63_THEAC were collected from the two other crystal forms (I222 and P42212) on beamlines 11-1 and 9-2 at SSRL at wavelengths of 1.00 and 0.9790 Å, respectively. MAD phasing for Q9HJ63_THEAC was carried out using the C2 crystal data and further refinement was performed using the I222 data at a higher resolution of 1.87 Å after molecular replacement with Phaser  or a MAR Mosaic 325 CCD detector (beamline 9-2). The data were integrated and scaled using either MOSFLM  at wavelengths corresponding to the high-energy remote . This server verifies the stereochemical quality of the model using AutoDepInputTool , 1(b) and 1(c) were adapted from PDBsum . Atomic coordinates and experimental structure factors for B8FYU2_DESHY at 1.45 Å resolution, Q9HJ63_THEAC at 1.87 Å resolution and Q2LQ23_SYNAS at 1.90 Å resolution have been deposited in the PDB and are accessible under codes 2glz, 2gvi and 3d00, respectively.The quality of the crystal structure was analyzed using the 3.3.1.a) was determined by MAD at 1.45 Å resolution. Data-collection, model and refinement statistics are summarized in Table 1A; residues 4–151 for chain B), 18 ethylene glycol molecules, one Zn atom, one Ni atom and 427 water molecules in the asymmetric unit. No electron density was observed for a few residues at the N- and C-termini of both chains  or for side-chain atoms of ValA3, GluA4, AspA43, ArgA117, GluA118, ArgA119, IleA151, GluB4, AspB43, HisB111, AspB113, ArgB117 and IleB151. The Matthews coefficient . The total β-sheet and α-helical contents are 24% and 58%, respectively. The monomer consists of a central five-stranded, mixed β-sheet (21345 topology) with one solvent-exposed face, while the other is covered by three α-helices. A distinctive feature of the structure is the protrusion of two helices (α4 and α5) and a connecting loop (residues 99–138) from the core of each molecule.The crystal structure of B8FYU2_DESHY  was initially determined by MAD from the C2 crystal form at 2.0 Å resolution. Molecular replacement was then used to determine the structure of the I222 crystal form at 1.87 Å resolution. Data-collection, model and refinement statistics are summarized in Tables 2V               M; Matthews, 1968I222 form was 2.87 Å3 Da−1 and the estimated solvent content was 56.8%. The Ramachandran plot produced by MolProbity . The total β-sheet, α-helical and 310-helical contents are 24, 58 and 2.5%, respectively. In addition to the N-terminal α+β core domain , which is similar to that of B8FYU2_DESHY, Q9HJ63_THEAC also has a C-terminal domain (CTD) with a treble-clef, zinc finger-like motif  was determined by MAD at 1.90 Å resolution. Data-collection, model and refinement statistics are summarized in Table 4R               cryst and R               free values of 23.3% and 26.8%, respectively. One possible explanation for the larger than expected R values is the anisotropy of the diffraction intensities, with a spread in the values of the three principal components of 21.4 Å2 and with diffraction intensity falling off more significantly in the a* and b* directions compared with the c* direction. No electron density was observed for residues A121–A126 or for side-chain atoms of GluA16, LysA17, AspA48, ArgA56, GluA95, LysA105, GlnA110, LysA118, LysA120, GluA128, ArgA129, LysA132, GluA136, LysA148, LysA150, GluA155, LysA156, LysA157, HisA158, LysA159 and LysA161. The Matthews coefficient  is composed of seven β-strands (β1–β7) and nine α-helices (α1–α9). The total β-sheet, α-helical and 310-helical contents are 18, 56 and 4.9%, respectively. Q2LQ23_SYNAS displays a similar architecture to Q2HJ63_THEAC, with a larger NTD (residues 1–154) and a smaller, treble-clef zinc-finger domain CTD coupled together through a nine-residue linker (residues 155–163). The linkers in Q9HJ63_THEAC and Q2LQ23_SYNAS separate the NTD and CTD domains so that the closest edges of the two domains are ∼20 Å apart.The crystal structure of Q2LQ23_SYNAS . The dimer has two prominent C-shaped grooves that extend along its surface parallel to the twofold axis; they are ∼15 Å wide and are exposed to solvent at either end . All crystal forms of Q9HJ63_THEAC  and Q2LQ23_SYNAS  show similar twofold-symmetric, domain-swapped dimers in which the NTD and the CTD of one polypeptide chain are separated by an 11-residue linker and the CTD is anchored to the NTD of the symmetry-related monomer. Analysis of the structures of the Q9HJ63_THEAC and Q2LQ23_SYNAS dimers using CASTp  that is large enough to accommodate a fairly large ligand.B8FYU2_DESHY, Q9HJ63_THEAC and Q2LQ23_SYNAS contain stable dimeric interfaces of 2030, 5860 and 4350 Å3.3.a). The metal ion is solvent-accessible and within coordination distance of His15, His17, Cys19 and Cys55 .A metal ion-binding site was identified at the bottom of the C-shaped groove in B8FYU2_DESHY . The I222 crystal form also showed four additional zinc-binding sites, which are likely to be attributable to the presence of zinc acetate in the crystallization experiments.X-ray fluorescence emission spectroscopy from the b). These side chains are conserved in B8FYU2_DESHY, in which the NTD metal ion-binding site occupies a similar position. In the I222 crystal form of Q9HJ63_THEAC, unexplained electron density near the zinc and Cys61 was modeled as an unknown ligand . The UNL is only 1.8 Å from the S atom of the conserved Cys61, which is consistent with a thioester bond between the protein and the UNL.In Q9HJ63_THEAC the zinc-binding site on the NTD is situated on a loop connecting the N-terminal α-helices (α1 and α2). The zinc is within coordination distance of His16, His18, Cys20 and Cys61 . Each dimer contains two symmetry-related clefts positioned ∼25 Å apart that are assembled from both subunits, including portions of the zinc-finger domain and its β-strand bridging the N- and C-terminal domains. In Q2LQ23_SYNAS no zinc is bound to the NTD. It is worth noting that two of the zinc-binding residues in B8FYU2_DESHY and Q9HJ63_THEAC are not conserved in Q2LQ23_SYNAS: His15 and Cys19 (B8FYU2_DESHY numbering) are replaced by Tyr and Ala, respectively . Instead, an occupied anion-binding site was identified in Q2LQ23_SYNAS  and was modeled as a chloride based on the electron density being within 3.5 Å of the polypeptide backbone N atoms of Arg56 and Gly82 and the presence of chloride in the crystallization reagent. The chloride is bound near the end of the central β-sheet facing towards the extended stretch of polypeptide connecting the NTD and the CTD on the symmetry-related subunit.This binding site and the UNL are located within an elongated cleft on the surface of the dimer that is approximately 30 Å long and 10 Å wide , the human integrin-linked kinase ankyrin-repeat domain in complex with the PINCH1 LIM1 domain , LIM domains 1 and 2 in complex with the LIM-interacting domain of LDB1 from mouse  and the heterodimeric core primase from Sulfolobus solfataricus . Recently, the structure of a prokaryotic homolog of the transcriptional regulator of Ros from Agrobacterium tumefaciens was reported in which an Asp also replaces a Cys as a zinc ligand in the Cys2His2 domain  and an NTD fused to a domain from the uncharacterized protein family UPF0066 (PF01980).a) contain two conserved sequence motifs. The first motif, with a consensus sequence FHGHxC , contains three residues that coordinate the bound metal and is located on a loop connecting α1 and α2 . The second motif contains Asp58, Gln61 and Thr67 (B8FYU2_DESHY numbering) and is located along the twofold-symmetry axis at the dimer interface.Pairwise structural comparisons of B8FYU2_DESHY, Q9HJ63_THEAC and Q2LQ23_SYNAS Fig. 3 revealedα atoms. Two conserved Cys residues on the first β-loop of the CTD coordinate zinc (i.e. the zinc knuckle). These loops are located between β8 and β9  and between β6 and β7  in Q9HJ63_THEAC and Q2LQ23_SYNAS, respectively. The remaining zinc ligands (i.e. the two other Cys residues in Q2LQ23_SYNAS and a Cys and an Asp in Q9HJ63_THEAC) are located near the C-terminal α-helix H10 .The overall fold of the zinc-finger domains of Q9HJ63_THEAC (residues 171–201) and Q2LQ23_SYNAS (residues 162–190) are similar, with an r.m.s.d. of 1.1 Å for 24 superposed C3.6.DALI . The low sequence identity between the NTD and the DALI hits suggests alternate functions for PF02663. In addition, four of the five strands in the β-sheet  and one of the α-helices .A 3.7.Methanoscarina barkeri could be a chaperone . However, in contrast to the structures of the three PF02663 proteins described here, the zinc-finger domain is at the N-terminal end of the protein and the motif for the zinc finger in thioredoxin-2 is a zinc ribbon distinct from the treble-clef motif in the PF02663 structures.The identification of a treble-clef, zinc-finger domain on Q9HJ63_THEAC and Q2LQ23_SYNAS indicates that some PF02663 family members may be involved in transcriptional regulation or protein–protein interactions. However, since the range of functions performed by zinc fingers is diverse, a more detailed functional annotation remains a challenge at present. It has been suggested that a PF02663 homolog in fmdE is co-transcribed with genes encoding the catalytic subunits of a key methanogenic enzyme. Genome-context analysis indicates that only a handful (13 of 208) of genes corresponding to PF02663 members are adjacent to and likely to be co-transcribed with genes encoding the catalytic subunits of molybdemum formylmethanofuran dehydrogenase. Sequence analyses, combined with the structure determinations described here, indicate that 12 of these genes are likely to be part of an fmd operon with a two-domain NTD + zinc-finger architecture, whereas an fmdE homolog from M. barkeri has a one-domain NTD-like architecture. However, most of the genes encoding PF02663 homologs, irrespective of domain architecture, are adjacent to genes encoding metal-ion transporters. These results indicate the absence of a strict correlation between domain architecture and gene context; nevertheless, the results do suggest a possible involvement in metal-ion transport.Previous investigations have established that in some organisms 4.The structures of three members of PF02663 enhance our understanding of the role of these proteins in microbes. Individual proteins within this family display differences in domain architectures, metal-ion binding propensities and dimer interactions. These structural differences suggest a broad range of potential functions for this group of proteins. The identification of a C-terminal zinc-finger domain in two of the structures suggests one possible role for this class of proteins as transcriptional regulators. The NTD together with the CTD might serve as part of the nucleic acid binding surface and/or serve as a signal-sensing domain for the binding of unknown effectors. The absence of a zinc-finger domain in some PF02663 homologs, such as B8FYU2_DESHY, provides some evidence for involvement in alternate processes. Further biochemical and biophysical studies should yield valuable insights into the relationship between structure and function for this interesting group of proteins.et al., 2010http://www.topsan.org/explore?PDBid=2glz for B8FYU2_DESHY, http://www.topsan.org/explore?PDBid=2gvi for Q9HJ63_THEAC and http://www.topsan.org/explore?PDBid=3d00 for Q2LQ23_SYNAS.Additional information about the proteins described in this study is available from TOPSAN (Krishna B8FYU2_DESHY, 2glz            PDB reference: Q9HJ63_THEAC, 2gvi            PDB reference: Q2LQ23_SYNAS, 3d00            PDB reference:


The appendage domain of the γCOP subunit of the COPI vesicle coat bears a striking structural resemblance to adaptin-family appendages despite limited primary sequence homology. Both the γCOP appendage domain and an equivalent region on βCOP contain the FxxxW motif; the conservation of this motif suggested the existence of a functional appendage domain in βCOP.Sequence comparisons in combination with structural prediction tools show that the fold of the COOH-terminus of Sec26p is strongly predicted to closely mimic that of adaptin-family appendages. Deletion of the appendage domain of Sec26p results in inviability in yeast, over-expression of the deletion construct is dominant negative and mutagenesis of this region identifies residues critical for function. The ArfGAP Glo3p was identified via suppression screening as a potential downstream modulator of Sec26p in a manner that is independent of the GAP activity of Glo3p but requires the presence of the COOH-terminal ISS motifs.Together, these results indicate an essential function for the predicted βCOP appendage and suggest that both COPI appendages perform a biologically active regulatory role with a structure related to adaptin-family appendage domains. S. cerevisiae, respectively), and (ii) the B-COPI sub-complex, composed of α-, β'-, and εCOP  with homology to clathrin [SEC28 (εCOP) [The intracellular transport vesicles of eukaryotic cells are responsible for ferrying cellular cargoes between membrane-bound compartments of the secretory and endocytic systems. Many of these vesicles are formed by a protein coat which functions both to select and trap cargo and to deform the membrane as part of the budding process. COPI-coated vesicles are responsible for the retrograde transport of cargo from the cis-Golgi back to the ER and have a role in intra-Golgi transport (reviewed in -4). The 8 (εCOP) .2-terminus which form the core of the complex together with the medium and small subunits [F-COPI and adaptin tetramers such as AP2 are composed of two large subunits (α and β2 in AP2), a medium subunit (μ2 in AP2), and a small subunit (σ2 in AP2). The structure of the AP2 holocomplex reveals two large subunits composed of helical trunk domains at the NHsubunits . Extendisubunits ,13; thessubunits ,14,15. Tsubunits ,17 and tThe adaptin-family appendages can be divided into two subdomains, an amino-terminal β-sandwich subdomain with a fold resembling that of immunoglobulins, and a COOH-terminal "platform" subdomain. The platforms of the γCOP and AP2 appendages each contain an FxxxW motif; in AP2 this motif has been demonstrated to mediate binding to a number of ligands important in the regulation of clathrin coat formation (reviewed in -20). A sCOPI vesicle formation is controlled by the small GTPase Arf1p which, in turn, is regulated by guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs) (reviewed in -28). TheIn this study we sought to examine if βCOP contained an adaptin-family appendage and if such a domain would be critical for COPI-coated vesicle formation. Using a combination of structural modeling, genetic, biochemical, and cell biological approaches, we provide supporting evidence that βCOP does indeed have an adaptin-family appendage and demonstrate that this domain is essential for βCOP function. We also identified the ArfGAP Glo3p as a downstream effector of Sec26p. This role of Glo3p is not dependent on its catalytic GAP activity, suggesting that this protein serves distinct functions in COPI vesicle trafficking that are independent from its regulation of the nucleotide bound state of Arf1p.2-terminal 730 residues are predicted to be primarily α-helical in nature with some unstructured content (data not shown). The accuracy rate (Q3) of PSIPRED is considered to be approximately 78% per residue [2-terminal helical trunk domain [Based on the known similarities in size, sequence, and structure of members of COPI to the AP2/clathrin coat, we predicted the Sec26p COOH-terminus to contain an adaptin-family like appendage domain . To eval residue ,47. Fromk domain , followek domain ,13,48. C2-terminal trunk domain of Sec26p with the other adaptin-family trunk domains, has been previously noted [mGenTHREADER is a protein fold recognition server. Input of the entire Sec26p sequence returns several "Certain" hits, i.e. matches with a confidence level > 99%. Included in this list are both the AP2 and related AP1 cores, specifically the αAP2, β2AP2, and γAP1 chains. Input of the sequence for residues 700–973  returns a single "Certain" (p < 0.0001) hit with a net score of 0.889, namely, the αAP2 appendage, which has 11.7% primary sequence identity to the Sec26p appendage. The next closest match is the γCOP appendage with a "Medium" certainty level (p < 0.01), with a net score of 0.439 and a percent identity of 16.2%. Third on the list, with a "Guess" certainty level (p >/= 0.1), is the β2AP2 appendage with a net score of 0.317 and a sequence identity of 12.9%. The sequence relationship of the NHly noted ,50. ThesIn addition to structural predictive methods, sequence alignments were also performed to predict the appendage domain in Sec26p. Alignments between βCOPs and γCOPs suggest the appendage of Sec26p to begin at approximately residue 667, whereas the alignment with αAP2s suggests a start residue of 729, closer to that predicted by the secondary structure predictions. The platform subdomain is predicted to start at residue 849 based on the alignments with γCOPs and at residue 845 based on alignments with αAP2s, both very close to the start site predicted by the secondary structural prediction models. The appendage domains include several residues which are conserved amongst appendage domains  coding for the trunk region alone was created  of the predicted platform subdomain also resulted in inviability  region which is non-lethal [To determine if the predicted Sec26p appendage domain was a critical functional unit, a truncated construct of e Figure , demonsty Figure , demonstn-lethal .sec26plat Δat endogenous levels as the only copy of SEC26, we wished to test if this construct could provide function when over-expressed. The truncated gene was placed under control of a copper-inducible promoter (PCUP1) for over-expression . The truncated constructs  reveal the dose-dependent nature of the dominant negative effect . A random PCR-derived error resulted in the trunk domain mutation sec26-1 . This construct was temperature sensitive, whereas other constructs containing the D903A D904A mutations were robustly wild type, suggesting that the phenotype was attributable to the T536A mutation.The phenotype difference between Sec26p and Sec21p appendage domain deletion highlighted the essential nature of this region for COPI function. We created point mutations of the Sec26p appendage domain to further dissect the action of this domain at a molecular level. Initially, targeted mutagenesis was performed on a subset of charged residues chosen based on sequence alignments of fungal species. Eight residues were mutated to alanine residues, singly or in pairs as listed: K667 and K669, N751, D823, E828 and D829, and D903 and D904. Silent restriction enzyme sites were incorporated to facilitate the identification of correctly mutated constructs. Constructs were transformed into the We next performed random mutagenesis on the Sec26p appendage to generate conditional lethal thermosensitive (ts) mutants. Error-prone PCR was performed on the appendage domain of Sec26p, leaving the trunk domain wild type. Tester strain transformants were screened for functionality after plasmid shuffling on 5-FOA plates incubated at 30 and 37°C. Thirty-eight unique ts mutants were generated from approximately 6000 transformants Table  and contSEC26 were due to loss of expression at the restrictive temperatures. Whole cell lysates were analyzed by Western blotting using an antibody generated against a recombinant fusion protein of the Sec26p appendage domain , was found to contain 5 mutations restricted to the predicted β-sandwich subdomain (E633D I733T D781V T768P F802L), with no mutations in the platform subdomain. Because both of the appendages of AP2 have been identified to have two binding sites [sec26FW.One mutant allele identified through random screening, ng sites ,14,21,22ng sites , we selesec26FW and sec26-2 alleles were integrated into the genome as the sole copy of SEC26 for use in these screens to facilitate recovery of the suppressor plasmids  controlled over-expression library [SEC26 was recovered multiple times, and GLO3 was recovered twice from the sec26-2 screen. Suppression by GLO3 over-expression is partial and does not restore full wild type growth. The PGAL1/10 GLO3 plasmid  was transformed into each of the appendage mutants and found to suppress a subset of them , another two sets showed either suppression or synthetic lethality (Group II and III), and a fourth set did not show either type of genetic interaction (Group IV). We made use of these groups as additional functional information to identify the residues most significantly contributing to function amongst the 181 mutated positions resulting from the random mutagenesis screen. Only 9 residues were shared by at least three groups  was co-expressed with GST-tagged full-length Glo3p in E. coli however, we were unable to demonstrate the existence of a direct physical interaction between Glo3p and the Sec26p appendage in vitro (data not shown).Our genetic data clearly demonstrate an interaction between Sec26p and Glo3p. In order to determine if this was mediated by a direct physical interaction with the appendage, we performed in vitro co-precipitation assays with recombinant proteins. HisCUP1. This was performed in glo3Δgcs1Δ double knockout cells to eliminate any contribution from the functionally overlapping ArfGAP, Gcs1p. We first verified that Glo3p tagged with GFP at the COOH-terminus is a fully functional construct by testing its ability to act as the only retrograde Arf GAP in a double knockout of glo3Δ and gcs1Δ, a genetic background where GLO3 becomes essential for viability , we created truncation and point mutants of GLO3 under control of the regulatable promoter PGAL1/10. The two Glo3p truncation mutants: glo31–147 and glo31–332 respectively includes the ArfGAP domain only , and the first two thirds of the protein . We also created a full length construct of Glo3p with the catalytic arginine mutated to lysine (glo3R59K) under control of PGAL1/10 . Constructs encoding GLO3 and glo3 mutants were transformed into selected sec26ts strains and plated on galactose-containing media for over-expression. Suppression required a full length version of the protein, but did not require catalytic activity . These results suggest that Glo3p has a second function that resides in its COOH-terminal domain. The COOH-terminus of Glo3p contains two repeats of the amino acid sequence ISSxxxFG (residues 388–395 ISSDQLFG and residues 420–427 ISSSSYFG) which are critical for its ability to suppress the growth defect of arf1-16 [sec26ts  [sec26FW, which contains mutations of the conserved FxxxW motif (to AxxxA) in the platform subdomain [sec26ts appendage domain alleles identified in this study contained a total of 276 mutations distributed amongst 181 out of a possible 380 positions. Given that there were three to twelve point mutations in each allele, the set also probably contains "noise", mutations in residues that do not contribute to the phenotype, in addition to individual point mutations and groups of mutations that together contribute to the overall phenotype. To identify a core set of residues that reliably contributed to the phenotype, we considered the subset of residues that appeared to be frequently mutated and intersected in terms of a genetic interaction with GLO3. Although this is an arbitrary designation, we used this intersection of phenotypes to provide a stringent filter to select functionally important residues from the experimental set of 181 mutated positions. These 9 residues could be expected to be involved in either receiving or transmitting βCOP functionality given that these residues appeared analogous to surface exposed residues of γCOP that might be expected to engage in protein-protein interactions.The appendage domain of the γCOP subunit of the COPI vesicle coat bears a striking structural resemblance to adaptin-family appendages despite limited primary sequence homology (12–16% identity). The adaptin appendages are integral to the regulation of clathrin-coated vesicles via FxxxW and other motifs. Both the γCOP appendage domain and equivalent region on βCOP contain the FxxxW motif; based on the conservation of this motif in βCOP, we predicted the existence of a functional appendage domain in βCOP  and one attractive possibility is that Glo3p combines GAP and effector interactions within the same polypeptide. Taken together with our suppression data, this strongly suggests that Glo3p has critical functions in addition to its GAP activity and that this function involves the COOH-terminus of Glo3p operating in conjunction with Sec26p and COPI. Glo3p contains two repeats of the amino acid sequence ISSxxxFG. This motif is critical for its ability to suppress the growth defect of arf1-16 [GLO3 to suppress sec26ts mutants. The mechanistic underpinnings of this motif are unclear at present; a search of the yeast proteome reveals the ISSxxxFG motif in other proteins of very divergent function . Further studies are required to decipher how this motif functions and if it could play a more general role. It is interesting to note that this motif is conserved in the human orthologs of Glo3p ARFGAP2 and ARFGAP3 [Our results do not address where coatomer and ArfGAPs interact physically in vivo, but do provide interesting insights into the multi-functional nature of Glo3p. What is most remarkable about this interaction is that n assays . Over-ex ARFGAP3 ,59. OutsIn these studies we sought to determine if βCOP contains an adaptin-family appendage domain similar to that determined for γCOP. Sequence comparisons and structural prediction tools suggest that the fold of the COOH-terminus of Sec26p is strongly predicted to closely mimic that of adaptin-family appendages. Experimental approaches, using both deletion and mutagenic studies illustrate the critical role of the predicted Sec26p appendage domain and in particular the residues in close proximity to the FxxxW motif that are expected to form a platform-like subdomain. The essential nature of the appendage, the presence of a conserved motif (FxxxW) in the predicted platform subdomain, the weak but significant sequence homology with other appendages, and the predicted secondary structure and fold collectively support the existence of an appendage domain on βCOP that plays an important regulatory role in COPI function.Yeast strains were cultured using standard media and conditions . PlasmidB. taurus γCOP appendage (PDB ID 1PZD) were downloaded from the RCSB Protein Data Bank [The protein sequence for Sec26p was submitted to the PSIPRED v2.5 server ,42,44 wiata Bank  and loadSEC26 truncations and mutants were created in pRS315 [sec26Δ tester strain . Non-essential KANR single knockout strains were obtained from Research Genetics, Inc., Huntsville, AL; other knockouts were created using standard techniques.All n pRS315  for evalSEC26 using primers designed to anneal beginning at the coding sequence for residue E594 and ending 579bp downstream of the stop codon, and was carried out as described by Wilson and Keefe [sec26Δ tester strain with a linearized plasmid containing a gapped SEC26 gene  for recombination to form the complete SEC26 ORF. Transformants were replica plated to two 5-FOA plates for plasmid shuffling and incubated at 30 and 37°C. Digital images of the plates were pseudocolored and overlaid to identify ts colonies. The plasmids were extracted and re-transformed into the tester strain to confirm thermosensitivity of the construct. Confirmed sec26ts alleles were sequenced at the Cornell Biotechnology Facility using Big Dye Terminator chemistry on an Automated 3730 DNA Analyzer .Random mutagenic PCR was performed on the appendage domain of nd Keefe . The mutsec26ts plasmids of interest were integrated into the genome. Plasmids were digested with XbaI and XhoI to obtain a 1.9kb fragment containing the mutant appendage, which was ligated into the integrating LEU2 vector pRS305. The resulting plasmid was linearized at a unique HindIII site located immediately upstream of the appendage start and transformed into yeast for a one-step gene disruption and replacement at the SEC26 locus. The integrated sec26ts strains were transformed with a library under control of the inducible galactose promoter  [SEC26. Novel suppressors were then sequenced as above and identified using the BLAST server at NIH.To facilitate suppressor plasmid recovery during the screen, the etscher)  and apprGLO3 following a short linker (GGPGG) and expressed under the control of its endogenous promoter in pRS316 . The haploid gcs1Δ ::KANR deletion strain was crossed to a glo3Δ ::HIS3 strain transformed with the Glo3p-GFP plasmid to generate a double knockout gcs1Δglo3Δ strain containing Glo3p-GFP. Diploid double knockouts were generated by crossing complementary haploid double knockouts . Live cells were analyzed with a Nikon Eclipse E600 microscope equipped with a 100X (1.4NA) lens and 1X optivar (0.08 μm/pixel). A Sensicam EM High Performance camera  was used for image capture using IPLab 3.6.5 software . Blind deconvolution was accomplished using AutoDeblur 9.3 software package  for 25 ± 5 z stacks with a 0.2 μm slice size for 30 iterations.GFP was fused to the COOH-terminus of E. coli and purified from inclusion bodies. Post-sonication pellets were resuspended in Buffer A  with 2% DOC and washed twice with rocking for 15 minutes at room temperature in between. The purified inclusion bodies were resuspended in equal volume 2× sample buffer  and resolved by SDS-PAGE. The band containing the fusion protein was excised, cut into slices containing ~100 μg fusion protein, and stored at -20°C. Pre-laying hens were injected intramuscularly with ~100 μg purified fusion protein suspended in PBS every two weeks for a total of three injections. The first two injections were given with 1 part by volume Freund's adjuvant and 2 parts 2% Tween 80 in sterile phosphate-buffered saline (PBS). IgY antibody was partially purified from the egg yolks once laying commenced by emulsification in 25 ml PBS per egg brought to 100 ml with chloroform, followed by centrifugation to separate the aqueous layer. 0.002% sodium azide was added to the IgY-containing aqueous extract which was stored at 4°C.GST fused to Sec26p residues E594-A862 in pGEX-4T-1  was expressed in 600 units for each sample in duplicate were harvested from log phase cultures. One set of samples (t = 0) was washed, and resuspended in 1 ml 10 mM sodium azide and kept on ice. The other set was washed, resuspended in 1 ml YP + 0.1% D-glucose and incubated at 40°C with shaking for 1 hour (t = 1) and processed as for the t = 0 set. Each sample was processed for two measurements, external invertase was measured on whole cells and total invertase was measured after glass-bead lysis according to standard methods [2 OD methods . Percent4 was added to the copper-inducible strains, and all cultures were incubated for 3 hours at 30°C to mid log phase (OD600 ~0.5–0.8). 10 OD600 units of cells was collected, washed in 500 μl ice cold TAZ buffer , resuspended in 50 μl TAZ buffer plus protease inhibitors  and subjected to glass bead lysis for two minutes at 4°C. 70 μl 2× sample buffer was added and the samples heated to 60°C for 10 minutes prior to SDS-PAGE gel electrophoresis. The proteins were transferred to methanol pre-wetted 0.45 μm PVDF membrane  at 250 mA for 2 hours in transfer buffer  with 2% methanol. Membranes were probed with the chicken anti-Sec26p appendage antibody described above. The antibody was incubated with the membrane overnight at room temperature at 1:1000 in Tris-buffered saline plus 0.2% Tween-20 (TBST) with 5% nonfat milk. After three ten-minute washes in TBST, blots were probed with alkaline phosphatase conjugated goat anti-chicken antibody  at 1:10,000 in TBST for 30 minutes, and then subjected to three additional washes in TBST. Membranes were washed in 100 mM Tris pH 9.5, 100 mM NaCl, 5 mM MgCl2 for 10 minutes and developed with phenylphosphate substituted 1,2 dioxetane  imaged in a Fujifilm LAS-3000 cabinet .For lysate preparation, cultures were grown to early log phase, 0.7 mM CuSOsec26ts alleles as the only copy of SEC26 were grown to mid-log phase and then shifted to 40°C for 1 hour prior to lysis. The cultures were pelleted and resuspended at 10 OD600 units/ml in ice cold TAZ  buffer. 10 OD600 units were collected, and resuspended in 100 μl TAZ buffer plus protease inhibitors and subjected to glass bead lysis for two minutes at 4°C. An additional 150 μl lysis buffer plus protease inhibitors was added, and 300 μl 3× sample buffer  was added. Samples were heated at 80°C for 10 minutes and 10 μl resolved on an 8% 1.5 mm SDS-PAGE gel. Western blotting was carried out as described above, except the chicken anti-Sec26p appendage antibody was used at 1:2000. Blots were re-probed without stripping with an anti-Elp1 antibody [To evaluate the expression levels of the Sec26p mutants, yeast expressing the antibody  as a celS-transferase; GFP, green fluorescent protein; Arf1, ADP-ribosylation factor-1; GEF, guanine nucleotide exchange factor; GAP, GTPase activating protein; ts, temperature sensitive; 5-FOA, 5-fluoroorotic acid; SCD, synthetic complete media; SCG, synthetic complete media with galactose as the sole carbon source; PBS, phosphate-buffered saline.The abbreviations used are: ER, endoplasmic reticulum; COPI, Coat Protein complex I; AP2, Adaptin Protein complex 2; GST, glutathione CJD performed the experimental studies and together with RNC, was involved in the study design, analysis and interpretation of data, drafting the manuscript and figure preparation, PBR created several yeast strains and plasmids used and performed the initial experiments identifying the βCOP appendage domain as critical for viability, GRH and RAC made intellectual contributions to the conception of the study. All authors participated in the design of the study, critiqued and approved of the final manuscript.


Hsp104, a yeast protein-remodeling factor of the AAA+ (ATPases associated with various cellular activities) superfamily, and its homologs in bacteria and plants mediate cell recovery after severe stress by disaggregating denatured proteins through a poorly understood mechanism. Here, we present cryo-electron microscopy maps and domain fitting of Hsp104 hexamers, revealing an unusual arrangement of AAA+ modules with the prominent coiled-coil domain intercalated between the AAA+ domains. This packing results in a greatly expanded cavity, which is capped at either end by N- and C-terminal domains. The fitted structures as well as mutation of conserved coiled-coil arginines suggest that the coiled-coil domain plays a major role in the extraction of proteins from aggregates, providing conserved residues for key functions in ATP hydrolysis and potentially for substrate interaction. The large cavity could enable the uptake of polypeptide loops without a requirement for exposed N or C termini. S. cerevisiae disaggregates chemically or thermally denatured proteins in an ATP dependent manner, and cooperates with the Hsp70/Hsp40 chaperone system to refold the proteins to their native state  separated by a variable middle region. X-ray crystal structures of subunits of the full-length, bacterial Clp/Hsp100 proteins ClpA , containing the 26 amino acid ClpP binding motif of ClpA, unfolds aggregated substrates and delivers them to the ClpP protease for degradation  with an N-terminal deletion mutant (Hsp104 ΔN)  . All datp104 ΔN) . AnalysiN728A forms a three-layered hexameric complex. A two-tiered ring structure capped by a smaller ring on one surface is evident in the raw images and more distinct in the class averages (Hsp104averages A. On theaverages B. In botT. thermophilus ClpB AMPPNP (The 3D reconstructions of full-length and ΔN Hsp104 have a diameter of 157 Å and a height of 129 Å and 95 Å respectively C and 1D.B AMPPNP . The meaB AMPPNP . On the B AMPPNP E. The enPSI+] prion (a self-templating Sup35 amyloid) when overexpressed . The short hinge region between the AAA+ domains (10 amino acids) requires NBD1 to be placed to the upper right of NBD2. Fitting the boomerang-shaped cross section of NBD1 . Next, we determined the hand of the full-length Hsp104 map by tilt experiments . The dom of NBD1 B into thSince the N termini of Hsp100 proteins share little sequence identity, homology modeling for Hsp104 residues 1-48 failed. Filling in with the ClpB X-ray structure, the N termini fit the density of the full-length Hsp104 EM map well and show tight packing in the ring C. The N-When the monomeric Hsp100 structures from the crystal and the EM fits are superimposed through the Cα atoms of NBD1, the most significant difference is the position of the coiled-coil domain . In the + domain points into the ATP binding groove of the adjacent domain to facilitate ATP hydrolysis. Since the overall dimensions of the structurally conserved AAA+ domains are very similar, the maximum diameter of an AAA+ hexamer assembled with this packing is limited. We found that none of the AAA rings of the above-mentioned hexameric protein complexes are wider than 130 Å in any nucleotide state observed. Accordingly the maximum pore size formed by the complexes is ∼25 Å.Hexameric crystal structures of p97, HslU and the LTag of SV40 show that the oligomeric packing of AAA domains is conserved . These f+ domains in Hsp104 differs substantially from that of p97. With an outer diameter of nearly 160 Å and a pore size of up to 78 Å in the NBD1 ring, the packing of the AAA modules is significantly expanded compared to that in p97. Furthermore, we observe well-separated domains with little contact to adjacent domains, whereas individual AAA+ domains in p97 interlock with each other and share large interaction surfaces. When viewed down the 6-fold axis from the N-terminal surface, Hsp104 AAA+ domains are rotated clockwise relative to p97's by ∼45° in NBD1 . Hsp104N728A and Hsp104 Δ1-157 (ΔN) were purified from E.coli BL21-Codon Plus®-RIL cells (Stratagene) as described . Hexamerization of wild-type or mutant Hsp104 proteins (0.2 μM monomer) was assessed by glutaraldehyde crosslinking as described  and the isogenic strain where the HSP104 gene is deleted (W303a Δhsp104) , then either maintained at 30°C (untreated) or incubated at 37°C for 30 min (preconditioned). Cells were then incubated at 50°C for increasing times. Heat shock was terminated by transferring tubes to ice. Cells were then diluted in ice-cold SD-ura, and various dilutions were immediately plated onto SD-ura and allowed to recover for 3 days at 30°C to assess viability. Colonies were counted using an aCOLyte automated colony counter (Synbiosis). Immunoblot analysis showed that each Hsp104 protein was expressed at a similar level under equivalent conditions (data not shown).W303a (Δhsp104)  were tra2, 1 mM DTT and 2 mM ATPγS. A 3.5 μl sample of the solution was applied to glow discharged, lacey carbon film on copper grids (300 mesh). After 30 s excess solution was blotted and the grid was flash frozen in liquid ethane. Cryo EM was carried out on a Tecnai F20 FEG operated at 200 kV under low dose conditions. Images were taken at a magnification of 50,000x with defocus ranging from 1.5–4.1 μm.Proteins were diluted to a final concentration of 0.3 mg/ml in 20 mM HEPES (pH 7.5), 20 mM NaCl, 10 mM MgClN728A) and 5353 particles (Hsp104 ΔN) were manually selected using the MRC program Ximdisp  at a sampling rate of 1.4 Å per pixel at the specimen level. A total of 9497 particles  between image tilt pairs of negatively stained complexes for all possible tilt axes based on the prediction of orientations from the untilted particles (The absolute hand of the Hsp104articles . The CCCarticles . The relarticles . The CCCwww.pymol.org). Automated fitting in Chimera optimised the fit for NBD2. The fit for NBD1 in Hsp104 ΔN was optimised by docking the AAA+ subdomains individually. All figures were prepared using PYMOL, except for Docking was done using an Hsp104 homology model based on known X-ray structures with pdb entry codes 1qvrA.pdb, 1qvrB.pdb, 1qvrC.pdb , 1ksf.pdb , 1r6b.pdb  and 1jbkA.pdb  generated with SWISS-MODEL . The AAA


The crystal structure of the first representative of the Pfam PF07336 (DUF1470) family reveals a two-domain organization that contains a new fold, termed the ABATE domain, at the N-terminus and a treble-clef zinc finger that is likely to bind DNA at the C-terminus. Jannaschia sp. strain CCS1, a member of the Pfam PF07336 family classified as a domain of unknown function (DUF1470), was solved to a resolution of 1.45 Å by multiple-wavelength anomalous dispersion (MAD). This protein is the first structural representative of the DUF1470 Pfam family. Structural analysis revealed a two-domain organization, with the N-terminal domain presenting a new fold called the ABATE domain that may bind an as yet unknown ligand. The C-terminal domain forms a treble-clef zinc finger that is likely to be involved in DNA binding. Analysis of the Jann_2411 protein and the broader ABATE-domain family suggests a role as stress-induced transcriptional regulators.The crystal structure of Jann_2411 from  The Pfam database  and a calculated isoelectric point of 6.6 and its crystal structure was determined using the semiautomated high-throughput pipeline of the Joint Center for Structural Genomics  and primers  corresponding to the predicted 5′ and 3′ ends. The PCR product was cloned into plasmid pSpeedET, which encodes an expression and purification tag followed by a tobacco etch virus (TEV) protease cleavage site (MGSDKIHHHHHHENLYFQ/G) at the amino-terminus of the full-length protein. The cloning junctions were confirmed by DNA sequencing. Protein expression was performed in a selenomethionine-containing medium, with suppression of normal methionine synthesis, using the Escherichia coli strain GeneHogs (Invitrogen). 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. After one freeze–thaw cycle, the cells were homogenized in lysis buffer  and passed through a Microfluidizer (Microfluidics). The lysate was clarified by centrifugation at 32 500g for 30 min and loaded onto 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 HEPES crystallization buffer  using a PD-10 column  and treated with 1 mg TEV protease per 15 mg eluted protein. The digested eluate was passed 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 to 12.5 mg ml−1 by centrifugal ultrafiltration (Millipore) for crystallization assays. Jann_2411 was crystallized using the nanodroplet vapor-diffusion method . The diffraction data were indexed in monoclinic space group C2  sodium azide. The molecular weight was calculated using ASTRA 5.1.5 software (Wyatt Technology).The gene encoding Jann_2411  was amplified by polymerase chain reaction (PCR) from genomic DNA using 2 Table 1. The oli2.2.1), inflection (λ2) and peak (λ3) of a selenium MAD experiment. The data sets were collected at 100 K with a MAR Mosaic 325 mm CCD detector  using the Blu-Ice data-collection environment  data. The refinement included phase restraints from SOLVE and TLS refinement with four TLS groups per chain. Data-collection and refinement statistics are summarized in Table 1Multiple-wavelength anomalous diffraction (MAD) data were collected at SSRL on beamline BL11-1 at wavelengths corresponding to the remote . This server processes the coordinates and data using a variety of validation tools including AutoDepInputTool  was adapted from an analysis using PDBsum . Atomic coordinates and experimental structure factors for Jann_2411 have been deposited in the PDB (http://www.pdb.org) under accession code 3h0n.The quality of the crystal structure was analyzed using the 3.3.1.a) was determined to 1.45 Å resolution using the MAD method. Data collection and refinement statistics are summarized in Table 1V               M; Matthews, 19683 Da−1 and the estimated solvent content was 50.9%. The Ramachandran plot produced by MolProbity . The first domain (residues 1–142) can be visualized as two subdomains  that share similar topology and secondary-structure elements, namely a helix–β-hairpin–helix motif , with an additional helix (H4 from the first subdomain and H5 from the second subdomain) linking the two motifs. We have therefore named this region the ABATE domain, representing the Alpha-Beta-hairpin-Alpha TandEm motif. The helices of the first subdomain (H2 and H3) are stacked perpendicular to helices H5 and H7 which, together with H6, form a helical bundle capped on one end by the 310-helix H8 and on the other by helix H4. In both subdomains, the β-hairpin is oriented orthogonally with respect to the long axes of the helices in the ABATE motif. Superposition of the two ABATE motifs (residues 9–46 for ABATE1 and 80–139 for ABATE2) results in an r.m.s.d. of 3.2 Å over 27 residues (3% identity), which is non-significant. Additionally, sequence alignment shows little residue conservation in this domain, with no strictly conserved residues observed between representative ABATE-family sequences . This arrangement of zinc-coordinating residues is typical of treble-clef zinc fingers  forms a treble-clef zinc finger Fig. 2. The zinThe above findings led to the re-evaluation of the Pfam DUF1470 family which, as a result of our study, will now be split into two entries in the next Pfam release . The original DUF1470 entry has been truncated and renamed to represent the ABATE domain, while a new Pfam family has been created for the C-terminal CGNR zinc-finger domain called zf-CGNR (Pfam accession PF11706).3.2.SCOP classifies Jann_2411 as an α+β protein with an unusual fold (http://scop.mrc-lmb.cam.ac.uk/scop/data/scop.b.e.dda.b.b.b.html). A search with FATCAT  and a histidine phosphotransferase domain (PDB code 1sr2)] involved less than half of the domain and displayed diverse functionalities. No single hit was found for the N-terminal domain in its entirety, leading us to propose that this domain represents a new fold. The C-terminal domain was structurally similar (main-chain r.m.s.d. of 2.5 Å over 40 residues with a sequence identity of 11%) to a plant homeodomain (PHD) finger from yeast (PDB code 2jmi), confirming the identity of this domain as a treble-clef zinc finger. Superposition of H9 onto the corresponding helix of the yeast structure revealed that the arrangement of the zinc ion and coordinating cysteines is conserved between the two structures .               PISA server . This arrangement results in the formation of a deep cavity  include a transmembrane protein of unknown function (Jann_2410) and the transcriptional regulator Jann_2412, a member of the Asr gene family. The Asr gene family is widespread in higher plants and most members of this family are up-regulated under a range of environmental stress conditions; their products are thought to function as transcriptional regulators  and plant pathogens ; they are around 180 residues in length and also contain the newly designated zf-CGNR (Pfam accession PF11706). Genome-location analysis of representative ABATE sequences shows co-occurrence with putative DNA-binding proteins, transcriptional regulators and membrane proteins implicated in ABC transport. In Streptomyces, several of the proteins co-occurring with ABATE homologs, such as sporulation-specific cell-division proteins and RNA polymerase sigma factors, are implicated in the control of aerial mycelium development (Dalton et al., 2007et al., 2008Other members of this newly defined ABATE protein family are found in plant symbionts (et al., 2000et al., 2006et al., 2001et al., 1996et al., 2005et al., 2007et al., 2007A number of molecular mechanisms are shared between symbionts and pathogens, especially those involving host colonization and adaptation to a particular ecological niche (Hentschel http://www1.jcsg.org/cgi-bin/models/get_mor.pl?key=3h0nA.The availability of additional ABATE sequences and structures should shed light on the evolutionary history of this protein family. The information presented here, in combination with further biochemical and biophysical studies, should yield valuable insights into the functional role of Jann_2411. Models of Jann_2411 homologs can be accessed at et al., 2010http://www.topsan.org/explore?PDBid=3h0n.Additional information about the protein described in this study is available from TOPSAN (Krishna 4.The first structural representative of the DUF1470 family revealed a two-domain organization, with the N-terminal domain presenting a new fold and the C-terminal domain consisting of a treble-clef zinc finger. The structure additionally allowed a re-evaluation of the Pfam signature and the Pfam assignment and suggests a role for this family in stress-induced transcriptional regulation.Jannaschia sp. strain CCS1, 3h0n, r3h0nsfJann_2411 from             PDB reference: