Interestingly, the chimeric protein could catalyse site-specific integration in bacteria with an efficiency comparable to that of TrwC, while the HUH domain of TrwC alone was unable to catalyze this reaction, implying that the Rep68 C-terminal helicase domain is complementing the TrwC HUH domain to achieve site-specific integration into TrwC targets in bacteria. Our results illustrate how HUH domains could have acquired through evolution other domains in order to attain new roles, contributing to the functional flexibility observed in this protein superfamily.Endonucleases of the HUH family are specialized in processing single-stranded DNA in a variety of evolutionarily highly conserved biological processes related to mobile genetic elements. They share a structurally defined catalytic domain for site-specific nicking and strand-transfer reactions, which is often linked to the activities of additional functional domains, contributing to their overall versatility. To assess if these HUH domains could be interchanged, we created a chimeric protein from two distantly related HUH endonucleases, containing the N-terminal HUH domain of the bacterial conjugative relaxase TrwC and the C-terminal DNA helicase domain of the human adeno-associated virus (AAV) replicase and site-specific integrase. The purified chimeric protein retained oligomerization properties and DNA helicase activities similar to Rep68, while its DNA binding specificity and cleaving-joining activity at  For the annealing reaction, the labelled oligonucleotide was mixed with the corresponding complementary strand and boiled for 5 min in a 1l beaker, then the water was left to cool down to <30°C. All labelled substrates were purified using illustra Microspin G50 columns .To generate the heteroduplex substrates we first labelled the top strand with .2 μl of the episomes were electroporated into SURE bacteria (Stratagene). The electroporated bacteria (30 μl) were diluted in 1 ml of LB and incubated at 30°C for 1 h. 100 μl of the electroporated bacteria were plated on LB/Amp plates to determine the number of AmpRibbon diagrams of the N-terminal part of TrwC (PDB ID: 1OSB) and Rep (PDB ID: 1M55), as well as the alignment of the two proteins, were generated using PyMOL Molecular Graphics System .In order to test the functional conservation of HUH endonucleases, we engineered a chimera between two distantly related HUH superfamily members: the Rep68 protein from the human virus AAV2 and the conjugative relaxase TrwC from the bacterial plasmid R388.Superimposition of Rep68 and TrwC  shows thWe generated a TrwC-Rep chimera by fusing the relaxase domain of TrwC (N293-TrwC), capable of sequence-specific DNA nicking and strand-transfer reactions , 41, to trwC/rep chimeric gene into a eukaryotic expression vector. Transfection of 293T cells with this vector showed that the chimeric protein exhibits the expected molecular weight and is stably expressed in human cells ) (oriTw(25+8) oligonucleotide, resulting in a mobility shift of 49% of the radiolabeled DNA (oriTw(25+8) competitor. The interaction was highly substrate-specific, as the TrwC/Rep chimera did not bind to a mutated binding site (IRmut) or the Rep68 substrate (AAV ori) ) . The Trweled DNA . The shiAAV ori) . These roriTw. The association constant (Kd) for TrwC/Rep chimera was 16 nM, which indicates a high affinity binding of the protein to its substrate , or AAVS1  were incubated as described in Materials and Methods. If endonuclease activity is present, the plasmid conformation will change from sc to an open circular (oc) form  form , which ion AAVS1 , 35. TheoriTw (34mer) substrate and subjected to analytical ultracentrifugation  was mobilized into the recipient bacteria by TraI, the conjugative relaxase of the RP4 conjugative system, which recognizes oriTp. The recipient bacteria harbour an oriTw-containing plasmid (pLA32) and another plasmid expressing either N293-trwC (pSU1600), trwC (pSU1621) or trwC/rep chimera (pLA131).Unexpectedly for a conjugative relaxase, TrwC is able to integrate the transferred ssDNA site-specifically into both an romosome , 43. To -5 (-5 integrants/donor cell)  . Fig 7C This positive result prompted us to assess the potential of the chimera to perform site-specific integration in human cells, using an episomal assay similar to the one developed to determine the requirements for AAV Rep-mediated site-specific integration into human host DNA . BrieflyoriTw in both orientations) were transfected with the suicide plasmid pCMS11, and either pLA117 (expressing trwC/rep chimera) or pLA119 (expressing trwC). As a control for transfection efficiency, C17 cells carrying these episomes were transfected with plasmid pIRES-eGFP, and an efficiency of around 15% transfected cells was obtained (not shown). As a positive control, C17 cells carrying p220.2::AAVS1(kb 0–0.5)  were infected with wt AAV at a high MOI to ensure 100% infection efficiency.To test TrwC and TrwC-Rep chimera for their ability to mediate site-specific integration, C17 cells carrying either pLA58 or pLA59  [R colonies screened respectively, reacted with the pCMS11 probe (The results are shown in iciency) . However11 probe . We alsoHUH endonucleases define a superfamily of proteins present across all biological kingdoms; it comprises proteins from bacterial plasmids, plant and mammalian viruses, and transposases from bacteria, archaea and eukaryotes. Their functions vary, ranging from bacterial conjugation to DNA transposition and viral replication by RCR . To achiIn this work, we focus on two of these members, the viral master replication protein AAV2-Rep68 and the bacterial conjugative relaxase R388-TrwC, both known to perform integration of ssDNA into a sequence-specific dsDNA target in human and bacterial cells, respectively. Both proteins have been well-characterized biochemically, rendering them suitable candidates to determine the requirements for their SSI activity through a domain swapping approach. To this end, we designed a TrwC/Rep chimeric protein composed of the N-terminal domain of TrwC, and the C-terminal domain of Rep68. The chimera contains the N293 relaxase domain of TrwC, which is able to perform sequence-specific binding and nicking on scDNA, and thus we expected the chimera to retain the HUH activities of TrwC. The chimera further contains the C-terminal SF3 helicase domain of Rep68. The two domains are joined by the interdomain linker of Rep68 that was shown to be required for Rep68 oligomerization. It is important to note that N293-TrwC is not sufficient to catalyse SSI in bacteria on its own. We wanted to test if the C-terminal domain of Rep68 could contribute to the function of the chimeric protein by allowing it to interact with DNA structures and proteins from the host cell that may be required to achieve SSI.oriTw sequences, and catalyzed nicking of scDNA plasmid DNA containing oriTw, but not AAVS1. Taken together, our biochemical data showed not only that the TrwC/Rep chimeric protein maintains a functional HUH domain, but also that the redirection of this new protein is highly oriTw-specific since it does not recognize control substrates, highlighting the specificity of HUH proteins for their target DNAs.The engineered chimeric protein was found to be stable when expressed in bacterial and human cells , which aSimilarly, the properties provided by the C-terminal domain of the chimera, namely DNA unwinding activity and oligomerization, were expected to be similar to the parental protein Rep68. However, some differences between the parental Rep68 and the TrwC/Rep chimera were apparent. The results of the EMSA assay suggested that different oligomeric species are formed by the TrwC/Rep chimera compared to Rep68 . Our work goes one step further to show cross-talk between functional domains of two distantly related HUH proteins in order to attain new functions. Future work using Rep68 mutations, or addressing interacting partners of the chimeric and parental proteins, will shed light into the requirements needed for a HUH endonuclease to become a site-specific integrase.


With the decreasing strain rates, the grain boundary/interface sliding gradually became prominent, which resulted in some superplastic deformation features, e.g., intensive strain-induced grain growth and interface migration, enhancing “wetting” of the γ grain boundaries, continuous weakening/vanishing of the local texture, etc. Meanwhile, the temperature played an insignificant role in the hot deformation behavior. The deformation mechanism was discussed in detail based on the microstructural observations and deformation kinetics.In this study, a Ti-40Al-10V alloy with quenching-tempering microstructure was prepared and was characterized by ultra-large β/B2 grains and submicrocrystalline γ laths within it. A definite Kurdjumov-Sachs orientation was identified between the β/B2 and γ phase. Isothermal compression tests were performed to examine the hot deformation behavior at various temperatures and strain rates. Based on the hyperbolic-sine equation, the deformation kinetics of the alloy were characterized by unexpectedly high activation energy (384 kJ/mol) and low stress exponent (2.25). For all the deformed samples, continuous dynamic recrystallization intensively occurred in the β matrix, accompanied by the simultaneous rotation of the γ laths. Moreover, a preferential orientation of <100> Thirdly, as the decreasing strain rate, the pole intensity is continuously weakened. At the lowest strain rate, the <111>γ fiber tends to be decomposed as that in the β phase.As for the γ phase, the pole figures shown in d by DRX ,26,27. I instead , which a instead . Therefo−1 under different temperatures. One can note that for the β matrix  TiAl alloys, for either the power-law creep or the power-law breakdown regime. However, the deformation behavior of the present alloy reflects some unexpected features from both kinetics and microstructure aspects, indicating unique deformation mechanisms.In general, the intragranular deformation is predominant during the dislocation creep of metallic materials including ordinary (αβ fiber is produced via the synergic effect of strain-induced GB migration and continuous DRX [2 + γ) TiAl alloys where the remnant α2/γ lamellar colonies aligned perpendicular to the deformation direction [2/γ lamellae. Therefore, it may be speculated that the habit plane for the present γ laths is still {111}γ [γ orientations align parallel to the CD, and hence a single {111}γ fiber is produced rather than the typical deformation texture. In addition, by using the proposed mechanism, i.e., deformation is mainly concentrated within β matrix accompanied by γ lath rotation, can also clarify the secondary hardening of the flow curve at highest strain rate , the predominant deformation mechanism is the intragranular deformation of β phase at all temperatures. The GBS, as well as the interface sliding, plays an insignificant role. However, the increasing temperature causes the strain-induced GB migration more readily and therefore the <100>β intensity is increased. Simultaneously, the rotation process of the γ laths is also accelerated by the increasing temperature, which leads to an increasing <111>γ intensity.In comparison with strain rate, temperature has an insignificant effect on the deformation behavior. As pointed out in Z regime. For all the deformation conditions, the rate-controlling mechanism is believed to be concentrated in the soft β phase. All the microstructure observations, as well as the change of the stress exponent  in the β matrix, whereas the GBS and interface sliding are predominant in the low exponent c, can beAs pointed out in the Introduction, the hyperbolic-sine law (Equation (1)) is widely used to describe the deformation kinetics of metallic materials, because it describes the dislocation creep behavior in the power-law and power-law breakdown regimes quite accurate . Howeverσ item is replaced by the sinh(ασ) item in order to straighten the stress-strain rate curves in a logarithmic scale . Secondly, the Qapp in the hyperbolic-sine law is an empirical variable which is just an approximation of the effective diffusion energy (QD in the power-law equation). Here we use a simple example to reveal the application conditions for the hyperbolic-sine law. There are two mathematical differences. Firstly, in the hyperbolic-sine law the Qapp is 153 kJ/mol, and the corresponding n value is 4.0. If uses the data at low stresses (power-law creep regime), the calculated Qapp and n value are 152 kJ/mol and 5.0, respectively. Concurrently, in the high-stress regime (power-law breakdown) the n is about 7.1 whereas the Qapp value is nearly unchanged (151 kJ/mol). Therefore, one can conclude that the Qapp value is almost independent on the applied stress from the power-law creep to power-law breakdown. This is believed to be a key factor for the application of the hyperbolic-sine law. Such a conclusion is also valid for (α2 + γ) TiAl alloys, where the apparent activation energy for the power-law breakdown is 370 kJ/mol and is nearly equal to that of power-law creep [The applied dislocation creep data of pure Al is quoted from Ref. . This craw creep .Qapp value is about 325 kJ/mol, while in the low strain rate regime it is only 245 kJ/mol. Note that both values are much lower than that calculated by using the entire dataset  TiAl alloys  was 384 kJ/mol, and the stress exponent value was determined to be ~2.25. Detailed analysis revealed that the Qapp values were 325 kJ/mol and 245 kJ/mol at high and low strain rate range, respectively, with corresponding stress exponent values of 2.7 and 4.5. The hyperbolic-sine law was believed to be invalid when it was not the case of dislocation creep, especially there was a transition in rate-controlling process.The flow curves were characterized by a single peak at the initial stage and then continuously soften to reach a steady-state flow. However, the flow curve at 0.5 sβ and <111>γ parallel to the compression axis was observed for β and γ phase, respectively. With the decreasing strain rates, the grain boundary/interface sliding gradually became prominent which resulted in some superplastic deformation features such as intensive strain-induced grain growth and interface migration, as well as continuous weakening/vanishing of the local texture, etc. Meanwhile, the temperature had slight effects on deformation behavior. After a detailed discussion, it was believed that the deformation mechanism was the intragranular deformation (or dislocation creep) in the β matrix under high strain rates, but gradually evolved to the grain boundary/interface sliding mechanism at low strain rate regime.For all the deformed samples, continuous dynamic recrystallization intensively occurred in the β matrix, accompanied by the simultaneous rotation of the γ laths. Meanwhile, a preferential orientation of <100>In this study, the deformation behavior of a Ti-40Al-10V alloy with Q&T microstructure has been examined by isothermal compression at various temperature and strain rates. It was found that, despite the ultra-coarse grain structure, the present alloy with a Q&T microstructure exhibited superplastic flow even at industrial wrought conditions, whereby the hot workability was expected to be significantly improved considering the natural brittleness of TiAl alloys. Such an alloy & microstructure design may provide a new strategy for fabricating complex-shaped but cost-effective TiAl products. The main conclusions for the present study are drawn below:


It is uncommon for a bronchial stump-related complication to develop during the remote postoperative period in a case of obstructive pneumonia owing to migration of the suture material. Here, we describe a case of bronchial obstructive pneumonia that developed owing to migration of the suture material in the airway 8 years after pulmonary resection.A 34-year-old woman underwent left lower lobectomy for a pulmonary carcinoid tumor (pT1bN0M0-stage IA) in 2010. She experienced obstructive pneumonia, and chest computed tomography revealed a mass protruding from the bronchial stump to the bronchial lumen in 2018. After treatment for pneumonia, flexible bronchoscopy showed the presence of a fibrous suture material (Teflon pledget) completely obstructing the left second carina. A week later, the Teflon pledget obstructing the bronchial lumen was removed using a flexible bronchoscope with the patient under general anesthesia. The procedure was completed without removing the small amount of granulation tissue because the bronchial lumen opened after removing the Teflon pledget. She has remained asymptomatic for 1 year after removal.In this case, the complication of obstructive pneumonia developed owing to migration of the non-absorbable suture materials used to suture the bronchial stump. Bronchoscopic management of this rare complication comprised endobronchial removal with the patient under general anesthesia. Given our experience with this case, we believe that such conservative management should allow for excellent results in most instances and avoid the need for reoperation. Bronchial fistula is a critical complication after pulmonary resection. Different techniques of reinforcing the bronchial stump to prevent this complication have been described, and the incidence of this complication has decreased in recent decades. Pledget sutures have been suggested for this purpose but have the potential to become infected and erode into the bronchus years after pulmonary resection. For example, there are a small number of reported cases of intrabronchial granuloma formation owing to the suture material used in pulmonary resection during the remote postoperative period , 2. HoweA 34-year-old woman underwent left lower lobectomy for a pulmonary carcinoid tumor (pT1bN0M0-stage IA). Postoperatively, there was no pulmonary fistula or bronchial stump fistula, and her clinical course was favorable. She had been doing well for 8 years after completing therapy until 42 years of age when she visited a clinic with the chief complaints of fever and sputum. Chest plain radiography showed left-sided pneumonia, for which antibiotic treatment was initiated. Although the blood test result showed improvement of inflammatory markers, chest plain radiography showed no improvement in pneumonia; therefore, she was referred to our hospital.Chest computed tomography (CT) showed a tumorous lesion nearly obstructing the bronchial lumen and the extension of pneumonia into the residual left lung Fig. . RecurreConsidering the risk of hemoptysis and bronchial stump fistula, the suture material obstructing the bronchial lumen was removed using a flexible bronchoscope with the patient under general anesthesia 1 week later Fig. b. LittleAccording to a copy of the 2010 operating report, the bronchial stump was sutured with five stitches using an absorbable monofilament suture (4–0 polydioxanone suture [PDS]). However, the left lower bronchus stump had hypertonicity. To decrease the tension at the suture site, a 4–0 PDS attached to the Teflon pledget  with two needles was placed in the central part of the stump. This procedure and our bronchoscopic findings indicated that the bronchial obstruction was secondary to the endobronchial migration of the Teflon pledget that had eroded through the bronchial wall.At 1 month after removal, bronchoscopy showed pedunculated granulation tissue, the biopsy result showed no malignant findings, and the bronchial lumen was still open Fig. c. At 4 mThe patient’s clinical course after the procedure was favorable. At 1 year after Teflon pledget removal, there were no recurrences of the pulmonary carcinoid tumor or obstructive pneumonia.In thoracic surgery, one of the important concerns that has led to an insistent demand for suture protection is bronchopleural fistula. Risk factors for the development of bronchopleural fistula include advanced cancer stage, preoperative chemotherapy or radiation therapy or both, previous steroid therapy, residual carcinomatous stump, postoperative mechanical ventilation, chronic obstructive pulmonary disease, diabetes mellitus, and malnutrition , 4. The Wound healing of the bronchial stump is characterized by granulation tissue formation with angiogenesis and proliferation of fibroblast cells. During this process, a foreign substance such as a suture can become buried in the connective tissue. Healing of the bronchial stump is not directly attributable to healing of the suture site, but it is achieved secondarily when the bronchial stump is covered with peribronchial tissue. The outer surface of the bronchial stump is covered by connective tissue within 2 weeks, and complete healing with mature fibrous tissue is achieved within 4 weeks . In accoThe absorbable suture, which was used in the present case, is safe because the tensile strength is maintained for 2 weeks until the anastomosis site and bronchial stump are covered by connective tissue, although there is a concern for suture failure owing to an early decrease in tensile strength . In the To prevent late complications, it is desirable to use biological materials such as fat pad, muscle fascia, and pericardium. However, in cases where the wound healing process is not compromised, late complications can be prevented even with non-absorbable suture materials. Regarding the mechanism of translocation of the suture material from the pleural space to the bronchial lumen in the present case, it was hypothesized that the suture with the pledget remaining in the pleural space had been buried in the submucosa of the bronchial wall during the wound healing process after the bronchus was cut. However, it is difficult to prove this hypothesis.We described about a patient who developed a late complication owing to non-absorbable suture material at 8 years after lobectomy. The results indicated that if a non-absorbable suture material has been used, surgeons should be aware of the risk of exposure of suture material in the bronchial stump during the remote postoperative period, although it is extremely rare. In such cases, the pleural side of the suture material is considered to be covered with connective tissue and fibrous tissue according to the mechanism of wound healing. Therefore, if the complication occurs, it is possible to rapidly remove the foreign material endoscopically.


Protein tyrosine phosphatase PTPN13, also known as PTP-BL in mice, is a large multi-domain non-transmembrane scaffolding protein with a molecular mass of 270 kDa. It is involved in the regulation of several cellular processes such as cytokinesis and actin-cytoskeletal rearrangement. The modular structure of PTPN13 consists of an N-terminal KIND domain, a FERM domain, and five PDZ domains, followed by a C-terminal protein tyrosine phosphatase domain. PDZ domains are among the most abundant protein modules and they play a crucial role in signal transduction of protein networks.Here, we have analysed the binding characteristics of the isolated PDZ domains 2 and 3 from PTPN13 and compared them to the tandem domain PDZ2/3, which interacts with 12 C-terminal residues of the tumour suppressor protein of APC, using heteronuclear multidimensional NMR spectroscopy. Furthermore, we could show for the first time that PRK2 is a weak binding partner of PDZ2 and we demonstrate that the presence of PDZ3 alters the binding affinity of PDZ2 for APC, suggesting an allosteric effect and thereby modulating the binding characteristics of PDZ2. A HADDOCK-based molecular model of the PDZ2/3 tandem domain from PTPN13 supports these results.Our study of tandem PDZ2/3 in complex with APC suggests that the interaction of PDZ3 with PDZ2 induces an allosteric modulation within PDZ2 emanating from the back of the domain to the ligand binding site. Thus, the modified binding preference of PDZ2 for APC could be explained by an allosteric effect and provides further evidence for the pivotal function of PDZ2 in the PDZ123 domain triplet within PTPN13. The  classes –11. This solvent . Recentl solvent , 14., that the binding characteristics of single domains differ from PDZ tandem and that allosteric effects can indeed regulate the binding properties of PDZ tandem domains [Tyrosine-protein phosphatase non-receptor type 13 (PTPN13), which is known as protein tyrosine phosphatase PTP-Basophil/Basophil-like (PTP-BL) in mice, is the central scaffolding component of a supramolecular protein complex that exhibits numerous domain-specific interactions , 15, and domains , 22, 23.Here, we describe the binding characteristics of the second and third PDZ domain of PTPN13, both individually and as a PDZ2/3 tandem domain through heteronuclear multidimensional nuclear magnetic resonance (NMR) spectroscopy. Our results reveal that PRK2 is an additional binding partner of the PDZ2 domain. Furthermore, we have discovered an allosteric effect due to an interaction of the PDZ3 domain with the opposite surface of the canonical PDZ2 ligand binding site, which is structurally broadcasted to the binding pocket of PDZ2. The analysis of the interaction between PDZ2 and PDZ3 as well as with their respective ligands sheds light on the pivotal role of the PDZ2 domain within the PDZ123 cassette of PTPN13 on a molecular level.15N isotopically-enriched PDZ2 and PDZ2/3 samples, respectively, and by monitoring the NMR chemical shift perturbations in a series of 2D 1H-15N heteronuclear single quantum coherence (HSQC) spectra with increasing ligand concentrations. It could be shown that all peptides bind to the canonical binding groove of PDZ domains , the PRK2 peptide only weakly binds to the PDZ2 domain (KD = 661 ± 71 μM)  in the PDZ2/3 tandem domain than for the single PDZ2 domain (KD = 286 ± 21 μM), while the extracted KD value for PDZ3 (KD = 2574 ± 770 μM) is even higher in comparison to the single PDZ3 domain (KD = 721 ± 148 μM). Binding of APC to the tandem PDZ2/3 domain occurs in the fast exchange NMR regime. At a final stoichiometric ratio between APC and PDZ2/3 of 30:1, no further chemical shift changes were observed for amide resonances originating from the PDZ2 domain. However, due to the extremely low affinity of PDZ3 for APC, PDZ3 was not fully saturated at the end of the titration and thus represented a fractionally APC-bound state (data not shown). NMR chemical shift perturbations extracted from 2D 1H-15N-HSQC NMR spectra for the PDZ2/3 titration with the APC peptide are observed for both domains. As can be seen in Figs. 1H-15N-HSQC NMR spectra of the PDZ2/3 tandem domain upon titration with the APC peptide. For example, NMR chemical shift perturbations were observed for G12, L19, T31, G63, H94, L95, L97 of PDZ2 and K152 as well as S231 of PDZ3 in the tandem domain upon titration with APC  and monitored their interaction by recording a series of 2D 1H-15N-HSQC NMR spectra  to the 1H-15N-HSQC NMR spectra could not be detected   Fig.  a, b. NM31) Fig. . This su31) Fig. . Finally31) Fig. .2  while the binding affinity of the APC peptide for PDZ3 is even lowered (KD ~ 721 μM vs KD ~ 2574 μM). The latter KD value indicates that significant binding of APC to PDZ3 in the tandem domain will most likely not occur under physiological conditions.Even though the binding behaviour of PDZ domains has been intensively studied, the functional consequences are still not completely understood. Previous studies show  that theZ domain –5. This Based on the results from the NMR chemical shift perturbation analysis and HADDOCK molecular docking calculations we conclude, that an allosteric effect modulates the APC-binding characteristics of PDZ2 from PTPN13 Fig. . AlthougOur data on the PTPN13 PDZ2/3 tandem domain are therefore consistent with and complement findings for the PDZ1/2 tandem, as the binding affinity for the APC peptide ligand is increased in the PDZ 2/3 tandem domain in comparison to the single PDZ2 domain  Table 1. ObviousThis modulation of binding affinities of PTPN13 for its ligands might also lead to the formation of supramolecular protein complexes through which PTPN13 exhibits its well established scaffolding functions in several cellular processes such as cytokinesis and actin-cytoskeletal rearrangement –37. InteTaken together, our study suggests that the PDZ2 of PTPN13 plays a central role in the triplet cassette PDZ123 and that PDZ2 is regulated by the adjacent PDZ domains 1 and 3 Fig. . This isBacterial expression plasmids pGEX-2 T-PDZ2 (PTPN13 residues 1347–1450), pGEX-2 T-PDZ3 (PTPN13 residues 1474–1580), and pGEX-2 T-PDZ2/3 (PTPN13 residues 1347–1580) were constructed by sub-cloning of PCR-generated PTPN13 cDNA fragments in-frame into appropriate pGEX-2 T vectors.E. coli BL21 (DE3) cells in isotope-enriched minimal media using 13C glucose and/or 15N ammonium chloride as carbon and nitrogen sources [As previously published, isotope-labeled proteins were prepared by growing  sources , 42–47.E. coli BL21(DE3) under ampicillin and chloramphenicol selection. In order to generate 15N isotopically-enriched proteins, cells were grown in minimal media [Microfluidics Corporation. Cell debris was removed by centrifugation at 10,200 g and at 4 °C for 45 min. Afterwards, the supernatant was incubated with Glutathione Sepharose 4B beads  at 20 °C for 60 min. The GST-tag was subsequently cleaved off through incubation with Thrombin protease  for 12 h at 4 °C. Then, the protein was eluted from the Glutathione Sepharose beads with phosphate buffered saline (PBS) at 20 °C. Protein containing fractions were pooled and concentrated in a Millipore concentrator with molecular weight cut-offs of 5000–10,000 Da, frozen in liquid nitrogen, and finally stored at − 80 °C until further use. The integrity of purified proteins was checked by SDS-PAGE and mass spectrometry (data not shown).GST-fusion proteins were expressed in al media . Proteinal media , 49. In 13C and/or 15N-enriched protein in PBS buffer at pH 7.4 including 10% D2O [www.bruker.com). The backbone assignments were obtained from the BioMagResBank accession numbers 15,199 (PDZ2/3) [15N-enriched PDZ2 at a molar ratio of 1:1 at 298 K by recording 2D 1H-15N-HSQC NMR spectra. In this study, the numbering scheme of the assigned chemical shifts for PDZ2 (5131) and PDZ23 (15199) has been shifted by one residue so that, for example, T30 according to the BMRB entry 5131 now equals T31. Amino-terminally acetylated dodecapeptides were commercially obtained from JPT Peptide Technologies (Germany). The peptide sequences KRHSGSYLVTSV (M = 1332.69 g/mol) and MFRDFDYIADWC (M = 1580.64 g/mol) correspond to the last 12 C-terminal residues of APC and PRK2, respectively. APC and PRK2 peptides were titrated with PDZ2, PDZ3, and PDZ2/3 domains that were isotopically enriched with 15N. The experiments were carried out at protein-to-ligand ratios of 1:0, 1:1, 1:3, 1:5, 1:10, 1:20, and 1:30 at 298 K and pH 7.4. For all binding studies that included a PDZ3 domain, Dithiothreitol (DTT) was added to the sample before carrying out the titration in order to avoid the oxidation of cysteines. The differences in backbone 1H and 15N NMR chemical shifts were monitored via 2D 1H-15N-HSQC NMR spectra. The weighted 1HN and 15N chemical shift differences were calculated according to the following equation [NMR spectra were acquired at 298 K on Bruker DRX 600 and AVANCE III HD 700 spectrometers. Typically, NMR samples contained 0.1–0.5 mM uniformly  10% D2O –44. All  10% D2O  and anal 10% D2O  or TopSp(PDZ2/3)  and 5131(PDZ2/3) , 44, 46.equation , 47:www.originlab.com) as previously published [D values was calculated from the individual fit of several significant chemical shift perturbations found for the amide resonances located in and/or next to the canonical PDZ binding cleft. For the PDZ2/APC titration amide resonances of G23, K50, A52, L73, Q80, and A81 of were used, for the PDZ2/PRK2 titration amide resonances of T21, G23, T30, Y43, G51, A52, S55, and D56 were used, for the ‘PDZ2 in PDZ2/3’/APC titration amide resonances of L19, D57, and Q81 were used, and, finally, for the ‘PDZ3 in PDZ2/3’/APC amide resonances of G159, F162, S163, K184, L223, and G225 were used.Averaged dissociation constants for the different complexes were calculated from least-squares fitting of the NMR chemical shift perturbations observed for several amino acids as a function of ligand concentration. NMR chemical shift perturbation data were analysed with the ORIGIN software package  software package was used to generate a model of the APC-bound PDZ2/3 tandem from PTN13 based on the differential NMR chemical shift perturbations outside the canonical binding cleft of PDZ domains between PDZ2/APC and ‘PDZ2 in PDZ2/3’/APC titration . For PDZ


Phytochromes are photoreceptor proteins that transmit a light signal from a photosensory region to an output domain. Photoconversion involves protein conformational changes whose nature is not fully understood. Here, we use time-resolved X-ray scattering and optical spectroscopy to study the kinetics of structural changes in a full-length cyanobacterial phytochrome and in a truncated form with no output domain. X-ray and spectroscopic signals on the µs/ms timescale are largely independent of the presence of the output domain. On longer time-scales, large differences between the full-length and truncated proteins indicate the timeframe during which the structural transition is transmitted from the photosensory region to the output domain and represent a large quaternary motion. The suggested independence of the photosensory-region dynamics on the µs/ms timescale defines a time window in which the photoreaction can be characterized (e.g. for optogenetic design) independently of the nature of the engineered output domain. Synechocystis phytochrome from the red- to the far-red-absorbing states extend to the seconds timescale. Interestingly, while the rapid structural changes are independent of the output domain the slower changes are not, providing useful insights on photosensory-region dynamics.Heyes et al. report that the conformational changes of a full-length  The photosensory activity of phytochromes results from their capacity to undergo a light-induced and reversible switching between two conformers, a red-light-absorbing Pr form and a far-red-light-absorbing Pfr form, which are characterized by distinct 3D structures and spectral properties. Phytochromes are generally soluble and dimeric proteins, with each monomer consisting of multiple domains. The protein consists of an N-terminal photosensory region that comprises three domains, namely a PAS, GAF, and PHY domain, and a C-terminal output domain, which is normally a histidine kinase domain. The light-sensing properties of phytochromes result from the presence of a bilin chromophore, either phytochromobilin in plant phytochromes, phycocyanobilin in cyanobacterial phytochromes or biliverdin in bacterial phytochromes, which is covalently attached to a conserved cysteine residue within the GAF domain. Due to their near-infrared spectral sensitivity, where the absorbance of other biological macromolecules is low, the phytochrome family is becoming an important target for optogenetic applications and they are the preferred templates for designing optical molecular tools for applications in mammals3.Phytochromes are light-sensing proteins that are able to transduce a light signal into a biochemical output in plants, bacteria and fungiZ/E isomerization of the C15-C16 double bond between the C and D rings of the tetrapyrrole on the picosecond timescale5 accompanied by deprotonation/reprotonation of the pyrrole nitrogens6. Isomerization induces rotation of the D ring, which is followed by a translation of the bilin chromophore within its binding pocket5. Photoisomerization of the chromophore is then followed by slower steps on the micro- to millisecond timescale, which have been proposed to involve global changes in protein structure, to form an active signaling conformation of the C-terminal region in the final Pfr state8. A recent comparison between the crystal structures of a dark-adapted form and a red-light illuminated form has provided a model for the structural changes in the photosensory module of a bacterial phytochrome9. It was proposed that a unique tongue region, extending from the chromophore binding domain to the adjacent PHY domain, undergoes a change in secondary structure from an anti-parallel β-sheet configuration in the Pr state to an α-helix in the Pfr state10. This change in secondary structure is thought to induce a strain along the dimer interface to force the opening of the neighboring PHY domains. Although this motion has only been observed in crystals of the photosensory region of a phytochrome9, it provides a plausible mechanism to transmit the light signal to the output domain in the full-length protein via a structural motion that triggers downstream signaling. While the initial Z/E photoisomerization of the bilin chromophore at the C15-C16 position has been confirmed by several studies, it is still largely unknown how isomerization alters the bilin conformation, changes the structure of the bilin binding pocket, and ultimately triggers a rearrangement of the interface between the chromophore binding domain and the adjacent domains that results in the secondary structure change in the tongue region. It is also unclear if these motions that have been observed in the photosensory domain10 are affected by the presence of the output domain in the full-length phytochrome.The mechanism by which phytochromes transmit the light signal from the chromophore through the photosensory module, and ultimately into the regulatory module to actuate signaling remains unclear. For most phytochromes, there is a general consensus that the absorption of a photon by the chromophore in the Pr form triggers a 3. This rational design process will ultimately require a detailed spatio-temporal understanding of the structural changes that occur in full-length phytochrome proteins. However, no 3D structure of a full-length phytochrome containing an output domain has been solved yet and it is unknown how structural changes in the photosensory module extend to the C-terminal regulatory module to trigger the signaling process. Time-resolved X-ray scattering studies remain the most attractive approach for tracking structural transitions of phytochromes in solution that follow the initial photochemical event. Recent time-resolved X-ray scattering studies have described the structural dynamics of a bacterial biliverdin-containing phytochrome in both the photosensory region on its own and in a full-length form11. The major structural events in the bacterial phytochrome were found to occur from tens of microseconds to a few milliseconds in both forms of the protein. It was proposed that the entire phytochrome protein undergoes a concerted structural rearrangement, resulting in the twisting of the output domains prior to the formation of the final Pfr state8. However, it is unclear if the structural changes are specific for the bacterial homologs or if the mechanism is conserved across the plant and cyanobacterial phytochromes, which undergo more complex reaction cycles.In order to fully exploit phytochromes as optogenetic tools it will be necessary to structurally reassemble the natural output domains with other biologically-relevant output modules12 and time-resolved13 small- Synechocystis sp. PCC6803. We detect changes in the scattering patterns from the small-angle region up to about 0.7 Å−1, and from the microsecond timescale to hundreds of milliseconds, where the signal is still evolving, in agreement with spectroscopic data. The results indicate that the kinetics of structural changes in the full-length cyanobacterial phytochrome extends to the seconds timescale with at least two identifiable intermediates and is more complex than that recently reported for a similar phytochrome from Deinococcus radiodurans, where the global structural change occurs in a single concerted motion within a few milliseconds8. Moreover, a comparison of TR-S/WAXS and spectroscopic data on the full-length protein with data collected for the photosensory region alone suggests that both localized and global changes on the µs/ms timescale are largely independent of the presence of the output domain, while those at longer times differ significantly in the presence of the output domain. Low-resolution modeling of the static small-angle data suggests that the differences in the small-angle scattering reflect a large motion of the output domains, allowing us to propose a model for the overall structural changes in the full-length cyanobacterial phytochrome.Here we use a combination of steady-state13 were carried out to study the kinetics of the structural transitions during the Pr to Pfr photoconversion for both the full-length Cph1 protein and the photosensory region (PAS-GAF-PHY) of Cph1 upon laser excitation at 630 nm. The experimental setup is shown schematically in Fig. −1)  that are similar to those reported previously on the same protein15.In order to correlate the temporal behavior of the structural changes with optical changes, transient absorption spectroscopy measurements were performed on the full-length Cph1 protein and the photosensory region (PAS-GAF-PHY) of Cph1 on the microsecond-to-second timescale. Kinetic transients were recorded at 720 nm following excitation with a nanosecond laser pulse at 630 nm to report on the formation of the Pfr state  and the photosensory region (time-resolved S/WAXS) are largely unaffected by the presence of the output domain up to the millisecond timescale. This separation between the photosensory region dynamics and the output domain on the µs/ms timescale indicates that in this time window the photoreaction can be characterized independently of the nature of the engineered output domain. It is only on slower timescales that the structural changes are propagated to the output domain in a process that is likely to be transmitted through the linker region20.The results of the present study will also have implications in the design of optogenetic tools based on phytochromes, where one of the most promising strategies is to replace the natural output domains with other domains that have the desired biological functionSynechocystis sp. PCC6803 were produced and purified as a phycocyanobilin-bound holoprotein using a dual-plasmid Escherichia coli expression system7. The Cph1 genes were synthesized (GenScript) and cloned into the pET21a expression vector (Novagen). BL21 cells containing the two plasmids were grown in 500 ml of Luria–Bertani medium to an OD600 of ~0.5 at 20 °C and expression of the genes was induced overnight after addition of 100 µM isopropyl β-D-thiogalactoside. The C-terminal His-tagged proteins were purified as described previously7.All chemicals were obtained from Sigma-Aldrich unless otherwise stated. The photosensory region (residues 1–514) and full-length Cph1 proteins from 2  corresponding to a power density of ~1 mJ/mm2. To maximize the overlap between the pump- and probe-illuminated volumes, orthogonal pump–probe geometry was employed  penetrated a 2-mm-diameter quartz capillary, 0.3 mm from its edge. In order minimize any X-ray radiation damage over a large sample volume and to recover the Pr state of the protein after the laser pump pulse the sample was continuously pumped (Gilson Minipulse 3) from the capillary to a ~1 ml reservoir under 735 nm LED constant illumination (ThorLabs) and then again to the capillary through a teflon loop tubing. The flow speed was set according to both the laser pump–X-ray probe time delay and the repetition rate in order to keep the sample in the pump–probe intersection area during a pump–probe sequence and also to refresh the sample between two consecutive pump pulses. A variable number of single X-ray pulses were selected from the synchrotron pulse train by means of a high-speed chopper and a millisecond shutter. The X-ray pulses scattered by the sample were collected with a 2D detector (Rayonix MX170-HS). Up to 25 patterns per time delay were acquired and averaged together to improve the signal-to-noise ratio. Laser-off images were also acquired with the X-ray pulse arriving 1 ms before the laser pulse and used as a reference to compute the TR-S/WAXS difference patterns. Images were azimuthally averaged and the peak of the undulator spectrum (0.8 Å−1) was used as the reference wavelength to convert the scattering angle to the momentum transfer q. All radial patterns were normalized to the water peak (2–2.2 Å−1) before calculating differences between laser-on and laser-off patterns. We also checked that the time evolution of the solvent heating peak at 1.8 Å−1 did not affect the scattering patterns in the q range where protein difference signal was present (0–0.7 Å−1). X-ray scattering data have been also collected on the full-length and photosensory region proteins after illumination with constant 625 and 730 nm LED light to produce Pfr and Pr stationary states, respectively.TR-S/WAXS experiments have been performed at the beamline ID09 of the European Synchrotron Radiation Facility (ESRF) in Grenoble, France. The protein samples (400 µM) were photolyzed with a circularly-polarized laser pulse  from a nanosecond laser  focused with cylindrical lenses to an elliptical spot approximately 2.0 × 0.25 mm7. Lifetimes were obtained from the average of at least five time-dependent absorption measurements by fitting to a triple exponential function. Reduced χ2 was 2 and 1.5 for the photosensory region and the full-length protein, respectively.Laser photoexcitation experiments on the photosensory region and full-length forms of Cph1 were carried out after all samples had been pre-illuminated with a 735 nm LED (Thorlabs) to ensure that the protein was in the Pr state prior to laser excitation. Photoconversion to the Pfr state was triggered by laser excitation at 660 nm (~20 mJ per pulse) in a cuvette of 1 cm pathlength. Absorption transients were recorded at 720 nm using an LKS-60 flash photolysis instrument (Applied Photophysics Ltd)m × n matrix A, with m the number of q values and n the number of time delays. The SVD algorithm provides the matrices U and V and the vector S, so that A = U × S × VT, where the column of matrix U are called left singular vectors, or basis patterns, the rows of VT are called right singular vectors, or amplitude vectors, and the elements of S are called singular values. The basis patterns are ordered following the high-to-low sorting of singular values. The aim of the SVD analysis of kinetic data is to identify a number of time-independent patterns containing all the relevant information out of the random noise and able to reproduce the dataset as a linear combination of such time-independent patterns. Visual inspection of basis patterns, amplitude vectors and singular values suggested that only the first three basis patterns are significant. Autocorrelation analysis of both basis patterns and amplitude vectors21 indicated three components containing significant information, with the rest containing essentially only random noise. A linear combination of the first three basis patterns was then used to reconstruct the entire dataset and to interpret and compare the kinetics of the photosensory region and full-length Cph1 protein.In order to unveil the kinetic structure of the time-dependent evolution of X-ray scattering patterns, singular value decomposition (SVD) analysis has been performed of the X-ray scattering dataset with a custom-made Python-based code. Briefly, the dataset of time-dependent difference patterns is arranged in a −1 using the GNOM software of the ATSAS package22. Ab initio envelope models of the Pr and Pfr states were derived using the DAMMIN software (ATSAS package22). First, 30 models were generated for each scattering curve, using a P2 symmetry on the basis of the known homodimeric structures of Cph116. DAMAVER software (ATSAS package22) was then used to remove outliers, align the remaining models and produce an averaged and filtered model and used as an input for a second DAMMIN analysis. The model produced by this second DAMMIN round with a minimized discrepancy from the scattering data was used to reveal the overall conformational motions responsible for the large difference signal in the SAXS region of the full-length Cph1 patterns  changes. The pair distance distribution functions P(r) was calculated from the absolute scattering curves on the q range 0–0.3 Å


Many Gram-negative bacterial pathogens directly deliver numerous effector proteins from the bacterium to the host cell, thereby altering the target cell physiology. The already well-characterized effector delivery systems are type III, type IV, and type VI secretion systems. Multifunctional autoprocessing repeats-in-toxin (MARTX) toxins are another effector delivery platform employed by some genera of Gram-negative bacteria. These single polypeptide exotoxins possess up to five effector domains in a modular fashion in their central regions. Upon binding to the host cell plasma membrane, MARTX toxins form a pore using amino- and carboxyl-terminal repeat-containing arms and translocate the effector domains into the cells. Consequently, MARTX toxins affect the integrity of the host cells and often induce cell death. Thus, they have been characterized as crucial virulence factors of certain human pathogens. This review covers how each of the MARTX toxin effector domains exhibits cytopathic and/or cytotoxic activities in cells, with their structural features revealed recently. In addition, future directions for the comprehensive understanding of MARTX toxin-mediated pathogenesis are discussed. To be a successful pathogen, host-invading bacteria produce various virulence factors. Examples range from catabolic utilization systems for host-derived nutrients that enable the in vivo growth of the pathogen to a series of cytopathic or cytotoxic molecules that disturb the host defense systems ,2,3,4. AVibrio cholerae as an accessory cytotoxic toxin for which the gene is physically linked to the cholera toxin prophage ,1611,16]6), the effector processing predominantly occurs in the host cell cytosol . O. OV. choX toxins   is responsible for this cytoskeleton disruption, and thus the region was assigned as an ‘Actin Cross-linking Domain’ . No. No2+ aride bond .Until recently, the cytotoxicity of ACD was believed to be the results of gradual reduction of the G-actin pool and the consequent depolymerization of the F-actin filaments in the intoxicated cells . HoweverV. cholerae mutant strain expressing the ΔACD MARTX toxin (ACD-domain deleted MARTX toxin) maintains the MARTX toxin-dependent cell-rounding activity, although the cell rounding occurs slowly. In parallel, they had carried out infection experiments with V. vulnificus and observed similar cell rounding [V. cholerae strain and the used V. vulnificus strain do not exhibit the actin cross-linking activity. Therefore, the results suggest that a certain region presenting in both the V. cholerae and V. vulnificus MARTX toxins can induce the host cell rounding in an ACD-independent manner. Among such regions, a domain corresponding to amino acids 2552–3099 (according to the sequence of MARTX toxin from V. cholerae N16961) actually caused host cell rounding when ectopically overexpressed [V. cholerae-mediated intoxication or anthrax toxin-mediated endocytosis (the LFN/PA system) [In contrast to ACD, which directly targets actin molecules, RID indirectly depolymerizes actin filaments by inactivating Rho-family GTPases. During the characterization of ACD, Sheahan and Satchell had noticed that the rounding . Notablyxpressed . Interes system) ,43, this system) .V. vulnificus RID have been disclosed [V. vulnificus MO6-24/O) for the cytopathic function of RID [ε-fatty acyltransferase transferring long-chain (more than 10 carbons) fatty acid from acyl-CoA to Lys residues in the C-terminal polybasic region of Rho-family GTPase proteins [Recently, the three-dimensional structure and molecular mechanism of isclosed . Overallisclosed . Certainisclosed . In the n of RID . When stproteins . Throughproteins ,44.Shigella flexneri type III secretion effector IcsB has recently been shown to carry out the same fatty acylation activity, although the sequence identity between the catalytic domain of RID and that of IcsB is very low (12–16%) [The (12–16%) ,47. InteV. cholerae MARTX toxin had revealed another distinct domain other than ACD and RID. Since the typical folding of alpha/beta hydrolase was deduced from the amino acid sequence of this domain, the latter has been named as the ‘Alpha/Beta Hydrolase’ domain [V. vulnificus ABH via the HHpred server [Streptococcus pneumoniae) and FGH  [P. haloplanktis FGH as a template, a typical alpha/beta hydrolase topology consisting of an eight-stranded β-sheet and surrounding α-helices is clearly shown (V. vulnificus MO6-24/O) are markedly present in a putative catalytic site [The CPD-mediated in vitro autoprocessing of the ’ domain ,24. No td server  indeed rlanktis) ,50. In tlanktis)  using P.ly shown . Furthertic site , suggestV. cholerae-mediated natural delivery of the ABH suggested that the domain might affects host cell signaling. This is because the solely delivered ABH significantly activated the host small GTPase Cdc42 in a catalytic His residue-dependent manner [In contrast to ACD and RID, ABH had not exhibited any distinctive cytopathic or cytotoxic phenotypes in transfection-based analyses, and thus its molecular function had remained elusive for around a decade. However, the t manner .sn1 position of glycerol phosphate in the PtdIns3P molecule. Since PtdIns3P is a crucial molecule for autophagosome formation and endolysosomal pathways [More recently, the Satchell group found that ABH is a phosphatidylinositol 3-phosphate (PtdIns3P)-specific phospholipase A1 . Certainpathways ,54, the pathways . This mopathways .V. vulnificus MO6-24/O, CMCP6, or BAA87 strains. Unlike with other MARTX effector domains, neither the solved three-dimensional structure nor the predicted model of DUF1 is available at this time. Importantly, no certain catalytic residues have been assigned or even assumed, and therefore it is still unknown if this domain functions as an enzyme or as a scaffolding protein. Although not exactly matched with DUF1, the ’RtxA1-D2’ region containing both DUF1 and the amino-terminal part of RID has been studied, which has given some insights into the function of DUF1 [V. vulnificus infection [V. vulnificus but not a MARTX toxin deficient one. Collectively, PHB1 has been assumed as an initial receptor protein for the early binding of the MARTX toxins and to complete a positive feedback route for the further bindings [DUF1 is a domain at the first position in the effector region of the MARTX toxin from the  of DUF1 . When ecnfection . To findbindings . Howeverbindings , indicatV. vulnificus MARTX toxin had revealed that it induces the apoptotic death of host cells [V. vulnificus but not a MARTX toxin-deficient nor a secretion defective mutant strain was treated onto the human epithelial cells. Consistent with this, cytochrome c was released from the mitochondria in the case of the wild-type strain treatment. However, the MARTX toxin effector domain responsible for this apoptotic cell death has only been investigated recently and was suggested as an MCF domain [An early study investigating the functions of the st cells . SpecifiF domain .Photorhabdus luminescens [The domain name MCF stands for ‘Makes Caterpillars Floppy-like’ since the amino acid sequence of MCF domain shows a significant similarity to that of insecticidal toxins Mcf1 and Mcf2 from inescens ,16. Wheninescens . Althouginescens ,61.V. vulnificus MO6-24/O) was substituted for Ala. In addition, the same catalytic residues were found to be critical for the MCF-mediated cell shrinking [Pseudomonas syringae type III secretion effector AvrPphB [When ectopically overexpressed in HeLa cells, the MCF domain showed amino-terminal autoprocessing. Indeed, such an event was not observed if a putative catalytic tripeptide residue  [N/PA system is cytotoxic to host cells [Before the discovery of its molecular targets in host cells, the RRSP domain had been called DUF5 meaning ‘domain of unknown function in the fifth position’ of the MARTX toxin from us CMCP6 . Intrigubacteria ,63. Bioiin (PMT) ,65,66. Ist cells ,67.One of the most important findings from previous studies is that DUF5 directly processes a peptide bond between Tyr32 and Asp33 in the switch I region of the cellular Ras and Rap1 proteins, and reflecting this activity, DUF5 has been renamed as the ’Ras/Rap1-Specific endoPeptidase’ domain . Notablyα atoms), although their amino acid sequences are largely different  [V. vulnificus CMCP6) is located . If the sequence was compared with that of the P. aeruginosa type III secretion effector ExoY (PaExoY), the identity was even higher (26%) and thus the effector domain has been named as the ‘ExoY-like’ domain (ExoY) [V. vulnificus, intracellular level of cyclic adenosine monophosphate (cAMP) was significantly increased. Consistent with this, the recombinant ExoY protein was able to convert ATPs to cAMPs in vitro, although the reaction was somewhat inefficient [In the amino acid sequence analyses, one of two newly found effector domains in the MARTX toxin of n (ExoY) . Indeed,n (ExoY) ,75. Morefficient .V. nigripulchritudo MARTX toxin increased more than 10,000-fold in the presence of actin molecules, although G-actins in this case [Since the substrate nucleotides are commonly present in both eukaryotic and prokaryotic cells, CyaA and EF have evolved to become activated only if the proteins interact with calmodulin (CaM), a calcium-binding protein found in eukaryotic cells ,77. Howehis case ,79. Unlihis case ,78. Therhis case .V. vulnificus ExoY domain generated by using the structure of PaExoY as a template. Similar to PaExoY, CyaA, or EF, the ExoY is likely to consist of two subdomains with a catalytic site between them. The three conserved CRs involving substrate binding and catalysis are indeed gathered near the site (V. vulnificus BAA87), which are the residues predicted to hold the α-phosphate of substrate ATP, and D3442 and D3444, which are the residues predicted to stabilize the β-and γ-phosphates of ATP via Mg2+ ions, are completely conserved in the CR1 and CR2 regions of ExoY, respectively. The residues predicted to bind to the adenine moiety of ATP, namely H3530, D3533, and N3536, are on the unstructured loop of the CR3 region , which stands for the domain of no known function [V. vulnificus BAA87) may have a structure similar to the P. syringae AvrPphB, a cysteine peptidase effector protein , il1r2 (interleukin-1 receptor type 2 precursor), and cxcr4 (CXC chemokine receptor type 4) are differentially expressed in a MARTX toxin-dependent way [In the meantime, the immunological consequences of effector domain functions should be considered. Indeed, MARTX toxins from roptosis . A genomdent way ,84. Howedent way ,86, fattdent way ,21,87.V. vulnificus MARTX toxin that functions as the pathogen’s primary virulence factor [V. cholerae MARTX toxin is considered as an accessory toxin helping colonization [V. vulnificus MARTX toxin) is much more virulent than one with fewer effector domains . However, this is not always true since a mutant V. vulnificus strain secreting ΔDUF1 or ΔABH MARTX toxin actually showed an increased virulence in mice [V. vulnificus MARTX toxin eventually lyses the host cells, while the same variant of V. cholerae MARTX toxin does not [The potency of MARTX toxins in the pathogenesis of bacteria is another subject for future study. In contrast to the e factor ,13,14,88nization . One mig in mice . This sudoes not ,22. OtheLast, the molecular mechanisms of pore formation and effector domain translocation need to be clearly uncovered for a comprehensive understanding of MARTX toxins. Do they require specific molecule(s) on the host cell plasma membrane for the cell binding and/or pore formation? Which portion of the repeats-containing region is necessary for that binding and pore formation? Can we use antibodies targeting that portion as prophylactic/therapeutic agents against MARTX toxin intoxication? How can we capture the structure of MARTX toxin translocons for a cryo-electron microscopic analysis? With the answers to these questions, we could expand our knowledge of toxin-mediated host–pathogen interactions and might be able to overcome MARTX toxin-triggered bacterial pathogenesis.


Cyclic nucleotide-binding (CNB) domains allosterically regulate the activity of proteins with diverse functions, but the mechanisms that enable the cyclic nucleotide-binding signal to regulate distant domains are not well understood. Here we use optical tweezers and molecular dynamics to dissect changes in folding energy landscape associated with cAMP-binding signals transduced between the two CNB domains of protein kinase A (PKA). We find that the response of the energy landscape upon cAMP binding is domain specific, resulting in unique but mutually coordinated tasks: one CNB domain initiates cAMP binding and cooperativity, whereas the other triggers inter-domain interactions that promote the active conformation. Inter-domain interactions occur in a stepwise manner, beginning in intermediate-liganded states between apo and cAMP-bound domains. Moreover, we identify a cAMP-responsive switch, the N3A motif, whose conformation and stability depend on cAMP occupancy. This switch serves as a signaling hub, amplifying cAMP-binding signals during PKA activation. Protein kinase A (PKA) is a cyclic nucleotide dependent protein kinase. Here the authors use single molecule optical tweezers and steered molecular dynamic simulations to follow in real time and quantitatively characterize the signals transduced by cAMP through the structure of the PKA regulatory subunit, and propose a model for PKA allosteric activation. In multi-domain assemblies, these signaling modules communicate and transduce ligand-binding signals to other functional domains, thereby enabling diverse responses to intracellular signaling cascades8. Cyclic nucleotide-binding (CNB) domains are ubiquitous and structurally conserved signaling modules that regulate the activities of protein kinases, guanine nucleotide-exchange factors, nucleotide-gated channels, and transcription factors in response to cyclic nucleotides1. To date, a general understanding of how the activity of CNB domains can be adapted to regulate a diverse array of protein functions remains rudimentary.Throughout evolution, nature has utilized structurally conserved protein domains as regulatory signaling modules9. The phosphorylating activity of the catalytic subunit is allosterically driven by two CNB domains of the regulatory subunit, termed CNB-A and CNB-B12. cAMP binding starts in the CNB-B domain and enables binding of a second cAMP molecule to the CNB-A domain, resulting in a profound conformational change that unleashes the activity of catalytic subunits10.Here we used optical tweezers in combination with steered molecular dynamic (SMD) simulations to study the mechanisms that link cyclic adenosine monophosphate (cAMP) binding and inter-domain communication with allosteric regulation of cAMP-dependent protein kinase A (PKA). PKA is an archetype of cyclic nucleotide-dependent protein kinases that is composed of regulatory and catalytic subunitsOur studies show that cAMP binding to the two CNB domains of PKA propagates a reorganization of inter-domain contact nodes that reshape the folding energy landscape of the protein. Changes in the energy landscape are unique to each CNB domain and arise from both ligand-binding and inter-domain interactions. We identify a division of labor among CNB domains: the CNB-B domain is responsible for initiating and triggering cAMP binding cooperativity, whereas the CNB-A domain induces strong inter-domain interactions that lock the entire protein complex into its active conformation. Moreover, we identify a cAMP-responsive structural element, the N3A motif, which switches in stability and conformation depending on cAMP occupancy and inter-domain contacts. Through mutagenesis and the use of cyclic nucleotide analogs, we show that this ligand-responsive switch is selective to cAMP and serves as a signaling hub, amplifying the cAMP-binding signal during the allosteric activation of PKA. Altogether, this study illustrates how each structurally conserved CNB domain has evolved to carry out unique but mutually coordinated regulatory tasks in a macromolecular assembly. Our work reveals new operating principles for ligand-directed protein allostery mediated by widely conserved signaling modules.14. The handle position determines the direction and region of the protein subjected to the force applied through the optical tweezers 17. We generated three PKA regulatory subunit constructs with unique pulling geometries to probe cAMP binding coupled to inter-domain interactions , resulting in a ~ 30-fold increase of τ0,F  and τ0,F increases by a factor of ~ 7  and a fourfold increase in τ0,F. The presence of the CNB-B domain induces a mechanical stabilization to the CNB-A domain of ~ 3 pN, resulting in Favg = 20.3 ± 1.4 pN (N = 1152) and a 160-fold increase in τ0. In the refolding reaction, the presence of the neighboring domain is highly asymmetric, decreasing τ0,U by 150-fold and 10-fold to the CNB-B and CNB-A domains, respectively  and unfolded states  at zero force for type-I and type-II constructs  = 1.2 Å between Cα atoms, Fig. Lc) might also reveal important differences in the native folded structures of the CNB domains upon binding cAMP. Although the mechanical unfolding of the CNB-B domain in all three types of constructs had a ΔLc of ~50 nm, corresponding to a fully folded domain  and another construct with handles flanking both CNB domains, except the N3A motif (D149C/S376C). The two constructs displayed two major unfolding rips corresponding to the CNB domains, but only the unfolding trajectory of the S110C/S376C construct revealed a small, reversible transition at ~11 pN with a ΔLc of 13 nm  simulations starting from the X-ray structure reveals that the unbound state (apo) during the relaxation undergone to a conformational rearrangement involving the loss of interactions between Trp260 and cAMP docked into CNB-A  revealed a two-step unfolding process instead of a single rip  and τ0,F by twofold to the cAMP-free CNB-A domain , indicating that as part of the regulatory subunit the CNB domains bind cAMP more tightly  show indistinguishable unfolding parameters compared with wild-type 30. Although the Ka for the mutant is significantly larger, its cAMP binding affinity is comparable to wild type. In silico studies proposed that the difference between intrinsic cAMP affinities and activation constants originate from mutational effects over the conformation of the protein. Our findings here show that the R241A mutant imparts cAMP-dependent functional deficiencies due to a disruption of the conformational dynamics of the N3A motif and its ability to serve as an efficient cAMP-responsive molecular switch, thereby impeding the PKA regulatory subunit to attain its final cAMP-bound conformation.To further dissect the role of folding dynamics and conformation of the N3A motif in inter-domain interactions, we conducted “force-clamp” experiments, wherein the protein was held at varying constant forces between 10 and 12 pN, and changes in extension due to unfolding and refolding of the N3A motif were monitored as a function of time Fig. . Analysiype Fig. . In addions Fig. , it is lons Fig. . Previou32 These studies have shown that cGMP binding to PKA is weaker than for cAMP at the level of individual CNB domains  = 4.2 nM, Kd,CNB-A(cGMP) = 820 nM, Kd,CNB-B(cAMP) = 2.8 nM, and Kd,CNB-B(cGMP) = 230 nM)34. Moreover, the activation constant of PKA by cGMP is ~140-fold higher than that of cAMP 32. Less understood, however, is the dependence of the conformational changes that the PKA regulatory subunit experiences upon binding cAMP vs. cGMP. Therefore, we investigated the contribution of the N3A motif dynamic switch mechanism towards cyclic nucleotide selectivity by mechanically manipulating the CNB domains individually (type-I constructs) or simultaneously (type-III construct) in the presence of cGMP. Both CNB domains bound to cGMP show unfolding parameters  that lie in between the values obtained with and without cAMP, indicating partial intra- and inter-domain stabilization effects , indicating that the N3A motif is not negatively coupled to cGMP, but instead unfolds as a single cooperative unit together with the rest of the domain. In agreement with this interpretation, the force-extension curves using a type-III construct with cGMP do not show the small, reversible transition characteristic of the N3A motif  and negative (destabilizing) coupling interactions that are fine-tuned to attain the final cAMP-bound conformation.The uncovered intra- and inter-domain communication network that is triggered by cAMP binding cannot be easily inferred from crystal structures22. In addition, our approach of selectively manipulating an individual CNB domain in the presence of the neighboring one allowed us to directly quantify interfacial stabilization effects due to cAMP binding. By using a type-III construct, we further determined that the N3A motif contributes 5.5 kcal mol−1 to the stability of the protein, allowing us to propose a folding energy landscape of the regulatory subunit when force is applied to both CNB domains simultaneously . Therefore, the dissociation from the catalytic subunit and the conformational rearrangement of the N3A motif to allow interactions with cAMP must be critical steps in the activation mechanism of PKA. Thus, our studies show that the N3A motif behaves like a dynamic switch, serving as a signaling hub that amplifies the cAMP-binding signal during the allosteric activation of PKA. By revealing the dynamic motions and stability changes that the N3A motif experiences as cAMP binds to the CNB-A domain and triggers inter-domain interactions with the CNB-B domain, our studies provide direct measurements on this conformational switch. Given the remarkable structural similarity between different regulatory subunit isoforms bound to the catalytic subunit (RMSD ~ 0.5–0.6 Å between RIα–RIIα or RIα–RIIβ)39, we proposed that the N3A motif plays a similar dynamic-switching role to activate other PKA isoforms. Moreover, as the N3A motif is found in many other cAMP- and cGMP-binding proteins40, the uncovered conformational switching mechanism may be a widespread strategy not only to ensure the completion of the allosteric activation process and provide ligand selectivity.Based on our studies, a critical element in the allosteric activation mechanism of PKA involves the dynamic switching of the N3A motif, which we show is important to stabilize the cAMP-bound conformation of the regulatory subunit. Previous structural and computational studies have identified substantial rearrangements and motions of the N3A motif during the allosteric signals triggered by cAMP binding11, further facilitating the dissociation of the catalytic subunit and enabling binding of the cyclic nucleotide to the CNB-A domain10. After cAMP binds to the CNB-A domain, the conformational switching of the N3A motif is triggered, breaking extensive surface interactions with the catalytic subunit and stabilizing the final cAMP-bound form11. Similar to unzipping “Velcro,” this mechanism efficiently peels off the two PKA subunits, where the strong inter-subunit interaction is the result of several smaller interactions that can be broken sequentially; hence, the dissociation of the two subunits does not require the crossing of a large free energy barrier but of many small ones. Within this allosteric regulation network triggered by cAMP binding, we identified the unique routes of domain communication that are disrupted either by R241A or cGMP  have been shown to decrease the Hill coefficient of activation of the PKA hetero-tetramer from 1.7 to ~1.0. The mutations S145G and R144S also located in the N3A motif, and that are related to Carney Complex disease have a lower Hill coefficient of 1.4. These mutational studies underscore the functional significance of the N3A motif in the allosteric communication networks and cooperative interactions triggered by cAMP binding36. However, communication across regulatory subunits in PKA hetero-tetramers formed with RII isoforms may be different, as the two N3A motifs in the crystal structure do not establish the same quaternary contacts seen in RI isoforms12.The allosteric networks we describe here may be amplified in the PKA hetero-tetramer composed of two regulatory and two catalytic subunits, where the potential cross-talk between PKA subunits is expanded20. The dissection of the folding energy landscape of the CNB domains showed that, when bound to the catalytic subunit, the CNB-B domain controls the stability of the CNB-A domain. This finding provides a thermodynamic foundation by which the CNB-B domain serves as the gatekeeper for cAMP binding to the CNB-A domain20. In this study, we use optical tweezers to directly interrogate another aspect of the activation mechanism of PKA, namely the forces that drive the cAMP-mediated activation and conformational changes in the PKA regulatory subunit. Remarkably, the mechanical fingerprints of the regulatory subunit and the underlying folding energy landscapes are unique and depend on whether the protein is in the heterodimeric PKA holoenzyme  or in the cAMP-bound conformation.In a recent study we showed how the CNB domains of the regulatory subunit (RIα) are thermodynamically coupled when bound to the catalytic subunit20. In contrast, the results of this study show that the N3A unfolding behavior is much more complex when bound to cAMP. Thus, the catalytic subunit and cAMP are competing for similar interaction regions in the regulatory subunit, suggesting a “tug-of-war model” in the activation of PKA21. Markov state models have suggested the existence of hybrid states, in which the regulatory subunit is bound to the catalytic subunit and partially occupied by cAMP43. Future studies will involve characterizing the interaction forces between CNB domains in the PKA hetero-tetramer and in hybrid states with partial cAMP occupancy.These unique mechanical fingerprints may emerge from the fact that the CNB domains have evolved to behave as molecular switches, changing in conformation upon external stimuli. A case in point is the N3A motif in the CNB-A domain that makes unique contacts depending on whether it is bound to cAMP or to the catalytic subunit. When bound to the catalytic subunit, the N3A motif unfolds together and simultaneously with the β-sandwich of the CNB-A domain, behaving as single cooperative unit44. The single-molecule approach exploiting optical tweezers in conjunction of molecular dynamics simulations presented here can be extended to map allosteric effects of disease mutations or inhibitor binding in other kinases or multi-domain assemblies49.In conclusion, we anticipate similar allosteric regulation mechanisms in other protein kinases with catalytic subunits that require the dissociation of regulatory signaling modules Bos taurus regulatory subunit gene isoform RIα containing residues 91–379 of the full-length sequence was used. To obtain isolated CNB domains, the sequence of the neighboring CNB domain was deleted by site-directed mutagenesis (QuikChange II Agilent). The isolated CNB-A domain contains residues 91–243. The isolated CNB-B domain contains residues 243–379. The mutations C345A and C360A were introduced in the CNB-B domain to prevent undesired reactions with the thiol-modified double-stranded DNA (dsDNA) handles. We have shown in a previous study that the double mutant C345A/C360A in the CNB-B domain does not alter the solution structure, stability, or the ability to form an inactive complex with the PKA catalytic subunit20. In addition, and compared with bulk studies, the CNB-B domain showed a similar binding affinity constant for cAMP either as an isolated domain or as part of the regulatory subunit,50. To manipulate each individual CNB domain (Type-I constructs), we introduced the mutations S110C/M243C and M243C/S376C for the isolated CNB-A and CNB-B domains, respectively. To manipulate either the CNB-A domain or the CNB-B domain selectively (Type-II constructs), we introduced into the regulatory subunit the mutations S110C/M243C and M243C/S376C, respectively. To manipulate both CNB domains simultaneously (Type-III construct), we introduced into the regulatory subunit the mutations S110C/S376C. All the protein constructs were expressed in BL21(DE3) (NEB) and purified as described previously51. Briefly, the protein was expressed in BL21(DE3)-competent cells overnight at 18 °C with 1 mM isopropyl β-d-1-thiogalactopyranoside. The cells were lysed in lysis buffer , pH 6.5) and the spin supernatant was precipitated with 40% ammonium sulfate before binding to a homemade cAMP-coupled agarose resin. The protein was eluted from the resin with cGMP (20 mM cGMP in lysis buffer) and run on a size-exclusion column to remove the excess cGMP. The protein is stored in gel filtration buffer The PRSET plasmid harboring the 14. Briefly, the purified target protein was concentrated to ~5 mg/mL in 10 mM DTT to reduce all cysteine residues. The protein solution was run through three Micro Bio-Spin columns  to remove the DTT before adding 10 mM 2,2′-dithiodipyriine  for 2 h at room temperature. The unreacted DTDP was removed from the modified protein using three additional Micro Bio-Spin columns. The DTDP-activated protein and two different 30 bp 5′-Thiol-modified dsDNA oligos were combined in a 1-to-1 molar ratio and incubated overnight at 4 °C. The resulting protein-oligo chimera was stored at −80 °C. Each 30 bp 5′-Thiol-modified dsDNA oligo used in the formation of the protein-oligo chimera has a unique non-palindromic overhang that is used to ligate 350 bp dsDNA handles modified with either biotin or digoxigenin in their 5′-end. The crosslinking reaction was done in the DNA crosslinking buffer . Single-molecule optical tweezers measurements of protein constructs in the apo state were obtained by gradient elution (using cAMP) of the protein-oligo chimera from a cAMP-coupled agarose resin. The first eluted samples have an initial cAMP concentration of 20 μΜ, which is further diluted to a final concentration of ~0.02 nM (100-fold < Kd) before using it in the optical tweezers experiment.We followed a protocol previously published by the Maillard laboratory52. Measurements were carried out in DNA crosslinking buffer. The protein sample with dsDNA handles were mixed with of 3.1 μm polystyrene beads (Spherotech) coated with anti-digoxigenin antibodies (termed AD bead) for 5 min at room temperature. The sample is diluted to 1 mL before applying it to the optical tweezers microchamber. The optical tweezer experiments were performed in DNA crosslinking buffer in a temperature-controlled room at 20 °C. A 2.1 μm bead coated with streptavidin (termed SA bead) is trapped on the tip of a micropipette, whereas the AD bead conjugated with the sample is trapped in the optical trap. To form a single tether, the AD bead in the optical trap was moved towards the SA beads on the micropipette tip. A single tether is confirmed by observing overstretching of the DNA handles at ~65 pN53.All data were collected in a MiniTweezers instrument−1, with a 10 s refolding time at 2 pN. Data were collected at a sampling rate of 200 Hz. For each protein construct in each experimental condition we collected 600–1200 trajectories from 6–12 different molecules. Rupture forces representing the cooperative unfolding of one or more protein domains and their associated extension changes were analyzed using a custom-built program implemented in MATLAB software. Unfolding force probability distributions obtained from force-extension curves were transformed to folded-state lifetimes as a function of force and analyzed using the methodology described by Dudko et al.19 (full details are provided in the Supplementary Methods). (2) Force-clamp experiments: the protein was stretched and then held at a desired constant force using constant-force feedback loop. Changes in position at a particular force were recorded at a frequency of 500 Hz for the R241A mutant and 1000 Hz for the wild-type protein. The data from force-clamp experiments was analyzed using a BHMM approach54.Data were collected in two modes as follows. (1) Force-ramp experiments: to mechanically manipulate the target protein construct, we moved the AD bead in the optical trap away and towards the SA bead on the micropipette tip, which results in force-extension curves. The experiment was conducted at a constant pulling velocity of 75 nm s55. The CHARMM27 force field56 was used for the protein and counterions, and the TIP3P57 for water. Parameters for cAMP were obtained with the CHARMM general force field58. The cAMP-bound state was modeled starting from the X-ray structure (PDB code: 1RGS)11. The apo state was obtained by removing cAMP from both the binding sites. Regulatory subunit of PKA with and without cAMP were solvated in a cubic box of 90 Å side. Systems included 24,200 water molecules and counterions were added to guarantee charge neutrality. The bonds between hydrogens and heavy atoms were constrained with the SHAKE algorithm59. The r-RESPA multiple time step method60 was employed where long-range electrostatic interactions are updated every 4fs and all the other interactions every 2fs. Periodic boundary conditions were used and the long-range electrostatics was treated with the particle-mesh-Ewald (PME) method61 using a grid of 81 × 81 × 81. The cutoff for non-bonded interactions was set to 10 Å and the switching function was applied to smooth interactions between 9 and 10 Å. Simulations were conducted in the NPT  ensemble. Temperature was set to 310 K through a Langevin thermostat62 and pressure was set to 1 atm through a isotropic Langevin piston manostat63. The systems were first minimized (2000 steps of conjugate gradient) and equilibrated for 800 ps with the atoms of protein restrained to their initial positions. Production runs for both systems where 230 ns long. The holo structure at 90 ns was used to model the R241A mutant. The R241A mutant with cAMP was minimized and equilibrated as above and production runs was 200 ns long.MD and SMD simulations were performed with NAMD (v2.12)64 and a threshold of 2 Å (Supplementary Fig. RMSD was evaluated over the Cα atoms considering all residues between 119 and 370, excluding the six residues of the unstructured N- and C-terminal tails. RMSD based clustering over Cα atoms was performed in the last 50 ns of simulation employing the hierarchical algorithm as implemented in Wordom65 simulations were performed starting from a set of conformations from the production phase. In particular, four conformations for each the cAMP-bound and apo states were selected from the last 50 ns of the simulation (Supplementary Fig. x-axis. To take in account the elongation of the protein during the pulling, the box was enlarged, by adding water molecules along the x direction, by 170 Å. Finally, the box for SMD simulations was 260 × 90 × 90 Å3 and the grid for the PME set to 256 × 81 × 81. The solvent was equilibrated for 400 ps harmonically restraining the protein in its original conformation and another 400 ps during which the restraints were progressively turned off. All SMD simulations were conducted in the NPT ensemble by using the same parameters employed to carry out the MD simulations. The final conformations were then used as starting point for the SMD simulations. The SMD simulations were performed by restraining the Cα atom of the first residue to its initial coordinates and applying the pulling constant velocity to a dummy atom attached via a virtual spring to the Cα atom of last residue: the spring constant was set to 0.5 kcal mol−1 Å−2 and the pulling velocity was set to 2.5 Å ns−1 along the x direction. Force as a function of the extension was averaged over each set of four SMD simulations. Clusters analysis was also performed for the four unfolding trajectories for each apo and cAMP-bound states. Snapshots of the trajectory where clustered, according to the end-to-end distances, are shown in Fig. SMDFurther information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Reporting SummarySource Data


We report one production and one comprehension experiment investigating the effect of animacy in relative clause attachment in Chinese. Experiment 1 involved a fill-in-the-blank task that manipulated the order of an animate noun phrase in a complex NP construction. The results showed that while low attachment responses exceeded high attachment responses overall (cf. Shen,  Previous studies have shown that languages have different relative clause attachment preferences  or NP2  (see A relative clause is ambiguous when a complex NP occurs in its head noun position as in (1), as it can be interpreted to modify either NP1 (rth) see .(1) A sample experimental sentence from Cuetos and Mitchell .NP the daughter of the colonel [RC who had had the accident]].The journalist interviewed  but not significant at all for the OR conditions [t1 = 0.36, n.s; t2 = 0.51, n.s.).The overall results are given in These results are compatible with the results in Experiment 1, where the animacy effect interacted with the default LA preference in Chinese. While all the conditions numerically showed an LA tendency see , the LA In summary, predictions were partially confirmed. An animate NP modification tendency interacted with relative clause type, but the NP ordering effect was significant only for SRs. On the other hand, there was no clear evidence that the animacy effect modulated relative clause attachments for ORs.The current study aimed to investigate a relative clause attachment preference in Mandarin Chinese by focusing on the interplay between animacy and structural information. To this aim, in Experiment 1, we first investigated whether animacy plays a role in relative clause attachment. The results showed more LA than HA responses elicited overall, confirming an LA preference in Chinese Shen, . HoweverThe results of the current study partially confirmed the animacy effect in relative clause attachment reported in Desmet et al. . That isWashington or Beijing) was considered an animate NP when some degree of agency was associated with it, but the same type of NP was considered inanimate when it was used as a locationExperiment 2 further showed that the animacy effect does not apply uniformly; rather, it is modulated by the relative clause type—specifically, the grammatical role that the head NP plays within a relative clause. This suggests that relative clause attachment in Chinese is constrained by the interplay of semantic  and structural  factors, among others. While the current study is limited in its scope and cannot account for parsing mechanisms underlying the LA bias in Chinese in general, the overall experimental results are not predicted by the Late Closure or the Predicate Proximity Principles, which predict a uniform attachment tendency across different types of relative clauses, regardless of the animacy of a potential head NP. In addition, our results may be incompatible with the Tuning hypothesis. As discussed briefly in the introduction, the Tuning Hypothesis predicts that initial structural decisions are based on language users' prior linguistic experiences (Mitchell and Cuetos, Now, we turn to the question of why animacy matters in the structural analysis of sentences with relative clauses in Chinese. As previously discussed, the importance of animacy has been demonstrated in the processing of relative clauses. For example, Wu et al.  showed tThe results of the OR conditions seem more complicated to account for. Given that head NPs in ORs in Chinese are more likely to be inanimate than animate Pu, , we initIn summary, the present study investigated the effect of animacy in relative clause attachment in Chinese. Experiment 1 confirmed the previous finding of an LA preference in Chinese Shen,  with morThe studies involving human participants were reviewed and approved by the IRB committees of Konkuk University and Nanyang Technological University. The patients/participants provided their written informed consent to participate in this study.NK conceived the study. DO and NK wrote the paper. DO, NK, and HC prepared experiments. DO and HC ran experiments. AZ prepared additional experiment and ran the experiment.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.