In this work, we have discovered a new function of the regulatory domain. We present a crystal structure of an Arabidopsis KCBP fragment showing that the C-terminal regulatory domain forms a dimerization interface for KCBP. This dimerization site is distinct from the dimerization interface within the N-terminal domain. Side chains of hydrophobic residues of the calmodulin binding helix of the regulatory domain form the C-terminal dimerization interface. Biochemical experiments show that another segment of the regulatory domain located beyond the dimerization interface, its negatively charged coil, is unexpectedly and absolutely required to stabilize the dimers. The strong microtubule bundling properties of KCBP are unaffected by deletion of the C-terminal regulatory domain. The slow minus-end directed motility of KCBP is also unchanged in vitro. Although the C-terminal domain is not essential for microtubule bundling, we suggest that KCBP may use its two independent dimerization interfaces to support different types of bundled microtubule structures in cells. Two distinct dimerization sites may provide a mechanism for microtubule rearrangement in response to Ca2+ signaling since Ca2+- binding proteins can disengage KCBP dimers dependent on its C-terminal dimerization interface.Kinesin-like calmodulin binding protein (KCBP), a Kinesin-14 family motor protein, is involved in the structural organization of microtubules during mitosis and trichome morphogenesis in plants. The molecular mechanism of microtubule bundling by KCBP remains unknown. KCBP binding to microtubules is regulated by Ca In vitro, active KCBP promotes formation of microtubule bundles while its negative regulation promotes dissociation of microtubule bundles Kinesin-like calmodulin binding protein (KCBP) is a molecular motor found in plants KCBP belongs to the kinesin family of molecular motors. Molecular motors of this family use the energy of ATP hydrolysis to drive a mechanical power stroke, leading to their directional movement along microtubules The motor head of KCBP is found near the C-terminus of its polypeptide chain. This structural organization places KCBP in the Kinesin-14 group of the kinesin family, together with its structural relatives, Drosophila ncd, yeast KAR3, and others 2+ ion sensor KIC 2+ ion coordinating EF hands, one of them being disabled by mutations, instead of four EF hands present in calmodulin. When bound to KCBP, these Ca2+-binding proteins cause the motor to detach from microtubules and thus negatively regulate its activity. The neck mimic both links the regulatory helix to the motor domain and plays an important role in the conformational response to nucleotide binding. The neck mimic docks along the motor domain, stabilizing the ATP-bound conformation of KCBP KCBP has another unusual structural domain that distinguishes it among kinesins, at the very C-terminus of the polypeptide chain. The C-terminal regulatory domain of KCBP consists of three structural features coil-helix-coil. These features are termed the neck mimic, regulatory helix, and negative coil, respectively However, the role of the third feature of the regulatory domain, the negative coil, in the function of KCBP is unclear. One possible role of the negative coil that was proposed Arabidopsis KCBP regulatory domain, namely, that it can self-associate, leading to a dimerization of Arabidopsis KCBP through its C-terminus. Here we present structural and biochemical data showing that the Arabidopsis KCBP dimers formed via association of the C-terminal regulatory domains exist both in crystals and in solution and that the negative coil is indispensable for maintaining dimerization of KCBP at its C-terminus. To address the physiological relevance of this unpredicted dimerization, we expressed the constructs of Arabidopsis KCBP with and without C-terminal regulatory domain and compared their biological properties in motility and microtubule bundling assays. Although the self-association of the C-terminal regulatory domain did not affect the biological function of KCBP in these assays, the geometry of the dimer structure suggest to us that KCBP may engage this feature to support Ca ion-dependent specific microtubule-based structures in cell.To determine the role of the negative coil in the function and regulation of KCBP, we initiated a series of structural and biochemical experiments. These experiments steered us to the discovery of a previously unrecognized feature of the Arabidopsis DNA constructs of KCBP (12–1261), KCBP (876–1261), KCBP (884–1253), and KCBP (884–1244) were cloned in pET28b using NcoI-EcoRI sites (generously provided by A.S.N.Reddy). The C1130N mutation was inserted in KCBP (876–1261) construct using QuikChange site-directed mutagenesis kit (Stratagene). All the pET28b KCBP constructs encoded a tag-free protein.The Arabidopsis DNA construct of KCBP (820–1225) was cloned into the vector pET32 Xa/Lic (Novagen) using the kit and protocols for ligation independent cloning (LIC). The plasmid encoded the N-terminal His6-Trx tag separated from the expression gene by a linker with the TEV-protease cleavage site.The Arabidopsis DNA construct of KCBP (884–1225) was cloned into pDEST17 (Invitrogen) using the kits and protocols for GATEWAY cloning technology. The forward PCR primer used for cloning was designed to insert the TEV-protease cleavage site between N-terminal His6 tag and the expression gene.The Arabidopsis KIC (1–135) was cloned into a modified pRSFduet plasmid (Novagen) adapted for Gateway cloning technology (Invitrogen) encoding the N-terminal His6 tag cleavable by TEV-protease.The full length Arabidopsis KIC (29–135) was cloned into the vector pET32 Xa/Lic (Novagen) using the kit and protocols for ligation independent cloning (LIC). The plasmid encoded the N-terminal His6-TRX tag separated from the expression gene by a linker with the TEV-protease cleavage site.The 600 ∼0.6–0.8. Protein expression was induced by adding 0.1 mM IPTG to the cell culture. After 3–16 h of expression at 25°C, the cells were harvested. The cell pellets containing the recombinant KCBP or KIC were subjected to lysis by sonication in the buffer containing 50 mM Tris (pH7.5), 50 mM NaCl, 2 mM MgCl2, 2 mM CaCl2, 0.1 mM ATP, 1 mM TCEP, and protease inhibitors mixture. The recombinant proteins carrying the His6-tag were purified from the soluble fraction of the cell lysate using the Ni-NTA beads (Amersham). The Ni-NTA bound proteins were eluted in the presence of 100 mM imidazole. To cut the tag peptide off, the protein samples were treated with TEV-protease while dialyzed overnight against the original imidazole-free buffer. Then, the sample was passed through the Ni-NTA beads again. The unbound fraction containing the tag-free protein was collected. The KCBP proteins expressed carrying no tag were purified out of the soluble fraction of the cell lysate using Calmodulin-Sepharose 4B (Amersham) as described in For protein expression the described constructs were transformed into E. coli competent cells BL21(DE3). The cells were allowed to grow at 37°C until OD2, 0.1 mM ATP, 1 mM TCEP, and either 1 mM EGTA or 2 mM CaCl2.Size-exclusion chromatography was done using Superdex 200 16/60 column (Amersham) and the AKTA chromatography system (GE biotech). The gel-filtration buffer contained 50 mM Tris (pH7.5), 150 mM NaCl, 2 mM MgClC1130NKCBP (876–1261) was purified using Calmodulin-Sepharose 4B and concentrated up to 10–15 mg/ml. Crystals were grown by using the vapor-diffusion method, in sitting drops under the following conditions: 10% PEG 3000, 100 mM imidazole (pH 8.0), 200 mM Li2SO4, at +4°C. Before data collection, the crystals were frozen in liquid nitrogen. 15% ethylene glycol was used as a cryo-protectant. Data collection was done at the Advanced Light Source  Beamline 8.3.1 (λ = 1.1 Å) by using a single crystal. Data were integrated and scaled by using HKL2000 software package. The crystal was of the primitive monoclinic space group P21 with cell dimensions a = 45.7 Å, b = 75.1 Å and c = 120.6 Å, and α = 90°, β = 91.45°, γ = 90°. The asymmetric unit contained 2 molecules of KCBP. The solvent fraction of the crystal is 46.9%, and the Matthews coefficient is 2.4 A3/Da. The structure of the ArabidopsisC1130NKCBP (876–1261) was determined by molecular replacement using CNS software Arabidopsis KCBP, chain B comprised residues 877–1077, 1085–1122, 1139–1239, and 1244–1252 of Arabidopsis KCBP. Two Mg2+-ADP, 98 water molecules, 4 molecules of imidazole, and 3 molecules of ethylene glycol were also modeled into visible electron density. The final model was submitted to PDB database (PDB ID 4FRZ).Before crystallization, the Arabidopsis 280 ∼0.25, OD280 ∼0.4, OD280 ∼0.5) were subjected to spin at 5 different speeds ranging between 3,000 rpm and 16,000 rpm. The sedimentation curves were registered at 281 nm, 5.8°C, and processed using UltraScan 3.0.Analytical ultracentrifugation was done according to the standard manufacturer protocol for the method of sedimentation equilibrium using Beckman Optima XL-A. Purified recombinant KCBP (884–1253) and KCBP (884–1244) at three different concentrations  was varied in the mixtures from 5 to 10 µM. KCBP (884–1253) or KCBP (884–1225) were added to the final concentration of 1–10 µM to achieve various molar ratios of KCBP vs tubulin from 1∶10 to 1∶1. When needed, KIC was added to the protein mixtures at the molar ratio of 1∶1 to KCBP and the buffer was then supplemented with 2 mM CaCl2. The samples were prepared at room temperature and 5 µl of the prepared sample was immediately placed between two glass cover slips and sealed to prevent evaporation. The sample was then analyzed using differential interference contrast microscopy.Taxol-stabilized microtubules were prepared as described in KCBP purified as described above was thawed, combined with an excess of porcine microtubules and 1 mM AMPPNP, and centrifuged at 100,000×g for 15 minutes at room temperature. 5 mM ATP and 200 mM NaCl was added to the resulting pellet to release the active fraction of KCBP and the mixture was centrifuged at 100,000×g for 10 minutes. Active KCBP was present in the supernatant. Motility analysis was performed with an ATP regenerating system and oxygen scavengers as described in To clarify the function of the negative coil in the structure of KCBP, we performed crystallographic studies to better characterize this element on a structural level.Solanum tuberosum (potato) Arabidopsis and obtained a different crystal lattice of P21 space group, with 2 KCBP molecules per asymmetric unit. For one of the two molecules, the regulatory domain was visible through its entire length  from e length . The linThe N-termini of two molecules display different degrees of order. One molecule in asymmetric unit has a short coil at the N-terminus while nine additional amino acids of the N-terminus in the second molecule are observed as a short α-helix, a fragment of the predicted helical neck domain. The differences in the structures of both N-and C-termini in two molecules of KCBP surely relate to the different environments in the crystal lattice.Arabidopsis KCBP crystals is that they interact with each other via the regulatory helices , lacking the regulatory helix and the negative coil. Then, we compared the Arabidopsis KCBP (a.a. 884–1253) with an intact regulatory helix and the truncated KCBP using size exclusion chromatography  lacking most of the negatively charged amino acids at the C-terminus but long enough, by 3 amino acids, to support the helical conformation of the preceding residues of the regulatory helix. The molecular weight of this truncated construct was compared with the molecular weight of KCBP (884–1261) with the intact regulatory domain by analytical ultracentrifugation using the method of sedimentation equilibrium. We found that a 1-component model best described sedimentation equilibrium for both constructs . The mol2+ nor by deletion of the first 35 N-terminal residues  , Table 22+, the KCBP-KIC complex, pre-formed and isolated using binding of the His-tagged KIC on Ni-NTA agarose, dissociated on Superdex-200 into the individual homodimers.In contrast to the stable dimers of individual KCBP and KIC, their heterodimer was not stable in solution enough to stay together during the gel-filtration. In the presence of Ca2+-KIC reversed the action of KCBP (884–1253), but not of KCBP (884–1225) which lacks the calmodulin/KIC-binding domain. Our results showed that the truncated construct of KCBP was sufficient to promote formation of the microtubule bundles. Thus, the regulatory domain and its self-associative properties are not required for the microtubule bundling by KCBP.To determine the effects of the dimerization via the regulatory helix on microtubule bundling properties of KCBP, we performed microtubule bundling assays in the presence of either KCBP (884–1253) or truncated KCBP (884–1225). Microtubule bundling was observed using differential interference contrast microscopy (DIC) . Both KCTo determine whether dimerization via the regulatory helix changes motility of KCBP, we performed motility assays using different constructs of KCBP. We used a construct of KCBP (a.a. 876–1261) containing the C-terminal regulatory domain but without the predicted dimerization domain at the N-terminus. We also prepared a construct of KCBP (a.a. 820–1225) with the predicted dimerization motif at the N-terminus but without the C-terminal regulatory helix. KCBP (a.a. 820–1225) eluted on gel-filtration as a dimer and its MW was measured as 72±1 kDa .The motility of KCBP (a.a. 876–1261) without the dimerization domain at the N-terminus could best be described as a back-and-forth movement . This moThe N-terminal dimer of KCBP without the regulatory helix, KCBP (820–1225), moved toward the minus end of the microtubules . WalkingThus, only the N-terminal dimerization domain is required for motility of KCBP. Deletion of the regulatory domain reduced, but did not abolish KCBP motility and did not affect the directionality of movement.Arabidopsis KCBP presented here and potato KCBP presented in our earlier works Arabidopsis KCBP dimers were recognized using both size exclusion chromatography and analytical ultracentrifugation. Deletion of the regulatory calmodulin binding helix and the following negative coil destroyed the dimerization interface resulting in free KCBP monomers. Our crystal structure of Arabidopsis KCBP ruled out a possibility of the negative coil swapping between two neighbor molecules. Thus, the interactions of the negative coil with the microtubule-binding surface of the motor core do not contribute to the dimer interface. Although the negative coil is not a part of the dimerization interface, deletion of just the negative coil was, to our surprise, sufficient to break the KCBP dimers apart.In this study, we have discovered a previously unsuspected self-association of KCBP into a dimer via its C-terminal regulatory domain. We have found that the negative coil of KCBP, a part of its regulatory domain, stabilizes KCBP dimers, which are formed by hydrophobic interactions between the residues of the calmodulin binding helices, another part of its regulatory domain. In our experiments, dimerization of KCBP via regulatory domain was observed both in crystals and in solution. In crystals, dimerization was observed for both Another function of the regulatory domain of KCBP discovered here, namely dimerization, may have an evolutionary origin. As was noted previously, the linker connecting the regulatory helix to the motor core and carrying the name of neck mimic is strikingly similar by sequence and structure to the neck linker of kinesin-1 Dimerization of KCBP via its regulatory domain was completely unexpected because its predicted dimerization domain is located on the opposite end of the polypeptide chain, N-terminal to the motor head. Having two distinct dimerization domains creates a possibility for KCBP to make continuous oligomeric structures. Two molecules of KCBP in the dimer formed via C-terminal helix are oriented such that their microtubule binding surfaces are near 90° relative to each other. This arrangement of KCBP molecules may be important for its physiological functions in orienting and bundling microtubules. In particular, KCBP is abundant in the plant-specific pre-prophase band and phragmoplast, and it functions in the formation and bundling of microtubules in these structures To establish the biological relevance of the regulatory helix self-association we performed microtubule bundling and motility assays. We found that deletion of the regulatory helix did not play a role in microtubule bundling and did not abolish motility of KCBP. The motor domain of KCBP by itself was sufficient to promote the microtubule bundling under the assay conditions of DIC. However, the structures of microtubule bundles formed by the KCBP motor domain by itself and by the KCBP motor plus regulatory domain may differ. Higher-resolution microscopy techniques would be required to resolve those differences. Low velocities demonstrated in motility assays by all tested constructs of KCBP indicate that this kinesin is likely involved in non-transport cellular events such as cytoskeleton organization. KCBP may function chiefly by aligning and bundling microtubules in a certain way.2+ ions, these Ca2+-binding proteins would bind to the regulatory helix of KCBP and break the dimers or higher order oligomeric structures if they do exist. Then, KCBP would be removed from microtubules in a complex with a regulatory protein.Dimerization of KCBP via its regulatory domain brought into consideration another possible role for its negative regulators, KIC and calmodulin. Activated by CaIn summary, we found that the negative coil of the regulatory domain is required for dimerization of KCBP via the regulatory domain. The dimerization interface formed by the regulatory helices is independent from the dimerization interface within the N-terminal domain of KCBP. We speculate that KCBP uses both dimerization interfaces either together or alternating them to support certain cytoskeletal structures.Figure S1Analytical ultracentrifugation sedimentation equilibrium data for KCBP. (A) KCBP (884–1244) and (B) KCBP (884–1253) were analyzed at three concentrations ranging from 5 to 10 µM at centrifugation speeds ranging between 3,000 rpm and 16,000 rpm at 20°C. Representative fits for each sample are shown. The solid red line shows the fit of the data to the ideal 1-component model, and the residuals of the fit are graphed to the right. The graphs were obtained using the program UltraScan3.(JPG)Click here for additional data file.Movie S1(AVI)Click here for additional data file.Movie S2(AVI)Click here for additional data file.


The mycobacterial cell wall presents significant challenges to mycobacteriophages – viruses that infect mycobacterial hosts – because of its unusual structure containing a mycolic acid-rich mycobacterial outer membrane attached to an arabinogalactan layer that is in turn linked to the peptidoglycan. Although little is known about how mycobacteriophages circumvent these barriers during the process of infection, destroying it for lysis at the end of their lytic cycles requires an unusual set of functions. These include Lysin B proteins that cleave the linkage of mycolic acids to the arabinogalactan layer, chaperones required for endolysin delivery to peptidoglycan, holins that regulate lysis timing, and the endolysins (Lysin As) that hydrolyze peptidoglycan. Because mycobacterial peptidoglycan contains atypical features including 3→3 interpeptide linkages, it is not surprising that the mycobacteriophage endolysins also have non-canonical features. We present here a bioinformatic dissection of these lysins and show that they are highly diverse and extensively modular, with an impressive number of domain organizations. Most contain three domains with a novel N-terminal predicted peptidase, a centrally located amidase, muramidase, or transglycosylase, and a C-terminal putative cell wall binding domain. Mycobacterium smegmatis and Mycobacterium tuberculosisM. smegmatis mc2155 CaudoviralesMycobacteriophages are viruses that infect mycobacterial hosts such as Actinomycetales. In response to this, mycobacteriophages are atypical in encoding a lipolytic enzyme, Lysin B, an esterase that hydrolyzes the linkage of the mycolic acids to the peptidoglycan-arabinogalactan complex As with all dsDNA-tailed bacteriophages, mycobacteriophages must ensure lysis of the host cell at the completion of the lytic cycle in order to release progeny phage particles Aside from the unique mycobacterial outer membrane, the cytoplasmic membrane and peptidoglycan contribute to the integrity of the mycobacterial cell just as in both Gram-negative and Gram-positive bacteria Staphylococcus aureusPhage endolysins are modular in their structures Bacillus subtilis and most Gram-negative bacteria including Escherichia coli [reviewed in Escherichia coli, M. tuberculosis virulence A previously developed taxonomic system for classifying peptide bridges places mycobacteria in group A1γ, which also includes 2) of phage Ms6, the full length product of 384 residues, and a 241 residue protein derived from use of an internal translation initiation codon Mycobacteriophages encode endolysins (Lysin As) that are required for lysis at the end of lytic growth Phage-encoded endolysins have considerable potential as potent antimicrobial agents – or enzybiotics – against a number of Gram-positive bacterial pathogens We describe here a bioinformatic analysis of the endolysins encoded by 224 sequenced mycobacteriophage genomes. They are highly modular in nature and most are composed of three domains: a C-terminal domain that is likely to be associated with binding to the cell wall, a central catalytic domain that is commonly a glycoside hydrolase  or amidase, and an N-terminal domain with putative peptidase activity. The prevalence of these peptidase domains, of which several variants have not been previously identified in phage endolysins, likely reflects the complexity of the peptidoglycan substrate with its high degree of crosslinking and its unusual interpeptide linkages. We also demonstrate hydrolysis and cell lysis by some of these endolysins including ones that lack known glycoside catalytic domains.−5 or 20% similarity respectively were used to define shared regions. A summary of the domains present in each of the endolysins is shown in Phage endolysins typically are composed of two domains: an N-terminal domain with catalytic activity directed at hydrolysis of the peptidoglycan cell well, and a C-terminal cell wall binding domain The most striking feature of the mycobacteriophage endolysins is their amazing diversity, encompassing enormous variation and differing in size by more than two-fold , 3. TherA second notable feature is the number and variety of the combinations in which these domains are assembled in the individual endolysins. With six possible types of N-terminal peptidase domain, five types of amidase/glycosidase domains, and four putative C-terminally positioned cell wall binding motifs, there are a least 120 possible combinations (assuming one of each of the three units), of which 25 are observed , 3. ThesMycobacterium, Corynebacterium, and closely-related species, all GH19-containing endolysins belong to phages infecting Gram-negative hosts, primarily Pseudomonas.Enzymatic activities that cleave the 1,4-β linkages between the N-acetylmuramic acid and N-acetylglucosamine sugars of the peptidoglycan backbone . Two of M. tuberculosis resuscitation promoting factor RpfA. Given the relative rarity of TG activity found thus far in mycobacteriophage and other phage endolysins, the resulting TG-containing endolysins are interesting variants, as most transglycosylase endolysins are found in phages infecting Gram-negative hosts, including the λ R endolysin Pseudomonas phage φKZ144 lysin The third enzymatic activity is a transglycosylase (TG) domain pfam06737), also a member of the lysozyme_like superfamily . There a7, also aAmidases that cleave N-acetylmuramoyl-L-alanine linkages connecting the sugar backbone to the peptide crosslink see , 3. HoweM. tuberculosisThere are a number of potential peptidase targets within peptidoglycan, both within the pentapeptide chain and at the interpeptide crosslinks  and the resuscitation-promoting factor interacting protein of M. tuberculosis, RipA (Rv1477). The crystal structures of RipA and the related RipB protein show a peptidase active site that likely cleaves the 4→3 linkage between the D-Glu and m-DAP residues Staphylococcus aureus – and these are generally defined as proteins of the large papain family , the putative endolysin catalytic domain is flanked on its C-terminal side by a second conserved domain, a position where other phage endolysins typically have a cell wall binding domain As described above, there are three types of segments in most of the mycobacteriophage endolysins, a central catalytic domain that targets either the sugar backbone or its linkage to peptide chain, a C-terminal domain that is presumed to be involved in cell wall binding, and an N-terminal domain with predicted peptidase activity. These are assembled into 25 distinct organizations (Org-A to Org-Y) , 3. HoweThe relationships reflecting domain distributions in the 25 different organizations and the genomes and clusters they represent can be presented using the NeighborNet function in Splitstree 32 sequence against a database of mycobacterial genomes identifies three full-length homologues  that share the same organization (Org-O), as well other genes that have partial matches   and the  matches . Specifi matches , 7, refl matches . Boomer The observation that some organizations lack the central catalytic domain  is consistent with the interpretation that the N-terminal domains are important for peptidoglycan hydrolysis. These are all relatively small proteins (<325 aa) and include the smallest of the mycobacteriophage endolysins, L5 gp10 and Saintus gp5, which are 292 and 225 residues respectively. Although the N-terminal peptidases are abundantly represented, there are three organizations  that lack an N-terminal peptidase domain, suggesting that it is not absolutely required. However, two of these (Org-R and Org-S) are notable in that they contain two central catalytic domains, with a GH19 in an N-terminal position to an Ami-2A or Ami-2B. There are also two examples of four-domain proteins. Timshel gp9 (Org-Y) appears to contain two N-terminal domains, N1 and N2, in addition to GH19 and C1 domains. Packman and Alma gp11 (Org-D) contain only one N-terminal domain (N4) but have two possible “C-terminal” domains, with C3 and C1 motifs flanking its central GH19 domain .et allysA gene (2) – whose primary product gp2 shares 99% amino acid identity with Fruitloop gp29 – encodes a second gene product (LysA271) resulting from translation initiation at codon 144. They also predicted that Lysin As with similar organizations can be expressed similarly, as with Boomer gp32, Che8 gp32 and Ramsey gp32. Although this is a reasonable extension from the Ms6 gp2 observations, we note that the putative translation initiation signals are predicted to be weak, raising the question as to whether these play a similar in vivo role. We note, however, as reported previously E. coli and the preparations analyzed by zymography with lyophilized Micrococcus luteus all show products smaller than the full length protein that can hydrolyze peptidoglycan. Internal gene starts may therefore indeed be a common phenomenon with lysA genes.Catalão E. coli with the primary product a protein of 439 amino acids and we confirmed that the start side we predicted bioinformatically lysA. While we cannot eliminate the possibility that the smaller product is generated by proteolysis, its apparent high specific activity of the truncated Ami2B-C2 form of Corndog gp69 suggests that the N3 N-terminal domain was inhibiting catalysis in this zymogram assay. Although observations of activity using zymograms are only poorly quantitative, we note a similar effect is observed with the Ms6 Lysin A protein To extend this we analyzed the Corndog gp69 expression further . The pro31 deletion mutant (unpublished observations). However, in the course of these experiments we noted that expression of the D29 endolysin (gp10) appeared to lead to cell death, even though the putative holin gene (11) was excluded from the expression clone. We therefore tested to what extent this occurred when other mycobacteriophage endolysins are expressed in M. smegmatis.Expression of phage endolysins alone in bacteria does not typically result in cellular lysis because the enzyme fails to reach its catalytic target in the cell wall, and activation of catalysis is triggered by the holin that provides a timely passage of the enzyme through the cell membrane lysA gene, we searched for the presence of potential transmembrane domains within all six possible translation frames of L5 gene 10; none were found and a second holin gene seems an unlikely possibility. We also note that none of the Lysin A proteins contain N-terminal SAR domains, including L5 gp10.Of the 12 different endolysins  that we tested , we obseWe have described here a detailed dissection of the endolysins encoded by mycobacteriophages. The complexity of these endolysins is truly amazing, with each being composed of exchangeable domains assembled into a large variety of organizations. Although a total of 15 different domains have been identified, these fall into three main types, and the majority of the endolysins have one of 4–6 domains from each of the three menus, in a common order – an N-terminal peptidase, a central non-peptidase catalytic domain, and a C-terminal motif presumed to be involved in cell wall binding. A subset of the endolysins departs from this and either lack one of the three units, or has multiple copies of them.M. smegmatis mc2155, but there are several lines of evidence suggesting that they may also infect other hosts, and that the broad diversity of the phages reflects a range of overlapping host preferences M. tuberculosis are restricted to the Cluster K and Subcluster A2 phages Actinomycetales.The prevalence and variety of the peptidase motifs in the mycobacteriophage endolysins is unusual and likely reflects the complexity of the cell wall of their mycobacterial hosts. A complication in the interpretation of this is that the range of potential hosts for the sequenced mycobacteriophages is not known. They all share a common host, Although nearly all of the mycobacteriophages encode both an endolysin and a Lysin B, there are a few examples in which the Lysin B gene is absent. These include Che12 and Packman , Arbiter, Ares, Hedgerow, Qyrzula, and Rosebush , Myrna , and Charlie and Redi . However, there is little or no correlation between the types of endolysins encoded by these , and thuThe intragenic mosaicism of these endolysins is especially striking and represents a microcosm of the relationships among the phage genomes as a whole The apparent holin-independent mycobacterial lysis by the L5 endolysin is noteworthy in that the activity demonstrates that enzyme can potently act on the cell wall even though it is predicted to contain only an N-terminal peptidase domain and a presumed C-terminal cell wall binding domain. Although lysis occurs efficiently in the absence of the holin gene, it is plausible that a high level of protein expression and interaction with the cell membrane could cause sufficient destabilization to enable access of the enzyme to its peptidoglycan substrate. It seems unlikely that lysis during an L5 infection would be entirely holin independent, because the genome encodes a good holin candidate (gp11) with two predicted membrane-spanning domains, although a holin-defective mutant of L5 is required to resolve this question.The mycobacteriophage endolysins have potential utility as antimycobacterial agents just as enzybiotics have been successfully developed for other gram-positive bacteria −5 and a ClustalW identity >20%. Domains were attributed potential functions based on hits to conserved domain families from the Pfam http://toolkit.tuebingen.mpg.de/hhpredLysin A protein sequences were subjected to BLASTp and PSI-BLAST to identify homologous regions of sequence from non-redundant sequences at NCBI (blast.ncbi.nlm.nih.gov). Multiple sequence alignments were performed using the ClustalW algorithm executed using the CLC Bio Main Workbench (v. 4.1.2) software package, which was also used for general sequence analysis and alignments. Distinct regions of homology were identified and grouped into domains with a threshold of e-value <1×10M. smegmatis mc2155 strains were cultured in Middlebrook 7H9 medium or grown on Middlebrook 7H10 agar supplemented with 10% Albumin Dextrose Complex (ADC), 0.2% succinate, 0.05% Tween-80, 1 mM Ca2Cl, carbenicillin (50 mg/ml), cyclohexamide (10 mg/ml) and kanamycin (20 mg/ml) as required. E. coli was grown in L-broth (LB) supplemented with carbenicillin (50 mg/ml) and kanamycin (20 mg/ml) as needed, with E. coli GC5 cells (Stratagene) used for cloning and E. coli BL21(DE3) cells (Stratagene) for protein overexpression.All E. coli and M. smegmatis was performed as described previously lysAs were cloned with primers bearing NdeI and HindIII restriction sites into pET21a (Novagen) for IPTG-induced expression of C-terminally His-tagged protein in E. coli BL21(DE3) cells, then purified by affinity chromatography using TALON Co2+ resin; after two washes with 10 and 20 mM imidazole, protein was eluted with 120 mM imidazole. Eluted fractions were concentrated using Vivaspin concentration columns  followed by dialysis against storage buffer  and stored at −20°C.Cloning and purification of Lysin As for expression in Micrococcus luteus cells as a source of peptidoglycan into the gel matrix. Zymograms were developed by renaturation overnight at 37°C in 25 mM Tris (pH 7.5), 1% Triton X-100 and 0.1 mM ZnSO4, then stained with 0.5% methylene blue with 0.01% KOH before destaining with water.Zymograms were performed as described previously M. smegmatis mc2155, lysAs were cloned into pLAM12 600 nm of 0.4–0.6, M. smegmatis with a pLAM12-based lysA construct was induced with 0.2% acetamide and incubated with shaking at 37°C. Protein expression was assessed by taking a 1 ml sample prior to induction and at the end of the 7 h period, separating the cells into a pellet and supernatant fractions by centrifugation, lysing the pellet by sonication, and loading amounts equalized with respect to the culture OD600 at the time of sampling onto an SDS PAGE gel. The procedure for the ATP release assay is similar to that described in Payne et al. (2009). Briefly, after reaching log-phase M. smegmatis was induced and diluted to an OD600 of 0.03 so that readings were within the measuring range of the luminometer. Every hour 100 µl samples were taken, combined with 100 µl of ENLITEN rLuciferase/Luciferin reagent (Promega), and luminescence recorded for a 10 second interval in a Monolight 2010 luminometer. The results were reported as the fold-difference in ATP-release compared to a control culture containing pLAM12. Growth inhibition and lysis was also tracked visually by following the OD600 of induced cultures incubating with shaking at 37°C for up to 36 hours as compared to uninduced controls.For expression in Figure S1Phylogenetic tree of Lysin A amidase domains. A neighbor-joining tree based on a ClustalW alignment of amidase domains found in Lysin As. Numbers indicate distance between nodes.(PDF)Click here for additional data file.Figure S2Cloned Lysin As for expression in M. smegmatis. The above 12 Lysin As were cloned into the acetamide-inducible pLAM12 vector. They were chosen to represent the diversity of domains, with each domain represented at least once. Organizations are listed to the right of the Lysin A name in parentheses.(PDF)Click here for additional data file.Figure S3Expression profiles of lysins induced in M. smegmatis. The above samples are from the cultures used in the ATP assay described in A.3.2. One milliliter samples were taken from induced and uninduced cultures with pLAM12 vector control, one of 12 Lysin As, or one Lysin B (D29 gp10). These were sonicated, centrifuged at 14,000 rpm to produce pellet and supernatant fractions, and separated on SDS PAGE gels. Red asterisks mark identifiable expressed protein. Red question marks indicate instances of uncertain protein expression.(PDF)Click here for additional data file.Table S1Coordinates of mycobacteriophage endolysin domains.(PDF)Click here for additional data file.


The quench process in high-temperature superconducting (HTS) wires plays an important role in superconducting power devices, such as fault current limiters, magnets, cables, etc. The superconducting device should survive after the overheating due to quench.We studied the evolution of the resistance of the YBCO tape wire during the quench process with 1 ms time resolution for various excitation voltages.The resistive normal zone was found to be located in a domain of about 1-4 cm long. The normal state nucleation begins in 40-60 ms after voltage is applied across the HTS tape. In subsequent 200-300 ms other normal state regions appear. The normal domain heating continues in the following 5-10s that results in a factor of 2–3 increase of its resistance.Formation of the normal domain during the quench process follows the same stages for different excitation voltages. Characteristic domain sizes, lifetimes and temperatures are determined for all stages. The quench process in high-temperature superconducting (HTS) wires plays an important role in superconducting fault current limiter operation. It occurs when current in a wire exceeds the critical value and as a result, the wire resistance becomes nonzero. The problem of quench stability is related to the heat transfer and is especially crucial for the Second Generation HTS wires on highly resistive substrates. We present here the results of studies of the normal zone generation.c = 91 K) and 1.5mu Ag layers. Measurements were performed with the tape immersed in liquid nitrogen. The AC (50 Hz) voltage step with the amplitude V0 was applied to the sample at the time t0. After that, during the subsequent 40s, we registered the current I and sample AC resistance Z with 1 ms time resolution.We studied the process of quench in HTS tapes using the experimental procedure described in  is expressed the following way  of the zone where the YBCO layer is in the normal state (T(K) > Tc) is given by the following expression:Equation (1) is experimentally proven to be valid in the range 0.5 V < Vc for t > t1, V0 (mV) is the applied voltage magnitude.here L (mm) is the length of the zone where T > T1 is the domain resistance at liquid nitrogen temperature and Z3 is the resistance at the maximum temperature, one can obtain the domain size (L1) and the size of zone with TM (L3) as function of V0:Using (1) and (2) and assuming that Z1 and L3 values versus V0 calculated from (3) and (4).Figure M to 90 K.From the above results we conclude that during the superconducting-to-normal state transition in HTS tape the normal phase is limited to a single domain. The domain nucleates in 40-60 ms after the voltage is applied. In the subsequent 5-10s the domain heats up; it results in 2–3 times increase of the resistance. Central part of the domain is about 20-30 mm long. Inside the both of the 3-5 mm long edges of the domain the temperature falls from the maximal temperature T


Previously, the iPfam domain–domain interaction data was integrated within the Pfam database and website, but it has now been migrated to a separate database. This allows for independent development, improving data access and giving clearer separation between the protein family and interactions datasets. In addition to domain–domain interactions, iPfam has been expanded to include interaction data for domain bound small molecule ligands. Functional annotations are provided from source databases, supplemented by the incorporation of Wikipedia articles where available. iPfam (version 1.0) contains >9500 domain–domain and 15 500 domain–ligand interactions. The new website provides access to this data in a variety of ways, including interactive visualizations of the interaction data.The database iPfam, available at  Systems biology is the examination of the organization and dynamics of cellular and organismal function ,2. ModerHigh-throughput experiments have provided large datasets of protein–protein interaction in a variety of organisms [e.g. ]. HoweveAs well as protein–protein interactions, it is important to capture other types of interactions at a similar level of detail. The Protein Data Bank (PDB)  is the cTo aid the detailed understanding of protein interactions, we have combined information from the Pfam database (which classifies protein sequence space into conserved evolutionary units)  and the iPfam has been expanded to include both DDI and domain–ligand interactions (DLI), whereas most similar, domain centric, resources exclusively provide either DLI  or DDI dIn the following section, we briefly describe the process of identifying domain interactions (both DDI and DLI). To identify Pfam protein family interactions in 3D structures, we have to take the Pfam data that is principally defined on UniProt sequences and transfer those annotations to the PDB atom coordinates, while taking account of the data in both resources being in flux.The first step of the new, independent, iPfam update cycle is to synchronize the underlying databases, PDB and Pfam. iPfam version 1.0 is built using PDB entries as of 26 August 2013 and Pfam version 27.0, released in March 2013. Due to the asynchronous data release of the source databases, Pfam entries are mapped to PDB entries using a two-stage process.The first stage depends on the high-quality Pfam–PDB mapping data provided by Pfam, which are constructed from a residue-to-residue mapping between PDB and UniProt called SIFTS . This apE-value match. Overall, this two-stage mapping approach resulted in 272 900 structural domains (from 6634 distinct Pfam entries) being identified in 87 386 different PDB entries.The second stage addresses the fact that many new PDB entries will have been added since the Pfam mapping was generated and will, therefore, not be annotated with Pfam domains using the first stage. To make iPfam more comprehensive, we supplement the Pfam sourced Pfam–PDB mappings with data from RCSB PDB , which uo and 140o. A van der Waals bond is determined if the distance between the two atoms is greater than the sum of the atom’s van der Waal radii (R) and less than R+ 1.5 Å. The list of atom properties  used in the calculations is available on the iPfam FTP site (atomData.csv).Having performed the Pfam–PDB mapping, each PDB entry containing a Pfam protein family is analyzed for interactions. This is achieved by taking each domain and testing to see if any atoms in the domain are in close enough proximity to form a bond with any atom from another domain and/or a small molecule ligand found within the structure. We define the following bond types—covalent, electrostatic , amino acid side chain and main chain (backbone) hydrogen bonds and van der Waals. All bonds are defined based on both the geometric (distance and angle) and chemical properties  of the atoms involved, . This iWe acknowledge that the lack of an interface threshold could be a source of some false-positive interactions, and we will incorporate interface area metrics in the future to provide users with as much discriminatory information as possible about the interface.We also do not try to discriminate between different small molecules in the DLI calculations. The presence of a co-crystallized ligand may be functionally important or simply an artifact due to its presence in the crystallization buffer. The aim of the iPfam database is to harvest as much of the high-resolution interaction information as possible from the few known structures, and to present this data in an accessible way that allows the user to make a judgment regarding the biological relevance of an interaction.The iPfam database is constructed from two continuously updating sources, PDB and Pfam, both of which are well established in their respective fields of 3D structure data and protein domain data. The simple, rich user interface in iPfam, combined with the popularity of the source databases, makes interaction data available to a wide audience that includes both bench biologists, looking at their favorite sequence, and computational biologists, requiring comprehensive interaction data for their own data mining purposes. Bringing both DDI and DLI interaction data into the context of protein families, via the unified framework in iPfam, will help researchers to better understand the relationship between ligand and domain binding at a molecular level. There is an increasing body of evidence showing that DDI and DLI interfaces often overlap, allowing the knowledge of one class of interaction to be used to derive hypotheses about both classes of interaction . Combini


The “Function to Find Domain” (FIIND)-containing proteins CARD8  and NLRP1  are well known components of inflammasomes, multiprotein complexes responsible for activation of caspase-1, a regulator of inflammation and innate immunity. Although identified many years ago, the role of the FIIND is unknown. Here, we report that CARD8 and NLRP1 undergo autoproteolytic cleavage at a conserved SF/S motif within the FIIND. Using bioinformatics and computational modeling approaches, we detected striking structural similarity between the FIIND and the ZU5-UPA domain present in the autoproteolytic protein PIDD. This allowed us to generate a three-dimensional model and to gain insights in the molecular mechanism of the cleavage. Site-directed mutagenesis experiments revealed that the second serine of the SF/S motif is required for CARD8 and NLRP1 autoproteolysis. Furthermore, we discovered an important function for conserved glutamic acid and histidine residues, located in proximity of the cleavage site in regulating the autoprocessing efficiency. Altogether, these results identify a function for the FIIND and show that CARD8 and NLRP1 are ZU5-UPA domain-containing autoproteolytic proteins, thus suggesting a novel mechanism for regulating innate immune responses. The NLR (nucleotide-binding domain and leucine-rich repeat containing) gene family is a relatively new class of innate immunity proteins that operate as intracellular surveillance molecules for sensing and responding to exogenous pathogens and endogenous “danger” signals derived from tissue injuryNLRP1 and CARD8 gene of humans have been correlated with autoimmune and chronic inflammatory diseasesNLRP1 protein differs from all the other NLRP proteins in the C-terminal region, which contains also a FIIND domain followed by a CARD domain, both located after the LRR. The acronym FIIND (domain with function to find) was coined several years ago to indicate a highly conserved protein region with unknown functionIn this report, we demonstrate that the FIIND is a previously unrecognized type of ZU5-UPA domain, which undergoes post-translational autocleavage. The findings extend intra-molecular autoproteolysis mediated by the ZU5-UPA motif to the NLR family of innate immunity proteins.During attempts to identify protein interaction partners of the FIINDs of CARD8 and NLRP1, we noticed the appearance of unexpected protein products when expressing constructs in HEK293T cells encoding the FIIND-CARD regions of CARD8 and NLRP1 . SpecifiConsidering that the smaller forms of these proteins could represent an artifact, we next checked the status of the endogenous CARD8 protein by performing immunoblot analysis of cell lysates. Thus, using an antiserum raised against the last 50 amino acids of CARD8, we tested several tumor cell lines of epithelial and hematopoietic cell lineages. In all cases, a prominent ∼28 kDa form of the CARD8 was present, typically  in addition to the expected full-length ∼60 kDa CARD8 protein, as well as other bands detected in some cell lines .To verify the identity of the smaller form of CARD8, shRNA-mediated gene silencing was used. For these experiments, human HCT116 cells were infected with two different shRNAs targeting CARD8 mRNAs or with a scrambled control, then lysates were prepared and CARD8 protein levels were analyzed by immunoblotting. As shown in We utilized mass spectrometry to analyze the smaller form of CARD8. To this end, we generated stable HEK293T cells expressing either CARD8 or GFP (negative control) with a N-terminal streptavidin binding peptide (SBP) tag. These proteins were purified by a streptavidin pull down followed by biotin elution, and analyzed by SDS-PAGE with Coomassie Blue staining . In the The two smaller bands were subjected to tryptic digestion and analyzed by mass spectrometry. As expected, both bands corresponded to fragments of CARD8. Two of the recovered peptides–VEPFYAVLESPSF and SLMGILLR–were of interest, as they could not have been generated by trypsin cleavage , thus idZO-1 and UNC5), followed by a UPA domain  and by a Death Domain (DD) foldSince the cleavage site was identified in the middle of FIIND, we searched for structural templates by applying bioinformatics tools. Using the entire CARD8 protein sequence for finding distantly related protein structures, HHpred software gave a significant hit with UNC5bBased on the reported alignment, a three-dimensional model of the protein was constructed with the Modeller tool and refined with the Relax program of the Rosetta suite, which improved the packing of side-chains and arrangement of secondary structure motifs. The resulting model was used to study both the cleavage site and the interface between the ZU5 and CARD domains. The region of CARD8 containing the FIIND and CARD can be divided into three domains. Following the nomenclature of the UNC5b template, these three domains consist of (1) the all-beta strand ZU5-like domain (residues Leu-162 to Thr-309), (2) the mainly beta strand UPA-like domain (residues Arg-310 to Pro-434), followed by a linker region of about 20 amino acids, and finally (3) the all-alpha helix CARD domain (residues Lys-455 to the end). In our model, the FIIND encompasses both the ZU5-like and UPA-like domains. The N-terminal ZU5-like domain is located between the UPA-like and the CARD domains. Similar results were obtained with the FIIND-CARD region of NLRP1 (refseq accession code NP_127497.1), with the exception that the UPA-like domain was aligned with less confidence by the HHpred algorithm. However, the location of the cleavage site within the ZU5-like domain can be predicted with high confidence.2, but is predicted to be less hydrophobic in CARD8. In this regard, the FIIND/CARD interface in CARD8 shows stronger electrostatic potential, including four possible salt bridges between the two domains: (1) Glu-247↔Lys-508, (2) Arg-202↔Glu-500, (3) Lys-272↔Glu-479, and (4) Glu-285↔Lys-509, where only the last of these is conserved in UNC5b (involving residues Glu-666↔Arg-913). One example of hydrophobicity change is given by amino acid Val-619 in UNC5b, which in our CARD8 model is replaced by Glu-247, an amino acid predicted to participate in a salt bridge in CARD8. Finally, we observed a considerable change in the cysteine composition between the CARD8 model and the UNC5b template. The UNC5b protein contains a total of 13 cysteine residues and one disulphide bond formed by Cys-663↔Cys-670. In contrast, the FIIND and CARD domains completely lack cysteine residues. The disulphide bond-forming cysteines have been replaced by two alanine residues and create a small hydrophobic patch. Cysteine residues are only found at the N-terminus of the CARD8 protein prior to the FIIND in a region, which, according to the Xtalpred serverThe distinct interface between the ZU5 and the DD in the UNC5b protein structure appears to be partially retained in our model of the CARD8 protein. The cleavage we observed experimentally occurs between residue Phe-296 and Ser-297 in the completely conserved SFS motif of the FIIND. These residues are located at the C-terminus of the ZU5-like domain just proximal to a long beta strand that has been shown to be crucial for fold stability of UNC5bIn contrast to enzymatic proteolysis, which is defined as an inter-molecular reaction involving an enzyme, autoproteolysis is characterized by a peptide bond disruption caused by an intra-molecular reactionin vitro translated wild-type CARD8 and S297A CARD8 (The similarities of the CARD8 (SF/S) and PIDD (HF/S) cleavage sequences, together with our three-dimensional model of the FIIND/ZU5-like domain, prompted us to investigate whether CARD8 is subject to an autoproteolytic mechanism analogous to PIDD. Additionally, we used mutational analysis to clarify whether the analogous Ser-1213 in the SF/S sequence of NLRP1 is required for its autocleavage. Substitution of Ser-1213 of NLRP1 or Ser-297 of CARD8 with alanine completely abrogated NLRP1 and CARD8 cleavage in HEK293T cells . Thus, t7A CARD8 , furtherThe importance of the conserved SFS motif in CARD8 was further investigated by mutagenesis of the Phe-296 residue. In both Nup98 and PIDD, the corresponding phenylalanine was described to play an important structural role and its substitution with histidine dramatically abolished autocleavageHaving verified the importance of the Phe-296 and Ser-297 residues in the SF/S motif of CARD8, we turned to the role of Ser-295, which substitutes for the functionally important histidine residue present in the HFS sequence of PIDD and Nup98. Both mutagenesis experiments and the three-dimensional structure showed that Nup98 mutations H862Q or H862A only slightly affected protein autocleavageTo find functionally relevant amino acids for the deprotonation and activation of the nucleophilicity of Ser-297, we searched the ZU5-like domain for amino acids with high conservation across different species . Only thFinally, we explored other conserved amino acids that could contribute to Ser-297 nucleophilic activation. Since in various Ntn hydrolases, such as glycosylasparaginase (GA) and Taspase1, an aspartic acid was demonstrated to be essential for autocleavageIn summary, our results show that CARD8 and NLRP1 join the repertoire of proteins capable of intra-molecular autoprocessing. We show that Ser-297 in CARD8 and the equivalent Ser-1213 in NLRP1 are responsible for the nucleophilic attack at the scissile peptide bond and therefore essential for protein autoprocessing. Our data also strongly suggest that His-270 in CARD8 is a likely candidate for Ser-297 activation. Furthermore, Ser-295 and Glu-279, together with His-252 and Phe-296, are also important for CARD8 autocleavage, presumably by providing the required structure-related conformational strain necessary for hydrolysis of the peptide bond between Phe-296 and Ser-297 . Elucida® cloning technology. For all other expression studies, FIIND and CARD domains of CARD8 (residues 131-537) and NLRP1  were cloned into a pcDNA3 vector modified to contain a N-terminal flag-tag, followed by a Tobacco Etch Virus (TEV) protease cleavage sequence, with a myc-tag and a flexible linker region of poly-Glycine. All constructs were verified by DNA sequencing. CARD8 antibody was purchased from Abcam (ab24186).Biotin (B4501) and shCARD8 lentiviral vectors (TRCN118329 and TRCN118331) were purchased from Sigma. For protein purification and LC/MS analysis, the FIIND-CARD region of CARD8  was cloned into a N-terminal streptavidin binding peptide (SBP) lentiviral vector using a GatewayHEK293T, HCT116, CaCo2, HT29, Colo205, WM-35, THP-1, U937 and Jurkat cell lines were purchased from the American Type Culture Collection (ATCC). PLB-985 cell line was kindly provided by Dr. Mary Dinauer .HEK293T, HCT116, CaCo2, HT29, Colo205 and WM-35 cells were cultured in DMEM media supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin. PLB-985, THP-1, U937 and Jurkat cells were cultured in RPMI 1640 media supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin. Media and supplements were purchased from Invitrogen.Pan troglodytes, Mus musculus, Canis familiaris, and Ailuropoda melanoleuca. For each organism, a single gene product was chosen from this hit list, giving priority to isoforms “1” or “a”, where available. Using the resulting 23 protein sequences, a multiple sequence alignment (MSA) was constructed with the Mafft v6.821bThe protein sequence of human CARD8  was selected as a query for a NCBI Blast online search. In this isoform of CARD8, the FIIND is comprised of residues from Leu-162 to Ala-444, followed by the CARD domain, which terminates the protein sequence at residue Leu-537. The resulting list of homologs included sequences for CARD8-like proteins and NLRP1 proteins from several species, including E-value 0.028) but the PsiPred secondary structure predictionThe complete CARD8 protein sequence was submitted to the HHpred serverBased on the CARD8/UNC5b alignment calculated by HHpred, we derived a three-dimensional model with Modeller v9.9Approximately 20 million of stably-transfected HEK293T cells were suspended in 2 ml of lysis buffer (10% (v/v) glycerol, 50 mM HEPES-NaOH pH 8.0, 150 mM NaCl, 2 mM EDTA, 0.1% (v/v) Igepal CA-650, 2 mM DTT, complete mini protease inhibitors cocktail, 10 mM NaF, and phosphatase inhibitors cocktail) and incubated overnight with magnetic streptavidin beads (Invitrogen). Biotin was used for elution at a final concentration of 10 mM.6 cells per well and transiently transfected with 2 µg of various expression plasmids. After 24 hours, cells were collected and lysed in 10% (v/v) glycerol, 50 mM HEPES-NaOH, pH 8.0, 150 mM NaCl, 2 mM EDTA, 0.1% (v/v) Igepal CA-650, 2 mM DTT, complete mini protease inhibitors cocktail, 10 mM NaF, and phosphatase inhibitors cocktail. Cleared lysates were normalized for total protein content and aliquots were subjected to SDS-PAGE/immunoblot analysis. Epitope-tagged proteins were revealed using anti-flag antibody (Sigma) and using Licor Odyssey system (LiCor).HEK293T cells were seeded into 6-well plates at a density of 1×10The shRNA lentivirus plasmids were packaged by transfection into 293T cells, as described in Krieg et al® II XL kit from Agilent Technologies (Cat# 200521). All primers were obtained using the QuickChange® primer design program. All plasmid constructions were verified by DNA sequencing. In vitro translation of wild-type (wt) and S297A CARD8 constructs were obtained using a TNT® T7 quick coupled transcription and translation system from Promega (Cat# L1170).All mutants were generated by site-directed mutagenesis using QuikChangeFigure S1MS2 spectra of band 1 and 2. MS2 spectra for peptide VEPFYAVLESPSF from Gel Band 1 with MH+ 1484.7313, m/z 742.8693 and MS2 spectra for peptide SLMGILLR from Gel Band 2 with MH+ 918.5447, m/z 459.7760. b ions (blue) and y ions (red) are corresponding to tandem CID fragmentation of peptides matching the theoretical fragmentation pattern. Yellow M shows the oxidation of methionine.(TIF)Click here for additional data file.Figure S2Complete multiple sequence alignment of FIIND domains. High residue conservation is found across the whole domain. Sequences are sorted by percentage identity to the first listed protein, Human_card8. The most remote homolog is given by the death effector domain of dog, Dog_ded, which shares 47% identical residues with Human_card8. In a certain sense the opossum FIIND domain presents an outlier since it is equipped with an insertion of 41 residues at the C-terminus. As this fragment is absent in Human_card8, secondary structure prediction is not shown in the ‘SSPred’ row.(TIF)Click here for additional data file.Figure S3Complete model of the FIIND and CARD domains of human CARD8 protein. The model is based on the structure of UNC5b, deposited in PDB with accession code 3g5b. The FIIND domain consists of two domains, at the N-terminus a ZU5-like domain (green) followed by a UPA domain (blue). The CARD8 protein is terminated by a CARD domain (magenta). The center of the figure displays in ball-and-stick mode the SFS motif given by residues Ser-295, Phe-296, and Ser-297. Furthermore, residues Glu-240, Glu-242, His-252, His-270, Glu-279, His-280, and His-333 were selected for mutagenesis studies, they are colored orange in the MSA ( the MSA  and are (TIF)Click here for additional data file.Figure S4Role of His-252 in stabilizing the protein structure. Residue His-252 is located at the end of a beta-strand, which is part of a five stranded beta sheet. The side-chain hydrogen bonds with the carbonyl oxygen of Glu-285, located in the last strand of the sheet. This hydrogen bond orients the side-chain of Glu-285 such that it forms a salt bridge to Lys-509 in the CARD domain in exactly the same way as in the template structure UNC5b. A second hydrogen bond is formed with the backbone nitrogen of a neighboring residue, Ile-254. As histidine is encountered in different protonation states, hydrogen atoms have been added as green spheres in this figure. Hydrogen bonds are emphasized by dashed lines, which connect the heavy atom acceptor with the hydrogen atom. Distances are given in units of Ångström. This figure was prepared with PyMOL.(TIF)Click here for additional data file.Figure S5Model of CARD8 autoprocessing. Upon protein folding, the CARD and the ZU5-like domains of CARD8 interact with each other. This causes the conformational strain necessary for protein autocleavage. The two cleaved fragments remain associated after cleavage.(TIF)Click here for additional data file.Methods S1Supporting Methods.(DOC)Click here for additional data file.


The repertoire of the antigen-binding receptors originates from the rearrangement of immunoglobulin and T-cell receptor genetic loci in a process known as V(D)J recombination. The initial site-specific DNA cleavage steps of this process are catalyzed by the lymphoid specific proteins RAG1 and RAG2. The majority of studies on RAG1 and RAG2 have focused on the minimal, core regions required for catalytic activity. Though not absolutely required, non-core regions of RAG1 and RAG2 have been shown to influence the efficiency and fidelity of the recombination reaction.Using a partial proteolysis approach in combination with bioinformatics analyses, we identified the domain boundaries of a structural domain that is present in the 380-residue N-terminal non-core region of RAG1. We term this domain the Central Non-core Domain .We show how the CND alone, and in combination with other regions of non-core RAG1, functions in nuclear localization, zinc coordination, and interactions with nucleic acid. Together, these results demonstrate the multiple roles that the non-core region can play in the function of the full length protein. Development of the adaptive immune system relies on the coordinated assembly of the genes encoding immunoglobulin and T cell receptor subunits in a process known as V(D)J recombination . In thisV(D)J recombination occurs in two distinct phases, the first of which relies largely on the RAG proteins. During the first phase of recombination, the RAG proteins assemble on the RSSs of the two gene segments to be recombined, forming a pre-cleavage synaptic (or paired) complex. The proteins first generate a single-strand nick 5' of the heptamer sequence of the RSS, producing a free 3' hydroxyl group on the coding gene segment. This hydroxyl group subsequently attacks the opposing strand in a direct transesterification reaction, generating a double-strand break at the coding gene:RSS border . Under pEarly studies of the RAG proteins identified the minimal regions of the proteins required for catalysis -17. ThesThe non-core regions of both RAG proteins, though considered "dispensable" for recombination, are well-conserved, suggesting that these regions have a functional role. Notably, a number of studies have shown that core RAG1 and core RAG2 were significantly less efficient in the recombination of both exogenous plasmid substrates and endogenous genetic loci when compared to their full-length counterparts ,17,31-36Multiple previous studies have shown that non-core RAG2 contains a PHD module , which bThe role of the N-terminal non-core region of RAG1 has been more elusive. There are three separate lines of study that indicate important roles of the non-core region in the function of the RAG1 protein. First, previous mutagenesis studies revealed regions in non-core RAG1 that significantly impacted recombination of exogenous plasmid substrates ,44, inclTo further our understanding of RAG1, we have utilized biochemical, bioinformatic, and biophysical approaches to identify the domain composition of the N-terminal non-core region of the protein. In this context, a domain is defined as an independently folded unit that can retain biological activity even if excised from the full length protein. Since domains can participate in key functions of the intact protein, such as providing binding surfaces for macromolecular interactions, it will be critical to determine the domain architecture of non-core RAG1 to obtain a detailed understanding of its function. Here, we have identified domain boundaries that yielded isolation of a structurally independent domain, termed the central non-core domain (CND), which consists of residues 87-217. We show that this domain interacts strongly with zinc ions, preferentially binds double-stranded DNA, and in conjunction with a neighboring non-core region contributes to nuclear localization of the full length RAG1 protein.E. coli. Expression of the non-core region in its entirety resulted in extensive proteolysis and aggregation of the protein. We therefore took a domain approach to characterize the non-core region of RAG1, with the goal that identification and characterization of independent structural domains would provide important insights into the function of non-core RAG1. In this study, we identified a structural domain in the N-terminal non-core region of RAG1, which we term the central non-core domain (CND). Our strategy is outlined in Figure To characterize the non-core region of RAG1, residues 1-380 of murine RAG1 were expressed as a maltose-binding protein (MBP) fusion construct in Noting that proteolysis frequently occurs at domain boundaries , we firsNext, we expressed the remaining region of non-core RAG1 as an MBP fusion construct (MBP-RAG1(87-380)) Figure . Though E. coli resulted in fusion protein that eluted from SEC in two peaks, with the peak eluting at Vo likely containing aggregated protein and the second peak containing more well-behaved protein  of non-core RAG1, there is no sequence homology to known protein domains. Difficulties remain in undaries . Howeverundaries . Here, wIt is well acknowledged that the full length RAG1 protein is problematic to work with; and as such it has not been feasible to show that the non-core domains are proteolytically resistant in the mammalian expressed full length protein. However, these bioinformatic results combined with the experimental studies on the bacterially-expressed non-core region Figure  stronglyIt is possible that the first 86 residues of full length RAG1 also fold into an independent domain; however, the low sequence conservation of this region suggests it does not play a central role in RAG1 functions. Moreover, the basic region, which links the CND and ZDD, is predicted to be disordered, consistent with our findings of multiple cleavage sites in this region Figure .Full length RAG1 likely functions as a dimer, or higher oligomer . Both thE. coli BL21 cells. Although the mutants were expressed in lon-deficient E. coli BL21 cells, each CND mutant underwent significant proteolysis during purification, consistent with the importance of these residues in the structural integrity of the CND (not shown).The CND contains multiple conserved cysteine and histidine residues, and it was previously suggested that this region of non-core RAG1 may coordinate zinc ions . Howeverm values from five separate experiments ranged between 55°C to 62°C yielding an average Tm value of 59 ± 3.5°C. The changes in CD spectra observed during thermal denaturation of CND were not reversible under the conditions used here. To determine the impact of zinc on the secondary structure of CND, apoCND was also monitored using CD spectroscopy under conditions identical to those used for the zinc-bound domain. The CD spectrum of apoCND at 20°C showed a significant loss of signal at 222 nm indicating a decrease in alpha helical content  spectroscopy (at 20°C) demonstrated that the domain was structured, containing 28 ± 4% alpha helical and 25 ± 2% beta sheet content Figure . Changest Figure . The CD t Figure , indicatPreviously, immunofluorescence studies showed the cellular localization pattern of full length RAG1, as well as various N-terminal non-core fragments . The nonTo analyze cellular localization properties of different RAG1 regions, we generated GFP fusion constructs of the CND, bZDD, and a combination of both domains (CND+bZDD). In addition, constructs of GFP fused to both full length and core RAG1 were produced. Each GFP fusion construct was transfected into HeLa cells and cellular localization of the expressed protein in fixed cells was visualized by fluorescence microscopy.GFP-full length RAG1 was localized primarily to nucleoli, consistent with previous results Figure  31,66,6,666,67. In cells expressing GFP-CND, the localization pattern was similar in all cells analyzed with the majority of the protein localized to the nucleus in each cell  Figure . The GFPThe relative mobilities of GFP fused to full length and non-core RAG1 domains in live HeLa cells were evaluated using fluorescence photobleaching experiments Figure . In fluoTo determine the extent that nucleoli-localized GFP fusion proteins could diffuse from these nuclear subcompartments, fluorescence loss induced by photobleaching (FLIP) experiments were performed with HeLa cells expressing GFP-(CND+bZDD) Figure . The celd) in the range of 0.8-3.0 μM. Furthermore, tagless CND demonstrated a binding affinity for the DNA substrate comparable to that observed for the MBP-fusion construct . Increasing concentrations of the isolated domains (fused to MBP) were incubated with a radiolabeled 12-RSS and the extent of complex formation analyzed by nondenaturing polyacrylamide gel electrophoresis. First, we determined that the CND bound to a ds DNA substrate containing the wild type (WT) 12-RSS Figure . The abit Figure , confirmEven though both the CND and bZDD could bind to the RSS substrate, neither domain could form sequence-specific interactions with the RSS. EMSA assays using nonlabeled WT or mutant RSS substrates as competitors demonstrated that the CND did not show significant specificity for the RSS nonamer Figure  or RSS hThe observed dissociation constants of the isolated CND and bZDD domains for the RSS are only of moderate affinity. If either domain recognized an as yet unidentified DNA sequence, it is likely the binding affinities would be measurably higher to this DNA sequence. To determine this possibility, a systematic evolution of ligands by exponential enrichment (SELEX) method was used . In thisRecent evidence suggests that full-length versus core RAG1 can more effectively retain the coding end hairpin (hp) in the post-cleavage paired complex containing both 12- and 23-RSS signal ends . It is pE. coli (not shown). Thus, the formation of the C-terminally truncated MBP-RAG1(1-380) and MBP-RAG1(87-380) fusion proteins during protein purification  strongly localized to nucleoli, with some protein present in the nucleoplasm, but absent the cytoplasm, consistent with previous reports ,66,67. TWithin the library of RAG1 constructs tested, the core region demonstrated the weakest nuclear localization pattern, with nearly equal distribution between the nucleus and cytoplasm in the majority of cells. These results correspond to previous findings with a RAG1 N-terminal deletion, termed Δ(13-330), which diffusely localized throughout the nucleus and cytoplasm .et al. ATP and T4 polynucleotide kinase. The top strands of the WT 12-RSS and the 16 bp ds DNA substrates were radiolabeled at the 5' end, and subsequently annealed to their respective complement. The indicated RAG1 domain was incubated with 1 nM 32P-labeled DNA substrate at 25°C. The binding buffer (Buffer A) contained 10 mM Tris, pH 8.0, 5 mM MgCl2, 2 mM dithiothreitol, 6% glycerol, and 100 mM NaCl. Reactions were resolved on a discontinuous 3.5%/8% nondenaturing polyacrylamide gel as described [Each DNA substrate used in EMSA experiments was radiolabeled at the 5' end using [γ-escribed , and ana32 P-labeled WT 12-RSS and 0-500 nM of either unlabeled WT 12-RSS or one of the three mutant 12-RSS substrates described above. Each reaction contained 1.5 μM MBP-CND.Competition assays were performed under the conditions described above. In these assays, purified MBP-CND was incubated in the presence of 1 nMConstructs encoding GFP-fused to different regions of RAG1 were produced, which yielded expression of GFP-core RAG1 (RAG1 residues 384-1008), GFP-CND (RAG1 residues 87-217), GFP-bZDD (RAG1 residues 218-380), and GFP-CND+bZDD (RAG1 residues 87-380). To produce these constructs, PCR products from the corresponding regions of the RAG1 gene, engineered with SmaI sites at both the 5' and 3' ends of the gene fragment, were sub-cloned 3' to the GFP gene in the mammalian expression vector pWAY5 . GFP-fulHeLa cells were grown on cover slips in six-well plates in Dulbecco's modified Eagle's medium (DMEM) supplemented with antibiotics and 10% fetal bovine serum. Each plasmid construct was transfected into cells using FuGene6 (Roche). Cells transfected with GFP-CND and GFP-bZDD were washed by PBS at 48 hours following transfection; whereas cells transfected with GFP-CND+bZDD were washed by PBS at 24 hours. Subsequently, the cells were fixed in 2% PFA for 20 minutes at room temperature. After three washes in PBS, cover-slips were mounted in UltraCruz mounting medium containing 4',6-diamidino-2-phenylindole (DAPI) (Santa Cruz Biotech).Fluorescence cell imaging was performed using a Zeiss LSM-510 META Laser Scanning Confocal Microscopy . GFP was excited at 488 nm, and emission wavelengths between 530 and 560 nm were collected for imaging. FRAP and FLIP measurements were performed as previously described ,82. In bhttp://phylogenomics.berkeley.edu/INTREPID/index.html) and Conseq (http://consurf.tau.ac.il/) servers were used to identify structurally and/or functionally important residues in the murine RAG1 protein sequence (either non-core only or full length sequence). Both methods collect and align homologous sequences of an input sequence, derive an evolutionary tree, and identify structurally and functionally important positions based on phylogenetic relations between sequence homologs [Phylogenetic Analysis: The Intrepid  and Disopred (http://bioinf.cs.ucl.ac.uk/index.php?id=806) servers were used to predict structurally disordered regions in murine non-core RAG1 or full length RAG1. The methods predict regions of disorder by searching for sequence patterns that are not typically found in the protein databank or by sequences that appear in the protein databank sequence records but with coordinates missing from the electron density map .Protein Disorder: The Drippred (ity map ( and httpRSS: recombination signal sequence; WT: wild type; MH: mutant heptamer; MN: mutant nonamer; NBD: nonamer binding domain; CND: central non-core domain; bZDD: basic zinc dimerization domain; MBP: maltose-binding protein; GFP: green fluorescence protein; MALLS-SEC: multi-angle laser light scattering coupled with size-exclusion chromatography; FAAS: flame atomic absorption spectroscopy; ICP-MS: inductively coupled plasma-mass spectrometry; SEC: size-exclusion chromatography; EMSA: electrophoretic mobility shift assay; CD: circular dichroism; FRAP: fluorescence recovery after photobleaching; FLIP: fluorescence loss induced by photobleaching; DAPI: 4',6-diamidino-2-phenylindole; DTPA: diethylenetriamine pentaacetic acid.Project planning and manuscript composition were performed by JLA and KKR. JLA performed the protein purification, SEC, and MALDI-TOF mass spectrometry experiments in Figure