E. coli Adenylate kinase (AKE), folds cooperatively. Domain interfaces have previously been implicated in the cooperative folding of multi-domain proteins. To understand their role in AKE folding, we computationally create mutants with deleted inter-domain interfaces and simulate their folding. We find that inter-domain interfaces play a minor role in the folding cooperativity of AKE. On further analysis, we find that unlike other multi-domain proteins whose folding has been studied, the domains of AKE are not singly-linked. Two of its domains have two linkers to the third one, i.e., they are inserted into the third one. We use circular permutation to modify AKE chain-connectivity and convert inserted-domains into singly-linked domains. We find that domain insertion in AKE achieves the following: (1) It facilitates folding cooperativity even when domains have different stabilities. Insertion constrains the N- and C-termini of inserted domains and stabilizes their folded states. Therefore, domains that perform conformational transitions can be smaller with fewer stabilizing interactions. (2) Inter-domain interactions are not needed to promote folding cooperativity and can be tuned for function. In AKE, these interactions help promote conformational dynamics limited catalysis. Finally, using structural bioinformatics, we suggest that domain insertion may also facilitate the cooperative folding of other multi-domain proteins.Having multiple domains in proteins can lead to partial folding and increased aggregation. Folding cooperativity, the all or nothing folding of a protein, can reduce this aggregation propensity. In agreement with bulk experiments, a coarse-grained structure-based model of the three-domain protein,  Most individual protein domains fold in an all or nothing fashion. This cooperative folding is important because it reduces the existence of partially folded proteins which can stick to each other and create disease causing aggregates. However, numerous proteins have multiple domains, independent units of folding, stability and/or function. Several such proteins also fold cooperatively. It is thought that strong interactions between individual domains allow the folding to propagate from a nucleating domain to neighbouring ones and this enables cooperative folding in multi-domain proteins. Here, we computationally study the folding of the three-domain protein AKE and find instead that the topology of the protein, wherein the two less stable domains are inserted into the more stable one, promotes folding cooperativity. When the more stable domain is folded, the ends of the inserted domains are constrained and this allows them to fold easily. In such a protein topology, strong inter-domain interactions are not needed to promote folding cooperativity. Interface amino acids which would have been involved in ensuring that the domains fit together correctly can now be tuned for binding or catalysis or conformational transitions. Thus, inserted domains may be present in multi-domain proteins to promote both function and folding. E. coli Adenylate kinase (AKE)   and findse (AKE) . In the Domain insertion makes protein topology more complex and this will likely slow folding The 214 residue enzyme, AKE, has become an important model for the study of both protein folding and conformational transitions Structure-based models (SBMs) capture the funnelled energy landscape of proteins E. coli Adenylate kinase while inter-domain interactions are optimized for conformational transitions.A dual-SBM which includes information from both the ligand free and the ligand bound structures of AKE has been developed to understand the conformational transitions of WT AKE f, where multiple transitions occur between the equally populated folded and unfolded ensembles and the best sampling of the transition region is achieved. The presence of a single free energy barrier separating the native and unfolded ensembles at Tf implies that the protein folds cooperatively fs, partially folded states get populated at temperatures between the lowest and the highest domain specific Tfs. Upon mutation, a domain specific decrease of Tf can result in the incomplete folding of that domain at the Tf of the whole protein and the population of partially folded states in the folded ensemble. This results in reduced folding cooperativity.We perform all simulations close to the folding temperature, Tv(T) vs. T) using the ratio of the van't Hoff enthalpy (ΔHvH) to the calorimetric enthalpy  f then the folding transition is cooperative. We make perturbations to WT AKE which change the free-energetic balance between domains and in turn the folding cooperativity. However, since the domains of AKE are of unequal size (CORE≫LID>NMP) the largest contribution to Cv(Tf) comes from the folding of CORE which is not perturbed much across simulations. Thus, the ΔHvH/ΔHcal is not a sensitive measure of the folding cooperativity of AKE and its mutants  of WT AKE. Using these correlation coefficients, we partition native contacts into three clusters  such thaBTf) separating the unfolded (Q∼0.2) and the native (Q∼0.8) ensembles. There is a slight dip at Q∼0.3 due to the population of states with only LID folded  and the 2-dimensional free energy surfaces (2DFESs) for WT AKE along several suitable reaction coordinates . The FEPD folded . The sinD folded . Fig. 3ED folded .Intrinsic fluorescence from single tryptophan (Trp) mutants has been used to study the refolding of AKE (24). These experiments show that regions of AKE near residues 41 (part of NMP), 86 and 73 (both part of CORE-N)  of both ΔCORE-NMPi  is higher than that of WT-AKE  and LID We computationally generate CPs of AKE by connecting the WT termini . New N- CP-NMPcut(Q∼0.8)/kBTf)/exp(−ΔGWT(Q∼0.8)/kBTf) = exp(−1.6)/1∼0.202±0.006. The FEP of CP-LIDcut shows that the barrier height is ∼2 kBT lower than that in WT (CP-LIDcut(Q∼0.8)/kBTf)/exp(−ΔGWT(Q∼0.8)/kBTf) = exp(−0.94)/1∼0.390±0.026. In both CPs, incomplete folding in the native state indicates lower folding cooperativity relative to WT. Together our results show that stabilizing interactions  are insufficient to maintain folding cooperativity and that the insertion of the LID and NMP domains into the CORE domain is necessary. In the next section, we study the conformational transitions of AKE and its mutants to understand why domain insertion is preferred over strengthening inter-domain interactions for promoting cooperative folding.On simulating CP-NMPcut, we find that its folding barrier is not significantly different from that of WT AKE . Howeverat in WT  and the at in WT . The folf where the proteins do not unfold  not only stabilizes the inserted domain but also couples their folding and brings their Tfs closer to each other Inserting a smaller domain with fewer stabilizing interactions and a lower TWe extract the structures of diverse families of multi-domain proteins with inserts  from the Pfam database f (or the thermodynamic stability) of individual domains The structural parameters that we calculate separately for the inserts and the discontinuous domains of each protein chain are the absolute contact order (ACO) fThe ACO  is the aThermodynamic stabilities of proteins can be calculated from their chain lengths fFinally, we calculate and plot the packing fraction . This isfs, the thermodynamic and the kinetic stabilities of the discontinuous domains are larger than those of their respective inserted domains . Thus, it is likely that domain insertion stabilizes the folded state and increases the folding cooperativity of at least some other multi-domain proteins with inserts. Further, both our folding simulations and the above structural bioinformatics analysis indicate that the design of singly-linked multi-domain proteins is likely to be fundamentally different from that of proteins with inserts. Thus, studies that investigate the connection between the folding and evolution of multi-domain proteins The overall trend that emerges is that the TDomain insertion naturally couples the folding of the inserted and the discontinuous domains of a protein and can, given the right order of stabilities of domains, make folding cooperative. However, domain insertion also makes the topology of the protein more complex and is likely to slow down its folding When the area of interaction between domains is small or almost non-existent, it may not be possible to tune inter-domain interactions for anything other than function and with domain insertion, these interactions are not necessary to promote folding cooperativity. If the area of interaction between the domains is large, it may be possible to promote folding cooperativity using only inter-domain interactions In order to understand if other multi-domain proteins with inserts have function at inter-domain interfaces, we use the 36 inserted domain Pfam families (1713 protein chains). We further classify these families into 38 sets of proteins such that the proteins within each set have the same pair of Pfam IDs for the inserted and the discontinuous domains. We use the function annotation database, Firedb A recent study Except for the antagonistic coupling between the folding of the inserted and discontinuous domains in protein switches Based on our simulations of AKE, we conclude that the insertion of a smaller and less stable domain into a larger and more stable one promotes folding cooperativity. Given that folding cooperativity is hypothesized to have evolved to reduce misfolding and aggregation, we expect inserted domains to be an important motif in multi-domain proteins. Further, domain insertion (and the resulting protein topology) allows the residues that promote folding cooperativity to be separate from the residues which are required for function. Using structural analysis of several multi-domain protein families, we hypothesize that domain insertion may promote folding cooperativity in at least some other multi-domain proteins whose interfaces are tuned to facilitate substrate binding, conformational transitions or domain mobility and we hope that future experimental studies will investigate this aspect of the folding of multi-domain proteins with inserts.The details of the C-α SBMs Figure S1Clusters of native contacts from kinetic simulations of WT AKE. (A) The CLANS visualization of the three contact clusters. The two clusters on the right are shown in the inset. (B) The native contacts that belong to each of the clusters shown in A are marked. X and Y axes represent residue number. Intra helical contacts have inherently low cross-correlation coefficients and are not part of any cluster. However, their tertiary contacts are present in the clusters, and therefore, their residues are included in the corresponding cluster. By comparing the contact clusters with the domain definitions we identify the three clusters as: LID , CORE-N  and CORE-C .(TIF)Click here for additional data file.Figure S2fs.Free energy profiles and 2DFESs of WT AKE and its topological variants at their T Rows: (A) WT (B) ΔCORE-NMPi (C) ΔCORE-LIDi (D) CP-NMPcut, and (E) CP-LIDcut. Columns: (1st) FEP as a function of Q  with error bars representing twice the square root of the variance, (2nd) 2DFES with RCs, QCORE and Q, (3rd) 2DFES with RCs, QCORE-N and QCORE-C (4th) 2DFES with RCs, QLID and Q, and (5th) 2DFES with RCs, QNMP and Q. In , the scaled RCs, Q*, Q*NMP, Q*LID, Q*CORE, Q*CORE-N, and Q*CORE-C are used as applicable and are defined in the Supporting Methods. Some of these 2DFESs are shown in BTfs and is dimensionless.d Q, and th 2DFES (TIF)Click here for additional data file.Figure S3Ratio of the van't Hoff to calorimetric entalpies for WT AKE and its topological variants. Folding cooperativity is well estimated by the ratio of the van't Hoff to calorimetric enthalpies. A ratio of 1 indicates cooperative folding and a ratio of 0 indicates non-cooperative folding. These ratios are computed from the equilibrium simulations of WT and the mutants of AKE at their respective Tfs. The error bars represent twice the square root of the variance. WT AKE has the highest cooperativity followed by ΔCORE-NMPi and ΔCORE-LIDi. The CPs, CP-NMPcut and CP-LIDcut have lower folding cooperativity as compared to WT.(TIF)Click here for additional data file.Figure S4A typical simulation trajectory and free energy convergence for WT AKE. (A) A time trace of the fraction of native contacts (Q) from a representative simulation of WT AKE at its estimated Tf. This trace shows transitions between the folded (Q∼0.8) and unfolded ensembles (Q∼0.2). (B) We plot the evolution of the free energy profile with increasing simulation time. The bottom panel shows the location of folding (U to N) or unfolding (N to U) transitions at the time that they occurred in the simulation trajectory. 29 such transitions were observed for WT AKE. The middle panel plots the difference , calculated by using simulation data up to the time point it, and the final free energy profile . The intermediate free energy profiles, F, were first reweighted to their respective folding temperatures, Tf(i)'s before the difference was calculated. All F are in scaled by their respective kBTf(i). The Tf(i)'s are plotted in the top panel. The middle and the top panels show that the free energy profile and the Tf have converged by the end of the simulation.(TIF)Click here for additional data file.Figure S52DFESs of inter-domain interface contacts in WT AKE. (A) QCORE-NMPi vs. Q shows that the CORE-NMP interface is significantly formed in the transition state . The formation of the CORE-NMP interface in the transition state is consistent with its inclusion in CORE-N .(TIF)Click here for additional data file.Figure S6A family-wise comparison of the structural features of the inserted and the discontinuous domains. Domains from 36 inserted domain Pfam families are found to be inserted in the structures of multi-domain proteins present in the PDB. For each such multi-domain protein chain from the PDB, (A–C) the absolute contact order (ACO), (D–F) the chain length and (G–I) the packing fraction are plotted for the discontinuous vs. the inserted domains. The plot of every such chain is shown in (TIF)Click here for additional data file.Table S1Temperatures and the energetic scaling factors for equilibrium simulations. Error on the Tf estimates is ±0.1 K or 0.0008314 (kBT units).(PDF)Click here for additional data file.Table S2Summary of the experimental results obtained from refolding kinetics of AKE compared with folding simulations of WT AKE (See ).(PDF)Click here for additional data file.Text S1Supporting methods.(PDF)Click here for additional data file.Text S2Supporting results.(PDF)Click here for additional data file.Text S3Additional supporting information. Contact lists for WT AKE, some of which are used to define the reaction coordinates (RCs).(DOCX)Click here for additional data file.Text S4List of all PDBs having insert-discontinuous domain pairs used in the structural bioinformatics analysis.(DOCX)Click here for additional data file.Text S5Supporting references.(PDF)Click here for additional data file.


Benign/malignant overlap exists on time-intensity curve (TIC) of dynamic gadolinium-enhanced breast MRI. This study presents a new method for TIC generation to decrease overlap and improve accuracy.MR images of 100 patients with enhancing breast lesions  obtained before and repeatedly after intravenous injection of Gd-DOTA were evaluated. Signal intensity of lesions (SIlesion) and breast (SIbreast) were measured and TIC obtained. Relative signal intensity of the lesion was calculated as (SIlesion-SIbreast)/SIbreast and plotted versus time to obtain relative TIC. Four parameters were evaluated for diagnosis of carcinoma: peak enhancement (PE), initial enhancement slope (S), time-to-peak (TTP), and washout ratio (WO). Comparison of parameter performance on TIC and relative TIC has been done by the Student’s T test and the Receiver-Operator Curve (ROC) analysis.On TIC, TTP has been the only discriminating factor. When threshold for carcinoma has been set at TTP≤2 min, sensitivity, specificity, positive predictive value, negative predictive value, and accuracy have been 36, 100, 100, 43, and 57% respectively. On relative TIC, accuracy of TTP has increased to 89%. Area under the ROC curve for TTP has improved from 0.77 for TIC to 0.86 for relative TIC (p<0.05). WO ratio has become a second discriminating factor on relative TIC with more washout in malignant lesions (WO=41±32) than benign lesions (WO= 11±19) (p <0.05). PE and S were not statistically significant on TIC or relative TIC.The use of relative TIC improves the discrimination of benign and malignant breast lesions.


Hemigrapsus sanguineus, has recently been observed occupying salt marshes, a novel environment for this crab species. As it invades this new habitat, it is likely to interact with a number of important salt marsh species. To understand the potential effects of H. sanguineus on this ecosystem, interactions between this invasive crab and important salt marsh ecosystem engineers were examined. Laboratory experiments demonstrated competition for burrows between H. sanguineus and the native fiddler crab, Uca pugilator. Results indicate that H. sanguineus is able to displace an established fiddler crab from its burrow. Feeding experiments revealed that the presence of H. sanguineus has a significantly negative impact on the number as well as the biomass of ribbed mussels (Geukensia demissa) consumed by the green crab, Carcinus maenas, although this only occurred at high predator densities. In addition, when both crabs foraged together, there was a significant shift in the size of mussels consumed. These interactions suggests that H. sanguineus may have long-term impacts and wide-ranging negative effects on the saltmarsh ecosystem.The invasive Asian shore crab,  Phragmites australis, and the green crab, Carcinus maenas), some of which have significantly changed the structure of communities where they were introduced ; C. maenas)). One recently introduced species of concern in New England is the Asian shore crab, Hemigrapsus sanguineus.Marine systems that have suffered from a high level of human disturbance are more susceptible to successful invasion by non-indigenous species. Anthropogenic disturbances such as predator removal, land-use changes, and eutrophication may alter an environment to the extent that native species lose their locally-adapted advantages. If this happens, non-native species benefit from the disruption of ecosystems and may be able to establish themselves in the community . Once esHemigrapsus sanguineus is now well established on the northeast coast of the United States, where densities rival or exceed those in their native Asia  rather than rocks for shelter (U. pugnax) at the edge of a Connecticut salt marsh, suggesting the native fiddler crab may be facilitating the invasion of H. sanguineus into a new environment. If H. sanguineus is in fact able to exploit this new environment, its range along the Atlantic coast may expand beyond its present boundaries. Concomitantly, Asian shore crab abundance could increase in areas within its current range as novel habitat becomes populated, which could have important consequences for a number of salt marsh resident species.l stress . The cral stress . Recentl shelter  , an important salt marsh engineer. Mussels are limited by attachment sites and benefit from the stability provided by cordgrass stems and root mass. As they filter water, ribbed mussels take up particulate nitrogen and convert it to ammonia, dissolved organic nitrogen, and semi-solid biodeposits, the bulk of which are excreted and quickly utilized by other marsh residents , a long established invader. As H. sanguineus comes into the marsh in greater numbers, competitive interactions between the crabs will likely increase. This may indirectly affect mussel populations; despite being a potential shared prey for both crab species, the presence of multiple predators rarely has an additive effect on prey  to investigate the emergent effects of multiple predators—the green crab, Carcinus maenas and the new invader, Hemigrapsus sanguineus—on ribbed mussel predation.The primary objectives of this study were: (1) to experimentally examine whether competition for burrows occurs between this invasive crab and the native fiddler crab, Hemigrapsus sanguineus were collected by hand along the shore of Stony Brook Harbor, Long Island, NY, and maintained in an indoor saltwater aquarium, filled with rocks to provide shelter, for no more than one month prior to use in trials. Crabs were fed Wardley Algae Discs  supplemented with fresh algae, Ulva lactuca. Uca pugilator were collected by hand from Flax Pond and Scallop Pond, Long Island, NY, and kept in a tank built to mimic natural conditions: a sloping layer of sifted beach sand leading to a pool of salt water approximately 4 cm deep on one end of the tank. The crabs were able to submerge themselves, forage on the dry sand surface, or excavate burrows down to moist bottom sand. Uca crabs were kept no more than one month prior to use in trials and were fed a ground mixture of Wardley Algae Discs and Tetrafauna Hermit Crab Cakes . Male and female crabs of both species were used, and each crab was used in only one trial. Equal number of males and females of each species were used to determine whether there was a difference based on the sex of either crab. In addition, carapace width was recorded to determine if size affected the outcome between the crabs. The average female carapace width of U. pugilator was 18.6 ± 0.61 mm and ranged in size from 17.5 to 19.2 mm. The male size range was 16.9–21.2 mm and averaged 19.41 ± 1.32 mm. In comparison, the average size of H. sanguineus females was 15.8 ± 2.0 mm and ranged in size from 12.0 to 19.2 mm. The average male sizes were 17.9 ± 2.4 mm and ranged from 13.7 to 21.2 mm.2 surface area arena. Near the arena edge, an artificial burrow 2.3 cm in diameter and 6 cm deep was made. A 125 W heat lamp was positioned 22 cm from the sand surface, providing thermal stress to promote the fiddler crab’s burrowing response and increasing desiccation stress for H. sanguineus  vinyl number tags, attached to the carapace with cyanoacrylate glue. This was done to provide a means of distinguishing the original crab from the one added. These experiments were conducted in such a way to compare differences of the success of H. sanguineus in possessing a burrow based on crab size (CW) and sex of the U. pugilator defender.To compare the competitive interactions of H. sanguineus collected from Old Ponquogue Marine Park, Southampton NY. Arenas were created using the top half of a 19-l bucket driven into the sand approximately 3–5 cm. As in the laboratory experiments, an artificial burrow 2.3 cm in diameter and 6 cm deep was made in the arena. One randomly selected fiddler crab was placed in the arena and given sufficient time to occupy the burrow, then a randomly selected H. sanguineus was added opposite the burrow. After 20 min, the burrow occupant was recorded and any new burrowing activity noted. Conspecific trials, using two U. pugilator crabs, were performed in the same fashion.To control for laboratory artifacts, these experiments were conducted in the field as well. Experiments were performed at the beach on Scallop Pond, Southampton NY using resident fiddler crabs and H. sanguineus and the green crab, Carcinus maenas, on ribbed mussels, a feeding experiment was performed using a fixed number of ribbed mussels and 6 different combinations of H. sanguineus and C. maenas crabs. The treatments were (i) one H. sanguineus; (ii) one C. maenas; and (iii) one H. sanguineus plus one C. maenas; (iv) two H. sanguineus; (v) two C. maenas; and (vi) two H. sanguineus plus two C. maenas . The use of these different density combinations allowed both the impact of increasing density of conspecifics on mussel consumption as well as interspecific effects. Hemigrapsus sanguineus and Carcinus maenas were collected from Old Ponquogue Bridge Marine Park, Southampton, NY. Additional C. maenas were collected by trawl within Shinnecock Bay (NY). Sizes of H. sanguineus ranged from 18 to 24 mm carapace width (CW), with an average CW of 21.0 mm, while the sizes of C. maenas ranged from 53 to 76 mm CW and averaged 62.1 mm CW, within sizes commonly observed in marine habitats around Long Island and similar to sizes used in other studies (To determine the emergent effects of  studies . Soft-shGeukensia demissa) was collected from Red Creek Pond, Southampton, NY. The mussels were measured and classed by size, from <1.0 to 6.5 cm in 0.5 cm increments, then counted. This was done to utilize the natural size distribution of ribbed mussels in the field  haphazardly arranged in a 3-tiered, flow-through sea table with unfiltered seawater. Water was directed into each individual container, and allowed to flow over the top for water exchange. The water level in the sea table was kept at 9 cm deep, enough to help maintain constant temperature but preventing water exchange between different containers, thus keeping them independent . All crax = mussel shell length in centimeters and y = whole tissue dry weight in grams . PCPH is a correction factor for mussels that can’t be consumed twice. Both the two-way interaction approach and the multiplicative risk model have been used separately in previous studies examining multiple predator affects. We chose to use both approaches to identify whether an emergent effect occurred and determine the direction of the effect. These statistical analyses were conducted using the Sigma Plot 11.0 statistical software package.For the burrow competition experiment, statistical comparisons of the final burrow occupant were made via Chi-square tests . To detep < 0.001). After a total of 38 trials, the burrow was occupied by H. sanguineus alone 14 times, by Uca pugilator alone 4 times, and by both crabs 20 times. In no trial was the burrow unoccupied at the end of 20 min. The success of H. sanguineus in the trials was not dependent upon the size (CW) (p = 0.545), nor sex (p = 0.633) of U. pugilator. Neither was it dependent on the size (CW) (p = 0.592), nor sex (p = 0.504) of H. sanguineus.There was a statistically significant difference in the crab species occupying the burrow after 20 min  and repeated approaches to the burrow (both sexes). On two occasions, U. pugilator males were observed using their dominant claw to pry H. sanguineus out of the burrow. In two trials, U. pugilator excavated a new burrow after being displaced from the original burrow by H. sanguineus.The behavior of H. sanguineus would also be aggressive when U. pugilator defended or attempted to re-occupy the burrow. H. sanguineus would often approach an occupied burrow with claws raised; if already inside the burrow, it could be seen stretching its chelipeds to occupy as much space as possible. On several occasions, it was observed that H. sanguineus would use its claws as pinchers to deflect attempts by U. pugilator to re-enter the burrow.The behavior of H. sanguineus and U. pugilator, the same behaviors witnessed in the lab trials were observed in the field. In 20 trials, the burrow was occupied by H. sanguineus 11 times (55%), by U. pugilator 5 times (25%), and by both crabs 4 times (20%). These results of the field trials were not significantly different from those in the laboratory (p = 0.40).In the outdoor trials with Carcinus maenas consumed 17% of the mussels offered to them, while only 8% were consumed by Hemigrapsus sanguineus foraging alone, likely due to a size threshold of the prey. When one of each species was present together, the combined consumption was 14%. However, the proportion of mussels consumed did not vary significantly across predator treatments at low predator density (p = 0.801). Increasing from single predators to conspecific pairs of C. maenas more than doubled the number of mussel prey consumed (62% consumed), but had no effect on H. sanguineus .When foraging alone, C. maenas × H. sanguineus interaction, p = 0.294; p = 0.646; p < 0.001; p < 0.001; There was not a significant emergent effect on prey survival when single individuals of the two predator species foraged together based on the additive experimental design  to a length of 5.5 cm, while H. sanguineus consumed the smallest size range of mussels, from <1.0 to 2.0 cm in length. In treatments when both predators foraged together, the lack of consumption on the larger mussel size classes was dramatic  were significantly different (p < 0.001), with smaller mussels being consumed in heterospecific groupings. While this was affected by the increased consumption of the smaller size classes by H. sanguineus, it also demonstrates that C. maenas did not consume the larger individuals when H. sanguineus was present.In addition to a change in the number of mussels consumed, the mean size of ribbed mussels eaten shifted from larger to smaller mussels when both predators foraged together . When fodramatic . A Mann–C. maenas consumed a significantly higher biomass of ribbed mussels than the low density C. maenas treatment. When H. sanguineus was present there was significantly less biomass consumed than when C. maenas was alone at the highest densities  than did U. pugilator alone (11% of the time). The outcome of these competition arena experiments were similar with respect to H. sanguineus occupancy in both the lab and in the field experiments (80% of field burrows were occupied by H. sanguineus). However, how the occupancy occurred was slightly different. In the lab arenas, H. sanguineus occupied the burrow alone ∼37% of the time, while it co-occupied the burrow in ∼53% of the trials. In the field, this pattern was almost opposite, which H. sanguineus in 55% of the burrows alone compared to sharing the burrow 20% of the time. While it is unclear why the lab and field arenas differed in how the burrow occupancy occurred, possibly due to behavioral differences between lab adapted and resident crabs used in the different experiments, the results show that H. sanguineus is able to use burrows created by U. pugilator. Therefore, fiddler crabs (Uca pugilator and U. pugnax) may act as facilitators to H. sanguineus invasion by providing a suitable desiccation refuge for the invaders.The burrow competition experiments performed in this study revealed that H. sanguineus will have on Uca sp. However, the presence of H. sanguineus in burrows may cause an emigration of Uca sp. from areas within the marsh, reduce the density of burrows, or interfere with reproductive activity known to occur within burrows. Any of these scenarios could negatively impact the salt marsh, as the importance of fiddler crab burrows to salt marsh functioning is well documented . The ecological consequences of this for the salt marshes of Long Island and elsewhere are as of yet unknown.Previous surveys show that g Island , and the10.7717/peerj.472/supp-1Figure S1The natural size distribution of the mussels that were used to create the 18 subunits of the same size distributions for the crab predation experiments.Click here for additional data file.


Pseudomonas aeruginosa exopolyphosphatase  catalyzes the hydrolysis of polyphosphates (polyP), producing polyPn−1 plus inorganic phosphate (Pi). In a recent work we have shown that paPpx is involved in the pathogenesis of P. aeruginosa. The present study was aimed at performing the biochemical characterization of this enzyme. We found some properties that were already described for E. coli Ppx (ecPpx) but we also discovered new and original characteristics of paPpx: (i) the peptide that connects subdomains II and III is essential for enzyme activity; (ii) NH4+ is an activator of the enzyme and may function at concentrations lower than those of K+; (iii) Zn2+ is also an activator of paPpx and may substitute Mg2+ in the catalytic site; and (iv) paPpx also has phosphotransferase activity, dependent on Mg2+ and capable of producing ATP regardless of the presence or absence of K+ or NH4+ ions. In addition, we detected that the active site responsible for the phosphatase activity is also responsible for the phosphotransferase activity. Through the combination of molecular modeling and docking techniques, we propose a model of the paPpx N-terminal domain in complex with a polyP chain of 7 residues long and a molecule of ADP to explain the phosphotransferase activity. The presence of this polymer has been detected in all kinds of living organisms, including higher organisms. Main enzymes involved in the synthesis of polyP are the polyphosphate kinases , which catalyze the formation of polyP from ATP (Ppk1) and GTP (Ppk2). Endo- and exopolyphosphatases are the most important enzymes involved in the utilization of polyP. In bacteria only exopolyphosphatases have been described /v = ([S]/Vmax) + (KM/Vmax); Michaelis-Menten (v = Vmax [S or M])/KM + [S or M]; and/or Hill ((v/V0 − v) = Log K0.5 + n log [metal ion]), where [S] and [M] correspond to substrate and metal ion concentrations, respectively. Protein concentration was determined by spectrophotometric measurement at 280 nm using the correspondent theoretical molar extinction coefficient calculated with the “ProtParam” tool [http://www.expasy.com/).The phosphotransferase activity was measured after incubation at 37°C for 30 min in 200  paPpx homologues through the use of the BLASTp algorithm resulted in the identification of an ortholog protein in E. coli (ecPpx) with a 41% identity and 58% similarity. Two solved structures of this protein are available: PDB: 2FLO, 2.2 Å resolution [The search ofsolution  and PDB:solution . Accordi paPpx in a closed conformation was constructed by comparative modeling using the ICM program [A homology model of program . For loo126, Asp149, Gly151, Ser154, and Glu156) was measured with VMD software, and in its place an atom of Mg2+, which is an essential cofactor, was added prior to the molecular dynamics.The final model was validated using ProSA , ANOLEA paPpx(1–314) in an open state was constructed using the atomic coordinates of PPX/GPPA phosphatase from A. aeolicus (aaPpx) in complex with the alarmone ppGpp [ paPpx and aaPpx [http://www.sbg.bio.ic.ac.uk/phyre2/). The model was obtained with 100% of confidence and 292 residues of a total of 314 were aligned. Tautomeric states of histidine residues in the model were assigned according to the local environment and Mg2+ ion was located in the active site similarly as we described for the full-length model. The obtained models were subjected to MD calculations to reach a minimum energy state. For this, the models were embedded in a 15 Å water box, and KCl 80 mM was used not only to mimic the optimal conditions for enzyme activity but also to neutralize the total charge of the system. The initial configuration of both systems was optimized using energy minimization followed by an equilibration through a molecular dynamics (MD) simulation in the NPT ensemble at 310°K for 1 ns, using a backbone restriction of 0.5 kcal/mol Å. NAMD program was used to perform all molecular dynamic simulations [The model of the N-ne ppGpp   model in open conformation as receptor. ICM [Docking studies were carried out as previously described , definintor. ICM  version http://pubchem.ncbi.nlm.nih.gov/) and the polyP ligand input was constructed by using ICM molecule editor. All ligand charges were assigned by ICM software. All of the structures were protonated and optimized using standard ICM protocols. The thoroughness parameter, which represents the length of the docking simulation, was set at 2.0, as recommended by software developers when metals are present in the docking binding site. For the docking of ADP, the gridbox size was 16.95 Å × 19.23 Å × 16.05 Å, and the center was located at the points (−5.602 × 7.844 ×  −4.172) Å. The binding site was determined by structural alignment with aaPpx and the region at which ppGpp is located was used. Thus, the docking pocket was composed of the residues Asn25, His28, Gly152, Gly224, Asp272, and Arg274. For the docking of polyP7, the N-paPpx-ADP complex was used as receptor. The binding site was one of those detected by the icmPocketFinder tool and was consistent with the S-shaped canyon described by [94; Asn95, Ser122, Gly123, Arg124, Glu126, Ile130, Asp149, Ile150, Gly151, Gly152, Gly153, Ser154, Glu156, Ser170, Gln172, Ser223: Gly224, Arg227: Ala228, Leu231, Gly268, Ile269, Lys270, Asp272, Arg273, Ile276, Glu300, Ala302, Leu303, Arg304, and Glu305. The gridbox size was 30.26 Å × 28.29 Å × 25.82 Å and the center was located at the points (−0.387 × 1.915 × −0.669) Å.Nucleotide ligand inputs were extracted from the PubChem database  were aligned using Clustal Ω [http://weblogo.berkeley.edu/).To find orthologous proteins,database . Sequenclustal Ω , 37. Reslustal Ω , 39  was cloned and overproduced as the full-length recombinant protein and also the two peptides representing the N-terminal and C-terminal domains. All recombinant proteins were purified and used to study some of the biochemical properties of the enzyme. The identification of N- and C-terminal domains was performed considering the crystallographic structure reported by [paPpx(1–506)), while the N-terminal domain contained the first 314 aminoacyl residues and the remaining 192 aminoacyl residues corresponded to the C-terminal domain (N-paPpx(1–314) and C-paPpx(315–506), resp.). The theoretical MW for paPpx(1–506), N-paPpx(1–314), and C-paPpx(315–506) were 56,419.33, 34,325.25, and 22,112.10 Da, respectively. Only paPpx(1–506) and N-paPpx(1–314) were enzymatically active while C-paPpx(315–506) lacked enzymatic activity. The specific activities of paPpx(1–506) and N-paPpx(1–314) were 1.49 and 0.38 μmol of Pi min−1μmol−1 protein, respectively. In order to determine the fragment responsible for the enzymatic activity of paPpx, we took into consideration the N-terminal construct obtained by [paPpx(1–303), which were found to be inactive.The exopolyphosphatase gene oforted by , 14. The C-paPpx3–506, res paPpx(1–506) and N-paPpx(1–314) are displayed in 2+ and K+ concentrations, paPpx(1–506) increased its affinity for polyP in the following order: polyP25 < polyP45 < polyP65 < polyP75. The decreasing KM(app) values were accompanied by a sharp rise in the catalytic efficiencies, with this behavior being more noticeable with the increment in the length of the substrate chain (paPpx since both KM(app) and catalytic efficiency were similar independently of the length of the polyP chain (75 demonstrated that the full-length enzyme presented KM(app) that was approximately 23-fold lower than the one of N-paPpx(1–314) . This behavior was partially reversed by an experiment of complementation, where a mixture of N-paPpx(1–314)/C-paPpx(315–506), in a 1 : 10 ratio, produced an active enzyme with higher affinity for the substrate polyP75. The KM(app) value measured in the mix was 11 ± 2 μM, which represented an increase of ≈2.3-fold.The kinetic behavior evaluated and apparent catalytic constants obtained forte chain . This tryP chain , Table 2Divalent Ions Dependence. We studied the behavior of the full-length paPpx(1–506) and N-paPpx(1–314) against different concentration of divalent ions such as Mg2+, Zn2+, Ca2+, and Mn2+ as effectors, in presence of a saturating concentration (8 μM) for the substrate polyP65. The activation of both enzyme variants by Mg2+ was similar and showed no inhibition at high concentrations of this ion ( ecPpx activity that found a sharp decrease in the activity with Mg2+ concentrations of 1 mM and higher.this ion . This reK2+0.5(app)Mg in paPpx(1–506) and N-paPpx(1–314) were 0.30 ± 0.02 mM and 0.28 ± 0.02 mM, respectively. Zn2+ was able to activate the enzyme only 20% compared to Mg2+, whereas the activation by Ca2+ and Mn2+ was negligible . These data, added to the fact that the interaction between paPpx(1–506) and Mg2+ occurs in the N-terminal domain, showed a clear preference of the enzyme for Mg2+ without inhibition by ion concentration up to 10 mM.The values of + was a nonessential activator of paPpx. This result is in good agreement with other studies performed on Ppxs [2+, we performed a saturation curve of the divalent ion with the full-length enzyme, with and without K+. The presence of K+ did not affect the affinity of the enzyme for Mg2+; (K2+0.5(app)Mg values were similar: 0.30 ± 0.02 mM (K+) and 0.33 ± 0.01 mM (no K+)).As expected, K on Ppxs , 15. To Monovalent Ions Dependence. Considering the activation produced by K+ in the activity of paPpx(1–506), we decided to test the effect of other monovalent ions. We observed that Li+ and Na+ presented no effects on enzyme activity while NH4+, K+, Rb+, and Cs+ were activators of paPpx(1–506). Taking into account the physiological relevance of NH4+ and K+, saturation curves of paPpx with these ions were performed in the presence of 8 μM of polyP65 and 5 mM of Mg2+. The curves obtained with NH4+ and K+ were sigmoid and reached their maximum activity at concentrations of 30 mM and 80 mM, respectively (K4+0.5(app)NH was 10 ± 0.4 mM and K+0.5(app)K was 42 ± 0.5 mM for K+.ectively . K0.5(a paPpx(1–506) by NH4+, we tested alkylammonium ions with different degrees of methylation as activators. We found that activation decreased as the number of methyl substituents increased. Considering the NH4+ activation as 100%, the percentages of activity with methylamine, dimethylamine, and trimethylamine were of 28, 9, and 5%, respectively. Tetramethylammonium was not an activator of paPpx(1–506).In view of the activation of A. aeolicus acts on a nucleotide (pppGpp) and the structure of this enzyme in presence of the product (ppGpp) (PDB: 2J4R) is available [ paPpx there could be enough space to bind a nucleotide molecule such as ADP; thus we decided to test the paPpx as a polyphosphate:ADP phosphotransferase. The production of ATP from polyP and ADP was measured in paPpx(1–506) and N-paPpx(1–314) with polyP lengths of 25 and 65 residues as substrates. Both variants presented phosphotransferase activity and, similarly to results obtained for the phosphatase activity, the N-paPpx(1–314) had a lower catalytic efficiency (KM(app) and the Kcat were in the same order of magnitude for the polyP25 or the polyP65.Since the Ppx/GPPA fromvailable , 26, we ficiency . The cat2+; however, the K2+0.5(app)Mg value was approximately half that observed for phosphatase activity. The fact that the phosphotransferase activity was insensitive to the addition of K+ or NH4+ (KM(app) value was in the order of 90 μM for both substrates and variants tested (data not shown).The phosphotransferase activity was dependent on Mgor NH4+  constitu paPpx by comparative modeling, using the atomic coordinates of the crystal structure of ecPpx [ paPpx(1–506) and was reported in what the authors named “closed conformation.” In second place, we modeled the N-terminal domain of paPpx in the “open conformation” based on the atomic coordinates of aaPpx [Initially, we modeled a full-lengthof ecPpx  (PDB: 1Uof aaPpx  (PDB: 2J paPpx and aaPpx (27%), the model was constructed by threading using the “one-to-one threading” option of Phyre Server [ paPpx). In the proteins of this family, movements of up to 30° were described to be related to the catalytic function of the enzymes. In aaPpx a rotational movement of 22.5° between both domains around a single hinge region was described, indicating the access to the active site, located at the interface between domains. Kristensen and collaborators [ paPpx in the open and closed conformations. A rotation of 24° between subdomains I and II was detected by DYNDOM server between both structures [123:R124 and R304:E305 as bending residues.Based on the homology in secondary structure and in spite of the low identity level betweennd aaPpx %, the moborators  describe paPpx is formed by residues E126, D149, G151, S154, and E156. We confirmed the role of these amino acids in the active site of paPpx(1–506) by performing nonconserved site-directed mutations, replacing individually each of these residues by alanine. The two activities, phosphatase and phosphotransferase, were measured and the release of both Pi and ATP production was severely affected  was carried out. First, docking was performed with 7 residues long polyP (polyP7) chain in presence of Mg2+. Among the pockets detected by the IcmPocketFinder tool there was one that covered all the cleft, which was postulated to be opened or closed with N-terminal domain movements. Considering that this region was consistent with the location of the active site and the putative polyP binding sites described by [paPpx-polyP7 complex was used as the receptor molecule to perform docking with ADP. The possible conformations adopted by the nucleotide seemed to be led by the Pi moiety which is always located in the same position, and the guanosine group showed some degree of rotation resulting in slightly different positions inside the same binding site. The location of the terminal Pi nearby the Mg2+ atom was consistent with the proposed catalytic mechanism. It was also possible to see the rest of the polyP chain running along the cleft. The final complex is shown in 95, Asn95, Arg98, Ser122, Gly123, Arg124, Glu126, Ile150, Gly151, Gly152, Gly153, Ser154, Glu156, Ser170, Leu171, Gln172, Ser223, Gly225 Tre225, Arg227, Ala228, Leu 231, Lys270, and Arg273. polyP was stabilized by H-bonds with Gly153, Ser154, and Gly224 and saline bridges with Arg227, Lys270, and Arg273. Meanwhile, in the ADP, the ribose established an H-bond with Arg304 and α and β phosphates established an H-bond with Ser24 and Asn25. By comparison with the approach of Alvarado and collaborators [paPpx(1–314) in the open conformation with both ligands coexisting in the cleft was aligned with the N-terminal domain of ecPpx, which had SO42− ions in the cleft. It is assumed that these ions describe a path for the polyP chain. The criterion for alignment was based on the active site residues, which remain in the same position regardless of the state of the conformation (opened or closed). This superimposition is shown in  aaPpx and it is noted that phosphates residues of 3′ are superimposable with the polyP residue while the 5′ phosphates residues are superimposable with 5′ phosphates of ADP  model and ecPpx. These results are shown in Figures  paPpx the electropotential was much less positive than in ecPpx, especially around the cleft.A key aspect that remains to be characterized in theuld bind , we perf P. aeruginosa [ ecPpx is composed of two independently folded domains: N- and C-terminal domains [ paPpx: paPpx(1–506), N-paPpx(1–314), N-paPpx(1–303), and C-paPpx(315–506).We have recently demonstrated the involvement of Ppx in the pathogenesis ofruginosa . This fi domains –15. Base paPpx was localized in the N-terminal portion formed by the first 314 amino acid residues. Consistently with this, Alvarado and collaborators [ ecPpx, formed by 320 amino acid residues, was responsible for the catalytic activity. However, Bolesch and Keasling [ ecPpx activity in the N-terminal portion after limited proteolysis with Staphylococcus aureus V8 protease (Glu-C). A possible explanation for the discrepancy between these results may lie in the construction of the N-terminal variants. The peptide 304EMEGRFRHQDVRSRTAS320 located in the carboxyl end of the N-terminal variant constructed by Alvarado and collaborators [ecPpx295–321 and N-paPpx301–326, we obtained a high degree of identity between these two enzymes  are conserved . Likely, the lack of this helix may prevent the proper folding of the domain or it may cause a disruption affecting the active site, resulting in loss of activity.Our results indicate that the catalytic moiety ofborators  also fouKeasling  did not borators  was abseborators . This pe enzymes . The anaonserved . Structuonserved . We proppaPpx(1–314) presented enzymatic activity, it had no preference for long polyP chains, and its KM(app) and catalytic efficiency presented roughly the same values for all tested substrates. These results strongly suggest that the C-terminal domain is important for the recognition and/or interaction with long polyP chains. These findings are in concordance with those reported by [ ecPpx is involved in the recognition and processivity of long polyP chains.Although N-orted by  that sta paPpx was dependent on Mg2+, as it was demonstrated for other Ppxs [ ecPpx showed that the maximum activity was achieved in presence of Mg2+ 1 mM [ Sulfolobus solfataricus that Mn2+ is needed as an activator in concentrations ≈1 mM and that Mg2+ is needed to a lesser extent [ Saccharomyces cerevisiae [2+, Mn2+, and Co2+, including Zn2+ to a lesser extent. Results of these reports suggest similar catalytic mechanisms. Our results related to the activation of paPpx by divalent cations are in good agreement with the work of Dudev and Lim [2+ binding sites are not often specific for Mg2+ and that Zn2+ may replace it in that location. We found that K+ and NH4+ are nonessential activators of paPpx. Our results considerably differ from those reported for the ecPpx [+ was necessary to achieve the maximal activity (175 mM versus 80 mM) and where ammonium sulfate was not considered as an activator of the E. coli enzyme. We believe that the lack of activation by ammonium sulfate may be due to the presence of sulfate ions which can bind to the enzyme and mimic the phosphate residues of the substrate, thus blocking the activity. The crystal structure reported by [The activity ofher Ppxs . Studiesg2+ 1 mM  while hir extent , 39. Furrevisiae  the requ and Lim , who demhe ecPpx , 37, wheorted by  also sup paPpx activity is dependent on Mg2+ and is stimulated by K+ in a similar manner to ecPpx. Our present study also adds a new perspective to previous results, with the finding that NH4+ can act as a direct activator of paPpx at lower concentrations than K+. It was demonstrated that bacteria may accumulate transiently polyP during the stationary phase and especially under conditions of nitrogen and Pi limitation and during osmotic stress [+ is the most prevalent cation in the cytoplasm and it becomes more concentrated as the osmolarity increases [+ is useful to activate directly the paPpx. As the NH4+ concentration is limited or in the presence of a nonpreferential nitrogen source , the activation of paPpx is at transcriptional level since the expression of the ppx gene is under the control of the global regulator NtrC [4+ directly stimulates the enzymatic activity and the bacteria obtain energy to start their growth.The whole data presented confirm thatc stress . It is ancreases . Therefotor NtrC . On the  paPpx could have a second function, such as the synthesis of ATP by transferring Pi from polyP to a molecule of ADP. Confirming what we previously hypothesized, we found that paPpx is also a polyP:ADP phosphotransferase. As the phosphatase activity, the phosphotransferase activity is located in the N-terminal domain and depends on Mg2+; however, it is insensitive to the addition of K+ or NH4+, suggesting that the catalytic mechanism is somehow different, probably acting nonprocessively.Taking all these reports together, it was evident that polyP levels are involved in the global energetic state of the cell. Thus, we wondered if in silico assays to complement our experimental data. We modeled the full-length paPpx in a closed conformation and the N-paPpx in an open conformation.Additionally, we performed E. coli, the active site was proposed to lay in a cleft between subdomains I and II [ paPpx has full conservation of the proposed active site residues from ecPpx that would be formed by Glu156, Asp149, Gly151, Ser154, and Glu126 and the glycine-rich loop consisting of residues Gly141-Ser154. The conserved Glu126 is proposed as the residue which activates a water molecule for the nucleophilic attack to the phosphodiester bond. The solved structures of ecPpx lack the Mg2+ bound to the enzyme. The putative Mg2+ binding site was suggested by comparison with the binding of aaPpx to a Ca2+ atom [ ecPpx, the acidic residues Asp149 and Glu156 are predicted to contribute to the coordination sphere of Mg2+ in paPpx [126, Asp149, Ser154, Gly151, and Glu156 residues in the active site by nonconservative site-directed mutagenesis in the full-length paPpx. The phosphotransferase activity of paPpx was also measured in the mutated variants of paPpx and no ATP production was detected. This result indicates that the active site of paPpx is the same for both activities: the phosphotransferase and the hydrolase.InI and II  and a glI and II . paPpx ha2+ atom . By anal ecPpx and aaPpx also presented pppGpp activity [ aaPpx , it is clear that there is a single active site for hydrolysis of both polyP and pppGpp. As it was suggested by [ ecPpx must undergo a conformational change from the closed to the open state to allow the entry of the nucleotide pppGpp. A phosphotransferase activity was never described for exopolyphosphatases or PPX/GPPA phosphatases; this work is the first reporting ATP production in a Ppx. Our results suggest that in paPpx the same active site is responsible for this activity, and we think that the transition from the closed to the open conformation occurs also in paPpx to allow the entry of ADP. The pppGpp molecule in the active site is occupying the space that would occupy both polyP and ADP  under nitrogen limitation, where ppx is activated by NtrC, mediated by a σ54-dependent promoter [i deficiency mediated by PhoB by a σ70-dependent promoter; (iii) in the presence of preferential nitrogen source (NH4+), where the transcription of ppx is inhibited but the existing enzyme can be directly activated; and (iv) under hyperosmolarity, where paPpx is rapidly activated by K+, the most prevalent cation in the cytoplasm. This enzyme with its two functions can release Pi for a direct synthesis of ATP or meet the nutritional needs where Pi is necessary either to generate more energy or to initiate metabolic processes.The regulation of the degradation of polyP is complex and dual, since it involves regulation at transcriptional and biochemical levels. Thus, bacteria have mechanisms which ensure thatpromoter ; (ii) un paPpx is an enzyme of remarkable relevance due to its implication in polyP metabolism and, consequently, in virulence and pathogenesis of P. aeruginosa. Despite the fact that several specific aspects related to the paPpx enzyme, such as the detection of the specific binding site of the polyP chain, remain to be characterized, our present work contributes to the understanding of activity of the enzyme and of some physiological aspects of P. aeruginosa as opportunistic pathogen.Together, the foregoing data and observations point out that


Gene expression is controlled through the recruitment of large coregulator complexes to specific gene loci to regulate chromatin structure by modifying epigenetic marks on DNA and histones. Metastasis-associated protein 1 (MTA1) is an essential component of the nucleosome remodelling and deacetylase (NuRD) complex that acts as a scaffold protein to assemble enzymatic activity and nucleosome targeting proteins. MTA1 consists of four characterised domains, a number of interaction motifs, and regions that are predicted to be intrinsically disordered. The ELM2-SANT domain is one of the best-characterised regions of MTA1, which recruits histone deacetylase 1 (HDAC1) and activates the enzyme in the presence of inositol phosphate. MTA1 is highly upregulated in several types of aggressive tumours and is therefore a possible target for cancer therapy. In this review, we summarise the structure and function of the four domains of MTA1 and discuss the possible functions of less well-characterised regions of the protein. The class I histone deacetylase (HDAC) corepressor complexes are multi-protein assemblies that contain one or more enzymes that modify chromatin, components with nucleosome or DNA binding activity and proteins that function to provide a scaffold to the complex. Five major HDAC-containing corepressor complexes have been described and include nucleosome remodelling and deacetylase (NuRD) , 2, COREMetastasis-associated protein 1 (MTA1) and its homologues MTA2 and MTA3 are essential components of the NuRD corepressor complex (reviewed in –10). TheOver half of the residues of MTA1 are predicted to be intrinsically disordered, and this has presented a major challenge in the structural characterisation of this protein , methyl-CpG-binding protein 2/3 (MBD2/3) and chromatin-helicase-DNA-binding protein 3/4 (CHD3/4). Deacetylase and helicase activities are combined in this complex. HDAC1 and HDAC2 are single domain deacetylase enzymes that remove the acetyl group from modified lysines resulting in an extra positive charge on histone tails. This leads to a more compact chromatin structure as well as removing binding sites for proteins that contain bromodomains . CHD3 anOther proteins within the core NuRD complex support and enhance these enzyme activities, as well as facilitating recruitment to chromatin. RBBP4 and RBBP7 (RbAp48/46) are ∼50 kDa WD40 domain proteins that interact with chromatin and have been shown to bind to histone H4 tails . p66α anMTA1 is an 80-kDa protein that has four conserved domains within the amino-terminal half of the protein. These are a bromo-adjacent homology (BAH) domain; an egl-27 and MTA1 homology domain 2 (ELM2); a Swi3, Ada2, NCoR and TFIIIB domain (SANT); and GATA-zinc finger domain (GATA)  but without any corresponding publication. More recently, Millard et al. determined the structure of the ELM2 and SANT domains from MTA1 bound to HDAC1, revealing the nature of the interface between the two proteins as well as highlighting a mechanism of regulation through inositol phosphate signalling . FinallyThe SANT domain is a relatively small domain of around 50 amino acids and is found in a number of proteins that are involved in transcription regulation . The domThe NMR structure of the MTA3-SANT domain confirmed the predicted helix-turn-helix arrangement Fig. . InspectMore recently, the structure of a longer fragment of MTA1 that includes both the ELM2 and SANT domains bound to HDAC1 has been solved by X-ray crystallography. This structure shows that helix 3 of the SANT domain forms a basic pocket at the interface between HDAC1 and MTA1, near the active site of the enzyme . This poSequence alignments show that the SANT domain of MTA1 is highly related to the first of two SANT domains from the nuclear receptor corepressor protein SMRT . The SMRT-SANT is a critical partner of HDAC3 and is essential for HDAC3 activity . The strThe residues coordinating IP4 binding in HDAC3 and SMRT are conserved in HDAC1 and MTA1-SANT, and a similar basic pocket is present at the interface between the two proteins. This pocket is formed through the contribution of a lysine and two arginine residues from HDAC1, and two lysine and two tyrosine residues from MTA1. The similarity in size and charge of the pocket suggested that an inositol phosphate molecule could be accommodated. However, in the crystal structure, an inositol phosphate molecule is not present. Native mass spectroscopy showed that the inositol phosphate is lost during purification and is replaced during crystallisation by ordered sulphate molecules (2 M ammonium sulphate was used as a precipitant). As with HDAC3:SMRT, the addition of IP4 to HDAC1:MTA1 enhances HDAC activity, and mutation of the IP4 coordinating residues inhibits activation .i.e. all class I HDAC corepressor complexes).Interestingly, the inositol phosphate interacting residues are not only conserved in SMRT-SANT and MTA-SANT domains but also in related corepressor proteins such as RCOR1-3, MIER1-3 and RERE. These SANT domain-containing proteins are recruited to distinct corepressor complexes that recruit HDAC1, and all could potentially form an inositol-binding pocket. Since HDAC1 and HDAC2 are highly homologous, the binding of inositol phosphates will be a common activating mechanism for HDAC1, 2 and 3 , although there are predicted helical regions at the carboxy-terminus. Circular dichroism experiments show that the ELM2 domain contains essentially no secondary structure when expressed in isolation [The ELM2 domain within corepressor proteins such as MTA1-3, RCOR1-3, MIER1-3 and RERE has been shown to be required for the recruitment of HDAC1 and HDAC2 to the respective corepressor complexes. The ELM2 domain is positioned immediately amino-terminal to the SANT domain. Structure prediction of the MTA1-ELM2 domain suggests that the domain is largely intrinsically disordered  positions the active site of the two HDACs on approximately the same face of the dimer. This orientation situates the two active sites approximately 90 Å apart and could allow the complex to simultaneously target more than one nucleosome.RBBP4 and RBBP7 are WD40-repeat proteins that share 92 % identity and are integral components of the NuRD complex . RBBP4 aSeveral studies have used GST pull-down assays to map the interaction between MTA1/2 and RBBP4/7. These have suggested that there may be three non-overlapping regions in the carboxy-terminus of MTA1 that mediate this interaction. One of these includes the GATA-type zinc finger domain , 46. MorDrosophila homologue Nurf55 bound to a histone H3 tail peptide. In this structure, a peptide corresponding to histone H3 binds at the top of the propeller [The second binding site was observed in the structure of the ropeller . There iMTA contains additional domains and motifs that remain to be structurally and functionally characterised. These domains have been identified through sequence alignment with other characterised proteins. Through sequence comparison and modelling, we can speculate on how these domains will look and how they will function.Hydrophobic cluster sequence analysis identified a single copy of the BAH domain in MTA1 that spans residues 1–164 . The BAH10 helices, β-strands and ordered loops. This diversity may be a requirement of each protein to perform different functions. Structure prediction suggests that MTA1 will fold to form a β-barrel core with two long insertions.Several structures of BAH domains have been solved and show that the core of the domain is largely β-sheet in character Fig. . Beyond The BAH domain of MTA1 is most closely related to the two BAH domains of Polybromo. The proximal BAH domain of Polybromo has a six-stranded β-barrel core, the centre of which is completely filled with hydrophobic residues . A key fThe BAH domains in RSC2, ORC1 and SIR3 have been shown to mediate interactions with chromatin. If this were also the case for the BAH domain from MTA1, this would provide an attractive explanation as to how chromatin is recruited to the corepressor complex allowing the HDAC to target acetylated lysines in the histone tails.The BAH domain from the yeast RSC2 protein has been shown to bind histone H3 . HoweverThe yeast SIR3 protein not only interacts with histone tails but the BAH domain also makes extensive contacts with the nucleosome –54. The The ORC1-BAH domain has also been shown to bind to histone H4. In this case, binding only occurs when the histone tail is dimethylated at lysine 20 (H4K20me2) . The dimThese structures support the BAH domain as a histone-binding domain but the diversity within the family only allows a tentative suggestion that the BAH domain of MTA1 could bind to histone tails. Indeed, the two BAH domains from the chromatin-associated DNMT1 methyltransferase have not been shown to mediate direct interactions with chromatin . InsteadWhilst it remains to be seen whether the MTA1-BAH domain directly recruits chromatin, the location of the domain immediately adjacent to the ELM2 domain means that it will be positioned very close to the active site of the HDAC enzyme . TherefoMTA1 contains a GATA-type zinc finger domain positioned carboxy-terminal to the SANT domain. GATA-type zinc fingers are found in many eukaryotic transcription factors where they contribute to sequence-specific DNA recognition . The zinSequence comparison shows that the GATA-type zinc finger in MTA1 is rather different from GATA1. It is likely that it retains DNA-binding activity, but given the lack of conservation of the relevant residues, it will probably either recognise a different sequence from GATA1 or, perhaps more likely, may bind to DNA without sequence specificity.MTA1 contains additional motifs that remain to be structurally and functionally characterised . The xPPTowards the carboxy-terminus of MTA1, there are five SPXX motifs and these have been proposed to be sites of MTA1 phosphorylation . The phoFurther MTA1 motifs include a methylation site, which has shown to be involved in recruiting CHD4 and two nuclear localisation motifs , 63.MTA1 is a key scaffold protein in the NuRD corepressor complex and plays a vital role in both assembling the complex and activating the HDAC enzymes. MTA1 directly contacts many of the core NuRD components. A number of these interactions occur within the structurally conserved domains of MTA1, but others are directed through intrinsically disordered regions that become ordered on binding and are key to forming a stable NuRD complex.2 from solvent exposure [Three domains of MTA1 have now been characterised by NMR and X-ray crystallography. The ELM2 and SANT domains have been shown to recruit HDAC1 and/or HDAC2 through an extensive interface that excludes 1,278 Åexposure . The extexposure . The staexposure , 41, 64.The other structurally characterised motif (KRAARR) of MTA1 lies towards the carboxy-terminus. This motif has been shown to interact with RBBP4 and modulates binding to histone H4 . This coIn addition to its role as a scaffold protein, MTA1 also plays an important role in activating HDACs through forming one half of the binding site for inositol phosphates that have been shown to serve as activator molecules for class I HDACs.Intriguingly, a many of the core NuRD complex proteins have the potential to interact with DNA and chromatin. The RBBP, p66, MTA and MBD proteins all have domains that are likely to interact with either histone tails, nucleosome or DNA. This permits the NuRD complex to use a large repertoire of modules to target nucleosome remodelling and deacetylase activity to chromatin. A full understanding of the activity and mechanism of the NuRD complex will require more challenging structural studies of the intact complex and how it interacts with its chromatin substrate. Such studies are likely also to provide further insights into strategies to target the complex therapeutically.


Toll-like receptors (TLRs) are crucial components of innate immunity that specifically recognize diverse pathogen-associated molecular patterns from pathogens. The continuous hydrogen-bond network (asparagine ladder) formed among the asparagine residues on the concave surfaces of neighboring leucine-rich repeat modules assists in stabilizing the overall shape of TLR ectodomains responsible for ligand recognition. Analysis of 28 types of vertebrate TLRs showed that their ectodomains possessed three types of architectures: a single-domain architecture with an intact asparagine ladder, a three-domain architecture with the ladder interrupted in the middle, and a trans-three-domain architecture with the ladder broken in both termini. Based on a phylogenetic analysis, the three vertebrate TLR architectures arose during early evolution. The 1428 vertebrate TLRs can be divided into eight families based on sequence and structural differences. TLRs ligand specificities are affected by their ectodomain architectures. Three-domain TLRs bind hydrophobic ligands, whereas single-domain and trans-three-domain TLRs mainly recognize hydrophilic ligands. Analysis of 39 vertebrate genomes suggested that the number of single-domain TLR genes in terrestrial vertebrate genomes decreased by half compared to aquatic vertebrate genomes. Single-domain TLR genes underwent stronger purifying selective pressures than three-domain TLR genes in mammals. Overall, ectodomain architecture influences the sequence and functional evolution of vertebrate TLRs. Gene duplications are believed to the primary driving force of evolutionary innovation. Functional divergence between duplicates is usually required for their long-term retention in the genome. The retained duplicates have three distinct fates: subfunctionalization, neofunctionalization, and subneofunctionalization1Toll-like receptors (TLRs) are important pattern recognition receptors that play a crucial role in innate immunity in vertebrates468TLRs are type I transmembrane glycoproteins that are expressed on the cell surface or in intracellular compartments. They generally consist of an N-terminal extracellular ligand-binding domain, a single transmembrane helix and a C-terminal intracellular toll–interleukin 1 receptor (TIR) domain that mediates signaling12131415161718In the present study, we investigated the architectures of the vertebrate TLR ectodomains that are responsible for recognizing ligands. Based on comparative analyses of a large amount of sequence and structural information, we tried to reveal structural divergences, phylogenetic relationships, ligand preferences, and distribution characteristics in the vertebrate genomes among the different types of ectodomain architectures to provide a full-scale perspective of TLR evolution among the three levels of sequence, structure, and function.The TLR ectodomains responsible for recognizing PAMPs possess more sequence and structural variability than the intracellular signaling domains. The typical LRR modules of ectodomains include 24 amino acid residues in which the conserved “LxxLxLxxN/CxL” motifs constitute the concave surface of horseshoe-shaped ectodomain . The con20Analysis of the known TLR-ECD crystal structures showed that the ectodomains of TLR3/5/8/9/13 possessed complete asparagine ladders; therefore, they were designated as possessing single-domain architecture (SD TLRs). However, the residues in the asparagine ladder positions in the middle of the ectodomains of TLR1/2/4/6 were substituted with other amino acids, indicating that the continuous hydrogen-bond networks were broken in the middle. Therefore, their ectodomains were divided into three distinctive regions: N-terminal and C-terminal subdomains with the asparagine ladder and a central subdomain lacking the asparagine ladder. Hence, the ectodomains of TLR1/2/4/6 were designated as having a three-domain architecture TD TLRs, . The cen22Pairwise comparisons of TLR crystal structures were performed with TM-align . The aveNext, the module compositions of the representative sequences of 28 types of primary TLR ectodomains in vertebrates were thoroughly analyzed. For those TLRs with unknown crystal structures, we modeled their structures using the threading approach . The resThe compositions of the asparagine ladders in these TLR ectodomains were subsequently determined based on module predictions . The degInterestingly, the asparagine ladders throughout the TLR11/12/19/20 ectodomains were broken in both the C-terminal and N-terminal subdomains, but they were intact in the central subdomain . This waAlmost all known vertebrate TLR sequences were acquired from the database . These 1Family 1 includes TLR1/1LA/1LB/2/2A/2B/6/10/14/18/24/25 as well as TLR27Family 3 includes only TLR3, whose ectodomain possesses the single-domain architecture and contains 23 LRRs. Family 4 contains only TLR4, whose ectodomain possesses the three-domain architecture and 21 LRRs. Family 5 includes TLR5/5S/5SL. Although TLR5S/5SL lack transmembrane and intracellular domains, both of their ectodomains possess the single-domain architecture and include 21 LRRs (similar to TLR5). Family 7 includes TLR7/8/9, the ectodomains of which also possess the single-domain architecture and 25 LRRs.Family 11 is unique because TLR11/12/16/19/20/26 and TLR13/21/22/23 are divided into two clades at the root of the phylogenetic tree. The asparagine ladders of the ectodomains of TLR11/12/16/19/20/26 are broken in the C-terminal and N-terminal subdomains. Thus, their ectodomains may possess a trans-three-domain architecture. In contrast, the ectodomains of TLR13/21/22/23 possess intact asparagine ladders. Thus, they may possess the single-domain architecture. Hence, we propose that the original family 11 is split into two families: the new family 11, including TLR11/12/16/19/20/26; and family 13, including TLR13/21/22/23.Phylogenetic analysis showed that the ectodomains of TLRs belonging to the same family nearly always contained the same number of LRR modules and possessed the same architecture . FurtherThe ectodomains of vertebrate TLRs mediate the recognition of PAMPs. Although only the ligand-binding specificities of partial TLRs have been studied to date and although there may be some overlap in the ligands recognized by different TLRs, different ectodomain architectures evidently affect the ligand-binding specificities of TLRs .TD family 1 is the biggest TLR family and includes approximately one-third of the total number of known vertebrate TLRs. Family 1 can be further divided into three groups based on phylogenetic relationships . Group A2526TLR3 in SD family 3 exists in all vertebrate taxa as a single gene copy in each species’ genome. Hence, family 3 is the most conservative TLR family. TLR3 responds to double-stranded RNA (dsRNA) by forming homodimers in intracellular vesiclesTLR4 in TD family 4 has been reported in Reptilia, Mammalia and Aves. TLR4 homodimers recognize lipopolysaccharides (LPS) on the cell surface with the help of two MD-2 coreceptorsTLR5 in SD family 5 is expressed on the cell surface and recognizes bacterial flagellin in the form of a homodimerTLR7 in SD family 7 has been identified throughout the major vertebrate taxa, whereas both the TLR8 and TLR9 genes are lost from the avian genomes. Both TLR7 and TLR8 mediate the recognition of single-stranded RNA (ssRNA); moreover, TLR8 homodimers were recently verified as being able to sense ssRNA degradation products1733Xenopus and TLR19, TLR20 and TLR26 have only been found in some species of teleostToxoplasma gondii3739The known members of TTD family 11 only exist in a few vertebrate taxa. Both TLR11 and TLR12 are only found in mammals, whereas TLR16 is unique to SD family 13 can be divided into two groups based on phylogenetic relationships: group A including TLR13/21 and group B including TLR22/23 . IntriguTLR15 is the sole member in family 15 and is unique to avian and partial reptilian lineages. TLR15 recognizes secreted virulence-associated fungal and bacterial proteasesTaken together, three-domain TLRs were found to mainly recognize hydrophobic ligands, the majority of which are microbial membrane components such as lipids, lipoproteins, and LPS. In contrast, single-domain and trans-three-domain TLRs chiefly respond to hydrophilic ligands, such as nucleic acids and proteins. Three-domain TLRs are mainly expressed on the cell surface, whereas single-domain and trans-three-domain TLRs are chiefly expressed in intracellular vesicles, such as endosomes. Furthermore, both three-domain and trans-three-domain TLRs have been reported to possess some TLRs that work in the forms of functional heterodimers, whereas all known single-domain TLRs have been reported to form homodimers to bind ligands.The distribution of TLR genes was analyzed in 39 vertebrate genomes . The resdN/dS value at each TLR locus in the genomes of Mammalia, Aves and Actinopterygii was estimated  to the number of synonymous substitutions per synonymous site (dS), it can assess the functional constraint differences acting on distinct TLR genes. The results showed that the mean dN/dS values at TLR loci in the three taxa ranged from 0.173 to 0.529, suggesting that the evolution of vertebrate TLR genes was mainly dominated by purifying selection and a low proportion of non-synonymous substitutions helped preserve the biological function of the TLRs. The dN/dS values of single-domain TLR loci were significantly lower than those of the three-domain and trans-three-domain TLR loci in mammalian genomes, suggesting that single-domain TLRs may have been subjected to stronger functional constraints  and Ensembl databases. All partial sequences (<1800 bp) and pseudogene sequences were excluded. The taxonomic designations of species from which each TLR gene sequence originated were obtained from the NCBI Taxonomy database. In total, 1428 TLR sequences from 221 vertebrate species were collected . The disI-TASSER was used to predict the structures of ectodomains and TIR domains for TLRs that had unsolved crystal structures 47. I-TASPairwise structural differences of TLRs were measured with TM-alignFirst, we identified the structural elements of the representative sequence for each type of TLR. The signal peptide, ectodomains, transmembrane region, and intracellular TIR domains were predicted by SignalP, SMART, and TMHMM5051A multiple sequence alignment of the full-length protein sequences of vertebrate TLRs was implemented using MAFFT dN) to the number of synonymous substitutions per synonymous site (dS), dN/dS, an indicator of the selective pressure acting on a protein-coding gene, at the TLR loci and the corresponding 95% confidence intervals were calculated with the Datamonkey web serverA multiple sequence alignment of the nucleic acid sequences of the TLR genes, based on their codons, was constructed by TranslatorX and MAFFTHow to cite this article: Wang, J. et al. Ectodomain Architecture Affects Sequence and Functional Evolution of Vertebrate Toll-like Receptors. Sci. Rep. 6, 26705; doi: 10.1038/srep26705 (2016).


The human genome encodes 19 cyclophilins, 18 FKBPs and three parvulins. Immunophilins are receptors for the immunosuppressive drugs cyclosporin A, FK506, and rapamycin that are used in organ transplantation. Pin1 has also been targeted in the treatment of Alzheimer’s disease, asthma, and a number of cancers. While these PPIases are characterized as molecular chaperones, they also act in a nonchaperone manner to promote protein-protein interactions using surfaces outside their active sites. The immunosuppressive drugs act by a gain-of-function mechanism by promoting protein-protein interactions in vivo. Several immunophilins have been identified as components of the spliceosome and are essential for alternative splicing. Pin1 plays roles in transcription and RNA processing by catalyzing conformational changes in the RNA Pol II C-terminal domain. Pin1 also binds several RNA binding proteins such as AUF1, KSRP, HuR, and SLBP that regulate mRNA decay by remodeling mRNP complexes. The functions of ribonucleoprotein associated PPIases are largely unknown. This review highlights PPIases that play roles in RNA-mediated gene expression, providing insight into their structures, functions and mechanisms of action in mRNP remodeling in vivo.The peptidyl-prolyl  Remodeling of messenger ribonucleoprotein (mRNP) complexes is a dynamic process that is essential for gene expression ,2. Althoi.e., the ability to lower the rotational energy barrier about the prolyl imide bonds, thereby facilitating cis-trans proline isomerization and inducing conformational changes in their substrates [cis-trans isomerization. Furthermore, as has been shown for the cyclophilin PPIL1, cyclophilins can utilize surfaces outside the PPIase domains to promote protein-protein interactions in mRNP complexes. Several cyclophilins also have accessory domains, such as RRMs, U-box, TPR domains, and WD40 repeats [The first cyclophilin to be discovered was CyPA , a smallbstrates ,8. Howev repeats  that areX-ray crystal structures and solution NMR structures are available for cyclophilins from different species, in the unliganded form, as well as complexed to peptide ligands. Some of the structural features are highlighted in the sections below. Although most cyclophilins are non-essential proteins, they have received attention as drug targets in a spectrum of diseases due to their diverse roles in signaling and control of gene expression pathways. Eight cyclophilins that participate in RNA-mediated gene expression, and in particular pre-mRNA splicing  are high10 helix that pack against the β-sheet  towards the substrate N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide. PPIL1 is also inhibited by cyclosporin A.The peptidyl prolyl isomerase-like protein 1 (PPIL1)  is a 166is 1.2 Å . The actis 1.2 Å . As a rein vitro and Surface Plasmon Resonance (SPR) experiments determined a binding constant (DK) of ~0.125 μM [i.e., strands β2, β7, and the loop preceding β5) and N-terminal residues 59–129 of SKIP [2 and it involves 26 residues from PPIL1 and 19 residues from SKIP. The interface is predominantly hydrophobic, although electrostatic interactions also play a role in complex stability.The SKIP-PPIL1 interaction is of medium affinity  of SKIP  shows thTrypanosoma brucei (T. brucei) has also been found to be part of a non-snRNP PRP19 complex that is comprised of PRP19, CDC5, PRL1, SPF27, PRP17, and SKIP as the core components [T. brucei. The identification of the PPIL1-SKIP complex in humans and trypanosomes indicates an essential role for this complex exists in spliceosome activation that is conserved in evolution.The precise biological role of human PPIL1 in facilitating the B → B* transition in the spliceosome is unclear. Since SKIP is an adaptor like protein that interacts with other spliceosomal proteins, the hypothesis is that PPIL1 may act as a chaperone or a PPIase to facilitate conformational changes in the 45 S activated spliceosome. Recently, PPIL1 from mponents . This PRPPIL2 or Cyp60 is a large ~60 kDa (520 amino acids) protein that has a central cyclophilin-like domain and also has an N-terminal U-box domain that exhibits E3 ligase activity . PPIL2 hα atoms of CyPJ and CyPA is 1.8 Å. There are small differences between the two proteins that correspond to surface insertions/deletions in four turns and loops. The most notable difference is a longer loop that is present corresponding to the segment Pro135-Asn145 that is stabilized by three backbone-backbone H-bonds. In addition CyPJ has a unique disulfide bridge between Cys18 and Cys25 that is not present in CyPA The active site of CyPJ is identical to that of CyPA.The PPIL3 gene produces two alternatively spliced cyclophilin like proteins PPIL3a and PPIL3b, both of which are ubiquitously expressed in human tissues . PPIL3b in vitro [HOXC8, HOXA9, CDKN1B, and C-MYC, by directly altering H3K4 methylation and H3 acetylation at the promoter regions of these genes [cis-trans isomerization about the His1628-Pro1629 peptide bond that lies in the linker sequence between the PHD3 finger and the bromeodomain of MLL1 [PPIE or Cyp33 is a nuclear cyclophilin that has an N-terminal RNA recognition motif (RRM) that can bind poly (A) and poly (U) tracts of RNA, but not poly (G) and poly (C) RNAs . RNA binin vitro . PPIE wain vitro . The proin vitro . The 1.8in vitro . The ovein vitro , howeverin vitro ,27. MLL1se genes . PPIE prse genes . Therefo of MLL1 . SolutioA. thaliana (AtCyp59) [AtCyp59 also binds the RNA Pol II Carboxy-terminal domain (CTD), facilitating in its dephosphorylation and slowing cell growth [PPIL4 is another RRM containing cyclophilin that has been found to be a component of the spliceosome ,30. It cAtCyp59) . AtCyp59l growth .α atoms of PPWD1 and CyPA have an r.m.s.d of 1.34 Å over 124 atoms. The main structural difference between the isomerase domains lies in the β1-β2 loop, which is shorter by five residues in PPWD1 as compared to CyPA. Intriguingly, the crystal structure reveals intermolecular interactions of the PPIase domain with an internal Gly-Pro peptide from another PPWD1 molecule in the asymmetric unit that binds in a trans-Pro conformer. PPWD1 is able to bind this peptide, but kinetic measurements demonstrate that it does not exhibit PPIase activity towards this peptide [vs. catalysis of PPWD1 are not yet understood. The role/s of the accessory WD40 domain in mediating protein-protein interactions in the spliceosome are unclear.PPWD1 is a large PPIase that has a C-terminal CyPA-like domain and has four WD40 repeats in the N-terminal domain. It has been found to be a component of the human spliceosome C complex ,30. The  peptide . In cont peptide . The seqkcat/KM value of 8.8 × 106 M−1·s−1 was measured for CyPA and a kcat/KM of 1.3 × 106 M−1·s−1 for PPIH towards the same tetrapeptide substrate [Cyclophilin H (CyPH) or PPIH was first reported to be a component of the U4/U6 small nuclear ribonucleoprotein particle (snRNP) by Lührmann and colleagues in 1998 . PPIH isubstrate . PPIase 10-helices, one that lies in the loop connecting strands β6 and β7 that is present in CyPA, and the second in a loop between helix α2 and strand β8 that is absent in CyPA. In addition, PPIH has a longer N-terminus (by seven amino acids), an extra amino acid in the loop connecting helix α2 and strand β8, and five additional amino acids in the loop connecting helix α1 and strand β3, compared to CyPA. The presence of an additional amino acid in the loop between helix α2 and strand β8 results in an altered conformation for residues in the loop, which adopts a short 310-helix. The presence of five amino acids in the loop connecting helix α1 and strand β3 introduces a binding cleft in the protein that is predominantly hydrophobic and may be important for protein-protein interactions. The active site superimposes perfectly with that of CyPA and the root mean square deviation (r.m.s.d) for 158 shared backbone Cα atoms of PPIH and hCyPA is 0.8 Å.The 2.0 Å crystal structure of PPIH  shows thdi-snRNP, Prp18 is involved in the assembly of the tri-snRNP (U4/U6.U5) and plays a role in the second trans-esterification reaction. hPrp4 and hPrp18 have a 31 amino acid region that is highly homologous in sequence and this sequence and binds PPIH in both proteins [α atoms of free and bound PPIH is 0.56 Å. The peptide binds a region on PPIH that is remote from the PPIH active site. The equilibrium dissociation constant (KD) for the PPIH-hPrp4 peptide was determined to be 1.97 μM by isothermal titration calorimetry. The hPrp4 peptide is disordered in the absence of PPIH but folds into short α-helices (α1 and α2) that are connected by a loop that has a short β-strand (β1) in the crystal structure of the complex. The β-strand of hPrp4 (residues Ile118-Phe121) interacts with strand β8 of PPIH via backbone H-bonds, thereby extending the β-sheet of the cyclophilin. The total buried surface area between PPIH and hPrp4 peptide is 878 Å2. The side chain of Phe121 of the hPrp4 peptide that lies at the edge of the β-strand inserts into a hydrophobic cavity of PPIH formed by Pro57, Ile58, Gly59, Tyr60, and Lys61. The structure reveals that PPIH interacts with other spliceosomal proteins, such as hPrp4 and hPrp18 utilizing a surface that is removed from the active site, thereby leaving the enzyme catalytically active to facilitate prolyl cis-trans isomerization on its substrates.PPIH interacts specifically with hPrp3 and hPrp4 (or U4/U6-60K)  and the proteins . In hPrpproteins . No largcat/KMk compared to CyPA [PPIG or Clk associating RS-cyclophilin (CARS-Cyp) was identified in a yeast two-hybrid screen using the Clk (CDC28/cdc2-like kinase) as a bait in 1996 . Clk kin to CyPA . Its PPIin vivo and in vitro [PPIG has been shown to interact directly with the phosphorylated RNA Pol II Carboxy-terminal domain (CTD) via its RS domains in vitro . Since tcis-trans isomerases that participate in protein trafficking, signaling, and transcription as foldases or chaperones as their normal function in the cell. However these receptors act by a gain-of-function mechanism when bound to drugs, binding the primary member of the FKBP family, FKBP12. The FKBP12-FK506 complex inhibits the activity of a calmodulin-dependent protein phosphatase, calcineurin [The immunosuppressive drugs FK506 and rapamycin that are used in organ transplantation bind protein receptors called FK506 binding proteins (FKBPs) in T-cells ,43,44,45cineurin , whereascineurin  that areHuman FKBP51 and FKBP52 show 55% sequence identity and have a common domain structure consisting of an N-terminal FKBP domain (Fk1), and FKBP-like domain (Fk2), and a TPR domain at the C-terminus. Several X-ray crystal structures and solution NMR structures of FKBP domains are now available . All FKBFKBP 4 and 5 have been identified as novel components of the RNA-induced silencing (RISC) loading complex in embryonic stem cells  and HeLaFkbp6 show male infertility. Surprisingly, the N-terminal PPIase domain of FKBP6 lacks prolyl isomerase activity, and does not interact with FK506, even though the overall fold of the PPIase domain is similar to that of the “active” FKBP12. The crystal structure shows that this active site of FKBP6 is quite different from FKBP12, explaining the lack of observed isomerization activity in enzyme assays. FKBP6 interacts with Hsp90 in mouse testes extracts, as well as HEK293T cells, and this interaction is dependent on the C-terminal TPR domain. The biological data implicate a role for FKBP6 in piRNA biogenesis via interaction with Hsp90, although the exact mechanism is not clear [Similar to FKBP4/5 above, FKBP6 associates with Hsp90 via its tetratricopeptide repeat (TPR) domain to deliver piRNAs to the Piwi protein Miwi2 in mice . Piwi prot clear .FKBP25 is a nuclear PPIase that participates in epigenetic regulation of gene expression, as well as ribonucleoprotein complexes. It is known to directly associate with the histone deacetylases HDAC1 and HDAC2  via the E. coli Par10, the founding member of this sub-family is ~10 kDa consisting of only the PPIase domain. In humans, the Parvulin family of cis-trans prolyl isomerases includes Pin1, Par14, and Par17. Pin1 is the most well characterized parvulin and is a drug target for treatment of cancer, Alzheimers disease, asthma, and inflammation. Pin1 regulates the protein and mRNA levels of several proto-oncogenes, cytokines, chemokines, and tumor suppressors implicated in disease. Pin1 can alter gene expression by affecting transcription or mRNA stability. It has also been implicated in regulating protein levels by altering the sub-cellular localization and ubiquitination of protein targets. The role of Pin1 in posttranscriptional mechanisms that control gene expression is summarized in greater detail below. In addition to Pin1, there is an additional locus in the human genome on Chromosome Xq13 that encodes two other parvulins: Par 14 and Par 17. Par14 and Par17 are less well characterized biologically, but have been implicated in chromatin remodeling and cell proliferation.Parvulins form the third family of PPIases that are conserved in prokaryotes and eukaryotes . Parvulicis-trans isomerization of proline residues in proteins that have a phosphorylated serine or threonine preceeding a proline. Pin1 frequently acts along with kinases and phosphatases to induce conformational changes in its target proteins thereby regulating their activity. Pin1 protein targets include several oncogenes and transcription regulators such as c-Myc [The peptidyl prolyl isomerase Pin1 is a regulator of the cell cycle and is required for progression of cells into mitosis ,59,60. Pas c-Myc , c-Jun [as c-Myc , p53 [63as c-Myc , beta-caas c-Myc , as wellas c-Myc  and Cyclas c-Myc  and seveas c-Myc ,69,70. Pas c-Myc ,72,73 byas c-Myc ,75,76.RPB7 and the CTD phosphatase FCP1 could rescue the growth defects of the Ess1 temperature sensitive mutations [The functional role of Pin1 in modulating transcription via interaction with the RNA Pol II CTD has been particularly well-studied. The first insight into the role of Pin1 in interaction with the RNA Pol II CTD came from the yeast orthologue of Pin1 called Ess1 ,78. Temputations , when prutations . These sin vitro and in vivo [cis-Pro in this study, leading to an apparent increase in the activity of the Ssu72 phosphatase, which is specific for cis-Pro containing phosphopeptides. In contrast, the phosphatase activity of Scp1 was not significantly affected by Pin1, since Scp1 recognizes only the trans-Pro isomer. Therefore, Pin1 can affect the response of phosphatases in vivo by regulating the conformation of the CTD by proline isomerization.In mammals, Pin1 has also been shown to interact with RNA Pol IIO  and can  in vivo . This de in vivo . Ess1 is in vivo . It does in vivo . The cro in vivo . Pin1 racis-trans prolyl isomerization therefore is not clear. Pin1 is capable of a bivalent interaction with its substrates [Numerous X-ray and NMR structures of full-length Pin1 and its sub-domains have been deposited in the Protein Data Bank , either bstrates  in whichth [cis-elements (ARE) in their 3' untranslated regions (3' UTRs) and play important roles in control of the immune response in inflammation and asthma. These ARE sequences bind ARE binding proteins (ARE-BPs) such as AUF1 and KSRP, that regulate mRNA turnover. Pin1 regulates mRNA turnover of GM-CSF mRNAs by binding AUF1 that is phosphorylated by ERK at Ser83-Pro84 so as to dissociate AUF1 from the GM-CSF mRNA, preventing AUF1-mediated mRNA degradation [th mRNA in vivo [in vivo [l-motif RNA-binding domain of all SLBPs [N-terminus of SLBP [Several recent studies indicate that the prolyl isomerase Pin1 regulates mRNA stability for a subset of eukaryotic mRNAs. Pin1 has been implicated in control of mRNA stability and turnover of the cytokine GM-CSF ,105,106,th ,108, andth  mRNAs, aradation . Pin1-me in vivo ,111. Oth[in vivo . We rece[in vivo , a rate ll SLBPs ,114 and ll SLBPs ,115, alt of SLBP . The Pin of SLBP .In addition to these targeted studies, Pin1 gene silencing combined with microarray based gene expression profiling and quantitative RT-PCR shows that cellular pathways, namely cell adhesion, phosphatidylinositol signaling, and leukocyte migration are predominantly perturbed in response to Pin1 gene silencing . All corE. coli Par10 than Pin1. The five amino acid insertion forms a loop that lies ~10 Å away from the active site and, therefore, is not likely to be involved in substrate recognition.Par14 has a C-terminal parvulin-like PPIase domain and an N-terminal basic domain whereas Par17 has a longer N-terminal helical extension compared to Par14. The PPIase domains of Par14 and Par17 have a five amino acid insertion near the C-terminus  that is not present in Pin1. Par14 can partially compensate for Pin1 depletion in mouse endothelial fibroblasts (MEFs)  in cell ε2-His42-Nδ1—H-Nδ1-His123-Nε2—HO-Thr118. The hydrogen-bonding pattern is consistent with hydrogen bonds reported for the two histidines in a recent NMR study of the related parvulin PrsA from S. aureus [Active site residues that are important for catalysis in Pin1 are different in Par14. In particular, the nucleophile C113 in Pin1 that plays an important role in catalysis is an aspartic acid (Asp74) in Par14. A recent 0.8 Å crystal structure of Par14  implicat. aureus . The rotin vivo [Although Par14 and Par17 are conserved across species, their functions remain obscure. In HEK293 cells, Par14 is present in both the nucleus and the cytosol ,123. On in vivo . Par14 iin vivo ,127 and PPIases from all three subfamilies play biological roles in a number of cellular processes, yet they are non-essential genes. This makes them attractive targets in the treatment of a number of diseases. PPIase inhibitors have been used in the clinic as immunosuppressive agents and hold promise as molecular targets for therapy in a number of diseases ,129,130.