The precise regulation of microtubule dynamics is essential during cell division. Thekinesin-13 motor protein MCAK is a potent microtubule depolymerase. The divergentnon-motor regions flanking the ATPase domain are critical in regulating its targetingand activity. However, the molecular basis for the function of the non-motor regionswithin the context of full-length MCAK is unknown. Here, we determine the structureof MCAK motor domain bound to its regulatory C-terminus. Our analysis reveals thatthe MCAK C-terminus binds to two motor domains in solution and is displacedallosterically upon microtubule binding, which allows its robust accumulation atmicrotubule ends. These results demonstrate that MCAK undergoes long-rangeconformational changes involving its C-terminus during the soluble tomicrotubule-bound transition and that the C-terminus-motor interaction represents astructural intermediate in the MCAK catalytic cycle. Together, our work revealsintrinsic molecular mechanisms underlying the regulation of kinesin-13 activity.DOI:http://dx.doi.org/10.7554/eLife.06421.001 Within a cell, there is a scaffold-like structure called the cytoskeleton thatprovides shape and structural support, and acts as a transport network for themovement of molecules around the cell. This scaffold contains highly dynamic polymerscalled microtubules that are made from a protein called tubulin. The constant growthand shrinking of the ends of the microtubules is essential to rebuild and adapt thecytoskeleton according to the needs of the cell.A protein called MCAK belongs to a family of motor proteins that can move alongmicrotubules. It generally binds to the ends of the microtubules to shorten them.Previous studies have found that a single MCAK protein binds to another MCAK proteinto form a larger molecule known as a dimer. Part of the MCAK protein forms aso-called motor domain, which enables this protein to bind to the microtubules. Oneend of the protein, known as the C-terminus, controls the activity of this motordomain. However, it is not clear how this works.Talapatra et al. have now revealed the three-dimensional structure of MCAK'smotor domain with the C-terminus using a technique called X-ray crystallography. Theexperiments show that the C-terminus binds to the motor domain, which promotes theformation of the dimers. A short stretch of amino acids—the building blocks ofproteins—in the C-terminus interacts with two motor molecules. This‘motif’ is also found in other similar proteins from a variety ofanimals. However, once MCAK binds to a microtubule, the microtubule triggers therelease of the C-terminus from the motor domain. This allows MCAK to bind morestrongly to the microtubule.The experiments also show that the binding of the C-terminus to the motor domainalters the ability of MCAK to associate with microtubules, which encourages theprotein to reach the ends of the polymers. Future work is required to see whetherother motor proteins work in a similar way.DOI:http://dx.doi.org/10.7554/eLife.06421.002 The Kinesin-13 protein family is a class of microtubule depolymerases that regulatemicrotubule dynamics. Kinesin-13 family members are essential for correct interphasemicrotubule organization, cell polarity, and chromosome segregation during mitosisdomain. The CT domain induces motor dimerization in solution, reminiscent of theself-interaction mechanism of Kinesin-1 with its C terminus . The cryKinesin-1 is regulated through an auto-inhibitory mechanism whereby one C-terminaltail binds at the interface of the two motors, such that it creates a second point ofattachment in addition to the coiled coil region. This limits the head movement ofone kinesin with respect to the other . We sougAbove, we demonstrated that the MCAK CT domain interacts with the MCAK motor domain.Full-length MCAK is a dimer in solution . TherefoWe next determined the affinity of the MCAK CT domain for the motor domain usingintrinsic fluorescence spectroscopy. We titrated increasing amounts of the CT domainwith 1 μM motor domain and measured the corresponding change in thefluorescence intensity of aromatic residues . The cha709QLEEQASRQISS720) using molecular replacement to aresolution of 3 Å with good stereochemical parameters , full-length MCAKE712C eluted as onecomplex, of similar size to MCAK (E712C ran similarly to MCAK on an SDS-PAGE gel (E712C domain. Under oxidizing conditions(without DTT), the motor and both the GST-CTE712C domain and untaggedCTE712C domain formed a covalent complex , the affinity ofMCAKE712C for microtubules was reduced and a fraction ofMCAKE712C did not bind microtubules, even at saturating microtubuleconcentrations  fraction would be in a lockedconformation and would not bind or only bind weakly. Cosedimentation of the motordomain in the presence of the CTE712C domain and DTT was similar to theMCAK motor alone with Kds of 0.44 and 0.64 μM respectively,indicating that the CTE712C domain did not interfere with the motor underreducing conditions  sample did not bind to microtubules , His-MCAKprotocol . Full-leprotocol . His-MCAEEEEE, a gene encoding the C terminus of MCAK was synthesised byLifeTechnologies and was subcloned into the MCAK vector, described in TGCGCCTGGCCATGCAGCTGGAAGAGCAGGCTAGCAGACAAATAAGCAGCAAGAAACGGCCCCAGTGACTCGAGCCC– 3′ and reverse: 5′ –GGGCTCGAGTCACTGGGGCCGTTTCTTGCTGCTTATTTGTCTGCTAGCCTGCTCTTCCAGCTGCATGGCCAGGCGCAAGGCCTTGATGGATCCGGG– 3′ were first annealed together as double stranded DNA. The insertwas then ligated into pGEX 6p1 . For the MCAKCT600 of 0.7–0.8for 16 hr at 18°C. Cells were lysed by sonication in lysis buffer  and clarified at 20,000 rpm for 1 hr at 4°C.His-tagged and GST-tagged proteins were subsequently purified usingNi-NTA–agarose beads and glutathione-sepharose beads, respectively ) according to the manufacturer's guidelines.MCAK constructs containing the motor domain were eluted with elution buffer .Cleavage of the GST tag was performed using the GST-3C protease overnight at4°C. Proteins were further purified using gel filtration chromatographypre-equilibrated in gel filtration buffer . Analytical gelfiltration chromatography was performed using either a Superdex 75 or a Superose 610/300 GL column . To purify the CT domain alone, we cleaved GSTand CT after gel filtration and a concentration step, and performed a glutathioneaffinity-purification third step to remove the GST and collect the CT domain. The CTdomain was then further purified by separating it from remaining GST using aconcentrator with a 3 kD-cutoff. Protein concentrations were determined with acombination of Bradford protein assays and densitometry of Coomassie-stained gelsrelative to a BSA standard. To visualize both the motor and CT domains on proteingels, 16% Tricine gels were used, according to the manufacturer's instructions.Protein expression was induced by addition of 0.5 mM IPTG to BL21(DE3) Codon pluscells transformed with respective constructs at ODMCAK motor domain was pre-treated with spectroscopy buffer  supplemented with 5 mM EDTA to remove any bound ADP. The protein was thendesalted into spectroscopy buffer using a Disposable PD-10 Desalting Columns . The experiment was performed with a modified protocolas previously described . A Cary 709QLEEQASRQISS720), generated by GL Biochem (Shangai)Ltd (China) or anti-His antibody to probe for the motor domain alone . The data from at least three independent experiments were fittedto a modified Hill equation to determine the apparent Kd.Full-length MCAK complex was diluted in S buffer  to50 nM, in absence of nucleotide to prevent MCAK-dependent microtubuledepolymerisation. To assemble an MCAK motor-CT domain complex, 50 nM of the motordomain and 100 nM of the CT domain were used. Microtubule binding assays wereperformed as described  using eqMicrotubule depolymerization assays were performed essentially as describedpreviously . MCAK waE712C and CTE712C domains were cross-linked to themotor domain by incubating them with the motor domain in a buffer containing 100 mMHEPES, pH 7.4, 150 mM NaCl but lacking DTT for 1 hr at 4°C. As a negativecontrol the motor domain and the CTE712C domain were incubated in theidentical buffer, supplemented with 5 mM DTT.The GST-CTTubulin (5 mg/ml) was first polymerized into MTs in the presence of 1 mM GTP andgradual addition of 0.05 µM, 0.5 µM and 2 µM taxol for 1 hr at37°C. The polymerized MTs were then treated with 100 μg/ml subtilisinand incubated at 37°C for 10  min to cleave β-tubulin tails and120 min to cleave both α- and β-tubulin tails. Each reaction was thenterminated with the addition of 3 mM PMSF. DM1A  and c-terminalβ-tubulin  were used to detect the α- and β-tails,respectively by western blotting. The subtilisin treated microtubules were thenpelleted at 28°C in a TLA100 rotor at 80,000 rpm for 10 min and themicrotubule pellets were resuspended in warm BRB80 buffer to obtain subtilisintreated microtubules. The cosedimentation assays were then performed as describedbefore.av) were determined from the equation Kav= (Ve − Vo)/(Vt −Vo), where Ve, Vo, and Vt represent theelution volume of the protein of interest, the column void volume and the total bedvolume of the column, respectively.Size-exclusion chromatography with on-line multi-angle light scattering (SEC-MALS)was performed using a GE Superdex 200 10/300 GL column on an ÄKTA FPLC system.MALS measurements were performed using a MiniDAWN in-line detector . MCAK motor domain and C terminus were at 2 mg/ml in 100 mMHEPES, pH 7.2, 150 mM NaCl. Protein concentration was monitored using a UV monitor at280 nm and a refractive index detector was set at 690 nm . Data were analyzed using Astra software  using the refractive index detector and arefractive index increment (dn/dc) value of 0.185 ml/g. Gel phase distributioncoefficients in a ratio of 1:2 for 1 hr at 4°C before setting up crystallization trials.Elongated rectangular crystals appeared by vapor diffusion after two days in sittingdrops using 24% wt/vol PEG 1500 and 20% vol/vol Glycerol as a precipitant. Crystalswere grown in MRC 2 Well Crystallization Plate  at 19°C. Crystals were cryoprotected in a solution containing 28% wt/volPEG 1500 and 30% vol/vol Glycerol and flash-frozen in dry liquid nitrogen.1 mM MCAK motor domain  was incubated with theCT peptide Diffraction data were recorded at Diamond Light Source on beamline ID24 at 100 K.Data were processed using XDS package  and SCALThe final model and the structure factor amplitudes have been submitted to theProtein Data Bank under the accession code 4UBF.Transfection of GFP-MCAK and mCherry-EB3 constructs in HeLa cells was performed usingEffectene  according to manufacturer'sinstructions. Images were acquired on a Nikon TIRF inverted microscope system with aperfect focus, with a 100× TIRF Apo 1.49 objective  using an AndorZyla technology Scmos camera. Imaging was carried out at 37°C. Images wereanalyzed using ImagePro software and OMERO. Linescan averages were calculated fromover 100 comets. reviewprocess). Similarly, the author response typically shows only responsesto the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of thepublished articles . An edited version of theletter sent to the authors after peer review is shown, indicating the substantiveconcerns or comments; minor concerns are not usually shown. Reviewers have theopportunity to discuss the decision before the letter is sent  and two reviewers. The editor has concluded that the paper may become suitablefor publication in eLife if the issues raised by the referees can beaddressed suitably. The reviewers, who also reviewed your original submission, havediscussed their concerns with each other and the editor, and the review report mergesall of the comments. The review report is appended below.Thank you for sending your work entitled “The C-terminal region of MCAK controlsits structure and activity through a conformational switch” for consideration atReview:The authors present the crystal structure of the motor domain of the microtubuledepolymerase MCAK in complex with a C-terminal peptide. The structure reveals that thispeptide promotes dimerization of the motor-domain and regulates the conformation ofMCAK's neck linker region. The structural data are well confirmed by biophysicalmethods and mutant studies.The second part of the paper uses this structure to work out the role of the C-terminalpeptide. The authors use covalent attachment of the C-terminal peptide to the motordomain to show that C-terminal peptide blocks MT binding. They then use a mutant (S715E)in which the C-terminal peptide can't bind. This leads to a high affinityinteraction with the microtubule and decoration along the MTs length. The authors usetheir data to support a model in which the C-terminal peptide reduces the affinity ofthe MCAK motor domain for the lattice of the microtubule, allowing it to diffuse to theends where its activity is required.eLife.Overall the data are better presented in this draft and the story is clearer. Theauthors have added some nice experiments in which they remove the tubulin tails and tiethe data into their final model. Nevertheless, there are several issues that requireattention in order for the manuscript to be suitable for publication inMajor experimental issues that need to be addressed:There is still lingering concern about the role of the C-terminal domain in thecatalytic cycle of MCAK.1) The authors show that the MCAK motor domain binds to microtubules with good bindingaffinity and that the modified (Cys containing) C-terminal peptide does not affect thebinding affinity under reducing conditions. They have already clarified that theCys-containing C-terminal peptide does not bind MCAK under reducing conditions, i.e. itdoes not trigger dimerization of MCAK. So the binding affinity for MTs they areobserving now is the binding affinity of the monomeric motor domain for MTs. Next, theauthors remove the reducing agent and observe that the fraction of MCAK bound to MTs hasdecreased. They claim that this is the expected behavior and illustrate the result inThe problem is that this experiment is a “remnant” from the theory thatbinding of the C-terminal tail to the motor domain is part of the catalytic cycle ofMCAK and from the authors' attempts to lock this putative intermediate. Thistheory is downplayed in the present version and the authors could try to do theexperiment in a simpler and more convincing setting. They don't need a covalentlinker. They have created a full length MCAK with the S715E mutant, which they claimprevents binding of the C-terminal peptide to the motor domain. They can simply measurethe binding affinity of this construct, which is because another segment of MCAK makesit be dimeric , in the presence of growingconcentrations of free C-terminal peptide. As there should be no intra-molecularcompetition from the C-terminal tail (due to the S715E mutation) this experiment willclarify the actual effects of the C-terminal peptide in the absence of other confoundingeffects, particularly the underlying monomer-dimer equilibrium of the MCAK motordomain.2) Concerning Other issues:3) The manuscript suffers from a number of minor mistakes which would need to becorrected (listed below). It also could be improved by a clearer and more concisewriting style. Parts of it are really hard to understand without reading it multipletimes. In particular, the discussion of the experiments in 4) 5) Figure 1–figure supplement 1: this figure looks suspicious. The intensity ofthe peptide bands in the M+CT peak are very high relative to the CT bands. Ifthey are staining equally it suggests a ratio of 20 peptides to 1 motor domain. Alsothere is a sharp drop off of intensity of the peptide band between fraction 10-11 andalso 15-16. This does not look like a gel filtration peak. The authors need to addressand possibly redo this gel. The fraction numbers on the gel need marking on the trace(which currently shows only volume).6) 7) 8) 9) 10) 11) In the first paragraph of the Results section, the term “upstream” isambiguous. Consider using N or C terminal. The manuscript would be clearer if theauthors maintained a consistent way of referring to the parts of the model. Sometimesthey use CT and others “C-terminal domain” or “CT domain” or“C terminus”.12) The authors should spell out clearly how they prepared CT alone (with no GST) andthen clearly identify when they are using GST-CT and when CT.13) In the second paragraph of the subsection “A conserved motif in theC-terminal region of MCAK is essential for the C terminus-motor interaction”, theterm “head group” is confusing. The authors should state more clearly thatthey are changing the size of the amino acid at position 715.14) In the subsection entitled “The C terminus-motor domain interaction isintramolecular”, the authors explain that removing the C-terminus of MCAK had noeffect in depolymerisation assays. They then state “It is possible that there isa trade-off between microtubule binding affinity and tubulin removal at ends”.This sentence needs to be expanded to make it clearer. As I understand it they thinkthere is no change in depolymerisation activity because of two counteracting effects(increase in activity and decrease in accessibility to the MT ends due to too muchlattice binding). The authors could also consider moving this lack of change in activityto the discussion. It made reading the paper difficult.15) The whole section on the intramolecular interaction is very difficult to follow andneeds rephrasing to make it clearer. The authors may consider removing it as it does notseem to add to the overall argument.16) In the second paragraph of the Results section: It may be easier to discuss thequantification of the binding affinity of the C-terminal peptide for the MCAK motordomain after describing the observation that the peptide causes dimerization of theC-terminal domain. The authors write: “…this measurement does not takeinto account any existing equilibrium between the motor domains ”. In fac17) At the end of the subsection headed “One MCAK C terminus binds to two ATPasedomains”: “…we could not conclusively determine whether…there were additional dimerization domains in the context of full length MCAK”.The authors demonstrate later in the manuscript that MCAK forms dimers independently ofthe C-terminal domain, so this sentence may be confusing.18) In the subsection “The MCAK C terminus binds at the interface between twomotor domains”: There are four MCAK motor domains in the asymmetric unit of thecrystals, two of which we are told are bound as a near-symmetrical dimer to a singlepeptide. The other two are not described, except for the explanation that they are notbound to the peptide because of a crystal contact. Are the two additional subunits thatare not bound to the peptide in a dimeric arrangement? And also: If one considers onlythe two MCAK subunits bound to the peptide, are the two motor subunits related by aperfect  2-fold axis? Clearly the presence of the peptide breaksthe symmetry, but limitedly to the motor domain, are they in a perfect dimer? If so, itshould be clearly stated.19) At the end of the first paragraph of “The C terminus-motor domain interactionis intramolecular”: “However it did raise the possibility that the CTdomain acts indirectly as an inhibitor and has an additional distinct cellularfunction”. “It” is a series of negative results discussed in theprevious lines and showing that a point mutant that prevents binding of the CT has noeffect in vitro and in vivo on microtubule depolymerization. It seems to me thatemphasis here should be given to the negative results (some of which are not shown)rather than on the authors' goal of proving the importance of their finding.20) In the second paragraph of the subheading “The C terminus-motor domaininteraction is intramolecular”, the authors hypothesize that the reason why theydo not see a covalent dimer form under oxidizing conditions with full length MCAK E712Cis that the linkage is intra-molecular. It is possible, but the alternative explanation,that the linkage is not formed at all in the context of the full-length protein, is moreplausible given the considerable number of residues lying in between the motor domainand the C-terminus.21) At the end of the subsection headed “A conserved motif in the C-terminalregion of MCAK is essential for the C terminus-motor interaction”:“data” is plural.22) In the beginning of the section entitled “The C terminus-motor domaininteraction is intramolecular”, the sentence starting with “It is possiblethat there is a trade-off…” is obscure.23) In the subsection headed “The C terminus stabilizes full-length MCAK insolution”, it is not clear what is the evidence that MCAK S715S is less stable.If this referred to the stability of the dimer, it would be useful that the authorsclarified how they reached this conclusion. As it stands, the inference that this mutantis less stable should be removed, as it is not justified. Major experimental issues that need to be addressed:There is still lingering concern about the role of the C-terminal domain in thecatalytic cycle of MCAK.1) The authors show that the MCAK motor domain binds to microtubules with goodbinding affinity and that the modified (Cys containing) C-terminal peptide does notaffect the binding affinity under reducing conditions. They have already clarifiedthat the Cys-containing C-terminal peptide does not bind MCAK under reducingconditions, i.e. it does not trigger dimerization of MCAK. So the binding affinityfor MTs they are observing now is the binding affinity of the monomeric motor domainfor MTs. Next, the authors remove the reducing agent and observe that the fraction ofMCAK bound to MTs has decreased. They claim that this is the expected behavior andillustrate the result inby showing that two equimolar populations of MCAK form, one of which can bindMTs and one of which cannot. This expectation is not well justified. How much of eachof these two populations is represented depends on the concentration of peptide andon the time the peptide is given to form a covalent bond with MCAK, so that tellingwhat is the expected result for this experiment is very hard unless all the reactionparameters are at hand. If it is true that the MCAK motor domain dimer:peptide has noor lower binding affinity for MT, peptide addition should result in progressivelylower MCAK binding as a function of the concentration of C-terminal peptide and thetime it is allowed to interact with MCAK.The problem is that this experiment is a “remnant” from the theorythat binding of the C-terminal tail to the motor domain is part of the catalyticcycle of MCAK and from the authors' attempts to lock this putativeintermediate. This theory is downplayed in the present version and the authors couldtry to do the experiment in a simpler and more convincing setting. They don'tneed a covalent linker. They have created a full length MCAK with the S715E mutant,which they claim prevents binding of the C-terminal peptide to the motor domain. Theycan simply measure the binding affinity of this construct, which is because anothersegment of MCAK makes it be dimeric , in thepresence of growing concentrations of free C-terminal peptide. As there should be nointra-molecular competition from the C-terminal tail (due to the S715E mutation) thisexperiment will clarify the actual effects of the C-terminal peptide in the absenceof other confounding effects, particularly the underlying monomer-dimer equilibriumof the MCAK motor domain.S715E-microtubule reaction, we gradually decreased the amount ofbinding to the microtubule, revealing that the CT domain indeed reduces the affinity ofMCAK for microtubules.We have now performed this experiment. When we titrate increasing amounts of free CTinto the MCAK2) Concerning: The authorsneed to do a control of MD no DTT, to show that the cross linker (rather than justlack of DTT) is causing the effects.E712C inabsence of DTT that affects MCAK function.We have performed these experiments in Other issues:3) The manuscript suffers from a number of minor mistakes which would need to becorrected (listed below). It also could be improved by a clearer and more concisewriting style. Parts of it are really hard to understand without reading it multipletimes. In particular, the discussion of the experiments inis really cursory: the inferences are stated and the reader is simply referredto the figure, for which the figure legend provides minimal information. This is akey figure describing crucial experimental data, and the authors should explain theexperiments, the resulting data and inferences completely. They may choose to breakthe figure up into multiple panels to achieve this end.We have now broken up 4): In the legendtothe authors write: “The star represents residual GST”. Only laterin the manuscript did this become clear. They need to explain that CT was made as aGST fusion and cleaved off. Inandthe order of the constructs is different. This is sloppy. The authors need tocorrect it.We have now stated that the tail domain (CT) is cleaved off from the GST and the GST isremoved (in the first paragraph of the Results and in the Methods section). We used asmaller chemically synthesized peptide for complex crystallization and we have indicatedthis in the text, that the peptide was chemically synthesized (please see the subsectionentitled “The MCAK C terminus binds at the interface between two motordomains”). We have also changed the order of constructs in 5) Figure 1–figure supplement 1: this figure looks suspicious. Theintensity of the peptide bands in the M+CT peak are very high relative to theCT bands. If they are staining equally it suggests a ratio of 20 peptides to 1 motordomain. Also there is a sharp drop off of intensity of the peptide band betweenfraction 10-11 and also 15-16. This does not look like a gel filtration peak. Theauthors need to address and possibly redo this gel. The fraction numbers on the gelneed marking on the trace (which currently shows only volume).mutants constructs on appropriate gels (Invitrogen 16% Tricinegels) and have displayed the corresponding gels and elution profiles in We apologize for this problem and we agree it was imperative to change the figure. Wehave now repeated the gel filtration of the M, M+CT andM+CT6): A number ofstructural elements are referred to in the text that need to be shown on this figure:e.g. α1 and β3 of the dimerization interface, L1 loop, the interactionbetween Glu172 and Ala241/A is missing, Lys286/B is missing, structural elements ofthe ATPase site . It would also be helpful to add anadditional overlay structure to the figure that shows that these elementsdon't change upon C-tail binding.We thank the reviewers for these excellent suggestions. They really help to understandthe functional aspects of our MCAK structure better. We have now included new figures withove7): A quadruplemutant (E711A/E712A/R716A/I718A) is shown in the figure but not discussed in thetext. It should be removed or talked about. If it is kept then the way it is writtenabove the gel needs to be improved as currently it is not clear that all 4 mutationswere made in the same construct.S715Abinds to the motor into We have now removed this from the figure and repeated the pulldown assay in 8):. The colorlines in this figure are mislabelled. The magenta line should be MD+CTsc noDTT (rather than MD). In the figure legend there is reference to 6C, which should be5D. The figure makes little sense otherwise. In this, and other figures, the axesshould be more clearly explained in the figure legend. References in the text to thefigures should also be readily understood by the reader. What is“Bmax”, referred to in the text but not apparent in thefigure?We thank the reviewers and have now corrected this mislabeled figure. We have alsorelabeled the graph axes to explain more clearly what they represent and removed thereference to “Bmax” in the text. We have also edited the references to thefigures.9): The cellimages are too small and bad quality. They need to be made brighter and larger inorder to show what is happening.We have now improved the presentation of the images to show the EB3 and MCAK stainingmore closely. We have also added linescan profile averages for the EB3 and MCAKlocalization on microtubule plus ends.10): This figure isnot currently mentioned in the text and needs to be.We apologize for this mistake and have now mentioned 11) In the first paragraph of the Results section, the term“upstream” is ambiguous. Consider using N or C terminal. The manuscriptwould be clearer if the authors maintained a consistent way of referring to the partsof the model. Sometimes they use CT and others “C-terminal domain” or“CT domain” or “C terminus”.We have now removed the term “upstream”. Throughout the text, we have moreconsistently labeled the C-terminus of MCAK construct as the CT domain. We apologize forthis inconsistency.12) The authors should spell out clearly how they prepared CT alone (with noGST) and then clearly identify when they are using GST-CT and when CT.We have now described in the Methods section more clearly how the CT domain is preparedand how the GST is removed. We have also carefully annotated the figures and have triedto avoid using GST-CT when possible.13) In the second paragraph of the subsection headed “A conserved motifin the C-terminal region of MCAK is essential for the C terminus-motorinteraction”, the term “head group” is confusing. The authorsshould state more clearly that they are changing the size of the amino acid atposition 715.We have now changed the term “head group” to “sidechain”.14) In the subsection headed “The C terminus-motor domain interaction isintramolecular”, the authors explain that removing the C-terminus of MCAK hadno effect in depolymerisation assays. They then state “It is possible thatthere is a trade-off between microtubule binding affinity and tubulin removal atends”. This sentence needs to be expanded to make it clearer. As I understandit they think there is no change in depolymerisation activity because of twocounteracting effects (increase in activity and decrease in accessibility to the MTends due to too much lattice binding). The authors could also consider moving thislack of change in activity to the discussion. It made reading the paperdifficult.We have now changed this sentence to: “However there are limitations to thisassay, in which we can only measure rate of microtubule depolymerization. It is possiblethat a change in MCAK microtubule binding affinity will have a counteracting effect onMCAK diffusion rate or the rate of tubulin removal at ends and that as a consequence.The overall depolymerase activity that our assay measures is unchanged, as previouslyshown .”15) The whole section on the intramolecular interaction is very difficult tofollow and needs rephrasing to make it clearer. The authors may consider removing itas it does not seem to add to the overall argument.We have now removed this section to streamline the paper.16) In the second paragraph of the Results section: It may be easier to discussthe quantification of the binding affinity of the C-terminal peptide for the MCAKmotor domain after describing the observation that the peptide causes dimerization ofthe C-terminal domain. The authors write: “…this measurement does nottake into account any existing equilibrium between the motor domains ”. Infact, the measurement results from the sum of two distinct interactions, thedimerization of the MCAK motor domain and the binding of the C-terminal peptide, anintrinsically cooperative interaction. It would be easier to clarify this if thedimerization had been already introduced.We apologize for discussing motor dimerization before we introduced the data relative toit in the paper and we thank the reviewers for this comment. We have now moved thesection quantifying the affinity of the peptide for the motor so that it follows thesection describing that the CT domain induces motor dimerization. We have clarified thatthe affinity measured represents the sum of two distinct affinities, of which CT domainbinding is cooperative.17) At the end of the subsection headed “One MCAK C terminus binds to twoATPase domains”: “…we could not conclusively determinewhether… there were additional dimerization domains in the context of fulllength MCAK”. The authors demonstrate later in the manuscript that MCAK formsdimers independently of the C-terminal domain, so this sentence may beconfusing.We have now included “from the above experiments” to highlight that thedata described in that paragraph does not indicate whether MCAK is still dimeric ornot.18) In the subsection “The MCAK C terminus binds at the interface betweentwo motor domains”: There are four MCAK motor domains in the asymmetric unitof the crystals, two of which we are told are bound as a near-symmetrical dimer to asingle peptide. The other two are not described, except for the explanation that theyare not bound to the peptide because of a crystal contact. Are the two additionalsubunits that are not bound to the peptide in a dimeric arrangement? And also: If oneconsiders only the two MCAK subunits bound to the peptide, are the two motor subunitsrelated by a perfect  2-fold axis? Clearly the presence of thepeptide breaks the symmetry, but limitedly to the motor domain, are they in a perfectdimer? If so, it should be clearly stated.We thank the reviewers for this excellent point. We have now changed the text to reflectthe structure better and have included this information and an additional figure of theCT binding site on the dimer that is not occupied by the CT domain.19) At the end of the first paragraph of “The C terminus-motor domaininteraction is intramolecular”: “However it did raise the possibilitythat the CT domain acts indirectly as an inhibitor and has an additional distinctcellular function”. “It” is a series of negative resultsdiscussed in the previous lines and showing that a point mutant that prevents bindingof the CT has no effect in vitro and in vivo on microtubule depolymerization. Itseems to me that emphasis here should be given to the negative results (some of whichare not shown) rather than on the authors' goal of proving the importance oftheir finding.S175E usingan assay first described by S715E. However, the sample heterogeneity was in the order of magnitudeof the changes recorded for the distinct mutants and transfection of GFP-MCAK rescuedgrowth lifetime, but not growth speed. Therefore we did not include this data.We have measured the activity of MCAK and MCAK mutant such as MCAKFinally, another paper reported20) In the second paragraph of the subheading “The C terminus-motordomain interaction is intramolecular”, the authorshypothesize that the reason why they do not see a covalent dimer form under oxidizingconditions with full length MCAK E712C is that the linkage is intra-molecular. It ispossible, but the alternative explanation, that the linkage is not formed at all inthe context of the full-length protein, is more plausible given the considerablenumber of residues lying in between the motor domain and the C-terminus.E712C for microtubules in absence of DTT . However, Cys287 (Cys283 in mouse) has also been reported to make adisulfide bond with Cys245 (Cys241 in mouse) in the murine MCAK crystal structure. Thusit is possible, that in the context of the full length MCAK and in absence of DTT, wegenerate at least two MCAK conformations of neck-motor complexes that make the analysisof this MCAKE712C mutant more complex.We believe that we do form the crosslink because we see a reduction in the affinity ofMCAK21) At the end of the subsection headed “A conserved motif in theC-terminal region of MCAK is essential for the C terminus-motor interaction”:“data” is plural.We apologize and have now corrected this grammatical mistake.22) In the beginning of the section entitled “The C terminus-motor domaininteraction is intramolecular”, the sentence starting with“It is possible that there is a trade-off…” isobscure.We have now clarified this sentence in the text.23) In the subsection headed “The C terminus stabilizes full-length MCAKin solution”, it is not clear what is the evidence that MCAK S715S is lessstable. If this referred to the stability of the dimer, it would be useful that theauthors clarified how they reached this conclusion. As it stands, the inference thatthis mutant is less stable should be removed, as it is not justified.S715E and only included the gel filtrationelution profile of MCAKS715E.The recovery rate from SEC-MALS was 33%, which implies some of the protein was notstable, but we did not include that data and we apologize for it. However, we have nowremoved the SEC-MALS data for MCAK


Huntington disease (HD) is an inherited, fatal neurodegenerative disorder caused by a CAG repeat expansion in the huntingtin gene. The mutant protein causes neuronal dysfunction and degeneration resulting in motor dysfunction, cognitive decline, and psychiatric disturbances. Currently, there is no disease altering treatment, and symptomatic therapy has limited benefit. The pathogenesis of HD is complicated and multiple pathways are compromised. Addressing the problem at its genetic root by suppressing mutant huntingtin expression is a promising therapeutic strategy for HD. We have developed and evaluated antisense oligonucleotides (ASOs) targeting single nucleotide polymorphisms that are significantly enriched on HD alleles (HD-SNPs). We describe our structure-activity relationship studies for ASO design and find that adjusting the SNP position within the gap, chemical modifications of the wings, and shortening the unmodified gap are critical for potent, specific, and well tolerated silencing of mutant huntingtin. Finally, we show that using two distinct ASO drugs targeting the two allelic variants of an HD-SNP could provide a therapeutic option for all persons with HD; allele-specifically for roughly half, and non-specifically for the remainder. HTT) gene Huntington disease (HD) is an autosomal dominant, fatal neurodegenerative disorder with a prevalence of up to 17 cases per 100,000, which makes it one of the most common inherited neurodegenerative disorders In 1983 the HD gene was mapped to the short arm of chromosome 4 and 10 years later the gene was isolated and cloned Hdh) gene in mice results in death at embryonic day 7–9 Two different gene-silencing approaches are currently under development for HD. The first and most straightforward strategy is to suppress the expression of both the wild-type (wt) and mutant protein. However, a general concern for total HTT silencing has been raised regarding the potential side effects of reducing wtHTT, whose beneficial activity for neuronal function and maintenance is well established Different approaches have been employed to achieve allele-specific silencing of mHTT by targeting disease-linked polymorphisms, including the CAG expansion HTT gene that are significantly enriched on HD alleles compared to wt alleles As an alternative therapeutic strategy, we and others have shown that targeting HD-SNPs using RNAi or antisense-oligonucleotides (ASOs) presents a promising approach towards achieving allele-specific treatment of HD patients ASO-mediated HTT knock down was demonstrated more than a decade ago using both phosphodiester and phosphorothiorated (PS) ASOs Out of the fifty HD-SNPs previously identified 50 values for wtHTT and mHTT. If there was less than 50% knock down at the highest ASO concentration tested and no possibility of calculating an IC50 value, then the highest ASO concentration evaluated was used to calculate the ratio, which is expressed as >x fold.The ultimate goal is to develop a panel of allele-specific ASOs that, in combination, will provide a therapeutic option to the majority of the HD patients. However, the purpose of this screen was to identify the most efficacious SNP sites and to develop the best possible ASO candidate. The selected HD-SNPs in the current study do not provide significant combinatorial advantage as they are all in high linkage disequilibrium with one another. To evaluate the activity at several SNP sites we used phosphorothioate substituted 19-mers containing five 2′-O-methoxy-ethyl  ribose sugars in each wing and a string of nine DNA residues in the gap (5e-9-5e gapmers) ). These 4 candidates were moved forward in the pipeline. The remaining ASOs showed limited HTT silencing and specificity that did not reach statistical significance with two-way ANOVA and Bonferroni post hoc test , specificity up to 3.1 fold, and no overt toxicity . These hoc test . None ofhoc test . Howeverhoc test . These f50,mHTT<200 nM) and moderate specificity (>5 fold). The most promising ASO series: A1, A2, and A3, is shown as an example in To further evaluate ASOs targeting these four SNPs and identify the ASOs with the most activity, we introduced S-constrained-ethyl  modifications to the wings of the 4 parent ASOs A1, B1, C1, and D1. Prior studies using these high affinity modifications in the wings found ASOs displaying improved potency without producing toxicity 50,mHTT<14 nM) , but with a small reduction in specificity (4.2 and 4.6 fold). Both ASOs induced spectrin cleavage above threshold and were therefore excluded. The strategy of changing and rearranging modifications of the wings with MOE and cEt nucleotides did not provide an ASO with a better profile than A3. Overall, our primary screen identified one tolerable candidate, A3, with good potency and moderate specificity. While it did not demonstrate the best specificity, we thought it would be easier to improve specificity with chemical modification than to improve potency. A3 was therefore used as the parent molecule for the subsequent SAR studies.In parallel, we wanted to investigate whether adding additional cEt modification to the wings of the ASO would lead to improvement in potency and specificity. To evaluate this, while maintaining the 9-base gap, three 19mer oligos, A4, A5, and A6, based on A1 were evaluated. First, we added two cEt modifications to the 5′ wing  while keeping the 3′ wing only modified with MOE chemistry. Next, we mixed the modifications in both wings , and finally replaced the MOE modifications with unmodified PS deoxynucleotides (represented by “d”) and alternating cEt modifications . All ASOs displayed excellent potency (ICT<14 nM) . A4 showT<14 nM) . Lastly,We have previously demonstrated that RNase H cleaves to the 5′ -ASO/3′-RNA side of the SNP 50 values for mHTT from 11–78 nM, which is comparable to previously evaluated ASOs, suggesting that the number of modifications is more important than their distribution  with robust specificity (>13 fold). However, at the highest dose of 1000 nM, A22 did cause a significant reduction in wtHTT expression of approximately 40%. Considering these data, the microwalk strategy did not provide sufficient improvement to specificity, and we therefore decided to move forward with investigation of shortening the gap of the oligo.To investigate these preliminary findings in more detail, we selected a subset of the ASOs with favourable properties, including A11, A20, A21, and A22, to be tested for potency, specificity, and toxicity in primary neurons . Our aimribution . We did 50,mHTT = 38 nM) and specificity (>26 fold). Unfortunately, A30 induced spectrin cleavage indicating that full cEt wings are not well tolerated for this specific sequence. Screening the remaining panel of ASOs, we found oligos with pronounced specificity (>56 fold) and high potency . However, the longer cEt modified ASOs (three out of five) were associated with toxicity, whereas the shorter oligos appeared more well tolerated with only one out of five inducing significant spectrin cleavage at the highest dose tested . Despite increased activity, specificity of mHTT silencing was maintained over increased treatment durations for 3 of 4 leads. ASOs A38, A39, and A40 showed minimal silencing of wtHTT, whereas there was greater reduction in wtHTT levels after longer treatments with A41  of HD patients are homozygous at 22 genotyped SNPs 50 values for mHTT  than was observed for A38 and A39, which demonstrates the impact of changing one of the 15 or 16 nucleotides in the oligo , which is one to two orders of magnitude higher than other current screening systems Primary neurons with the appropriate genetic background including human transgenic wt and mutant HTT and without the presence of endogenous murine Htt are an ideal system for rapid Our data demonstrate that initially selecting multiple sites for evaluation is critical, since ASOs at all SNP sites are not equally active. This is most likely caused by secondary and tertiary RNA structures that can either prevent binding of the oligo to the target RNA or sterically hinder the recruitment of the RNase-H enzyme to the ASO:RNA duplex in vitro systems using SiRNA, LNA oligos, single-stranded RNA, unmodified or modified RNA duplexes 50 values by free uptake into primary neuronal cells and impressive specificity, against rs7685686_A suitable for in vivo validation. Furthermore, our findings provide some insight into advantageous oligo design that can be used as a starting point for sequential screening of secondary and tertiary ASO candidates.After improving the ASO design and incorporating cEt modifications in combination with MOE chemistry, we find the potency of our ASOs to be in the lower nanomolar range comparable to what has been observed in other The steps described here are the initial process towards the long term goal of constructing a panel of ASOs to provide allele-specific silencing to all HD patients. We are currently in the process of re-populating our ASO pipeline using relevant HD-SNP targets that will add additional patient coverage. We believe that screening at these complementary sites will be faster and more efficient using information garnered from this screen. Despite this increased efficiency, building a full panel of allele-specific ASOs will take significant time. Another concern that has been raised is that some people with HD may not currently be targetable with this approach. Previous genetic population studies indicate that a minority of HD patients are homozygous at all investigated HD-SNPs. Warby et al. explored a panel of 22 SNPs and found that 7 out of 67 HD patients were homozygous at these SNPs As a start, our lead ASOs targeting rs7685686_A, could provide an allele-specific therapeutic option for 48.7% of HD patients. In addition, they could provide a non-specific HTT silencing option for 44.9% of HD patients that are homozygous (rs7685686_A/A). This means that one of our lead ASOs could potentially provide a therapeutic option to 93.6% of people with HD. Since, we have found that rs7685686 is an accessible SNP site, we have explored the possibility of targeting the opposite allele at the same SNP site (e.g. ‘G’ vs. ‘A’) to provide a therapeutic option for the remaining 6.4% of patients. Targeting rs7685686_G would provide an allele-specific therapeutic option to 3.8% and a non-allele-specific option to 2.6% of HD patients.50,mHTT of 150 and 134 nM) and were well tolerated in our screens. Overall, these findings show that two ASOs targeted to the two allelic variants of a single SNP could provide a therapeutic option for all HD patients, where roughly half would receive an allele-specific therapy and the remaining patients would receive a non-specific therapy. This strategy could potentially provide benefit during the time it takes to develop a complete allele-specific ASO panel. While there are safety concerns for long-term reduction of wtHTT, in short term, a non-specific HTT silencing therapy would likely be preferable to untreated HD.With this strategy in mind, we designed two ASOs, X1 and X2, that are analogous to our leads, A38 and A39, and evaluated them in primary neurons from YAC128 mice. ASOs X1 and X2 showed good activity . Animal experiments were performed with the approval of the animal care committee at the University of British Columbia.Consent and access procedures were in accordance with institutional ethics approval for human research (UBC certificate H05-70532). Publically available human fibroblasts cell lines were obtained from NIGMS Human Genetic Cell Repository at the Coriell Institute for Medical Research . Quantitative RT-PCR reactions were run on the ABI 7900HT instrument using the Quantitect Probe RT-PCR kit following the manufacturer’s instructions. Total RNA content measured by Ribogreen was used for normalization.Fibroblast line GM04022, which is heterozygous at SNP rs7685686_A, was used to measure the Mice were housed under a 12 hour light and dark cycle in a clean facility with free access to food and water. Hu18/18 and Hu97/18 ATTGCCCCGGTGCTGAGCG -3′ and reverse primer: 5′ - GCGGGCCCAAACTCACGGTC-3′) yielding product sizes of 351bp and 588bp for the YAC18 and BACHD alleles, respectively. For the YAC128 embryos, two PCRs at each of the YAC arms were used to confirm the presence of the full YAC insert. Actin was used as positive PCR control. Left YAC arm PCR (forward primer: 5′ - CCTGCTCGCTTCGCTACTTGGAGC-3′ and reverse primer: 5′ - GTCTTG CGCCTTAAACCAACTTGG-3′) yielding a product size of 230bp. Right YAC arm PCR (forward primer: 5′ - CTTGAGATCGGGCGTTCGACTCGC-3′ and reverse primer: 5′ - GTCTTGCCGCACCTGTGGCGCCGGTGATGC-3′) yielding product size of 170bp. Actin PCR (forward primer: 5′ - AGCCTCAGGGCATCGGAACC-3′ and reverse primer: 5′ - GGAGACGGGGTCACCCACAC-3′) yielding product size of 450bp.Embryos were collected on day 15.5–16.5 of gestation. Brains were extracted and transferred to Hibernate E (Invitrogen) for 24 hrs, allowing maintenance of neuron viability until genotyping was completed. Tail tissue for genotyping was collected from each embryo, and DNA was extracted using the QuickLyse Miniprep Kit (Qiagen). For Hu97/18 embryos, a PCR across the CAG expansion was used to distinguish between the two human HTT transgenes  and fed with 200 µl fresh medium every fifth day post treatment. Images were taken with EVOS XL Core Imaging System from Life Technologies with a 10X objective. Size marker was added to the images using a calibration grid slide (250 uM grids) from MBF Bioscience. As a positive control for spectrin cleavage, we used camptothecin, a topoisomerase inhibitor, to induce apoptosis. At DIV8 increasing concentrations of campthothecin were added to Hu97/18 neurons and spectrin cleavage was evaluated after 24 hours of stress.Embryonic brains were removed from Hibernate E, and the forebrains microdissected in ice-cold Hank’s Balanced Salt Solution  to remove the hippocampi, isolating the cortex and striatum, which was used to set up neuronal cultures. The tissue was minced and digested with 0.05% Trypsin-EDTA (Invitrogen) at 37°C for 8 minutes, and trypsin was subsequently neutralized with 10% Fetal Calf Serum  in Neuro Basal Medium . Cells were resuspended in complete culture media (NBM+), NBM containing 2% B27 (Gibco), 100 U/ml PS, and 0.5 mM L-Glutamine (Gibco), and treated with DNAse I (153 U/µl) (Invitrogen). Tissue was triturated 5–6 times with a 5 ml serological pipette, and cells were counted and seeded at 1.2×10Cortical and striatal neurons were collected from the culture dish on DIV 8, 12, or 17 by scraping in ice cold PBS and pelleting by centrifugation at 2400 g for 5 min at 4°C. Dry pellets were then stored at −80°C. Proteins were extracted by lysis with SDP+ buffer and 20–40 µg of total protein was resolved on 10% low-BIS acrylamide gels and transferred to 0.45 µm nitrocellulose membrane as previously described 50 values were compared using the extra-sum-of-squares F test and the F distribution and degrees of freedom F  and the associated p-values have been calculated. Allele specificity was calculated by dividing the IC50 for wtHTT by the IC50 for mHTT. If the IC50 for reducing wtHTT was greater than the highest ASO concentration tested, then allele specificity was calculated by dividing the highest ASO concentration tested by the IC50 for mHTT reduction and expressed as >fold. Two way ANOVA with Bonferroni post hoc test have been performed to determine if mHTT expression is different from wtHTT levels at each individual dose of oligo tested. Analyses were performed using GraphPad Prism Ver.5. Differences were considered statistically significant when p<0.05.Data are expressed as means±SD. Results were analysed using non-linear regression with normalized response and a variable curve. The ICFigure S1Spectrin cleavage assay. To enable a successful triage and exclusion of toxic ASOs, we measured the level of the 120 kDa spectrin cleavage fragment normalized to calnexin loading control, and then to the untreated sample. Camptothecin induced spectrin cleavage was used as a positive control. Representative Western blots and spectrin quantification from a non-toxic and a toxic ASO are shown. n = 4–6 per data point. Data is presented as mean ± SD. The red dashed line represents the toxicity threshold.(TIF)Click here for additional data file.Figure S2Selection of favourable SNP targets – HTT levels. Hu97/18 neurons were treated with 5e-9-5e ASOs targeted to 10 HD-SNPs and HTT protein level was analyzed. HTT levels were normalized to calnexin and then to the untreated sample for each allele. Representative images are shown. n = 4–6 per data point. Data are presented as mean ± SD. The PS backbone is represented by black; MOE modifications are illustrated by orange. The SNP is illustrated by the underlined nucleotide.(TIF)Click here for additional data file.Figure S3Selection of favourable SNP targets – Spectrin cleavage. Hu97/18 neurons were treated with 5e-9-5e ASOs targeted to 10 HD-SNPs and spectrin cleavage was analyzed. The 120 kDa fragment was normalized to calnexin and then to the untreated sample. HTT membranes were reprobed for spectrin. Representative images are shown. n = 4–6 per data point. Data are presented as mean ± SD. The # denotes two ASOs that induced rearrangement of the neurons. The PS backbone is represented by black; MOE modifications are illustrated by orange. The SNP is illustrated by the underlined nucleotide. The red dashed line represents the toxicity threshold.(TIF)Click here for additional data file.Figure S4Altered neuronal morphology after treatment with some ASOs. Treatment with ASOs E1 and G1 caused marked morphological changes at the highest doses tested (1000 nM) resulting in rearrangement of neuronal cell bodies into an organized network. Representative images are shown of treated and untreated neurons. Black arrows indicate cell bodies grouped together connecting to other cell clusters. Images were taken with EVOS XL Core Imaging System from Life Technologies using the 10X objective. A calibration grid slide with 250 uM grids from MBF Bioscience was used to add a size marker to the images.(TIF)Click here for additional data file.Figure S5Targeting 4 SNPs using two different cEt motifs – HTT levels. Hu97/18 neurons were treated with ASOs with cEt modified wings and HTT protein was analyzed. HTT levels were normalized to calnexin and then to the untreated sample for each allele. Representative images are shown. n = 6–10 per data point. Data are presented as mean ± SD. The PS backbone is represented by black; MOE and cEt modifications are illustrated by orange and blue, respectively. The SNP is illustrated by the underlined nucleotide.(TIF)Click here for additional data file.Figure S6ASO screen at 4 SNPs using two different cEt motifs – Spectrin. Hu97/18 neurons were treated with ASO with cEt modified wings and spectrin cleavage was analyzed. The 120 kDa fragment was normalized to calnexin and then to the untreated sample. HTT membranes were reprobed for spectrin. Representative images are shown. n = 6–8 per data point. Data are presented as mean ± SD. The PS backbone is represented by black; MOE and cEt modifications are illustrated by orange and blue, respectively. The SNP is illustrated by the underlined nucleotide. The red dashed line represents the toxicity threshold.(TIF)Click here for additional data file.Figure S7Wing SAR study. Hu97/18 neurons were treated with ASO with cEt modified wings and HTT protein and spectrin cleavage was analyzed. HTT levels were normalized to calnexin and then to the untreated sample for each allele. The 120 kDa fragment was normalized to calnexin and then to the untreated sample. Membranes were probed for HTT and reprobed for spectrin. Representative images are shown. n = 4–6 per data point. Data are presented as mean ± SD. The PS backbone is represented by black; MOE and cEt modifications are illustrated by orange and blue, respectively. The SNP is illustrated by the underlined nucleotide. The red dashed line represents the toxicity threshold.(TIF)Click here for additional data file.Figure S8Shortening the gap to 7 nucleotides – HTT levels. Replacing PS-nucleotides with RNase H resistant nucleotides and shortening the gap increases selectivity by preventing cleavage at secondary cleavage sites and restricting cleavage to the main site next to the targeted SNP. Hu97/18 neurons were treated with ASOs and HTT protein was analyzed. HTT levels were normalized to calnexin and then to the untreated sample for each allele. Representative images are shown. n = 6–10 per data point. Data are presented as mean ± SD. The PS backbone is represented by black; MOE and cEt modifications are illustrated by orange and blue, respectively. The SNP is illustrated by the underlined nucleotide.(TIF)Click here for additional data file.Figure S9Shortening the gap to 7 nucleotides – Spectrin cleavage. Hu97/18 neurons were treated with ASOs and spectrin cleavage was analyzed. The 120 kDa fragment is normalized to calnexin and then to the untreated sample. HTT membranes were reprobed for spectrin. Representative images are shown. n = 6–8 per data point. Data are presented as mean ± SD. The PS backbone is represented by black; MOE and cEt modifications are illustrated by orange and blue, respectively. The SNP is illustrated by the underlined nucleotide. The red dashed line represents the toxicity threshold.(TIF)Click here for additional data file.Table S1Summary of ASO RNA screen in human fibroblasts. MOE and cEt modifications are annotated by e and k, respectively. The SNP is underlined.(DOCX)Click here for additional data file.


Neisseria meningitidis 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (NmeDAH7PS) adopts a homotetrameric structure consisting of an extensive and a less extensive interface. Perturbation of the less extensive interface through a single mutation of a salt bridge (Arg126-Glu27) formed at the tetramer interface of all chains resulted in a dimeric DAH7PS in solution, as determined by small angle X-ray scattering, analytical ultracentrifugation and analytical size-exclusion chromatography. The dimeric NmeDAH7PSR126S variant was shown to be catalytically active in the aldol-like condensation reaction between d-erythrose 4-phosphate and phosphoenolpyruvate, and allosterically inhibited by l-phenylalanine to the same extent as the wild-type enzyme. The dimeric NmeDAH7PSR126S variant exhibited a slight reduction in thermal stability by differential scanning calorimetry experiments and a slow loss of activity over time compared to the wild-type enzyme. Although NmeDAH7PSR126S crystallised as a tetramer, like the wild-type enzyme, structural asymmetry at the less extensive interface was observed consistent with its destabilisation. The tetrameric association enabled by this Arg126-Glu27 salt-bridge appears to contribute solely to the stability of the protein, ultimately revealing that the functional unit of NmeDAH7PS is dimeric. Entry into the pathway is controlled by the allosteric regulation of DAH7PS by the pathway end products Phe, Tyr and Trp, or other intermediates of the pathway .Measurements were performed at the Australian Synchrotron SAXS/WAXS beamline equipped with a Pilatus detector . The wavelength of the x-rays was 1.0332 Å. The sample–detector distance was 1600 mm, which provided an NmeDAH7PS and NmeDAH7PSR126S  in the buffer . Scattering from glucose isomerase at an initial concentration of 10 mg/ml was collected to calibrate the mass of scattering particles. Data were collected in 2 second intervals. 2D intensity plots from the peak of the SEC run were radially averaged, normalised to sample transmission and background subtracted.Data were collected from a 2 mm glass capillary (Hampton Research) and the temperature was 25°C. Scattering data was collected from wild-type I) was plotted versus q. Extrapolation of the DAH7PS I(q) profiles to zero angle [I(0)] and comparison with that of glucose isomerase standards indicated a molecular mass consistent with the NmeDAH7PS tetramer or dimer. All samples were devoid of an increase in intensity at low q (increase is indicative of aggregation). Radius of gyration (Rg) did not vary significantly over the concentrations measured, and all Guinier plots were linear for q·Rg < 1.3. The data sets for structural analyses were recorded with 302 data points over the range 0.0110 ≤ q ≤ 0.4 Å−1. 1D profiles were background subtracted and Guinier fits were made using PRIMUS. Indirect Fourier transform was performed using GNOM  and all solutions were degassed in a vacuum. Protein concentration was measured by UV absorption immediately before titrations were started. The titrations were comprised of 28 injections, one 2 μL injection followed by 27 x 10 μL injections of Phe. The initial datum point was routinely deleted to allow for diffusion of ligand across the needle tip during the equilibration period. A heat of dilution experiment was measured independently and subtracted from the integrated data before curve fitting in Origin 7.0. For the binding of NmeDAH7PS to Phe, 15 μM of NmeDAH7PSWT and NmeDAH7PSR126S were used and the syringe contained 1.2 mM Phe; and the data were fitted with the two-site sequential-binding model supplied by Micro-Cal.Binding of NmeDAH7PSR126S was expressed and purified as previously described for the wild-type enzyme [N-oxide (TMAO), 600 μM MnSO4 and 15–20% (w/v) PEG 2000MME. Crystals were grown by hanging drop vapour diffusion over 500 μL of crystallisation buffer and the crystallisation trays were incubated at 20°C. Crystals began to form in 48 hours and were fully formed within seven days. Crystals were flash frozen using liquid nitrogen in a cryoprotectant solution containing reservoir solution and 20% (v/v) PEG400.1/2≥0.5 ensuring the data was complete in the highest resolution shell [NmeDAH7PSR126S were the same as those previously identified for the wild-type enzyme . NmeDAH7PSR126S had an expected mass of 77,300 Da, and showed similar thermal stability properties to the wild-type protein, with a melting temperature measured by differential scanning calorimetery of 47°C (NmeDAH7PSWT displayed a melting temperature of 49°C) (The variant protein of 49°C) .NmeDAH7PSWT and NmeDAH7PSR126S enzymes was carried out using both analytical size-exclusion chromatography (SEC) and analytical ultracentrifugation (AUC). SEC analysis revealed a clear difference in the solution behaviour of the two enzymes .Sedimentation velocity experiments were carried out on of 8.1 S . The calof 5.7 S . This prNmeDAH7PS R126S in solution. The scattering profile collected for the NmeDAH7PSR126S variant was compared to that collected for the wild-type enzyme (Rg) calculated from the scattering pattern of NmeDAH7PSR126S variant using the indirect fourier transform function, (P(r)), was ~ 26 Å and the maximum diameter of the scattering particle, Dmax, was calculated as ~ 95 Å using GNOM [NmeDAH7PSR126S crystal structure (Rg of ~ 25 Å and Dmax of ~ 86 Å). Similarly, CRYSOL [NmeDAH7PSR126S tetramer and dimer generated from the crystal structure. These data clearly show the NmeDAH7PSR126S variant adopts a dimeric structure in solution comparable to that of the NmeDAH7PSR126S tight-dimer  was employed to confirm the quaternary structure of e enzyme , Table 2ing GNOM . These v, CRYSOL  was usedht-dimer .NmeDAH7PSR126S was observed to be catalytically active with parameters that are comparable with those of the wild-type enzyme . The NmeDAH7PSR126S displays a lower KmE4P (13.4 μM) than the wild-type enzyme (37 μM). However, there is a noticeable decrease in specific activity for the dimeric variant. This implies that tetramerisation may have some influence on catalysis by NmeDAH7PS.e enzyme . The appNmeDAH7PSWT and the NmeDAH7PSR126S was carried out by measuring the activity of the enzymes over a 24 hour period. NmeDAH7PSWT lost ~10% catalytic activity, whereas the dimeric variant was far less stable and was observed to retain less than 60% of its activity after 24 hours . Data collection and refinement statistics are shown in NmeDAH7PSWT (PDB code 4HSN), which crystallised in the monoclinic (P1211) space group [The crystal structure of the NmeDAH7PSR126S structure (PDB code: 4UCG) contains four chains interacting to provide a homotetramer, resembling the arrangement of wild-type NmeDAH7PS.The asymmetric unit for NmeDAH7PSR126S tetramer has two distinct interfaces between the chains. The tight dimer interface remains extensive, burying 14% of the surface area of the monomeric unit, but is smaller than NmeDAH7PSWT (18%). The tetramer interface, on which the R126S substitution is located, is far smaller, burying 3.8% of the surface of the monomeric unit in NmeDAH7PSWT and this is reduced to 3.4% for the NmeDAH7PSR126S. Superposition of the NmeDAH7PSR126S homotetramer on the NmeDAH7PSWT protein yields an RMSD of 0.61 Å (1328 Cα atoms), whereas superposition of NmeDAH7PSR126S dimeric units on those of the wild-type protein gives RMSD values between 0.18–0.25 Å. These values are consistent with the small decrease in rotation of ~2°of the dimeric units with respect to each other at the tetramer interface, and this change may also account for the difference in space group between the NmeDAH7PSR126S and NmeDAH7PSWT structures.Like the wild-type enzyme, the NmeDAH7PSR126S crystal structure displays a theoretical solvation free energy gain (ΔG) for the formation of the tight dimer interface of -16.1 and -16.5 kcal/mol, correlating to the chain A-chain B and chain B-chain D interfaces respectively [NmeDAH7PSWT enzyme, where the ΔG values are -20.1 kcal/mol and -18.1 kcal/mol. The lower values are indicative of a weakening of the tight dimer interface in NmeDAH7PSR126S.According to PISA server calculations , the NmeG for formation of the tetramer interface in NmeDAH7PSR126S are -6.5 and -9.1 kcal/mol for the chain A-chain C and chain B-chain D interfaces respectively Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.


Siphovirus belonging to the order Caudovirales, has a flexible, three-fold symmetric tail, to which three L-shaped fibres are attached. These fibres recognize oligo-mannose units on the bacterial cell surface prior to infection and are composed of homotrimers of the pb1 protein. Pb1 has 1396 amino acids, of which the carboxy-terminal 133 residues form a trimeric intra-molecular chaperone that is auto-proteolyzed after correct folding. The structure of a trimer of residues 970–1263 was determined by single anomalous dispersion phasing using incorporated selenomethionine residues and refined at 2.3 Å resolution using crystals grown from native, methionine-containing, protein. The protein inhibits phage infection by competition. The phage-distal receptor-binding domain resembles a bullet, with the walls formed by partially intertwined beta-sheets, conferring stability to the structure. The fold of the domain is novel and the topology unique to the pb1 structure. A site-directed mutant (Ser1264 to Ala), in which auto-proteolysis is impeded, was also produced, crystallized and its 2.5 Å structure solved by molecular replacement. The additional chaperone domain (residues 1263–1396) consists of a central trimeric alpha-helical coiled-coil flanked by a mixed alpha-beta domain. Three long beta-hairpin tentacles, one from each chaperone monomer, extend into long curved grooves of the bullet-shaped domain. The chaperone-containing mutant did not inhibit infection by competition.Bacteriophage T5, a  Caudovirales (tailed phages) [Myoviridae, Podoviridae and Siphoviridae. Around half of all known bacteriophages are Siphoviruses and have long, flexible, non-contractile tails. The well-studied Siphovirus T5 has a three-fold symmetric tail-tube, to the end of which three long, L-shaped fibres are attached [Bacteriophages are ubiquitous viruses of bacteria. They are important biological model systems and have been applied in detection and control of pathogenic bacteria ,2. More  phages) . This orattached ,5. Theseattached ,7.Escherichia coli (E. coli) host strains, binding to the O8- or O9-type O-antigen of the bacterial lipopolysaccharide (LPS) [The L-shaped tail fibres are responsible for the initial recognition of certain de (LPS) . The strde (LPS) ,10. In ade (LPS) , and leade (LPS) ,13.Here, we present the structure of the carboxy-terminal domain of the bacteriophage T5 L-shaped tail fibres, solved in the absence and presence of the intra-molecular chaperone domain and show that only the processed, mature protein can inhibit phage infection by competition.w/v) PEG 2000 and were briefly soaked with the same solution including 20% glycerol before data collection.Production and crystallization of pb1(970–1263) was described before . Upon exw/v) PEG 4000, 10 mM iron(III) chloride and 20% (v/v) glycerol and were rapidly cryo-cooled directly in liquid nitrogen.Crystallization trials for native pb1(970–1263) in the presence of mannose were also performed . The purified protein (at 15 mg/mL in 20 mM Tris-HCl pH 8.5) was incubated in the presence of 2 mM mannose for 2 h at room temperature. Crystals were obtained in a drop where the reservoir contained 0.1 M Tris-HCl pH 8.5, 8%  in the presence of SYPRO Orange . Reaction volumes of 30 μL were prepared in 200 μL capped PCR tubes with 30 μg of protein and 1× SYPRO Orange from the supplied 5000× stock solution. Samples were heated from 4 °C to 94 °C with a ramp rate of 1 °C/min; the fluorescence was measured at every 0.5 °C increment.E. coli F. T5oad was amplified on the E. coli F strain and purified by centrifugation on cesium chloride gradients [E. coli F bacteria were harvested in the mid-log phase, washed in phosphate-buffered saline and resuspended at a cell density of 1 × 109/mL in T5-TBS buffer . Aliquots (0.05 mL) of cell suspension were incubated for 20 min at 0 °C with concentrations (0.015 to 1.5 μM) of purified pb1(970–1263) or pb1(970–1396). Estimating that a bacterial cell contains about 106 LPS molecules [E. coli F. In these experimental conditions, we confirmed that adsorption of T5oad to E. coli B, which has a semi-rough LPS, lacking the O-specific chain, was less than 1%. The presence of 8.5 μM of bovine serum albumin did not affect the adsorption of T5oad to E. coli F.The O-antigen dependent bacteriophage T5oad mutant  was usedradients . E. coliolecules , this coData collection for selenomethionine-modified pb1970–1263) has been described and statistics reported previously . Data fo3 has beeSamples (5 μL) of purified pb1(970–1293) and pb1(970–1396) proteins were applied to glow-discharged carbon-coated grids and negatively stained with 2% uranyl acetate solution. Images were recorded on a FEI Eagle 4k CCD camera in low-dose conditions with a Tecnai G2 electron microscope  operating at 200 kV. Frames were recorded at a detector magnification of 67,873× (2.21 Å/pixel sampling rate). Electron microscopy of bacteriophage T5 tails was described before .Image processing was performed using XMIPP  and defoE. coli cells led to production of the non-proteolyzed version of the protein, which we call pb1(970–1396). Substitution of a conserved serine to alanine was previously described to inhibit the auto-proteolytic cleavage of a similar intra-molecular chaperone domain [The proteins pb1794–1263) and pb1(970–1263), corresponding to two carboxy-terminal  fragments of the L-shaped tail fibres of different size, were expressed, purified and crystallized as described before . A singl94–1263 ae domain . Pb1(970E. coli F to successfully infect its host [E. coli F bacteria is expected to inhibit infection with T5oad. The inhibitory effects of purified pb1(970–1263) and pb1(970–1396) are shown in We expected the carboxy-terminal end of the L-shaped tail fibres to be the specific domain that binds to the O-antigen of the LPS. To confirm this, we measured the inhibition of phage T5oad adsorption by the carboxy-terminal domain of pb1. Unlike wild-type T5, the T5oad mutant is strictly dependent on the binding of the L-shaped fibers to polymannose O antigen of its host . Thus, aet al. [A dataset to 2.5 Å resolution of the seleno-methionine derivative of pb1(970–1263)  collecteet al.  for natiWhen the monomers in the pb1(970–1263) structure are superposed, they align with a root mean square difference (r.m.s.d.) of only 0.3 Å over 269 Cα-atoms; even loop residues superpose very well. The same is true for the pb1(970–1396) structure and here the r.m.s.d. is between 0.3 and 0.5 Å when 392–394 Cα-atoms are superposed. When the trimer of pb1(970–1263) is superposed onto the equivalent domain in pb1(970–1396), the r.m.s.d. is only 0.5 Å for 824 superposed Cα-atoms.The structure of pb1(970–1263) resembles a bullet A. We divThere are no proteins in the protein data bank (PDB) with the same topology as the pb1 bullet-shaped domain, although the topologies of some bacteriophage tailspikes, such as the podovirus P22 tailspike (PDB entry 1TSP) , partialThe second interdigitated region ir2  again forms a short triple beta-helix, with the beta strands in parallel disposition. This is followed by a second triangular domain td2 (residues 1161–1238). Like the first triangular domain, this domain is also a beta-prism formed by three concave beta-sheets, each containing five anti-parallel beta-strands (S through W). At the distal end of the mature protein, a third interdigitated region (ir3) is located, made up of residues 1239–1263, which form a tapered triple-helical structure. The taper makes the end of the structure pointed. In the mature, chaperone-free structure, the third interdigitated region does not contain any regular beta-structure, although there are many inter-chain main-chain hydrogen bonds. In the structure with the chaperone, two short beta-strands are present in the third interdigitated region, forming a four-stranded beta-sheet with the C′ and F′ beta-strands of the chaperone domain . GeneralSerine 1264, the residue responsible for the auto-proteolytic cleavage of the carboxy-terminal domain from the bullet-shaped domain, is located between the bullet-shaped and intra-molecular chaperone domains. In the structure where Ser1264 has been substituted with alanine, the chain continues with a loop (residues 1265–1276) winding around the other three, subsequently forming a hairpin (residues 1277–1322). This first hairpin contains two alpha-helices and two beta-strands (A' and B') and is oriented side-ways, nestled under the aforementioned loop of amino acids 1265–1276. Three 50 Å-long hairpins . The intra-molecular chaperone domain (ic) ends in a central triple-helical coiled-coil, to which each monomer contributes their carboxy-terminal alpha-helix (formed by residues 1371–1394).E. coli phage K1F endosialidase (PDB entry 3GW6) [Bacillus phage GA-1 (PDB entry 3GUD) [Bacillus phage phi29 (PDB entry 3SUC) [The structure of the intra-molecular chaperone domain shows similarity to the equivalent domain of the ry 3GW6)  and can ry 3GUD)  is also ry 3SUC)  reveals 3 Å2 of 23 × 103 Å2 for pb1(970–1263) and 19 × 103 Å2 of 35 × 103 Å2 for pb1(970–1396). The estimated free energy gains upon trimerization are 285 and 380 kcal/mol for pb1(970–1263) and pb1(970–1396), respectively. Thermofluor unfolding experiments were performed on both pb1(970–1263) and pb1(970–1396). In these experiments, a sample of the protein is incubated in the presence of a hydrophobic fluorescent dye and the temperature is slowly raised. When the protein unfolds, the dye increasingly binds to hydrophobic residues of the protein and the fluorescence increases. The experiments showed that pb1(970–1263) unfolds at 62 to 64 °C, while pb1(970–1396) unfolds between 64 and 65 °C, indicative of stable proteins.Analysis of the quaternary structure of both pb1(970–1263) and pb1(970–1396) shows that more than half of each surface monomer is buried in the trimer: 13 × 10The extensive intertwining of the monomers in the trimer and close association in other regions leads to the formation of numerous hydrogen bonds, van der Waals interactions and salt bridges  and 25 for pb1(970–1396)). The central, three-fold symmetric, longitudinal cavity between the three monomers is surrounded by hydrophobic and aromatic residues, for example Ile1134, Ile1149, Phe1044, Phe1077, Phe1174 and Trp1226 A,B. The i.e., avoid staggered interactions between the three monomers). The interactions also suggest that the fibre-distal part of the chaperone may be more rigid, and the fibre-proximal part more flexible, facilitating interaction with the fibre.In the chaperone, the bottom part of the centre of the chaperone coiled-coil is also hydrophobic, with Phe1383, Leu1387, Ile1390 and Leu1394 contributing to the core C. The to3 Å2 large and the association is accompanied by a free energy gain of just −12 kcal/mol. The interface contains almost exclusively hydrophilic interactions. Salt bridges are formed by the Glu1355 residues from the top of the tentacle with Arg1150 and Arg1222 projecting from the sides of the bullet-shaped domain. These residues are from three different monomers. After cleavage of the three Thr1263-Ser1264 peptide bonds, the chaperone must be released; therefore, the interaction between the bullet-shaped domain and intra-molecular chaperone domain must not be overly strong.Although the interface between the bullet-shaped domain and the intra-molecular chaperone domain appears extensive C, it is E. coli phage K1F, a mechanism has been proposed for the auto-proteolytic reaction [For the intra-molecular chaperone of the endosialidase of reaction ,42. All reaction ,42, whilreaction .Purified T5 tails were analyzed by negative stain electron microscopy A as repoi.e., between the white and black asterisks in Like other bacteriophage fibres and tailspikes ,40,43,44E. coli phage K1F [Bacillus phages GA-1 [E. coli phages K1E, 63-D, K1–5, K5, Bacillus phages PZA, B103 and Zymomonas mobilis phage ZM4 [Elongated trimeric beta-structured proteins have evolved different strategies to fold correctly. Some fold endogenously when expressed in bacteria, like the phage P22 tailspike  and the hage K1F , Bacilluges GA-1  and phi2ges GA-1  and preshage ZM4 ,51. Intehage ZM4 , althougIn phage T5 pb1, the folding may start from the distal part of the chaperone with the association of the three helices of the short triple coiled-coil. The chaperone domains fold and the upward-pointing tentacles are formed. These tentacles then interact with the bullet-shaped domain of the fibre, confining the chains of the bullet-shaped domain while they fold. Once the carboxy-terminal triple-helix is correctly formed, proteolysis can take place as described above ,42, leadEscherichia coli LPS. The minimal unit described to bind to T5 fibre is a tri-mannoside [Bacteriophage T5 binds to the polymannose O-antigens of annoside . Many prannoside , but aroSiphoviridae phage T5 tail fibre, with and without its carboxy-terminal intra-molecular chaperone domain. The structures reveal an extensively intertwined, beta-structured receptor-binding domain and an alpha/beta-structured intra-molecular chaperone domain, with long beta-hairpin fingers enveloping its folded substrate. Once receptor-binding sites have been identified, the structure may form the basis of rational design of phage-based fibres for specific recognition of target bacteria, together with the structures of the receptor-binding domains of fibres of Myoviridae phages [Podoviridae phages [We have solved the structure of the carboxy-terminal domain of the e phages ,54 and Pe phages .


Network biology currently focuses primarily on metabolic pathways, gene regulatory, and protein-protein interaction networks. While these approaches have yielded critical information, alternative methods to network analysis will offer new perspectives on biological information. A little explored area is the interactions between domains that can be captured using domain co-occurrence networks (DCN). A DCN can be used to study the function and interaction of proteins by representing protein domains and their co-existence in genes and by mapping cancer mutations to the individual protein domains to identify signals.The domain co-occurrence network was constructed for the human proteome based on PFAM domains in proteins. Highly connected domains in the central cores were identified using the k-core decomposition technique. Here we show that these domains were found to be more evolutionarily conserved than the peripheral domains. The somatic mutations for ovarian, breast and prostate cancer diseases were obtained from the TCGA database. We mapped the somatic mutations to the individual protein domains and the local false discovery rate was used to identify significantly mutated domains in each cancer type. Significantly mutated domains were found to be enriched in cancer disease pathways. However, we found that the inner cores of the DCN did not contain any of the significantly mutated domains. We observed that the inner core protein domains are highly conserved and these domains co-exist in large numbers with other protein domains.Mutations and domain co-occurrence networks provide a framework for understanding hierarchal designs in protein function from a network perspective. This study provides evidence that a majority of protein domains in the inner core of the DCN have a lower mutation frequency and that protein domains present in the peripheral regions of the k-core contribute more heavily to the disease. These findings may contribute further to drug development.The online version of this article (doi:10.1186/s13336-015-0016-6) contains supplementary material, which is available to authorized users. Domains are distinct functional or structural units in a protein. Most domains correspond to tertiary structure elements, and are able to fold independently. All protein domains exhibit evolutionary conservation and many either perform specific functions or contribute in a specific way to the structure of their proteins. Domains may exist in a variety of biological contexts, wherein similar domains can be found in proteins with different functions. Many proteins are composed of one or more domains that can fold independently into a stable core structure -3.Many complex systems have been analyzed as networks by representing the system as nodes and interactions between them as edges. Studies on complex networks including the network of co-authorships, sexual contacts and the world-wide-web (WWW) reveal that their structure and growth is governed by a set of generic organizing principles ,5. NetwoThe development of high-throughput, whole-exome/genome DNA sequencing has made it possible to evaluate normal and tumor tissue samples in a single study. These studies have revealed the connection between somatic mutations and cancer susceptibility, initiation and development . A centrSeveral studies have been conducted on protein domain co-occurrence networks (DCN). These studies represent domains as nodes and their co-occurrence in a protein are denoted as edges. The networks have also been shown to possess a scale-free property -24. IncrThe definition of k-core was first introduced by Seidman  to charaK-core decomposition is a network analysis approach that helps in understanding interesting structural properties that are not otherwise captured by many other network topological parameters. The basic principle behind the k-core is decomposition to identify particular subsets of the network called k-cores. Each k-core is obtained by a recursive pruning method ,35,36. THomo sapiens was constructed using the Ensembl database (version 72) that provides a comprehensive source of stable automatic annotation of individual genomes [The DCN for  genomes .H. sapiens consisting of 1929 nodes and 5171 edges as visualized by Cytoscape [To ensure correct coordinates in our version domains present in any protein sequence of the human proteome were determined using the program PfamScan . Domain ytoscape .Figure 1k connections with other protein domains . This is done by first identifying all nodes whose degree  is less than k. After removing these, the network is re-analyzed to determine if the removal of these nodes has resulted in other nodes  having now less than k connections. If such nodes are identified, then they are removed, and the process is continued, until no more nodes can be removed. The resulting sub-network is called the k-core of the network.To identify the core-periphery organization of the domain co-occurrence network, we subjected it to core decomposition. The cores of different orders of a network can be obtained by iteratively removing all nodes which have less than k-core, we compared them with the mean and variance of the corresponding values obtained for a randomized ensemble. Each randomized k-core in the ensemble is obtained by random selection without replacement of Nk domains from the DCN, where Nk is the size of the empirically determined k-core. The randomized ensemble for every DCN considered was generated by constructing 100 such randomized k-cores.To determine the statistical significance of the properties calculated for members of an empirically determined plus [. It consists of a database of PFAM alignment phylogenetic trees for known protein domains and their families. This database was constructed using a relational database which comprises of information regarding the functional categories, metabolic pathways, protein–protein interactions, disease associations, gene expressions, three-dimensional structures, as well as estimates from an evolutionary analyses of selective pressures.The evolutionary conserved protein domains were identified using the database PANDITplus . It conshttp://tcga-data.nci.nih.gov/tcga/) using mutation files from the hgsc.bcm.edu_COAD.IlluminaGA_DNASeq.1 and hgsc.bcm.edu_COAD.SOLiD_DNASeq.1 directories downloaded on March 30th, 2013. The silent and RNA mutations were filtered out from the data set as they were assumed unlikely to affect the cancer development. Somatic mutation counts for ovarian cancer were found to be 20,878. For breast and prostate cancer the values were found to be 35,558 and 23,349.Somatic mutation data for ovarian, breast and prostate cancer were obtained from TCGA data portal . This probability (p) was normalized using the signal to noise ratio of the Bernoulli distribution, which resulted in a normalized score, z and is given by“locfdr” package [The  package  from R w package . The locHomo sapiens was constructed and its statistical properties were determined. From the degree distribution plot .Cancer mutations obtained from TCGA data portal were mapped to the individual protein domains. The normalized mutation score for each protein domain was also calculated (for details see Methods). To study the mutations in domain co-occurrence network, the normalized mutation scores were assigned to all the nodes  in the network. In order to understand the nature of mutation in the S.cerevisiae, C.elegans, D.melanogaster, M.musculus and H. sapiens have been preserved during evolution. This high conservation of inner core domains across species development may explain why they are also less mutated in comparison to the peripheral protein domains in cancer [As shown in Figure n cancer .Figure 42) between them were also found to be positive. Interestingly, the outlier of the inner core is found to be more significant as it comprises of lower mutation rates.From the randomized simulations, we observed that the inner core domains had lesser rates of mutation compared to the peripheral cores. To verify if, the inner core significantly differed from the other cores , every domain’s normalized mutation rates were plotted against the k-core values for the three types of cancers as shown in Additional file Significantly mutated domains in all of the three cancer types were identified using the local false discovery rate. On comparing the significantly mutated domains among the cancer types, we found several domains common between ovarian, breast and prostate cancer Figure . The sigAs discussed in the previous section, core domains were found to be less mutated in comparison to the peripheral domains. We also investigated the presence of significantly mutated domains in each core of the domain co-occurrence network. We calculated the percentages of significant domains in each core of the DCN as shown in Figure On analyzing genomes studies have shown that more than 70% of eukaryotic proteins comprised of multiple domains. Domain-domain interactions are now becoming an upcoming trend of interest across numerous studies -51. StudBy constructing and analyzing domain co-occurrence networks we gain new and fundamental insights into the qualitative arrangement and evolutionary utilization of the proteome. Domain databases like Pfam and Interdom provide comprehensive domain information but mapping cancer SNPs to the individual domains may help identify cancer targeted protein domains rather than just the proteins. Domains with high relative rates of mutation in three hormonal cancer types were identified along with their common domains. Recent studies from Liu et al, 2014 revealed that the PDZ and LIM protein domain promotes breast cancer cell migration, invasion and metastasis . These tHomo sapiens DCN and the observed mutations included all the three cancer SNPs. The expected and observed mutations in different k-cores were calculated and plotted as shown in Figure Chi-squared goodness of fit test was employed to validate whether the observed and expected mutations are statistically significant. The expected mutations were calculated from the Analyzing mutations in domain co-occurrence network helps in identifying crucial protein domains that aid in the progression of cancer disease. Highly connected protein domains are found to be evolutionarily conserved in the domain co-occurrence network. This implies that protein domains in the inner core are more conserved than the domains in the peripheral region. Significantly mutated protein domains which were identified further contributed to determining the disease target protein domains in all the three cancer diseases. Comparing the mutational landscape of somatic mutations in the protein domain co-occurrence network with the random counterparts, our findings revealed that there is a statistically significant difference between them.The functional annotations obtained for all the significantly mutated domains were seen to be involved in all the three cancer diseases. Polymorphisms in inflammation-related genes, including those in the Toll-like receptor (TLR) signaling pathway, are hypothesized to be involved in prostate carcinogenesis . This ToA recent study done by Pasta A et al , showed