The regulation of a series of cellular events requires specific protein–protein interactions, which are usually mediated by modular domains to precisely select a particular sequence from diverse partners. However, most signaling domains can bind to more than one peptide sequence. How do proteins create promiscuity from precision? Moreover, these complex interactions typically occur at the interface of a well-defined secondary structure, α helix and β sheet. However, the molecular recognition primarily controlled by loop architecture is not fully understood. To gain a deep understanding of binding selectivity and promiscuity by the conformation of loops, we chose the forkhead-associated (FHA) domain as our model system. The domain can bind to diverse peptides via various loops but only interact with sequences containing phosphothreonine (pThr). We applied molecular dynamics (MD) simulations for multiple free and bound FHA domains to study the changes in conformations and dynamics. Generally, FHA domains share a similar folding structure whereby the backbone holds the overall geometry and the variety of sidechain atoms of multiple loops creates a binding surface to target a specific partner. FHA domains determine the specificity of pThr by well-organized binding loops, which are rigid to define a phospho recognition site. The broad range of peptide recognition can be attributed to different arrangements of the loop interaction network. The moderate flexibility of the loop conformation can help access or exclude binding partners. Our work provides insights into molecular recognition in terms of binding specificity and promiscuity and helpful clues for further peptide design. Signal transduction and DNA repair in the cellular communication pathway require specific molecular recognition to engage upstream and downstream regulation In a short time, the past 10 years, almost 100 FHA structures from different protein families have been deposited in the protein data bank (PDB) from both NMR and X-ray studies. All FHA domains share similar structural characteristics. The domain spans approximately 100 amino acid residues to fold into a twisted β sandwich of two large β sheets; each sheet contains five and six β strands . Some FHet al. indicated that the KI-FHA domain from kinase-associated protein phosphates (KAPPs) was natively rigid on the binding subsite. According to 15N NMR relaxation of nanosecond-scale motions, the fluctuations of the loop region were reduced, whereas the KI-FHA domain conferred peptide binding. The residues involving peptide recognition were also less movable in both free and bound states Despite sharing low sequence identity, FHA domains perform their function by grasping specific pThr substrates. Previous studies showed that the phosphate group of peptides interacts with the conserved and non-conserved residues around loops 2, 3 and 4 via hydrogen bonds, salt-bridges or both to form charge attractions et al.To date, diverse FHA domains have been widely reported. The first structure of the Rad53-FHA1 domain in complex with the target peptide Pad9 was solved by Durocher et al. studied phosphopeptide binding of the Polo-Box domain through molecular dynamics (MD) simulations, energy calculations and fluid solvation theory. The authors concluded that a phosphoresidue could generate major interactions with domain and water molecules to stabilize the charge of the phosphate group at the domain–peptide interface et al. studied thermodynamic properties of protein–peptide interactions in the PDZ domain systems. The binding of the PDZ domain and peptide was mainly driven by favorable non-polar attractions. The entropic and dynamic aspects also play an important role in recognition Although experimental studies have provided information about protein flexibility and rigidity, we still need to learn the correlation between dynamic movement and binding specificity or promiscuity. To address this question, computational works offer a powerful tool for deep insights Our goal in this study was to understand how signaling domains achieve peptide binding specificity and promiscuity via the structure of loops. We performed all-atom MD simulations with multiple FHA modules to capture domain motions and map the detailed conformational changes before and after peptide binding, including the system of Rad53-FHA1, Dun1-FHA and Ki67-FHA domains. To obtain the best knowledge of loop dynamics in the binding site, we carefully inspected the fundamental residues in loop–loop interaction networks. In addition, we examined the entropic contributions quantitatively to suggest the driving force of binding. Our work provides insights into how the intrinsic dynamic properties of a domain act as a transducer in response to promiscuous peptide recognition.We selected the free FHA domain from three different protein families, Rad53, Dun1 and Ki67, to study domain motions in a non-bound state. The initial coordinates were taken from the PDB codes 1G3G, 2JQJ and 1R21 et al. were applied + were placed on the basis of the Columbic potential to keep the whole system neutral. We also used Particle Mesh Ewald (PME) to consider the long-range electrostatic interactions −1 to maintain the temperature of 300K, and the hybrid Nose-Hoover Langevin piston method was used to control the pressure at 1 atm. The SHAKE procedure was used to constrain hydrogen atoms during MD simulations To study the protein dynamics in free and bound states, we performed MD simulations for non-bound FHA domains and FHA–phosphopeptide complexes using the Amber10 and NAMD2.6 simulation package We aimed to deeply understand how modular domains can exhibit various molecular recognitions with similar folding and how a slight change in loop structure leads to the binding of numerous partners. To study the domain motions of analogous architectures, we performed MD simulations on the FHA domain from different protein families, Rad53, Dun1 and Ki67. We chose these systems because both free protein and complex structures were available and they bound to novel motifs that had rarely been observed in other signaling domains. We used MD simulations with FHA complexes, free proteins, and proteins and peptides from complexes to investigate the dynamics of protein–peptide systems in the free and bound state.Although MD simulation has been a common way to study protein dynamics, the choice of force field is always a key issue in initiating a correct simulation. The ff03 and ff99sb parameters are widely used in the Amber package To confirm that simulations could reach a steady state, we checked the root mean square deviation (RMSD) shown in Although not all loops of the FHA domain interact with the peptide directly, the interactions formed between the six FHA loops could be in charge of peptide binding. We observed that the residues around the loop region formed an extensive network through hydrogen bonds, salt bridges or van der Waals attractions. From MD trajectories, we could identify loop interaction networks from different FHA domain families . The keyIn general, loops 3 and 4 are major pThr differentiated loops; loops 2, 5, and 6 are cooperative loops, stabilizing the whole system and balancing the remaining peptide sequence. For the Rad53-FHA1 domain, the binding loops 3 and 4 interact directly with pThr. These two loops also interact with loops 2, 5 and 6 to form a symmetric structure. Thus, the FHA domains feature a well-defined loop region relative to other signaling-related modules. Moreover, the loop interactions of the Dun1-FHA domain are similar to those of Rad53-FHA1; the only exception is loop 1. Like Rad53-FHA1, the Dun1-FHA domain uses two loop interactions (loops 3 and 4) via conserved residues Ser, Ser+1 and Asn-1 for pThr residue recognition.Compared to Rad53-FHA1 and Dun1-FHA, the Ki67-FHA domain shows different interactions on the binding surface. We did not observe loop–loop interactions between loops 1 and 6, 3 and 6, and 4 and 6; instead, we found interactions between loops 2 and 3, 3 and 4, and 4 and 5 . AlthougAlthough FHA domains have low sequence similarity  The snapshots are taken during 50 ns MD simulations. The thick blue and orange represents the peptide coordinate at 0 ns (after minimization and equilibrium). We observed that the peptide C-terminus tends to form a helical structure in ff03 simulation. (b) The snapshot at 0 ns (after minimization and equilibrium). The key residues, Arg83, Asp+3 and Thr+5 are shown in bond form. (c) and (d) show the conformation simulated by ff99sb and ff03, respectively. Salt-bridges and H-bonds are colored in blue and red, respectively.(TIF)Click here for additional data file.Figure S2The root-mean-square-deviation (RMSD) plot. Blue, red, green and orange represents Rad53-FHA1 (PDB: 1G6G), Rad53-FHA1 (PDB: 1K3Q), Dun1-FHA (PDB: 2JQL) and Ki67-FHA (PDB: 2AFF), respectively.(TIF)Click here for additional data file.Figure S3Correlation map of Rad53-FHA1 (a), Dun1-FHA (b) and Ki67-FHA (c) bound domain. We extracted only loop region here. The columns separated by the red lines represent six loops of each FHA domain. Red letters are the conserved residues.(TIF)Click here for additional data file.Figure S4The key domain-phosphopeptide interactions of Rad53-FHA1 (A), Dun1-FHA (B) and ki67-FHA (C) complex. We showed the overall structure in (1) and detail interactions in (2). Blue and pink indicates FHA domain and peptide, respectively. The dash lines show atom interactions.(TIF)Click here for additional data file.Table S1List of significant residues related to loop-loop interactions from different FHA domain families. Rad53-FHA1 and Dun1-FHA domain form well-organized loop interactions in both bound and free state; however, Ki67-FHA exhibit open-palm conformation due of lacking the interactions between two large β stands, therefore, the interactions between loop 3 and 6, 4 and 6, 1 and 6, and 1 and 3 disappear.(DOCX)Click here for additional data file.Table S2The entropic changes before and after phosphopeptide binding. TΔSX = TΔSX, bound state – TΔSX, free state.(DOCX)Click here for additional data file.Text S1Detail sequence alignments of signaling domains.(DOCX)Click here for additional data file.Text S2The five conserved residues of the FHA domain.(DOCX)Click here for additional data file.Text S3Details of peptide binding.(DOCX)Click here for additional data file.


Cullin-RING ubiquitin ligases are the largest E3 ligase family in eukaryotes and are multiprotein complexes. In these complexes, the Cullin protein serves as a scaffold to connect two functional modules of the ligases, the catalytic subunit and substrate-binding subunit. To date, eight members of the Cullin family proteins have been identified. In the Cul3 ubiquitin ligases, Bric-a-brac/Tramtrack/Broad complex (BTB) domain-containing proteins function as a bridge to connect Cul3 and substrates. While the BTB domain is responsible for Cul3 binding, these proteins usually contain an additional domain for substrate interaction, such as MATH, kelch, Zn finger, and PAM, Highwire, and RPM-1 (PHR domain). With the existence of a large number of BTB proteins in human, the Cul3 ubiquitin ligases ubiquitinate a wide range of substrates involving in diverse cellular functions. In this review, we will discuss recent advances on the functions of Cul3 ubiquitin ligases in cancer development, progression, and therapeutic response and the dysregulation of Cul3-mediated ubiquitination events in human malignancies. In particular, we will focus on three Cul3 substrate adaptors, kelch-like ECH-associated protein (Keap1), kelch-like family member 20 (KLHL20), and speckle type BTB/POZ protein (SPOP), with the intent to highlight novel targets in cancer therapy. The ubiquitin–proteasome system controls a wide range of physiological processes and disease conditions, including cancer. In this system, the addition of ubiquitin moiety to the lysine residue of protein is mediated by a cascade of enzymatic reactions involving E1 activating enzyme, E2 conjugation enzyme, and E3 ubiquitin ligase, in which substrate specificity is conferred by E3 ubiquitin ligase , 5. StruWith the existence of a large number of substrate adaptors, Cul3 ubiquitin ligases have recently been shown to participate in diverse cellular processes, such as cell cycle regulation, protein trafficking, development, and stress responses. In human, functional alterations of this family of ubiquitin ligases are associated with several disease states, such as muscle diseases, metabolic disorders, and cancers . This reKelch-like ECH-associated protein is a Cul3 substrate adaptor containing BACK and kelch-repeat domains in addition to the BTB domain –11. Keap−/− mice are more prone to chemical carcinogen-induced tumor formation in the stomach, bladder, and skin , phosphogluconate dehydrogenase (PGD), transaldolase 1 (TALDO1), and transketolase (TKT), are Nrf2 targets. Nrf2 also activates other metabolic genes, such as malic enzyme 1 (ME1), phosphoribosyl pyrophosphate amidotransferase (PPAT), methylenetetrahydrofolate dehydrogenase 2 (MTHFD2), and isocitrate dehydrogenase 1 (IDH1). These enzymes support NADPH generation, purine production, and glucose flux, thereby providing cancer cells with energy and building blocks of DNA and RNA for accelerating proliferation.Although the chemopreventive function of Keap1–Nrf2 pathway protects normal cells from carcinogenesis, once tumor is formed, cancer cells hijack this pathway for acquiring survival and growth advantage to cope with stressed conditions. For instance, increased Nrf2 expression in cancer cells decreases their sensitivity to a variety of chemotherapeutic agents as well as ionizing radiation, whereas Nrf2 knockdown sensitizes them to cancer therapy –24. A si−/− mice show increased tumor formation at 8th week, after the administration of carcinogen urethane, but become less advanced in malignant progression at 16th week  in cancers, especially in lung cancers, are also uncovered by large-scale omic project , which contains an STGE motif that is similar to the ETGE motif in Nrf2. Through this motif, p62 functions as a pseudosubstrate of Keap1 by competing with Nrf2 for Keap1 binding –51. InteDysregulation of Keap1–Nrf2 pathway in cancer can also be mediated by metabolites. In the hereditary type 2 papillary renal cell carcinoma, homozygous loss-of-function mutation in the fumarate hydratase leads to the accumulation of fumarate, a metabolite of the Krebs cycle. The excessive fumarate forms adduct on the cysteine residues of Keap1, thereby preventing Nrf2 ubiquitination to promote tumorigenesis , 57.via p62-dependent degradation may also be used as a strategy to modulate the activity of Nrf2.Given the dual roles of Keap1–Nrf2 pathway in cancer, manipulation of this pathway could in principle offer therapeutic benefits. For instance, compounds that activate Nrf2 may be used for cancer prevention, whereas Nrf2 inhibitors could be used as adjuvants in chemotherapy to overcome chemoresistance. Among the Nrf2 activators, many are naturally existing phytochemicals. The prototype and most studied agent is SFN found in cruciferous vegetables. SFN has been shown to exert chemopreventive effect against several cancer types, such as colon, skin, lung, and stomach cancers . To dateKelch-like family member 20 possesses the same domain architecture as Keap1, that is, an N-terminal BTB domain, followed by a BACK domain and six kelch repeats. Similar to Keap1 and many other KLHL proteins, KLHL20 binds to Cul3 through its BTB domain to function as a substrate adaptor of Cul3 ubiquitin ligase . This prDeath-associated protein kinase was originally discovered based on its involvement in interferon (IFN)-induced cell death . InteresPML gene was identified at the break point of the t(15:17) chromosome translocation of acute promyelocytic leukemia, which results in the generation of oncogenic PML–RARα fusion protein  unravels an additional layer of the regulation of KLHL20-mediated PML ubiquitination . HIF-1 ativation . Due to tivation . Thus, ttivation . Thus, KSince HIF-1α is frequently upregulated in tumors through hypoxia-dependent or -independent mechanism , KLHL20 In addition to the regulation of its expression level, the activity of Cul3–KLHL20 E3 ligase can be regulated in tumors by an inhibitor . InteresSpeckle type BTB/POZ protein comprises an N-terminal MATH domain, a BTB domain, a 3-box domain, and a C-terminal nuclear localization sequence. Similar to other BTB proteins, SPOP serves as a substrate adaptor of Cul3 ubiquitin ligase, and substrate binding is mediated by its MATH domain, which binds to a SPOP-binding consensus (SBC) motif φ-π-S-S/T-S/T  on the substrate . The linbona fide substrate of SPOP-based Cul3 ubiquitin ligase and an SBC motif in the hinge region of AR mediates its interaction with SPOP  , the mosA significant number of recent studies shed light on the biological functions of Cul3 E3 ligases that regulate tumor development, progression, and therapeutic response. In particular, Keap1, KLHL20, and SPOP are the most reported Cul3 substrate adaptors for their impacts on various cancer types. These three proteins mediate Cul3-dependent ubiquitination on multiple substrates to influence on tumor initiation, progression, and therapeutic response Figure , which cBoth the authors collected and reviewed literatures and wrote the manuscript.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.


Piper nigrum. The suppressive effect of NJ on the CAR-dependent transcriptional activity was found to be species specific, in the descending order of hCAR, rat CAR, and mouse CAR. The unliganded hCAR-dependent transactivation of reporter and endogenous genes was suppressed by NJ at concentrations higher than 5 μmol/L. The ligand-binding cavity of hCAR was shared by NJ and CITCO, because they were competitive in the binding to hCAR. NJ interfered with the interaction of hCAR with coactivator SRC-1, but not with its interaction with the corepressor NCoR1. Furthermore, NJ is agonist of human pregnane X receptor (hPXR). NJ is a dual ligand of hCAR and hPXR, being an agonist of hPXR and an inverse agonist of hCAR.The constitutive androstane receptor  is very important for drug development and for understanding pharmacokinetic drug–drug interactions. We screened by mammalian one hybrid assay among natural compounds to discover novel ligands of human constitutive androstane receptor (hCAR). hCAR transcriptional activity was measured by luciferase assay and mRNA levels of CYP2B6 and CYP3A4 in HepTR-hCAR cells and human primary hepatocytes were measured by real-time RT-PCR. Nigramide J (NJ) whose efficacy is comparable to those of hitherto known inverse agonists such as clotrimazole, PK11195, and ethinylestradiol. NJ is a naturally occurring cyclohexane-type amide alkaloid that was isolated from the roots of  The constitutive androstane receptor  and pregnane X receptor  are two important members of the nuclear receptor superfamily, playing a key role in response to xenochemical stimuli. The functions of CAR and PXR are also very important for drug development and for understanding pharmacokinetic drug–drug interactions, because they regulate the expression of various drug-metabolizing enzymes and transporters, such as CYP2B, CYP3A, CYP2C, glutathione S-transferases, sulfotransferases, UDP-glucuronosyltransferase 1A1, OATP2, MRP2, and MRP3  using mammalian one-hybrid assay  and GAL4/DBD-hCAR/LBD(+3a.a.) were constructed as previously reported . In this cell line, hCAR was expressed only in the presence of tetracycline (Tet) , cells were transfected with the appropriate expression and reporter plasmids, and with pGL4.74  as an internal standard. After overnight incubation, the cells were treated with individual compounds and solvent control [0.1% dimethyl sulfoxide (DMSO)] for 24 h. Luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega). The firefly luciferase activity was normalized against that of Renilla luciferase. Primary screening for hCAR ligands was conducted using a single luciferase reporter assay as follows: cells were transfected with pcDNA-GAL4-hCAR/LBD or pcDNA-GAL4-hCAR/LBD(+3a.a.) and pG5luc reporter plasmids.CYP2B6 (5′-AAG CGG ATT TGT CTT GGT GAA-3′ and 5′-TGG AGG ATG GTG GTG AAG AAG-3′), CYP3A4 , and β-actin (5′-TCC TCC TGA GCG CAA GTA CTC-3′ and 5′-CTG CTT GCT GAT CCA CAT CTG-3′).Total RNA was isolated from whole cell lysates using ISOGEN II , and cDNA was synthesized using a ReverTraAce qPCR RT Kit . Quantitative reverse transcription-PCR (qRT-PCR) was conducted using a THUNDERBIRD™ SYBR qPCR Mix (Toyobo) according to the manufacturer's protocol and the 7500 fast system SDS software . Specific primers were used to target μg), the VP16-hCAR expression plasmid (0.05 μg), pGL4.74 (0.01 μg), and the expression plasmid for GAL4-SRC-1/RID or GAL4-NCoR1/RID (0.05 μg) using PEI Max reagent. The following day, the cells were treated with NJ or solvent control [0.1% DMSO] for 24 h. Luciferase activity was measured using the Dual-Luciferase Reporter Assay System.HepG2 cells were transfected with the pG5luc reporter plasmid  were plated and cultured according to the manufacturer's protocol. Cells were plated on 24-well collagen-coated culture plates with Seeding Medium MIL212. After 6 h, the medium was exchanged for Incubation Medium MIL214. The next day, cells were treated with each compound for 24 h. The mRNA expression level was measured by the abovementioned qRT-PCR.P < 0.05.Statistically significant differences were determined using the one-way ANOVA analysis followed by Tukey's multiple comparison test as the post hoc test, and differences were considered statistically significant at Screening for hCAR ligands was carried out using the assay system established in our previous study . HepG2 cells were transfected with the PBREM-reporter plasmid and the hCAR, mCAR, or rCAR expression plasmids  expression, while it slightly repressed the expression of CYP2B6.Finally, we tested whether NJ could attenuate hCAR-dependent Piper nigrum  play key roles in drug sensing and mobilization of drug-metabolizing cascades by regulating the expression of drug-metabolizing enzymes represented by cytochrome P450s, such as CYP2B6, CYP2C9, and CYP3A4. Unlike PXR and other nuclear receptors, CAR expressed in the nuclear compartment of cultured cell lines retains basal (or constitutive) transcriptional activity in the absence of any ligands. On the basis of this phenomenon, CAR ligands are further subdivided into agonists and inverse agonists. In this study, NJ was found to be a novel potent inverse agonist of hCAR. NJ is a natural occurring cyclohexane-type amide alkaloid, isolated from the roots of Only a few inverse agonists of hCAR have been reported previously. In this study, NJ caused significant deactivation similar to known potent inverse agonist of hCAR, EE2, and PK11195 in HepG2 cells Fig. .Given the rather limited number of known NJ structural analogs, structure–activity considerations remain highly preliminary. Some of the previously reported known cyclohexane-type amide alkaloids did not show such inverse agonist activity as NJ did. Inactive nigramide C is differentiated from active NJ only by the existence of 3,4-methylenedioxyphenyl in C-5 of the cyclohexene ring, suggesting that the C-5 alkane substitution is essential for the potent inverse agonist activity. Nigramide H, which has reverse substitutions in C-2 and C-5 compared with NJ, exhibited weak activity.CYP2B6 and CYP3A4, in HepTR/hCAR cells. Most of the known inverse agonists of CAR also act as agonists of PXR, for example, PK11195, S07662, and CLT are known as potent PXR agonists . Thus, the repression of the expression of CYP2B6 and CYP3A4 can be attributed to functional hCAR. Furthermore, we observed NJ-mediated repression of PB-induced CYP3A4 and CYP2B6 mRNA expression in human primary hepatocytes -dependent expression of the endogenous target genes, CAR-dependent transactivation in the nucleus requires interactions with cofactors. Because CAR has constitutive transactivity, unliganded CAR can interact with coactivators such as SRC-1, TIF2, and PGC-1 (Muangmoonchai et al. In conclusion, we have disclosed a novel inverse agonist of hCAR, NJ, which is classified into a different structural category from the known hCAR inverse agonists, and has the most potent inverse agonist activity among hCAR inverse agonists hitherto known. The discovery of NJ as a potent inverse agonist of hCAR will provide further insight into the molecular basis of hCAR function.


Aspergillus oryzae strain S2 had produced two α-amylase isoforms named AmyA and AmyB. The apparent molecular masses revealed by SDS-PAGE were 50 and 42 kDa, respectively. Yet AmyB has a higher catalytic efficiency. Based on a monitoring study of the α-amylase production in both the presence and absence of different protease inhibitors, a chymotrypsin proteolysis process was detected in vivo generating AmyB. A. oryzae S2 α-amylase gene was amplified, cloned and sequenced. The sequence analysis revealed nine exons, eight introns and an encoding open reading frame of 1500 bp corresponding to AmyA isoform. The amino-acid sequence analysis revealed aY371 potential chymotrypsin cleaving site, likely to be the AmyB C-Terminal end and two other potential sites at Y359, and F379. A zymogram with a high acrylamide concentration was used. It highlighted two other closed apparent molecular mass α-amylases termed AmyB1 and AmyB2 reaching40 kDa and 43 kDa. These isoforms could be possibly generated fromY359, and F379secondary cut, respectively. The molecular modeling study showed that AmyB preserved the (β/α)8 barrel domain and the domain B but lacked the C-terminal domain C. The contact map analysis and the docking studies strongly suggested a higher activity and substrate binding affinity for AmyB than AmyA which was previously experimentally exhibited. This could be explained by the easy catalytic cleft accessibility.We previously reported that  It is often called the TIM-barrel and is generally formed by a set of sub-sites that bind the glucose monomers.8 TIM-barrel. Domain B was a small domain which lied between the strand β3 and helix α3 of the TIM-barrel while domain C was located at the C-terminal end and structured as eight antiparallel β-sandwich fold. A single conserved calcium binding site was identified in the structure of AmyA and found to bridge the A and B domains as in the case of all α-amylase structures. The presence of the calcium may have been important for stabilizing the loop conformation which formed a large lip over the substrate binding groove [1, AmyB and AmyB2proved that the two essential domains of the enzyme catalysis including domain A and domain B were well conserved which explained the conservation activity in the truncated isoforms. Indeed, domain A and B may have played a major role in enzyme catalytic and in the stability of substrate binding process, respectively [http://www.expasy.org/tools/) including theoretical pI and instability index showed a close similitude g groove . The threctively .The elimmilitude .The instability index classified these enzymes as stable proteins. To investigate the proteolysis effect on the tertiary structure of an enzyme, a contact map was used for the visualization and comparison of network of contacts among amino-acid residues belonging to the native and truncated AmyB protein .1, AmyB and AmyB2. The highest amounts of the pink and green dots showed the advanced difference in the compared structures. These results indicated that the changes in the tertiary structure of the truncated AmyB2were the lowest, and therefore the most stable conformation structure among the other truncated enzymes.A contact map was a particularly useful 2D representation of a protein 3D structure that could reflect the stability of the tertiary structure of the enzyme. The black dots showed the common contacts, the pink dots showed the contacts unique to the native structure and the green dots showed the contacts unique to the truncated enzyme structure. As shown in The enzymes were docked with maltotriose. The binding sites of maltotriose on the native enzyme and the truncated one are shown in 2, respectively. Therefore, seeing that AmyB exhibited more binding affinity than AmyA, it was more active.This result agreed well with our experimental studies showing that the affinity of AmyB to soluble starch (Km = 2.60 mg/mL) was about 2 folds higher than the native type (Km = 4.7 mg/mL). This observable fact could also be explained by the easy accessibility of the substrate to the catalytic cleft in the absence of domain C which lacked its common form in GH 70[Klebsiella pneumoniae CGTase from C-terminal led to a truncated one with a similar activity of the native form [Lactobacillus amylovorus were sufficient for catalytic activity and specificity [B. subtilis X23 α-amylase did not affect the catalytic activity, the specificity pattern, the transglycosylation ability, the pH-activity and stability, the optimum temperature, and the binding ability to the starch [Bacillus CGTase generated an enzyme with more activity in starch than that of the native enzyme. It also led to a reduced thermal stability [Bacillus played a role in the thermostability of α-amylases [Indeed, in addition to the catalytic residues interaction with the substrate, 7 other aminoacids comprising Y75, H80, R204, Y252, D340, P341, R344 were also involved in the AmyB-maltotriose complex. However, AmyA-maltotriose complex exposed just H80 and D340 . The ato in GH 70. This do in GH 70. The beg in GH 70–52, 53. ive form . Furthercificity . The elie starch . Howevertability . Furthertability  and Iefutability  reportedamylases .We previously demonstrated that in addition to AmyA and AmyB already monitored in submerged culture, this strain produced an AmyB oligomeric form, in particular, a dominant tetrameric form named AmyC .The elec1, AmyB and AmyB2 of 40 kDa, 42 kDa, 43 kDa that were revealed by a zymogram with a high acrylamide concentration. We demonstrated that AmyA isoform was encoded by an open reading frame of 1500 bp that was deduced from the cloned gene containing nine exons and eight introns. The deduced amino-acid sequence of the ORF revealed three potential chymotrypsin cleaving sites at Y371, Y359, and F379 likely to be AmyB1, AmyB, and AmyB2 C-Terminal end respectively. The molecular modeling study showed that AmyB conserved the (β/α)8 barrel domain and domain B but lacked the C-terminal domain C. The contact map analysis and the docking studies confirmed the high catalytic efficiency of AmyB as it was exhibited experimentally. This observable fact could be explained by the easy substrate accessibility to the catalytic cleft in the absence of domain C.This study was undertaken to describe, for the first time, a chymotrypsin proteolysis generating three α-amylase isoforms AmyB“This article does not contain any studies with human participants or animals performed by any of the authors.”S1 FileThe effect of temperature on the stability of the purified AmyA (A) and AmyB (B) at 40°C (■), 50°C (▲), 60°C (♦), and 70°C (X). (C) The effect of pH on the stability of the purified AmyA (▲) and AmyB (■). The residual activities were determined after the incubation of the enzymes at various pH values with the buffer solutions at 0.1 M (ranging between pH 3–12) and at 4°C for 48 h.(TIF)Click here for additional data file.S2 FileExons and introns are shown with capital letters and a low case letter, respectively. The signal sequence is shown in blue and both the start and the stop codons are shown in red.(TIF)Click here for additional data file.S3 FileThe electrostatic potential maps of AmyA (A) and AmyB (B) .(TIF)Click here for additional data file.


A recent analysis of the genomes of Chinese and Florida lancelets has concluded that the rate of creation of novel protein domain combinations is orders of magnitude greater in lancelets than in other metazoa and it was suggested that continuous activity of transposable elements in lancelets is responsible for this increased rate of protein innovation. Since morphologically Chinese and Florida lancelets are highly conserved, this finding would contradict the observation that high rates of protein innovation are usually associated with major evolutionary innovations. Here we show that the conclusion that the rate of proteome innovation is exceptionally high in lancelets may be unjustified: the differences observed in domain architectures of orthologous proteins of different amphioxus species probably reflect high rates of gene prediction errors rather than true innovation. Yunnanozoon lividum and Haikouella lanceolatum) and from the middle Cambrian (Pikaia gracilens)234Lancelet is the extant representative of basal chordates , which diverged from other chordate lineages (urochordates and vertebrates) about 550 Myr agoBranchiostoma floridae, Branchiostoma lanceolatum and Branchiostoma belcheri diverged ~100–200 Myr ago they are remarkably similar, suggesting that their networks of developmental genes were practically unchanged. In harmony with this assumption the expression pattern of genes implicated in different developmental functions of B. lanceolatum and B. floridae is conserved: the different species of amphioxus appear to be very similar, not only morphologically, but also in the genetic programs directing the development of their structural featuresExtreme conservation of the morphology of lancelets is also supported by the fact that although B. belcheri and the Florida amphioxus, B. floridae are markedly differentet al. have compared presence–absence status of protein domain combinations in proteins of various species and concluded that − in contrast to vertebrates − lancelets evolved rapidly and continuously, ultimately acquiring threefold more domain combinations than any vertebrate. According to their estimates lancelets gained new domain pairs at a rate of >10 per Myr, which is 10- to 100-fold higher than that observed in other metazoan lineagesA recent study, however, has suggested that the proteomes of the Chinese amphioxus, B. belcheri and B. floridae as well as thirteen other metazoan species the authors have selected a set of 729 protein-coding genes and compared the rate of their divergence in the different lineages. In harmony with earlier data of Delsuc et al.et al.910Lingula. In fact, the abundance of Lingula fossils from the Silurian, with morphology very similar to that of extant species, inspired Darwin with the idea of ‘living fossils’. Luo et al.Lingula genes associated with basic metabolism, such as ribonucleoprotein complex biogenesis and RNA processing, show the slowest evolutionary rate among lophotrochozoansInterestingly, the unusually high rate of domain architecture alteration is not accompanied by a high amino acid substitution rate. Out of the orthologs present both in Whereas the low rate of amino acid substitution appears to be in harmony with the low rate of morphological evolution in lancelets, the exceptionally high rate of creation of novel proteins (with novel domain combinations) would suggest that major changes in the complexity of the lancelet proteomes are not coupled to significant changes in phenotype. It must be emphasized that this seems to contradict the observation that high rates of creation of novel proteins are usually associated with major evolutionary innovations such as those that accompanied the appearance of metazoa and vertebratesIn the present work we provide a possible explanation for this contradiction. We suggest that the conclusion that the rate of protein innovation is exceptionally high in lancelets may be unjustified: we demonstrate that the differences in domain architecture of orthologous proteins of the two amphioxus species reflect gene prediction errors rather than true innovation.B. belcheri were predicted by Huang et al.de novo gene prediction, homology-based and transcriptome-based prediction using cDNA alignments, protein alignments, RNA-seq alignments, ab initio data sets and RNA-seq-based predictions14151617ab initio gene prediction methods.Gene models of B. floridae is underpredicted and/or that the number of genes of B. belcheri is overpredicted.Taken at face value, this difference in the number of predicted genes of the two lancelets is surprising since it would suggest that the Chinese lancelet invented or that the Florida lancelet has lost thousands of unique protein-coding genes. Since this dramatic difference in the number of unique genes is not reflected in major phenotype differences of the two species, a more plausible explanation is that the number of genes of B. belcheri and B. floridae are only available at the scaffold level, therefore genes may be fragmented onto multiple individual contigs and this is known to lead to significant errors in the apparent number of genesIt is important to point out that the draft genomes of B. floridae and B. belcheri a high proportion of gene models is mispredicted.The problem of gene prediction errors appears to be even more severe in the case of lancelet genomes: our analysis of the predicted proteomes of various metazoa with MisPred tools have shown that the rate of misprediction of lancelet genes is significantly higher than in the case of vertebrate genomes22et al.B. belcheri and B. floridae in an analysis of the presence–absence status of various domain combinations in different metazoa species. In harmony with earlier data13Huang In our earlier work we have pointed out that in genome-scale studies of protein domain architecture evolution it should not be ignored that a significant proportion of metazoan sequences is mispredicted and that this may seriously affect the validity of the conclusions drawn from such analysesB. belcheri proteome (the Branchiostoma.belcheri_HapV2_proteins.fa dataset) that contained at least two Pfam-A domains and identified their orthologs (if any) in the B. floridae proteome by the reciprocal best-hit method. The rationale of this approach is that when sequences from two complete proteomes are compared orthologs give reciprocal best hits, i.e. protein sequence A of species 1 finds protein sequence A’ of species 2 as its best hit and vice versa. The most important limitation of the reciprocal best match approach is that it may lead to erroneous conclusions if the proteomes compared are incomplete: paralogs or epaktologs may be identified as each other’s best match, instead of the missing orthologs26To estimate the actual contribution of gene prediction errors to the exceptionally high (apparent) rate of protein innovation in lancelets, we have randomly selected 100 proteins of the B. floridae genome was predicted to contain far fewer genes  than B. belcheri  and since one-third of B. belcheri proteins had no ortholog in the B. floridae proteomeB. floridae proteome is less complete than the B. belcheri proteome. Since the mean identity of orthologous proteins of the Chinese and Florida lancelets was calculated to be 81.2%, in the present work we used a cut off-value of 60% amino acid sequence identity as the minimum requirement for orthologs. Using these criteria we have found that 93 of the selected B. belcheri proteins had orthologs in the predicted proteome of B. floridae  of the 93 ortholog-pairs of the two lancelet species had non-identical domain architectures. The rate of domain architecture differences in Chinese and Florida lancelets thus appears to be an order of magnitude higher than that observed in the case of high quality UniProtKB/Swiss-Prot sequences of Metazoan species As shown in B. belcheri and B. floridae differed in domain architecture our working hypothesis was that domain architecture deviation is more likely to reflect errors in gene prediction than true innovationB. belcheri and B. floridae were identical with those of their Swiss-Prot orthologs, but in 15 cases only the B. belcheri protein, in 8 cases only the B. floridae had the same domain architecture as their Swiss-Prot orthologs. Note that in 22 cases both orthologs proved to deviate from the domain architecture of their orthologs in the Swiss-Prot database.In cases where orthologous proteins of To decide whether the domain architecture deviation reflects an error in gene prediction (and not true change in gene structure) we have subjected the ‘erroneous’ member(s) of the orthologous pair to the FixPred protocol to correct the prediction errors. FixPred protocol attempts to correct erroneous sequences in several steps, starting with the simplest solutions , progressing to more time-consuming gene-predictionsB. belcheri and B. floridae reflect errors of gene prediction, as evidenced by the fact that in all cases the FixPred approach found genomic evidence for the identity of the domain architectures of orthologs of the two lancelet species  is orthologous with the N-terminal part of XP_002607180.1 .The difference between the domain architectures of WFIKKN orthologs of B. belcheri is a full-length orthologue of calcium-binding mitochondrial carrier proteins, containing an EF-hand domain and three Mito_carr domains (B. floridae orthologue (XP_002602990.1) are 98% identical in the aligned region, but the B. floridae protein has a long C-terminal extension with a Peptidase_M2 domain homologous with angiotensin-converting enzyme.The 330210_PRF0 protein of  domains . The 330B. floridae protein. Such drastic truncation of a globular domain violates another of the basic dogmas that MisPred employs to detect gene prediction errors22B. floridae ESTs with the XP_002602990.1 protein identified several ESTs  that originate from the putative ‘fusion’ region but they argue against this fusion: they match either the N-terminal or the C-terminal fusion partners part but not both parts of the chimeric XP_002602990.1 protein. ESTs BI386388.1 and BW904813 identified the stop codon of the calcium-binding mitochondrial carrier protein of B. floridae, permitting the correction of its sequence  is orthologous with the C-terminal part of XP_002614044.1.Protein 127590_PFF0 of B. belcheri reflects an error in gene prediction rather than true change in domain organization. It should be noted that the Branchiostoma.belcheri_v18h27.r3_ref_protein.fa dataset is exempt from this error: predicted protein 212090F.t4 is a full-length agrin.Comparison of the domain architectures of 27590_PFF0, 127600_PFF0 and XP_002614044.1 with those of their orthologs in the Swiss-Prot database revealed that the domain architecture of XP_002614044.1 is typical of agrin, a basement membrane protein highly conserved both in protostomes and deuterostomesB. belcheri was found to be orthologous with the C-terminal part of XP_002613924.1 of B. floridae  and C-terminal parts (protein 293740_PRF0) of VPS13D were not located on a single scaffold. In the case of B. floridae all parts of the VPS13D protein are encoded on the same scaffold , but genome annotation identified the N-terminal part as an independent protein. .In view of the high conservation of this domain architecture of Vacuolar protein sorting-associated protein 13D the most plausible explanation for its fragmentation in both B. floridae and B. belcheri derived from their VPS13D proteins differ markedly in domain architecture reflects errors in gene prediction in both species and not proteome innovation.In other words, the fact that orthologous pair of proteins of B. belcheri protein (containing two Pfam-A domains) had no orthologue in the B. floridae proteome but orthologs from other metazoa were present in the high quality Swiss-Prot database our working hypothesis was that this reflects an error of genome annotation rather than true loss of a gene. In such cases we subjected genomic sequences of B. floridae to gene prediction with GeneWiseB. belcheri protein as input to find evidence for the presence of the ‘missing’ gene.If a B. belcheri that has a unique domain architecture characteristic of Ryk receptor tyrosine kinases. Ryk receptor tyrosine kinases have a WIF domain in their extracellular ligand-binding region that serves to bind Wnts35B. floridae. In harmony with the conservation of Ryk in Bilateria, analysis of the B. floridae genome and transcriptome provided evidence for a functional Ryk gene that encodes a protein with the same domain organization as that of B. belcheri  that contains the exons of a hypothetical Ryk receptor tyrosine kinase, permitting the construction of a gene model of the Ryk gene of B. floridae. Tblastn searches of the dataset of B. floridae ESTs with the predicted protein identified three ESTs  that provide full support for the gene model, confirming that B. floridae has a functional Ryk gene (B. belcheri but not in that of B. floridae reflects an error in gene prediction (i.e. the gene model of Ryk is not represented in the predicted proteome of B. floridae).This approach may be illustrated with the case of protein 247370_PRF0 of belcheri . Our anaRyk gene . In otheB. belcher-B. floridae ortholog pairs listed in Many of the For example, the first category contains proteins that appear to be evolutionary precursors/variants of vertebrate-specific proteins  or Pfam-A domain Neur_chan_memb (269420_PRF0). However, given the similarity of the two genomes and the similarity of gene-finding techniques, the identity of domain architecture of lancelet orthologs does not guarantee that they are valid Branchiostome-specific innovations. In some cases the innovation is an artefact: error convergence in genome annotation may yield similarly erroneous predictions. This danger may be illustrated with the case of protein 018300_PFF0 of B. belcheri and its B. floridae ortholog, XP_002588697.1 that contain a Lectin_C domain and five tandem fn2 domains. The co-occurrence of a Lectin_C domain and fn2 domains, however is refuted by ESTs that match the region linking these two domain types  that originate from this region but they argue against the fusion of the Lectin_C domain to the fn2 domains: they match either the Lectin_C domain  or the fn2 region (BW803184) but not both parts of the chimeric XP_002588697.1 protein. ESTs BW716515, BW784251, BW722680 identified the stop codon of the upstream gene encoding the Lectin_C domain, whereas EST BW803184 defines the N-terminal secretory signal peptide of the protein containing fn2 domain(s) encoded by a downstream gene  includes ortholog-pairs with identical but novel domain architectures  that arein types . Tblastneam gene .Cephalochordates are frequently referred to as ‘living fossils’ since they closely resemble fossil chordates from Cambrian strata. This extremely low level of morphological evolution is congruent with a low rate of evolution: cephalochordates were shown to be evolving more slowly than the slowest evolving vertebrate known, the elephant sharkB. belcheri and B. floridae by Huang et al.In view of the low amino acid substitution rate and low level of morphological evolution in cephalochordates, it was surprising that the analysis of the proteomes of This hypothesis, however, fails to provide an explanation for the apparent contradiction between the extreme morphological conservation of lancelets and the high rate of proteome innovation.In our earlier work we have pointed out that creation of novel proteins are inextricably linked to major evolutionary innovations and increased organismic complexity such as those that accompanied the evolution of metazoa and vertebrates13In the present work we provided evidence that the conclusion that the rate of protein innovation is exceptionally high in lancelets may be unjustified: the differences in domain architecture of orthologous proteins of various amphioxus species are more likely to reflect gene prediction errors than true innovation.http://mosas.sysu.edu.cn/genome/download_data.php). One hundred proteins of B. belcheri were randomly selected from the dataset Branchiostoma.belcheri HapV2_proteins.fa and their equivalents were identified in other releases of predicted proteins (Branchiostoma.belcheri v15h11.r2_protein.fa and Branchiostoma.belcheri v18h27.r3_ref_protein.fa). Orthologs of the selected proteins were identified by the reciprocal best-hit method using NCBI’s non-redundant database of B. floridae proteins and the high quality, manually curated Swiss-Prot database.We have used the genome data of Chinese lancelet et al.The domain architectures of proteins (defined as the linear sequence of Pfam-A domains) were determined using Pfam Secretory signal peptides of proteins were predicted with SignalP 4.0 .


Angelman syndrome, a severe neurodevelopmental disease, occurs primarily due to genetic defects, which cause lack of expression or mutations in the wild-type E6AP/UBE3A protein. A proportion of the Angelman syndrome patients bear UBE3A point mutations, which do not interfere with the expression of the full-length protein, however, these individuals still develop physiological conditions of the disease. Interestingly, most of these mutations are catalytically defective, thereby indicating the importance of UBE3A enzymatic activity role in the Angelman syndrome pathology. In this study, we show that Angelman syndrome-associated mutants interact strongly with the proteasome via the S5a proteasomal subunit, resulting in an overall inhibitory effect on the proteolytic activity of the proteasome. Our results suggest that mutated catalytically inactive forms of UBE3A may cause defects in overall proteasome function, which could have an important role in the Angelman syndrome pathology. By attaching ubiquitin molecules to their substrates, E3 ligases have direct control over the functions and, in many cases, protein turnover of these substrates. In addition, loss of function in a number of E3 enzymes has been shown to have an important role in the development of severe physiological conditions such as certain cancers and neurological disorders.3 A representative instance of the latter is Angelman syndrome (AS), a severe neurodevelopmental disorder, with clinical features of mental retardation, developmental delay, ataxia and epilepsy.5 The principal protein affected in AS is the E3 ubiquitin ligase E6-associated protein (E6AP/UBE3A), the gene being found on chromosome 15q11-13. UBE3A was initially identified as an interacting partner of high-risk HPV-16 and -18 E6 oncoproteins,7 but was subsequently found to be linked to the development of AS. AS develops mainly due to genetic defects that lead to the loss of expression of the maternal allele of the UBE3A gene in the hypothalamus.9 Between 65 and 75% of AS patients have been diagnosed with the deletions of 15q11-13, 3–7% of patients show uniparental disomy and ~3% of cases have been found with imprinting defects, such that the functionally defective maternal copy of the gene is expressed in the brain.5 In addition, there are also 5–11% of individuals with AS whose sequence analyses show UBE3A mutations. Most of these have in-frame deletions that would be predicted to result in protein truncations,11 but a number of those patients have milder mutations, such as point mutations, that do not affect the expression of the full-length protein.13 The majority of these mutations however are defective in ubiquitin ligase activity, indicating that the loss of enzymatic activity of UBE3A is important in promoting the development of AS.14Ubiquitination is a highly specific process that involves a group of proteins responsible for adding ubiquitin molecules to cellular substrates, thereby resulting in the modulation of numerous cellular pathways.α, α-Synuclein, p27, promyelocytic leukemia (PML) tumor suppressor, annexin A1, amplified in breast cancer 1 (AIB1) and HHR23A,22 characterization of their interactions with UBE3A have only partially contributed to an understanding of the molecular mechanisms behind the development of AS pathology. In addition, UBE3A has also been shown to interact with other components of the proteasome degradatory pathway, including the ubiquitin ligases HERC2, Ring1B and EDD,25 and recent studies demonstrated a direct interaction between UBE3A and the proteasome itself.27 Whether any of these interactions might also be involved in AS development is an open question. Thus, although many proteins are known to be targeted by UBE3A for proteasomal degradation, much less is known about UBE3A interactions with the proteasome itself, or how these interactions might affect substrate turnover, or whether perturbations in this association can contribute to AS development.Studies have demonstrated that ubiquitin ligase activity of UBE3A has a role in the proteasome-dependent degradation of several cellular substrates, and it can be reasoned that defects in the regulation of some of these substrates can contribute to AS development. However, although a number of UBE3A target proteins have been identified, including Sox9, C/EBP28 Among several subunits that are part of the 19S regulatory particle complex, there are two major ubiquitin receptors, Rpn10/S5a and Rpn13.31 The S5a subunit mediates the targeting of ubiquitin substrates to the proteasome by binding ubiquitin conjugates through a ubiquitin-interacting motif (UIM)32 and loss of this activity of S5a results in decreased proteolytic activity of the proteasome.35 It has also been shown that S5a is regulated by mono-ubiquitination, which inhibits its ability to interact with ubiquitin-conjugated substrates, and also leads to decreased proteasome activity.31 Recent studies have shown that UBE3A can directly ubiquitinate the S5a subunit, and that its Drosophila ortholog, Ube3a, mediates ubiquitination of the Drosophila S5a homolog, resulting in its subsequent degradation.27 Structural studies have indicated that a number of AS-associated UBE3A point mutations occur in the HECT domain, which most likely lead to the expression of catalytically defective proteins.14 We were therefore interested in investigating whether catalytically defective AS-associated point mutants can still interact with the S5a subunit and, furthermore, in determining whether they can exert any inhibitory effects on the proteasomal turnover of ubiquitinated substrates. We show here that AS-associated UBE3A mutants interact more strongly with S5a, with one of the consequences being a general inhibitory effect on the overall proteolytic activity of the proteasome. These results suggest that perturbation of overall proteasome function may be an important element in the development of AS, which thus shows many similarities with other proteasomal neurogical defects.The 26S proteasome is a complex cellular machine that contains a 20S central core, a hollow tube composed of multiple proteasome subunits, which contain proteolytic sites. On each end of the 20S proteolytic core, there is an ATP-dependent 19S regulatory particle, which is involved in capturing the ubiquitinated proteins.36 indicating that S5a is a potential proteolytic target of UBE3A.27 As mass spectrometric analyses identified multiple 19S regulatory subunits (data not shown)37 as potential interacting partners of UBE3A, we were first interested in investigating if any of those other subunits, along with S5a, could also directly interact with UBE3A. To do this, we performed a series of GST pull-down assays using a panel of GST-tagged 19S regulatory subunits. HEK293 cells were transfected with the wild-type FLAG-tagged UBE3A and pull-down assays were performed on the cell extracts 24 h post transfection. Interaction assays were performed using GST fusion proteins of the S2, S4, S5a, S6a, S6b, S8, S9 and S10b subunits, or GST alone for control. The results obtained are shown in Recent studies have shown that UBE3A can directly interact with the S5a proteasomal subunit or can mediate between this subunit and other molecules,in vitro38 and two are AS-associated point mutants, L502P and E550L, which are defective in ubquitination of HHR23A.14 We transfected wild-type and mutant UBE3A expression plasmids into HEK293 cells, and, 24 h post transfection, the cells were extracted and interaction assays performed using the GST–S5a fusion protein. Bound UBE3A was detected by Western blotting and the results in Since UBE3A is an E3 ubiquitin ligase, it was of interest to determine whether its catalytic activity had a role in the interaction with S5a. To do this we analyzed three catalytically inactive mutants of UBE3A. One mutant, C833A, was artificially generated and is defective in forming a thio-ester bond with ubiquitin On the basis of these results, we were next interested in investigating whether the increased interaction between the AS-associated mutants and S5a might have any effects on the overall activity of the proteasome. To do this, HEK293 cells were transfected with UBE3A expression plasmids or the empty vector alone as control. After 24 h, cells were extracted in 0.5% NP40 and homogenized, and the levels of UBE3A expression were verified by western blotting . The proWe then performed a similar analysis where we assessed the effects of E6AP and the two AS-associated catalytically defective point mutants, L502P and E550L, on proteasome activity. The assays were performed as above, but this time the inputs in the analysis were based on the equal protein concentrations from each transfection, rather than being based on equalizing the levels of E6AP expression. The results in The above results suggest that UBE3A can exert a direct effect on the overall proteolytic activity of the proteasome, probably via its interaction with the S5a subunit. To investigate this in more detail, we decided to analyze the effects of UBE3A upon specific ubiquitin/substrate combinations: first, we used p53 and Mdm2. To do this, p53 null H1299 cells were transfected with p53 in the presence or absence of Mdm2 and exogenously added UBE3A wild type or mutants, and total cell extracts analyzed by western blotting. The results in 39 We were therefore interested in investigating if the UBE3A mutants have the same inhibitory effect on HPV-16 E7-induced degradation of these proteins. HEK293 cells were transfected with p130 or p107 in the presence or absence of HPV-16 E7, and the wild-type and mutant UBE3A, and total cell extracts analyzed by western blotting. As can be seen in We then performed a similar analysis on a different set of substrates in a different cell type. It has been previously shown that high-risk HPV-16 E7 oncoprotein mediates proteasome degradation of the pocket proteins p107, p130 and pRb.The above results suggest that UBE3A can affect the overall activity of the proteasome. However, we also wanted to determine whether depleting endogenous UBE3A would affect the ability of Mdm2 to degrade p53. H1299 cells were transfected with siRNA directed against UBE3A or luciferase as a control, and, 48 h later, the cells were further transfected with p53 with or without Mdm2. After a further 24 h, the levels of p53 and UBE3A were then analyzed by western blotting. The results in To identify a potential mechanism by which the catalytically inactive mutants of UBE3A inhibit S5a function, we monitored their effects on the levels of S5a ubiquitination. HEK293 cells were cotransfected with plasmids expressing HA-ubiquitin and S5a, together with plasmids expressing either UBE3A wild-type, C883A, or L502P. Twenty-four hours post transfection, cells were collected in E1A lysis buffer, and complexes were immunoprecipitated using anti-HA-conjugated agarose beads. HA-ubiquitin-bound S5a was then detected by western blotting with anti-S5a antibodies. The results in 8 Although most AS cases display a complete loss of UBE3A, there are a number of cases having only point mutations that yet still develop AS pathology.13 However, all of these mutations render UBE3A catalytically inactive.14 Being an E3 ubiquitin ligase, UBE3A has an important role in the regulation of a number of cellular substrates, and it is reasonable to expect that some of these may be relevant for the development of AS.22 However, UBE3A interacts with other components of the proteasome degradatory pathway including other ubiquitin ligases and elements of the proteasome machinery itself, indicating that there may be other means by which UBE3A loss might contribute to the development of AS.26 Of particular relevance is the overall functional integrity of the proteasome, loss of which is a common feature in many neurogical disorders. Here, we show that AS-associated catalytically inactive mutants of UBE3A have an increased capacity to interact with the proteasomal subunit S5a. Furthermore, we show that one potential consequence of this increased association is an overall reduction in proteasome activity. These results suggest that loss of functional UBE3A expression, in addition to perturbing the turnover of its known cellular substrates, can also have more far reaching effects on general proteasome function and hence on the normal cellular homeostasis.Loss of functional UBE3A expression is associated with the development of AS.27 Proteomic analyses indicated that UBE3A might associate with other proteasomal components, suggesting a potentially more complex pattern of association. To investigate this, we performed interaction assays between UBE3A and a panel of purified proteasome regulatory subunits. In contrast to the proteomic analyses, we found that UBE3A exclusively interacts with the S5a subunit, and no interaction with the other subunits was observed. This supports previous studies showing a direct interaction between UBE3A and S5a, and suggests that the other potential proteasome component interacting partners of UBE3A identified in proteomic screens are indirect interactions. When we compared wild-type UBE3A with catalytically inactive mutants, a number of interesting features were observed. First, the synthetic catalytic mutant, C833A bound to S5a in a manner similar to the wild-type UBE3A. This alone suggests that the interaction between UBE3A and S5a does not require UBE3A catalytic activity. Most interestingly, the two AS-associated mutants E550L and L502P, which are also both defective in catalytic activity, interacted much more strongly than the wild-type protein with the S5a subunit. One potential explanation for this comes from the crystal structure of the Hect domain of E6AP, which happens to be an L-shaped molecule made of two lobes.13 Both of the Angelman-associated mutations, L502P and E550L, are located in the amino-terminal lobe of the HECT domain and their mutations could affect the overall structure of the HECT domain which might lead to defects in HECT domain activities. One consequence of this could be a locking of the HECT domain into a fixed conformation, such that the UBE3A/S5a turnover is blocked, resulting in an apparent increase in association, and thereby blocking access of other substrates to the proteasome.A number of studies have shown that UBE3A can interact with the S5a proteasome subunit leading to its polyubiquitination.in vivo degradation assays. In three cases, p53 degradation by Mdm2, and p130 and p107 degradation induced by HPV-16 E7, all were found to be increased in the presence of wild-type UBE3A. This was further confirmed by siRNA ablation of UBE3A, which also resulted in decrease in the degradation of p53 by Mdm2. In contrast, in each case, the catalytically inactive mutants of UBE3A all had an inhibitory effect on proteasomal activity. Interestingly, this inhibition was reversed by the addition of ectopically expressed S5a, suggesting that the block in the proteasome function was at the level of S5a. As UBE3A appears very closely linked to S5a function, it seems likely that catalytically active UBE3A might be able to promote ubiquitin recycling at the proteasome, and thereby provide a general stimulatory effect on overall proteasome function. Loss of UBE3A or acquisition of mutations inhibiting proteasome activity may occur through different mechanisms. Previous studies have demonstrated that UBE3A increases the degradation of chaperone-bound substrates, and UBE3A expression is increased under various stress conditions, with its overexpression protecting against endoplasmic reticulum stress-induced cell death.40 Therefore, it has been suggested that UBE3A also functions as a cellular quality control ubiquitin ligase. On the basis of this, our results suggest that under conditions where expression of UBE3A is lost or where there is simple catalytic inactivation of the UBE3A, such as with the C833A mutation, there could be an accumulation of chaperone-bound substrates, resulting in protein aggregation and inhibition of proteasome function. In contrast, with the AS-associated catalytically inactive mutants that we have analyzed, which have a greatly increased capacity to associate with S5a, this might be expected to directly block the functioning of the S5a subunit. In either case, the net result is an increase in the levels of S5a polyubiquitination. Interestingly, the inhibition of proteasome activity with MG-132 results in a pattern of S5a ubiquitination very similar to that seen with the catalytically inactive UBE3A mutants (data not shown), further supporting the idea that proteasome function can be blocked by decreased ubiquitin turnover on the S5a subunit. Ultimately, this suggests the importance of a functional UBE3A for optimal proteasome function, and therefore implies one way in which lack of either the protein (ablated expression) or its function  can contribute not only to AS pathogenesis, but possibly also to the pathogenesis of other neurological diseases which occur due to protein aggregation.Indeed, using proteasome activity assays, we showed that ectopically expressed UBE3A upregulates overall proteasomal proteolytic activity, whereas the catalytically inactive mutants of UBE3A have an inhibitory effect on the proteasome. The inhibitory effects on proteasome function were also confirmed using a series of specific ubiquitin ligase/substrate 41HEK293 cells and H1299 cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Transfections were done using calcium phosphate precipitations.45 as have the GST fusion proteins (pGEX-4T-2) of S2, S4, S5a, S6a, S6b, S8, S9 and S10b proteasome subunits.46 HA-tagged pCDNA-3 p130 and p107 expression plasmids were kindly provided by James DeCaprio.The pCDNA-3 UBE3A (isoform I) and C833A (isoform I), His-tagged pCDNA-4 L502 (isoform III) and E550L (isoform III), pCDNA-3 Mdm2, Flag-tagged pCDNA-3 p53, Flag-tagged and HA-tagged CMV HPV-16 E7 and pCDNA-3 pS5a plasmids have been described previously,γ-tubulin (Sigma), rabbit anti-α-actinin (Santa Cruz), anti-β-galactosidase  and appropriate secondary antibodies conjugated to horseradish peroxidase .The following antibodies were used: anti-Flag mouse monoclonal antibody M2 , mouse anti-UBE3A , anti-HA monoclonal antibody 12CA5 , mouse anti-p53 DO-1 , mouse anti-47 After the final wash, the bound proteins were detected using SDS-PAGE and western blotting. The purity and intensity of all fusion proteins was determined by Coomassie Brilliant Blue R (Sigma) staining.Using cellular extracts, pull-down assays were performed by incubating GST fusion proteins immobilized on glutathione agarose beads with cells extracted in E1A buffer  plus protease inhibitor cocktail set I  for 1 h at 4 °C on a rotating wheel. After the incubation the beads were washed once briefly, followed by three additional washes with E1A buffer for 10 min on a rotating wheel at 4 °C, as described previously.2 or 10-cm2 dishes in SDS lysis buffer, and protein detections were done as described previously .Total cellular extracts were prepared by directly lysing cells from 6-cm2 dishes and transfected using Lipofectamine RNAiMax  with siRNA against luciferase  as a control and siRNA against E6AP (Dharmacon).For transient small interfering RNA (siRNA) experiments, H1299 cells were seeded in 6-cm20 and cell lysates were processed according to the manufacturer's instructions . Equal protein amounts of each lysate were added in duplicates to a pair of wells of an opaque white microwell plate. The volume of each plate was brought to 100 μl with assay buffer. In addition, 10 μl of the positive control was added to paired wells. The volume of these wells was also brought to a total of 100 μl by addition of assay buffer. At the same time, the standard curve was prepared by adding 0, 2, 4, 6, 8 and 10 μl of AMC standard to a series of microplate wells. The volume was adjusted to 100 μl/well with assay buffer to generate a standard range of 0, 20, 40, 60, 80 and 100 pmol per well of AMC. Then 1 μl of the proteasome inhibitor was added to one of the paired wells (samples and positive control only) and 1 μl of assay buffer to the other well, followed by mixing. In the next step, 1 μl of proteasome substrate was added to all the wells except the standard curve wells, followed by mixing. The microwell plate was then protected from light. The samples were then incubated at 37 °C and the proteasome activity was assessed using a fluorescent microplate reader  measuring the chymotrypsin-like activity of cell lysates releasing free AMC for 60 min at Ex/Em=350/340.Twenty-four hours post transfection, cells were homogenized with 0.5% NP40 in dH2 dishes in E1A buffer , followed by immunoprecipitation using anti-HA-conjugated agarose beads for 2 h. The beads were then washed and poly-ubiquitinated proteins detected by SDS-PAGE and western blotting.HEK293 cells were transfected with the appropriate plasmids. After 24 h, cellular extracts were prepared by lysing 10 cm


Phytophthora, a genus of filamentous oomycetes, contains many devastating plant pathogens, causing multibillion-dollar damage to crops, ornamental plants, and natural environments. The genomes of several oomycetes including Phytophthora species such as the soybean pathogen P. sojae, have been sequenced, allowing evolutionary analysis of small RNA-processing enzymes. This study examined the evolutionary origins of the oomycete small RNA-related genes Dicer-like (DCL), and RNA-dependent RNA polymerase (RDR) through broad phylogenetic analyses of the key domains. Two Dicer gene homologs, DCL1 and DCL2, and one RDR homolog were cloned and analyzed from P. sojae. Gene expression analysis revealed only minor changes in transcript levels among different life stages. Oomycete DCL1 homologs clustered with animal and plant Dicer homologs in evolutionary trees, whereas oomycete DCL2 homologs clustered basally to the tree along with Drosha homologs. Phylogenetic analysis of the RDR homologs confirmed a previous study that suggested the last common eukaryote ancestor possessed three RDR homologs, which were selectively retained or lost in later lineages. Our analysis clarifies the position of some Unikont and Chromalveolate RDR lineages within the tree, including oomycete homologs. Finally, we analyzed alterations in the domain structure of oomycete Dicer and RDR homologs, specifically focusing on the proposed domain transfer of the DEAD-box helicase domain from Dicer to RDR. Implications of the oomycete domain structure are discussed, and possible roles of the two oomycete Dicer homologs are proposed.Gene regulation by small RNA pathways is ubiquitous among eukaryotes, but little is known about small RNA pathways in the Stramenopile kingdom.  Gene regulation through the action of small RNAs is ubiquitous among eukaryotes and is well characterized in plants, animals, and fungi but not in other eukaryotic lineages  duplex. The miRNA guide strand, similar to siRNAs, is chosen through thermodynamic selection and loaded into a RISC complex. In plants, DCL1 performs both processing steps of the pri-miRNA in the nucleus  in non-metazoans] and RNA-dependent RNA polymerase. Briefly, siRNAs originate from an inverted repeat transcript, sense/anti-sense transcription of the same template, or the action of an RNA-dependent RNA polymerase (RDR) on an ssRNA template  homologs, which include Dicer-like (DCL) proteins, are well conserved across many eukaryotic kingdoms, yet the number of homologs within a taxonomic group is quite variable. Dicers have been studied most extensively in animals, plants, and fungi, and limited numbers of Dicer homologs have been identified bioinformatically in other eukaryote lineages. RDR has also been conserved across eukaryotic kingdoms, although very few animal RDR homologs have been identified, and variation in the number of homologs among different groups is also observed. A study by Cerutti and Casas-Mollano  reconstrPhytophthora studies have provided evidence for functional RNA interference pathways, both post-transcriptional gene silencing and transcriptional gene silencing. Initial reports of transgene inactivation , one RDR, and five AGO homologs. A study of the expression of these and other possible small RNA-related proteins provided evidence for DCL1 and AGO1,2 playing a role in transgene silencing  using TBLASTN searches. Various queries were used, including full-length sequences or RNase III/RDRP domain sequences from species such as Homo sapiens, Trichoplax adhaerens, Arabidopsis thaliana, and Phytophthora sojae. Species with partial or fully-sequenced genomes were selected from all eukaryotic supergroups with the exception of Rhizaria. DCR and RDR homologs identified in previous phylogenetic analyses  was searched by querying specific organisms for the presence of annotated RNase III, DEAD-box helicase, or RDRP domains. Research in different organisms resulted in different naming conventions (DCR and DCL); when already annotated we retained the provided name, otherwise we used the DCR designation. Oomycete homolog sequences were identified at the VBI Microbial Database (currently housed at: eumicrobedb.org) and the P. infestans database at the Broad Institute (www.broadinstitute.org/annotation/genome/phytophthora_infestans/MultiHome.html). Peronospora tabacina homolog sequences were originally provided by David Zaitlin, and the genome has recently been published . Protein domains were identified using a search of the Pfam database , DCL2 (KT387803), and RDR (KT387804).Dicer and RDR homolog sequences were initially identified in the NCBI database , and mRNA was isolated from 250 μg total RNA with an Oligotex column  cDNA was synthesized with the GeneRacer RACE Ready kit (Invitrogen) following the manufacturer's guidelines. Genscan-predicted coding sequences from genome database queries were analyzed with the Primer3  and DCL2-F/DCL2-R (standard manufacturer protocol), and Phusion® High-Fidelity DNA Polymerase (NEB) respectively. The PCR amplicons were in-frame fused with GFP by inserting into the blunt site Stu I in the plasmid pYF2-GFP  was used to examine the expression and subcellular localization of DCL1-GFP and DCL2-GFP fusions. Living hyphae were picked from liquid V8 cultures after 2-3 days growth. Samples were stained with DAPI  for 20 min in the dark before microscopic examination Hardham, . Images mafft.cbrc.jp/alignment/server). For broad species analyses, protein sequences of specific conserved domains were aligned as follows: individual RNase IIIa and RNase IIIb domains, RNase IIIa and RNase IIIb domains concatenated with linking sequences deleted, DEAD-box helicase domains, and RDRP domains. To reduce errors introduced by the presence of partial sequences and to moderate the computational load, amino acid positions which were absent in a majority of the analyzed species were removed from the alignment .Condensed domain alignments were used for Bayesian phylogenetic analyses using MrBayes-3.1.2 . We isolated cDNA from P. sojae mycelium and amplified overlapping segments across the length of each gene, including the use of 5′ RACE and 3′ RACE, to confirm the predicted gene structures and annotate the 5′ and 3′ UTR regions , 55.6% (DCL2), and 52.2% (RDR). Thus the genes follow the general trends of Phytophthora genes, where only one-third possess an intron (average length of 79 bases), of which the average is 1.5 introns per gene, and transcribed sequences have a higher GC content of 58%  and WS41 , and only small changes in relative expression levels were detected . The fusion genes were then introduced into P. sojae transformants. Examination of three independent P. sojae transformants overexpressing DCL1-GFP or DCL2-GFP by confocal microscopy revealed that both Dicers showed strong nuclear localization  prediction using pSORT II , which has recently been described as a noncanonical Dicer that may function as a homodimer in an endogenous siRNA pathway . On the other hand, the two RNase IIIb domains clustered tightly together, with affinity to microsporidial sequences.While all the oomycetes tested, from Dictyostelium discoideum  was basal to the RDRα animal clade. The current study refined the placement of Phytophthora, Hyaloperonospora, Peronospora, Pythium, and Albugo homologs as basal to the RDRγ clade along with Hydra homologs, while the Saprolegnia and Ectocarpus homologs clustered with fungi in the RDRα clade . Only one homolog with a complete sequence covering that region had more than the change at the second residue—Mucor circinelloides RDR2 (DPDED). In the case of Albugo laibachii RDR (–DSE), the sequence is not clear at that site due to unresolved intron placement. More significant deviations from consensus sequences were observed, however, in the DEAD-box helicase domain  observed in canonical DEAD-box helicase domains from DCR homologs, the oomycete DCL1 homologs had numerous substitutions in both sequences. Another commonly observed variation was DEVH at the second motif, seen in Dictyostelium RDR homologs and Chondrus crispus DCL1. Supplementary Figure To analyze the potential for the oomycete DCR and RDR homologs to possess their canonical functions, conserved residues shown to be important for catalytic activity were compared within the domain alignments  had this arrangement; those from S. parasitica (Saprolegniomycetidae) did not. Among other eukaryotes, only RDRs from Dictyostelium (Amebozoa), and from Hydra and Nematostella  had this arrangement. The DEAD box domains from all of the RDR homologs, including the peronosporomycetes, formed a single clade separated with 100% support from the DEAD box domains of the Dicer homologs. This observation suggests that the RDR-associated DEAD box domains have an ancient common origin that is distinct from the Dicer DEAD box domains. This inference is supported in part by the observation that the peronosporomycete and Hydra RDRs also form a cluster within the RDRγ subgroup based on the RDRP domains of the proteins. The Dictyostelium and Nematostella RDRs also fall within the RDRγ subgroup, but do not cluster unambiguously with the peronosporomycete and Hydra RDRs. If the DEAD box domain-containing RDRs all have a common origin, then either the DEAD box domains must have been lost from some RDRs  or the DEAD box-containing RDRs may have been entirely lost from some eukaryotic lineages .The origin of the DEAD-box helicase domain in oomycete RDR homologs is not clear. RDR homologs with DEAD-box helicase domains were relatively uncommon among the eukaryote genomes examined. Among the oomycetes, only species in the class Dictyostelium and Nematostella RDRs coincides with the loss of DEAD box domains from a subset of the Dicer homologs encoded in each of those genomes (DCL2's in the case of the peronosporomycetes). However, the significance of this coincidence is not clear as in Saprolegnia DEAD box domains are absent from its DCL2 as well as all its RDRs. Furthermore, many other eukaryotes that contain Dicer homologs lacking a DEAD box domain also lack RDRs with DEAD box domains.The presence of DEAD box domains in the peronosporomycete, Dictyostelium, Hydra (not shown), and Nematostella only have Dicer homologs that lack DEAD box domains. Furthermore the branch lengths of the DEAD box domains of the peronosporomycete DCL1's are unusually long, whereas the branch lengths of the DCL1 RNase III domains are not exceptionally long. Whether this indicates the DCL1 DEAD box domains are currently degenerating or evolving a novel function is unclear. The oomycete DCL1 homologs are also unique in that they are the only DEAD-box domain-containing proteins that lack a predicted Helicase-C domain. Although the oomycete DCL1 sequences are highly divergent from other DCR homologs, it is unclear what the effect of these changes would be on the catalytic activity of the domain. In the DE[C/A]H box sequence, for example, switching the aspartic acid and glutamic acid residues may not have a huge impact on function, and a tryptophan in the third, most variable, position has unknown significance. As well, even though the other DEAD-box helicase domains in the analysis have a histidine in the fourth position, other members of the helicase family contain aspartic acid in the fourth position. Therefore the oomycete DCL1 homologs could potentially retain their function. Without biochemical or mutagenesis studies it is unclear whether the divergence observed for the DEAD-box helicase is related to function of the domain.Of the organisms that contain a DEAD-box helicase domain in the RDR homolog, peronosporomycetes are unique in that they also have a DEAD-box helicase domain in one of the Dicer homologs ; D. discoideum, Martens et al. ; and the USDA Agricultural Research Service (CRIS number 5358-22000-03900D).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.


Plasmodium falciparum erythrocyte membrane protein 1) encoded by the polymorphic multi-copy var gene family plays an important role in parasite biology and the host-parasite interactions. Sequestration and antigenic variation is an essential component in the survival and pathogenesis of Plasmodium falciparum and contributes to chronic infection . The DBLα domain of PfEMP1 is a potential target for immuno-epidemiological studies and has been visualized as a vaccine candidate against severe malaria. Extensive var gene diversity (DBLα domain of PfEMP1) is an important aspect of pathogenesis in parasite lines. Specific host receptors like heparin, heparan sulphate, blood group A and complement receptor 1 have been reported to bind DBLα domain. Although heparin has been experimentally shown to disrupt the parasite-host interaction and effectively disrupt rosetting, the binding sites for the DBLα domain and mechanism behind heparin-mediated rosette inhibition have not been elucidated.The variant surface antigen PfEMP1 (3D structures and epitopes of DBLα domain in 3D7 and in two parasite isolates have been predicted and compared. Docking studies on DBLα domains with human GAG receptors (heparin and heparan sulphate) to predict the strength of association between the protein–ligand interactions were performed.The DBLα domain structures showed extensive diversity and polymorphism in their binding sites. The linear as well as conformational epitope prediction analysis predicted several B cell epitopes at variable positions in different DBLα domain variants . The docThus such an analysis may lead to the development of novel interference strategies to block red blood cell invasion and provide protection against malaria and provide an insight into the complexities of host parasite interactions.


Chlorobium tepidum RocCOR tandem reveals conformational heterogeneity of the Roc domains and the influence of LRRK2-analogous PD-mutations.Kinetic data for leucine-rich repeat (LRR) kinase 2 (LRRK2) confirms that dimerization is essential for efficient GTP hydrolysis and that Parkinson's disease (PD) mutations cause decreased activity. Investigation of the  Chlorobium tepidum with site-directed spin labelling and pulse EPR distance measurements to follow conformational changes during the Roco G-protein cycle. Our results confirm that the COR domains are a stable dimerization device serving as a scaffold for the Roc domains that, in contrast, are structurally heterogeneous and dynamic entities. Contrary to other GAD proteins, we observed only minor structural alterations upon binding and hydrolysis of GTP, indicating significant mechanistic variations within this protein class. Mutations in the most prominent member of the Roco family of proteins, leucine-rich repeat (LRR) kinase 2 (LRRK2), are the most frequent cause of late-onset Parkinson's disease (PD). Using a stable recombinant LRRK2 Roc-COR-kinase fragment we obtained detailed kinetic data for the G-protein cycle. Our data confirmed that dimerization is essential for efficient GTP hydrolysis and PD mutations in the Roc domain result in decreased GTPase activity. Previous data have shown that these LRRK2 PD-mutations are located in the interface between Roc and COR. Importantly, analogous mutations in the conserved C. tepidum Roc/COR interface significantly influence the structure and nt-induced conformational changes of the Roc domains.Ras of complex proteins (Roc) is a Ras-like GTP-binding domain that always occurs in tandem with the C-terminal of Roc (COR) domain and is found in bacteria, plants and animals. Recently, it has been shown that Roco proteins belong to the family of G-proteins activated by nucleotide (nt)-dependent dimerization (GADs). We investigated the RocCOR tandem from the bacteria  The Roco family comprises large multi-domain proteins that are characterized by the presence of a Ras (rat sarcoma)-like GTP-binding (G) domain called Ras of complex proteins (Roc) that always occurs in tandem with a C-terminal of Roc (COR) domain –3. Roco The most prominent member is LRRK2 that has been found to be mutated and activated in individuals suffering from familial Parkinson's disease . Previously we have shown that Roco proteins belong to the class of G-proteins activated by nt-dependent dimerization (GADs) ,6. This In contrast, GADs show reciprocal complementation of their active sites and seem not to require GAPs and GEFs, as they appear to contain the elements necessary for the nt-regulated switching cycle . They exChlorobium tepidum RocCOR (CtRocCOR) unit  mutants were expressed and purified as previously described for the corresponding wild-type protein  experiments at low temperature (50 K) the buffer was supplemented with 5% glycerol (v/v). We omitted the use of deuterated solvents  to safely exclude possible isotope effects on nt-binding and conformational changes and/or shifts of the conformational equilibrium induced by nt-binding. For the different nt-bound states either 1 mM GDP, 1mM 5′-guanylyl imidodiphosphate (GppNHp) or 1 mM GDP, 1 mM AlCl3 and 10 mM NaF was added respectively. Spin concentrations have been determined by double integration of room temperature continuous wave (cw) spectra and comparison with reference samples of known spin concentration and have been used to calculate spin-labelling efficiencies that have been found to vary significantly between the different RocCOR mutants (40%–100%). For all EPR experiments, the protein concentrations were 50–100 μM.The spin label  methanethiosulfonate  was covalently attached to the cysteine residues of the RocCOR mutants. In brief, the protein in buffer  with a home-made EPR spectrometer equipped with a Bruker dielectric resonator (MD5), with the microwave power set to 0.4–0.6 mW and B-field modulation amplitude adjusted to 0.15 mT. Samples were loaded into EPR glass capillaries .DEER measurements were accomplished at X-band frequencies (9.3–9.4 GHz) with a Bruker Elexsys 580 spectrometer equipped with a Bruker Flexline split-ring resonator ER 4118XMS3 and a continuous flow helium cryostat ESR900 (Oxford Instruments) controlled by an Oxford Intelligent temperature controller ITC 503S. Measurements were performed using the four-pulse DEER sequence ,32:νobs). Time t′ is varied, whereas τ1 and τ2 are kept constant. The dipolar evolution time is given by t=t′–τ1. Data were analysed only for t > 0. The resonator was over-coupled to Q ≈ 100; the pump frequency νpump was set to the centre of the resonator dip and coincided with the maximum of the nitroxide EPR spectrum, whereas the observer frequency νobs was ∼65–75 MHz higher, coinciding with the low-field local maximum of the spectrum. All measurements were performed at a temperature of 50 K with observer pulse lengths of 16 ns for π/2 and 32 ns for π pulses and a pump pulse length of 12 ns. Proton modulation was averaged by adding traces at eight different τ1 values, starting at τ1,0=200 ns and incrementing by ∆τ1=8 ns. Data points were collected in 8-ns time steps. The total measurement time for each sample was 24–48 h. Data analysis was performed with the software package DeerAnalysis2013  was performed on π/2. A 16 ns MD simulation was carried out in YASARA, utilizing the Amber03 force field . Comparison with the experimental distance distribution  for the RocCOR dimer applying a RLA approach  to accouribution b shows cP(d)] (P(d) of the MD-RLA from the respective P(d) of the experimental data for the apo state. Grey-shaded areas indicate the remaining contributions of the distance distributions. It has to be noted that deconvolution of the experimental distance distributions is complicated due to the combined effects of spin-label rotamer distribution, protein conformational heterogeneity and experimental uncertainties. Furthermore, as discussed above for the DEER data for position 928, we cannot exclude a possible influence of background-correction artefacts. Nevertheless, the broadness and multimodality of the remaining contributions attributable to conformational states different from the crystal structure indicate the presence of at least two additional conformations. This is most obvious for position 542 where the difference distance distribution exhibits two clearly distinct populations. This leads us to the conclusion that the Roc domains in the RocCOR dimer in solution are characterized by at least three conformational states in equilibrium.The experimental distance distributions for all three spin-label mutations in the Roc domain  in the aP(d)] e were cax or GDP (P(d)] (P(d) for the apo state from P(d) obtained in the presence of the respective nts. As an additional indication for the average direction of the inter-spin distance changes upon nt binding, we calculated the means of the experimental distance distributions ] e were caibutions .x (–0.13 nm). Inspection of the distance distributions and ΔP(d) plots suggest that, bearing in mind the experimental error and the limitations of Tikhonov regularization, this can be explained by de-population of the conformational state characterized by the crystal structure and increased population of the conformational states characterized by distance distributions with mean distances of ∼ 2 and 4 nm respectively that have been identified in the apoP(d)–MD-RLAP(d) plots. Thus, our data show a clear influence of the bound nt on the occupancy of the conformational states. Remarkably, in the GppNHp- and GDP-bound states the population of both states increases, whereas in the GTP hydrolysis state only the state with short inter spin distances shows an increased population, resulting in the shortest mean inter-spin distance observed. Furthermore, the width of the short-distance peak, being significantly decreased compared with the distribution in the apo state in the 1.5–3.5 nm range, indicates reduced backbone and/or label dynamics, being in line with a fully assembled active site (bearing in mind that the putative arginine finger Arg543 is next to the label position) and thus a more rigid Roc dimer. Interestingly, in the GppNHp- and GDP-bound states, the width of the short-distance peak is even smaller than for GDP·AlFx, but the mean distances are ∼0.2 nm larger. This implies that nt binding, in general, causes reduced backbone and/or side chain dynamics at and near the nt-binding pocket.For S542R1, located directly in the dimer interface of the two Roc domains d, we obsx (–0.28 nm) and GDP (–0.37 nm). This is also reflected in the ΔP(d) plots, which reveal increased population of conformational states characterized by shorter distances for the latter nts and population of a conformational state with inter-spin distances around the mean distance in the apo state. Nevertheless, for C600R1,as well as for T476R1, the correlation between the distance contributions in the apoP(d)–MD-RLAP(d) plots with the changes in population from the nt–P(d)apoP(d) plots is not as clear as for S542R1, possibly owing also to the increased signal-to-noise ratio for these datasets. The mean distances for T476R1 exhibit the smallest variation upon nt binding and the shortest mean inter-spin distance is observed in the apo state. This indicates that the Roc dimer does not show ‘open’ and ‘closed’ states like they have been observed for MnmE [The mean inter-spin distance for C600R1  also remfor MnmE  in cours1441 to cysteine, glycine or histidine. Previously it has been suggested that the LRRK2 R1441C mutation has reduced GTPase activity [Spodoptera frugiperda Sf9 cells. A multiple turnover radioactive charcoal assay was used to measure the GTPase activity of the LRRK2 fragment. In this assay, the proteins are mixed with γ32P-labelled GTP and the subsequent Pi release is measured over time. The GTPase kinetics of the Roc-COR-kinase fragments of LRRK2 follow Michaelis-Menten kinetics. For the wild-type protein, its kcat of phosphate release is 0.8 min−1 and the Km of GTP is 343 μM. Consistent with the previous studies, the LRRK2 Roc (R1441C) PD-mutation results in a decreased GTPase activity (kcat of the R1441C mutant is ∼2-fold lower (0.37 min−1) than that of the wild-type protein, whereas the Km value is ∼1.5-fold higher (541 μM) compared with wild-type. Unfortunately the quality and the amount of the purified LRRK2 Roc-COR-kinase fragment were not sufficient for detailed biophysical studies. Furthermore, the fragment contains 15 cysteines, thus making it a very difficult target for spin labelling with standard approaches.The most prominent PD-mutations in the LRRK2 Roc domain are Argactivity . Howeveractivity  [21,37–41441LRRK2 in CtRocCOR is Tyr558, whereas the less frequently PD-related mutation Ile1371LRRK2 corresponds to Leu487CtRocCOR  is comparable to that found for mouse full-length LRRK2 (210 μM) and for recombinant human LRRK2 Roc domain (553 μM) [kcat) of the recombinant Roc-COR-kinase fragment is ∼20-fold slower compared with homologous expressed mouse full-length LRRK2 (13.8 min−1), but 40-fold faster than that of monomeric recombinant human LRRK2 Roc domain (0.02 min−1) [The results of the DEER distance measurements show that the COR domains in the RocCOR tandem dimer serve as a scaffold for the Roc G-domains by forming a constitutive dimer through interaction of their C-terminal subdomains ,25. Furtth LRRK2 0 μM and (553 μM) . In contth LRRK2 0 μM and 2 min−1) , suggest2 min−1) ,25, dimeCtRocCOR crystal structure. Given the large experimentally observed distance distribution widths of ∼3 nm for all positions and under all conditions tested in the present study, the theoretical mean distances lie well within these distance intervals, as can be seen from the difference distance distributions in CtRocCOR, as comparison with theoretical distance distributions calculated from the LRRK2–Roc dimer structure  dimer. In contrast with that, we could not identify ‘open’ and ‘closed’ conformations for the G-domains in the RocCOR dimer. Our observations indicate that the Roc domains remain associated throughout the whole GTP hydrolysis cycle, as can be seen from comparison with the mean inter-spin distances reported in x the mean inter-spin distances and distribution widths especially for S542R1 appear to be decreased, suggesting a more rigid assembly of the G domains compared with the other states. Thus, mutual complementation of the G-domain's catalytic centres by protrusion of the in prokaryotes conserved arginine finger (Arg543) into the active site of the other protomer to promote GTP hydrolysis would take place in RocCOR via subtle domain movements, e.g. relative rotation of the two domains and local conformational changes in the Roc domains rather than by large scale conformational changes and domain association. Furthermore, these observations indicate that, in contrast with other GADs like MnmE and hGBP1 where the isolated G domains suffice to form a functional dimer, the COR dimer scaffold is not only necessary to keep the Roc G-domains in close vicinity, but also for fine-tuning of their conformational equilibrium to favour states that are competent for nt-exchange and GTP hydrolysis respectively. Consequently, our findings also provide a rationale for regulation of the GTPase activity by neighbouring domains in Roco proteins.Furthermore, our DEER data support the notion based on the absence of defined electron density for the second Roc domain in the RocCOR dimer and on proteolytic digestion experiments  that theR domain . We furtelevance ,21. Our elevance ,28. We oelevance . A slighelevance . In thisC. tepidum RocCOR structure revealed that the position analogous to this LRRK2 PD-mutation site is located in the hydrophobic Roc/COR interface that is highly conserved between bacteria and man [CtRoc domain that are located in the Roc/COR interface and that have been shown to have the strongest effects on GTPase activity [We confirmed that the LRRK2–R1441C PD-related mutation results in decreased GTPase activity. The  and man . We showactivity  significactivity ,43. ThisCtRocCOR, do not affect nt binding, but do results in impaired GTPase activity [C. tepidum have a strong effect on the dynamics of the Roc domains and that this is the primary cause of the decreased GTPase activity of both proteins [Although we cannot exclude that the LRRK2 RocCOR domain has a different structure and activation mechanism than bacterial Roco proteins, it seems rather unlikely. Recent data have shown that LRRK2 has a similar low nt affinity and hydrolysis activity to that of the bacterial Roco proteins ,22. Likeactivity ,37,38. Wproteins ,37–40.