Nuclear lamins are type V intermediate filament proteins which form an elasticmeshwork underlying the inner nuclear membrane. Lamins directly contribute tomaintain the nuclear shape and elasticity. More than 400 mutations have beenreported in lamin A that are involved in diseases known as laminopathies. Thesemutations are scattered mainly in the lamin rod domain along with some in itsC-terminal domain. The contribution of the rod domain towards the elasticity oflamin A molecule was hitherto unknown. Here, we have elucidated the significance ofthe 1B and 2B domains of the rod in modulating the elastic behavior of lamin A bysingle-molecule force spectroscopy. In addition, we have also studied the networkforming capacity of these domains and their corresponding viscoelastic behavior. Wehave shown that the 1B domain has the ability to form a lamin-like network andresists larger deformation. However at the single-molecular level, both the domainshave comparable mechanical properties. The self-assembly of the 1B domaincontributes to the elasticity of the lamin A network. A-type lamins comprising of lamin A and lamin C aretranscribed from LMNA gene where lamin C is the splice variant of lamin A. B-typelamins, lamin B1 and lamin B2, are encoded by the LMNB1 and LMNB2 genesrespectively11314in vitro studies, it was shown that polarized arraysof the dimers interact in an antiparallel fashion to form apolar tetramericprotofilaments. The interaction of four lamin protofilaments leads to the formation of~10 nm filaments18LMNA gene have been linked to these diseases, which arecollectively known as laminopathies2021LMNA gene alter theregulation of gene expression during differentiation of tissues of mesodermaloriginThe nucleus of the metazoan cell maintains its 3D morphology and rigidity with the helpof filamentous elastic scaffold underlying the inner nuclear envelope146http://www.dmd.nl/lmna_home.html#protein)a and d positions of the heptad were occupied by anonpolar residue . On the other hand, 2B domain contains only fourheptad repeats. Lamin A forms a coiled-coil dimer through the intermolecularhydrophobic interactions of the coiled-coil regions and these dimers further canform the higher order structure by head-to-tail association. Different laminfragments in both coil 1 and coil 2 regions can form the half-lamin ormini-laminLike all other IFs, lamin A proteins have the same basic tripartitestructure3-1B-(Cys) and (I27)3-2B-(Cys) which would behenceforth referred to as (I27)3-1B-1C and (I27)3-2B-IC.Upon unfolding of these disulfide-linked dimers, we would expect the resultantunfolding pattern of corresponding six I27 domains and a coiled-coil unzippingof the lamin domain. We ensured the dimer formation by running the proteins in anon-reducing SDS-PAGE gel (see 3436c)reflecting the flexibility and heterogeneity in the local structure at the dimerlevelc) based on the dimer formed from 126 amino acidsin the lamin fragment used in the SMFS experimentsWe have thus shown that both 1B and 2B domains can assemble into higher orderstructures. Now it would be interesting to investigate their mechanicalresilience at their single-molecule level. We performed pulling experiments onlamin 1B and 2B by single-molecule force spectroscopy (SMFS) to understand themechanical properties of their dimers. To demonstrate distinct coiled-coilunzipping, we engineered tandem titin I27 domains3233 gel see . When puWe observed similar results for 2B domain also shown in . Lamin 2cal) byintegrating the area under a DSC thermogram which is described by the followingequation.In order to corroborate the mechanical unzipping of the 1B and 2B at dimer level,we studied thermal unfolding in the bulk. We performed differential scanningcalorimetry (DSC) from 10 °C to85 °C at a scanning rate of30 °C/hr. Both the unfolding events were endothermic. Wecalculated the calorimetric enthalpy  = ɤosin (ωt), so that the resultant stress becomesσ(t) = σosin(ωt + δ).This resultant stress can be analyzed by decomposing the two waves of the samefrequency (ω); one is in phase and another 90° out ofphase. The stress can be written in the form of the complex dynamic modulus.It is now challenging to unravel the effect of the distinct assembly mechanismsof 1B and 2B on their corresponding viscoelastic behavior. In retrospect,detailed studies from our group had already established the viscoelastic natureof the full-length lamin A protein in the context of some of its mutationsimplicated in DCMwhere, σ (t), G′ and G″ represent the appliedstress, shear and loss moduli respectivelyThe ratio of the loss modulus to the storage modulus gives an idea about theviscoelastic nature of the fluid. We performed the oscillatory shear experimentswith the preassembled half-lamin molecules. We applied constant oscillatoryshear of strain amplitude 1% at an angular frequency of 5 rad/s for1000 sec on the network formed by the half-lamins at variousconcentrations. Interestingly, we noticed that after 100 sec, boththe storage and loss modulus of the networks reached the saturation level  where 1B domain covers 81-218residues. We noted a seven residue heptad repeat skip from residue120–126 in the 1B domain from theoretical structural predictions ofCOILS/PCOILS. Similarly for the 2B domain also, we observed the same four residues(SRRL) insertion at 334–337 positions. This is in agreement with thecrystal structure data where four residues insertion was shown at position 327 andwas termed as stutter defectThe filamentous elastic scaffold inside the cell consisting of nucleoskeleton andcytoskeleton has a major contribution in maintaining the cellular 3D morphology6474851When we studied the network forming ability of these short lamin fragments toultra-structural details by scanning electron microscopy, we observed that lamin 1Bdomain formed a network much similar to full-length lamin ATo explore the mechanical resilience of the 1B and 2B domain we used the SMFStechnique. Formation of the dimer at the molecular level plays a pivotal anddeterministic role in the higher order assembly of lamins which proceed through thehydrophobic association of the helical rod region. We investigated the mechanicalproperties of the 1B and 2B domains at their dimer level. Surprisingly 1B dimer wasunzipped at slightly lower force (~70 pN) compared to thelamin 2B dimer (~80 pN). Despite the longer length of 1Bdimer, the interhelical interactions were comparably stronger for the 2B domain.However, both the domains were unfolding via several intermediate pathways whichsuggest that they are flexible in nature. End-to-end contour length increase for the1B domain was ~82 nm which essentially fitted intoPCOILS/COILS heptad repeat calculations. Based on these predictions, lamin 1B formcoiled-coil dimer from residue 85–210 amino acid and this partcontributes to a major extent in the resistance for unzipping of the 1B domain.However, both 1B and 2B domain were unfolded via several pathways. Also, the contourlength analysis for both the coiled-coil unzipping events confirmed the presence ofstutter in 2B and a heptad skip in 1B. From the heptad repeat analysis, we showedthat both 1B and 2B domains possess a heptad skip at 120 position for the 1B and at334 for the 2B domain. Due to this heptad skip the dimers are mechanically unstableat that position, so in the presence of the unzipping force molecular resistance forthe 1B dimer emerged from the residues 85–119 and 127–210and for 2B it was from residues 305–333 and 338–390. Hence,both the 1B and 2B dimers unfolded via an intermediate pathway. Crystal structuredata for 2B also confirmed the heptad repeat skipIn conclusion, we can say that the unfolding behavior of the 1B & 2B domainsat the single molecule level as well as bulk level would reflect directly on theviscoelasticity which in turn would be the reason to perturb the rigidity of thelamina, a major hallmark in all laminopathies. To address the viscoelastic behavior,we performed the rheological experiments. Earlier it was shown that full-lengthlamin A network could resist deformation up to 500% of the strainTo the best of our knowledge, this work presents the first report in the lamin fieldcharacterizing the mechanical behavior of different lamin fragments from the singlemolecule level to the highly polymerized state. We conclude that the mutations inlamin A, particularly in 1B and 2B, perturb polymerization to a different extent andthis might possibly lead to attenuated elasticity of nuclear lamina, which leadsdeformed and fragile nuclei as observed in the majority of laminopathies.We investigated the heptad repeats of the lamin 1B and 2B domains usingPCOILS/COILS server. We took 71–242 amino acids as 1B which includedboth the N-terminal and C-terminal linker region and 305–390 aminoacids as 2B domain. We used MTIDK matrix and window size as 14.31B 1 C for 1B and (I27)3 2B 1Cfor 2B domain. We fused lamin 1B and 2B domain to the three I27 cassettescontaining pQE80L vector. We introduced three I27 domains by iterative cloningmethod as described previously. Forward primer 5′CGCGGATCCGCGAGCCGCGAGGTGTCCGGCATC 3′ and reverse primer5′ CGGGGTACCCCGTTAGCAGGAAGATCTTCCCGCCAGCCGGCTCTCAAACTCAC3′ were used to amplify lamin 1B domain using Phusion High-FidelityDNA Polymerase . Similarly, for lamin A 2B,5′ CGCGGATCCGCGCAGCTCAGCCAGCTCCAGAAGC 3′ and5′ CGGGGTACCCCGTTAGCAGGAAGATCTTCCTAGCCTCTCCTCCTCGCCCTCC3′ were used as forward and reverse primer respectively. In both theforward primers, BamH1 was used as a restriction enzyme and BglII and Kpn1 siteswere introduced in the reverse primers. PCR products of 1B and 2B domains weredigested with BamHI-HF and KpnI-HF  and gelpurifications were performed using Gel Extraction Kit .pQE-80L vector containing three I27 cassettes was similarly digested with BglIIand Kpn1-HF restriction enzymes  and ligations weredone using T4 DNA Ligase  at16 °C overnight. For only 1B and 2B domains, we followedthe same path but instead of using pQE80L (I27)3 vector we used onlypQE80L vector. K97E and E161K were generated as reported earlier600 for 4 hr at 37 °C.Cell pellets were lysed with 25 mM Tris-HCl (pH 8.5),250 mM NaCl, 1% Triton-X 100. All the proteins were purified usingHis-trap column  followed by gel filtration withSuperdex 200 column  and resolved on 10% and/or20% SDS-PAGE followed by Coomassie staining. Final proteins were dialyzed using25 mM Tris-HCl (pH 8.5), 250 mM NaCl buffer. Proteinconcentrations were checked with Bradford reagent .To apply the mechanical unzipping force to different coiled-coil lamin domains,we used (I27)All the protein samples assembled in 10 mM Tris-HCl (pH 8.5),250 mM NaCl, and 1 mM DTT containing buffer were placedon coverslips, dried in vacuum and coated with gold. All the images were takenin Hitachi S530 Scanning Electron Microscope (Japan) between 4000-6000xmagnifications at 25 kV.We performed mechanical unzipping experiments using single-molecule forcespectroscopy (SMFS) on a custom-built atomic force microscope (AFM) as describedpreviouslyAll the force-extension (F-X) traces were fitted with the worm-like chain modelusing the B and T denote persistence length, contour length,Boltzmann constant and absolute temperature respectively. All the F-X traceswere analyzed using Igor Pro 6.02 .where, p, L, kThermal unfolding studies of all the proteins were performed using differentialscanning calorimetry (DSC). For these denaturation studies,100 μM and 20 μM samples wereused for lamin 2B and 1B domains respectively. All the experiments wereperformed in 25 mM Tris-Cl pH 8.5), NaCl buffer with a scanningrate of 30 °C/hr at ~30 psipressure in a VP-DSC Microcalorimeter . Forboth the samples temperature ranges were10–85 °C. All the thermograms were analyzedwith in-built VP Viewer software with Origin 7.0. The lamin 2B domain unfoldingwere fitted with the independent two-state unfolding model whereas lamin 1Bdomain unfolding was fitted with the independent non-two-state transitionmodels. In the independent two-state model, molar heat capacity varies with thetemperature by the .5, NaCl The molar heat capacity in non-two-state transition varies by the p (T) is the molar heat capacity; KA (T) and KB(T) are the equilibrium constants for the unfolding events,∆HA (T) change in the calorimetric enthalpy,Tm is the temperature of the transition point,∆Hm molar enthalpy change at the transition point.∆Hc is the change in the calorimetric enthalpy and∆Hv is the van’t Hoff enthalpy. For thetwo-state unfolding events calorimetric enthalpy is equal to thevan’t Hoff enthalpy. The thermograms were fitted using Marquardtmethods based on the non-linear least squares guessing the each parameter andthen fittings were improved by the several iterations.CRheological measurements were performed in a rheometer where the lower platewas fixed and shear deformations were applied by the upper cone plate. Weapplied the shear force on the lamin protein for 1000 sec at aconstant 1% strain and at an angular frequency of 5 rad/sec for thebuild-up experiments. Appropriately diluted the lamin protein samples wereapplied between the two plates in assembly buffer containing 25 mMTris-Cl (pH 8.5), 250 mM NaCl at 25 °C. Forthe build-up experiments, we used 1, 5, 10 and 20 μM ofboth lamin 1B and 2B protein and 20 μΜ ofboth the proteins were used for the network yielding experiments where 0 to 500%strains were applied at a constant angular frequency of 5 rad/sec.All these experiments were performed as described previouslyHow to cite this article: Bera, M. et al. Significance of 1B and 2Bdomains in modulating elastic properties of lamin A. Sci. Rep.6, 27879; doi: 10.1038/srep27879 (2016).


Saccharomyces cerevisiae and Bos taurus. This RING-like domain is possibly related to those of other vesicle membrane-associated complexes, such as CORVET, HOPS and SEA, and likely mediates interactions with Dsl1p and assist in coat oligomerization.The C-terminal domain of α-COP, an essential subunit of the COPI coatomer complex, is composed of an all α-helical region and a small β-sheet domain. We show that this β-sheet domain is a Really Interesting New Gene (RING)-like treble clef zinc finger. The zinc-binding residues are substituted by other aminoacids in many homologs including the structurally-characterized proteins from The online version of this article (doi:10.1186/s13062-015-0099-9) contains supplementary material, which is available to authorized users. Bos taurus; PDBid 3MV3_A from Saccharomyces cerevisiae)  complex, which coats the vesicles involved in retrograde transport of proteins from the Golgi apparatus to the endoplasmic reticulum (ER), and in intra-Golgi protein trafficking, comprises of a coatomer and ADP-ribosylation factor 1 (ARF) GTPase . The coaae) Fig.  4–7]. T–7. TBos ret1-3 allele of α-COP (S1188F mutation in the β-sheet domain) causes instability of α-COP and the coatomer, defects in cellular transport and temperature sensitivity in the organism [The C-terminal β-sheet domain of α-COP is necessary for its interaction with- and incorporation of- ε-COP into the coatomer, for maintaining normal levels of ε-COP, and has also been shown to interact with the β’-subunit . The yeaorganism . The CTDorganism , 10.Currently, Pfam  and ConsTrypanosoma cruzi (gi|407411736) and Colletotrichum orbiculare (gi|477532069)  Fig. . Other cConsistent with the observed pattern of conservation for the zinc-coordinating residues in the α-COP β-sheet domain, which is particularly high for the second metal-coordinating site, sequence-profile-similarity-based searches using HHpred  and FFASStructurally, the β-sheet domain of α-COP consists of two β-hairpins, a small α-helical turn, and a loop with a tight turn Fig. . Albeit α atoms , where a water molecule participates to stabilize zinc binding). There could also be compensatory chelating sites owing to oligomerization. Hence, it may be better to state 'This RING finger has three of the four classical Zn-binding residues mutated' or ‘Sequence analysis suggests that this RING finger may not have zinc-binding function …’5. line 247: “This RING finger has lost its ability to bind to zinc ions …” This phrase appears too speculative since there are atypical zinc-chelating residues (like 3His-Asp as in adiponectin receptors (These lines have been edited in the revised manuscript.Response: 6. line 276: Specific reference to PYMOL visualization maybe avoided.We have retained the reference to PyMOL in the methods section in the Additional file 1 as we used it extensively for doing manual structure analysis.Response: Dr. Eugene KooninThe finding that you report clearly is valid and of certain importance. At the same time, it is highly specific, so I think you made the right choice of describing your finding in a Discovery Note. However, the present manuscript is far too long and detailed for this format, and this could, I believe, discourage Editorial Board members from handling the submission. My suggestion is that, if you remain interested in Biology Direct as the publication venue, you substantially shorten the manuscript by dropping non-essential details and references.We have edited the manuscript to suit the submission criteria for a Discovery Note.Response:


Xbp1 transcript, or suppress gene expression. This referred suppression of gene expression is mediated through degradative cleavage of a select cohort of cellular RNA transcripts, initiating the regulated IRE1-dependent decay (RIDD) pathway. The review first discusses the anti-viral mechanism of the OAS/RNaseL system and evasion tactics employed by different viruses. This is followed by a review of the RIDD pathway and its potential effect on the stability of viral RNAs. I conclude with a comparison of the enzymatic activity of the two RNases followed by deliberations on the physiological consequences of their activation.The mammalian genome has evolved to encode a battery of mechanisms, to mitigate a progression in the life cycle of an invasive viral pathogen. Although apparently disadvantaged by their dependence on the host biosynthetic processes, an immensely faster rate of evolution provides viruses with an edge in this conflict. In this review, I have discussed the potential anti-virus activity of inositol-requiring enzyme 1 (IRE1), a well characterized effector of the cellular homeostatic response to an overloading of the endoplasmic reticulum (ER) protein-folding capacity. IRE1, an ER-membrane-resident ribonuclease (RNase), upon activation catalyses regulated cleavage of select protein-coding and non-coding host RNAs, using an RNase domain which is homologous to that of the known anti-viral effector RNaseL. The latter operates as part of the Oligoadenylate synthetase OAS/RNaseL system of anti-viral defense mechanism. Protein-coding RNA substrates are differentially treated by the IRE1 RNase to either augment, through cytoplasmic splicing of an intron in the  Recent reports, however, have demonstrated transmission of an apoptotic signal between cells that are in contact through gap junctions, although such a signaling from an virus infected host cell to an uninfected one is not known yet  dsRNA segments are detected by dedicated pathogen associated molecular pattern receptors (PAMPs) or pattern recognition receptors (PRRs) in the host cell, like RIG-1, MDA5, and the IFIT family of proteins . The traFigure 2A; Figure 1; in vitro concentrations, RNaseL could oligomerize even in the absence of 2–5A , a middle catalytically inactive pseudo-kinase (PK) and a C-terminal RNase domain  although there seems to exist a bias towards cleavage of viral RNA . RNA seqFigure 1; Figure 1; trans-autophosphorylation by the kinase domain allosterically activates the RNase domain  along with a battery of ER-lumen resident enzymes  catalyse/mediate the necessary folding, disulfide-bond formation, and glycosylation reactions . A pertue domain . In facte domain . Once ace domain . Dependie 1; Phe .Xbp1u (u for unspliced) mRNA transcript at two precise internal positions within the open reading frame (ORF) generating three segments, the terminal two of which are ligated by a tRNA ligase in yeast and by an unknown ligase in mammalian cells, to produce the Xbp1s (s for spliced) mRNA transcript  and the ERAD pathway genes  whereas RIDD reduces flux of polypeptides entering the ER lumen (Casp2) controlling miRNAs, constitutes the pro-apoptotic function of IRE1 . The possibilities of such a direct anti-viral function are encouraged by the fact that all these viruses encode at least one protein which, as part of its maturation process, requires glycosylation and disulfide-bond formation. Such a necessity would entail translation of the mRNA encoding such a protein, which in case of positive-sense single-stranded RNA viruses would mean the genome, in association with the ER-membrane (Figure 1; cis, promoting formation of such subdomains (Figure 1; Infection of mammalian cells by a multitude of viruses induce an UPR which is sometimes characterized by suppression of signaling by one or more of the three sensors; . Among tbdomains . The facbdomains . Since tbdomains . Most ofTable 1 and Figure 2; Figure 2C; IRE1 and RNaseL, in addition to biochemical similarities in protein kinase domain and structural similarities in their RNase domain, share the functional consequences of their activation in initiating cellular apoptosis through JNK signaling (In the context of a virus infection, the pathway leading from both these proteins have the potential to lead to cell death. Notwithstanding the fact that this might be an efficient way of virus clearance, it also portends pathological outcomes for the infected organism. Future research would probably lead to design of drugs targeting these proteins based on the structural homology of their effector domains, regulating the pathological denouement of their activation without compromising their anti-viral or potential anti-viral functions.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Ellis Reinherz and I were riding in a luxury Boston Coach to Long Island in New York State driven by a professional driver. This was not a vacation trip to one of the best leisure places in the United States. We were heading toward the National Laboratory at Brookhaven, where a powerful X-ray source is available in a facility called a synchrotron. The aim of our trip was to use a strong X-ray to shoot the crystals of the T cell receptor (TCR) we had just managed to grow after considerable effort for three-dimensional structure determination. In 1996, this was one of the hottest projects in structural biology as well as in immunology.Nature articles was in the New York Times in 1990. It is interesting to add that it was not until ten years later in 2001 when I was already on the faculty of Dana-Farber Cancer Institute and Harvard Medical School that Ellis and I published another structure of CD4  cell to express the protein, because these cells can produce glycoproteins. Then another headache struck. Glycosylation is notoriously heterogeneous. In order to grow crystals of diffraction quality, the prerequisite is to have a chemically homogeneous protein sample. Fortunately, scientists around that time developed an engineered CHO, termed CHO lecStanley, . At thatBob Sweet was in charge of the National Synchrotron Light Source (NSLS) beamline X12C at Brookhaven. I actually met Bob personally more than ten years ago when I paid a visit to David Eisenberg’s lab at UCLA in 1980. Bob was David’s postdoctoral fellow then who hosted my visit. We had a very interesting talk on his project. With Bob as a host at NSLS, I felt very comfortable. He was friendly and introduced us to his facility, after Ellis and I safely arrived and most of crystals seemed to survive. On the site also was a group from Albert Einstein College led by Jim Sacchettini, another X-ray crystallographer with whom Ellis had previously established a collaboration. That was the very early days when protein crystallographers just began to freeze protein crystals to collect diffraction data at around −140°C. To protein crystallographers, the synchrotron radiation source was like a double-edged sword. On one hand, the synchrotron X-ray source was so powerful, it had made possible the data collection of those weakly diffracting crystals like TCR. On the other hand, the X-ray was so strong that it quickly damaged these fragile crystals before any complete data set could be finished. To collect a data set at −140°C under cold stream generated from liquid nitrogen was invented early in 1990’s to circumvent the problem. X-ray damage was no longer a headache. Bob had a well set-up facility for all these purposes.I would never have expected to almost lose all my crystals brought to Brookhaven that day. I thought I was well prepared for the data collection because I had already carried out all the experimental procedures at home in Boston. The well-developed crystals were safely harvested from the tiny droplet into a large well with so-called cryo-protectant solution. These incubated crystals could then be fished up using a small nylon loop supported on a metal cap, and quickly flash frozen by mounting the cap on the diffractometer equipped with cold stream from liquid nitrogen, ready to shoot with X-rays. The problem arose when the cap was mounted on the machine by the magnetic force. On Bob’s machine, the magnet was so strong that the moment a crystal was put on, the force that attracted the cap on the machine shook the crystal off from the nylon loop, no matter how careful I was. We only had three crystals had left. The atmosphere, not the crystals, seemed frozen. Ellis suggested that everyone leave the room. He then turned to me and said: “Jia-huai, I am going to buy lunch for you. Just relaxed”. That did help reduce the tension! I used my right hand to fish crystal, at the same time I used my left hand to help hold the cap more smoothly and set the crystal on. A beautiful diffraction pattern showed up. This was our first 2.8 Å data set of TCR crystal! That would lead to our 1998 published study  sites, these two groups had published their structures of TCR alone and in complex with pMHC (Garboczi et al., The ecto-domains of α and β subunits of TCR are both made up of two Ig-like domains: a variable domain and a constant domain. There are two obvious deviations of the two TCR constant domains from a canonical Ig-like domain. First is the strange constant domain of α subunit. It could hardly be designated as an Ig-like domain, which normally consists of two β sheets packing face-to-face. The TCR α constant domain does have an inward β sheet facing the constant domain of β subunit. But the outward face is no longer a β sheet. One of the strands curves into a helical conformation, whereas the other two strands just loosely hang around. When our structure got published, Ian Wilson told me that he felt so much relieved to see that our α constant domain was just like theirs since the Wiley group could not trace this outside sheet in their structure. We still have no idea even today why this domain appears so poorly structured. This became even more of a mystery when Jamie Rossjohn published his pre-TCR structure (Pang et al., As a structural biologist, I was also fascinated by the second deviation of TCR structure from the normal Ig-like domain, the unusually long loop between the F strand and G strand, stretching out of the constant domain of the β subunit. I noticed that this was a common feature shared in the other two published structures. We now know that this is a very conserved structural feature of αβ TCR molecules and evolved with the molecular speciation of CD3γ and CD3δ from a common precursor molecule several hundred million years ago (Kim et al., “This is my fourteen-year dream”, said Ellis to me on the way back from that Brookhaven trip. He was referring to the day he and his colleagues at Dana-Farber Cancer Institute discovered the human TCR in the early 1980s. We both were so excited and kept chatting about how to proceed that we didn’t notice the downpour going on outside the coach. We finally realized that the poor driver missed our exit to Boston on the highway and we were on the way to Maine! The trip took more than two hours longer it should be, yet the sweet memory is still kept vividly present in my brain ever since.


Cdk5 is essential for neuronal differentiation processes in the brain. Activation of Cdk5 requires the association with the mostly neuron-specific p35 or p39. Overactivation of CDK5 by cleavage of p35 into p25 is thought to be involved in neurodegenerative processes. Here, we have tested an approach to inhibit pathological Cdk5 activation with a Tat-linked dominant-negative fragment of p25. It reduced cell death induced by staurosporine and showed a tendency to alleviate manganese-induced cell death, while it did not protect against 6-OHDA toxicity. Our results suggest that the Tat technique is a suitable tool to inhibit dysregulated CDK5. In contrast to other Cdks, Cdk5 does not require the association with a specific cyclin, but the neuron specific protein p35 for activation. This Cdk5/p35 complex is essential for neurite outgrowth, cortical development, and other differentiation processes . Cellulaand vivo . Moreoveand vivo . Vice veand vivo . The cleand vivo . p25 is and vivo . To dateand vivo , or appland vivo . In our and vivo ,12. One and vivo . Here, wE. coli and purified via Ni-affinity chromatography as described [Tat-p25 constructs were generated from expression vectors in escribed ,15. S.F.escribed . 3 diffeescribed . Amino aE. coli strain BL21(DE3) pLysS . After sonication in 8M urea, we pelleted the bacterial debris and purified the supernatant by metal-affinity chromatography using a Ni-NTA matrix . We removed salt by gel filtration on Sephadex G-25M  The desalting buffer consisted of Tris 10mM pH6.0; 50% Glycerol, 0,02% Tween-80 and 0,1% Pluronic. We confirmed the identity of proteins by Western blotting  followed by PCR amplification with the primers AGTCATCCATGGTCAAGAAGGCCCC GCACCC/GTCATGAATTCATTCTTCAAGTCAGAGAACA (for N122); AGTCATCCATGGCCA ATGTGGTCTTCCTCTACAT/GTCATGAATTCATTCTTCAAGTCAGAGAACA (for N200) and AGTCATCCATGGCTCAGCCTCCCGCCCCCCC/GTCATGAATTCTGGGTCGGCATTGATCTGCA (for N279) to add XhoI and NcoI restriction sites and cloning into the vector pRSET A . The vectors used to isolate Tat-fusion proteins are derivatives of this vector. Tat-p25  were expressed in blotting . The calSH-SY5Y cells were plated at a density of 20,000 cells/well of a 96 well dish. Cells were maintained in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum (PAA), 1 mM L-glutamine, 100 U penicillin/mL, and 100 µg of streptomycin sulfate/mL at 37 °C. 24 h after plating, cells were treated with 10 µM retinoic acid. 2 was used at 100 µM concentration. Western blots revealing CDK5 expression after MnCl2 treatment were analysed by densitomety (Alpha Innotech Software Version 3.1.2.). Values measured for CDK5 band intensities were normalized by dividing them by the corresponding intensities of the same blot probed with an anti-actin antibody. To induce apoptotic cell death, cells were treated with 400 nM staurosporine for 24 h. 6-OHDA toxicity assays were performed as described [Exactly 24 h after retinoid acid treatment, in all toxicity assays, Tat constructs were applied. Exactly 1 h after protein application, toxic agents were added. 24 h after toxin application, cell survival was assessed by WST viability assay according to the manufacturer’s protocol . MnClescribed , with 802, as a paradigm for oxidative stress and found that Cdk5 expression was increased up to 24 h after treatment  induced apoptosis in their paradigm [et al. demonstrated CDK5-mediated mitochondrial fragmentation upon staurosporine-treatment of neuronal cells [We then were interested how the TAT-p25 constructs would influence cell survival in other cell death assays, which have been shown to be influenced by the Cdk5/p25 pathway. Zhang paradigm . Moreoveal cells . We obseTo test whether inhibition of Cdk5 would be beneficial in another model for oxidative stress, we tested the p25 mutants in a 6-OHDA toxicity assay; this drug is often applied in models for Parkinson’s disease . None ofIn our approach we addressed two challenges of therapeutic Cdk5 inhibition: first, we aimed to block its pathological activation, rather than is normal function, by blocking the binding site for p25, and possibly other off-site targets. Secondly, we addressed the problem of drug delivery by using Tat-linked proteins. in vitro studies we addressed different paradigms of cellular stress believed to play a role in neurodegenerative diseases. We applied manganese, which can induce neurodegeneration, including Parkinsonism [et al. [in vitro binding studies or in assays directly assessing the effect on CDK5 enzymatic activity, the previously described functions may not be alienable from bovine to mouse. Moreover, the results obtained using viral transduction and Tat-mediated transduction may not be immediately comparable; the gene expression from different viral constructs may be variable and different from intracellular protein concentrations reached after Tat-mediated transduction [E.coli-expressed p25 fusion protein may be different from p25 directly expressed and folded in the cell to be examined. In future studies, assays to test the impact of the different Tat-p25 fragments on Cdk5 enzymatic activity could address that issue.Using insonism ,22. Manginsonism ,24,25. Oinsonism . It has insonism ,28. Our insonism  showed a [et al. . The celsduction ,30. MoreNeither overexpression of a dominant-negative CDK5, nor the CDK5 inhibitors roscovitine and butyrolactone-1 prevented staurosporine-induced cell death ,32. ConsIn a third paradigm, 6-OHDA, which is often used in models for PD, different p25 constructs did not have a significant effect on cell survival; however, there was a trend towards protection with the control constructs. The main apoptotic stimulus by 6-OHDA is believed to be delivered via activation of the ERK pathway ,35. Cdk5In conclusion, the effects of virally or cell-penetrating peptide-mediated delivery of proteins may be different. Moreover, the effect of dominant-negative p25 delivery depends on the experimental model examined: For instance, while it blocks staurosporine-induced cell death, it does not protect against 6-OHDA-induced toxicity.


Hydra, the hypostome, is a key body part for developmental control and the nervous system. We herein examined genes specifically expressed in the head region of Hydra oligactis using suppression subtractive hybridization (SSH) cloning. A total of 1414 subtracted clones were sequenced and found to be derived from at least 540 different genes by BLASTN analyses. Approximately 25% of the subtracted clones had sequences encoding thrombospondin type-1 repeat (TSR) domains, and were derived from 17 genes. We identified 11 TSR domain-containing genes among the top 36 genes that were the most frequently detected in our SSH library. Whole-mount in situ hybridization analyses confirmed that at least 13 out of 17 TSR domain-containing genes were expressed in the hypostome of Hydra oligactis. The prominent expression of TSR domain-containing genes suggests that these genes play significant roles in the hypostome of Hydra oligactis.The head region of  Hydra, a member of the phylum Cnidaria, has a simple diploblastic body plan with an oral-aboral axis including a head surrounded by tentacles, a body column, and a foot  [Hydra oligactis. Although several studies using SSH have been performed on Hydra (regenerating tissue [Hydra.The importance of the hypostome in the development and nervous system of on (SSH)  was usedg tissue , buddingg tissue , interstg tissue , immunosg tissue , 25, devg tissue , and speg tissue ), regionThe DNA sequences of the cDNA clones obtained by SSH were used to search for homology in the nucleotide, protein, and conserved domain databases. Of the clones obtained by our SSH procedure, 25% showed homology to genes containing the thrombospondin type-1 repeat (TSR) domain, many of which are considered to play roles in cell-cell and cell-extracellular matrix interactions , 29.Hydra oligactis was cultured in hydra culture medium  at 19°C and fed 6 days a week with hatched Artemia cysts. Experiments were performed after a 2-day starvation period.Total RNA was extracted from tissues using TRI® Reagent . In order to obtain head region tissues, we cut animals (approximately 200 polyps) just below the base of the tentacles, retained a piece of the tissue around the mouth, and then removed the tentacles. The body region tissue was a piece of the body just adjacent to the head region, the size of which was similar to the head region tissue.Tester cDNA was generated from 1 μg of total RNA from the head region tissues using the SMART cDNA synthesis kit  according to the manufacturer’s instructions. We used 1 μg of total RNA from the body region tissues for driver cDNA.E. coli clones were examined by colony hybridization with driver cDNA as the probe in order to remove transformants that contained cDNAs for the mRNA expressed in the body region. Clones showing no hybridization signal were individually selected and grown in medium. Plasmid isolation and the determination of cDNA sequences were conducted according to standard procedures.SSH was performed using the PCR-Select cDNA Subtraction Kit (Clontech) according to the manufacturer’s protocol. The resulting subtracted cDNAs were cloned into the pBluescript II KS+ vector . Following blue/white selection, white DH5α A homology search for the DNA sequences of 1414 clones was performed on the National Center for Biotechnology Information (NCBI) nucleotide database using the Basic Local Alignment Search Tool nucleotide program (BLASTN). The Conserved Domain Database (CDD) search (CD-search) at the NCBI site  was usedin situ hybridization experiments. Digoxigenin (dig)-labeled cRNA probes were generated by in vitro transcription using T3 or T7 RNA polymerase with PCR-amplified cDNA clones as templates. Hydras were relaxed with 2% urethane and fixed in 4% paraformaldehyde at 4°C overnight. After fixation, fixed animals were dehydrated and rehydrated in a graded series of methanol diluted with PBS containing 0.1% Tween 20 (PBT). After the proteinase K treatment and acetylation, the tissues were refixed in 4% paraformaldehyde for 30 min. Tissues were rinsed and then heated to 80°C in order to eliminate endogenous alkaline phosphatase (AP) activity. Hybridization was performed with dig-labeled cRNA probes (0.4μg /ml) in the hybridization solution (HS) containing 50% formamide, 5 x SSC, 0.1% CHAPS, 0.02% yeast tRNA, 1 x Denhardt’s solution, 0.01% heparin, and 0.1% Tween 20 at 55°C for approximately 60 hr. After hybridization, tissues were washed with HS at 55°C, 75% HS-25% 2 x SSC, 50% HS–50% 2 x SSC, and 25% HS–75% 2 x SSC for 10 min each, and then twice with 2 x SSC with 0.1% CHAPS for 30 min. The hybridized probes were detected by AP-conjugated anti-dig Fab fragments  diluted 1:2000 in maleate buffer  containing 20% sheep serum. Excess amounts of the antibody were removed by washing with maleate buffer. Hybridization signals were visualized with BCIP and NBT substrate solution  at 4°C in complete darkness for approximately 10 min ~ 2 hr. Stained tissues were dehydrated with ethanol and mounted in Euparal .The protocol originally described by Grens et al. (1996)  was usedHydra oligactis, we constructed a subtractive cDNA library containing head-specific genes by SSH. We determined the DNA sequences of 1414 subtracted clones, and then analyzed the sequences obtained using a local BLASTN program . Fourteen percent (195 clones) of the analyzed clones showed no similarity to any sequence in the NCBI nucleotide database (nr-nt). The remaining 1219 clones were grouped into 540 genes based on the results of the BLASTN search. In order to identify genes selectively expressed in the hypostome of Hydra magnipapillata, the genome of which has been sequenced and analyzed [Hydra magnipapillata Wnt1 (No.35) and Wnt7 (No.41). In addition to Wnts, we found a predicted cDNA sequence for Hydra magnipapillata brachyury-like protein , which is expressed in the hypostome, possibly under the control of the Wnt pathway [Most of these nucleotide database records are predicted cDNA sequences of analyzed . In thes pathway . FurtherWe next searched the CDD (CD-search) using the deduced amino acid sequences of the nucleotide database records. We found that 11 out of the top 36 records in HyTSR1 (AM182484) and TSR-containing protein 2 precursor , were predicted.Our BLASTN analyses revealed that 358 cDNA clones encoding TSR domain proteins were derived from 17 genes. We tentatively classified these genes into five groups according to the domain structures , even thHyTSR1, which contains 48 TSR domains, was previously reported to be exclusively expressed in the hypostome, specifically in granular mucous gland cells [Hydra oligactis. XM_004211723 contained at least 28 TSR domains, and was intensely expressed in the endoderm cells of the head region, most likely in non-epithelial cells, possibly gland mucous cells or neurons HyTSR1, wons Figs  and 3B. ons Figs . In contons Figs .Tsrp2, AM233513) gene, had previously been cloned and analyzed [Tsrp2 was composed of 11 TSR domains and a single galactose-binding lectin domain. The cDNA clones for Tsrp2 were the most abundant among the clones obtained by our SSH procedure , which had one or two TSR domains with the von Willebrand factor type A (vWA) domain in common . The vWAThe fifth group was classified by the existence of the zinc-dependent metalloprotease domain. Although it comprised three genes , the domain structure of XM_002166845 appeared to markedly differ from those of the other members, which share the cell-wall integrity and stress-response component 1 (WSC) domain and Meprin, A5 protein, and protein tyrosine phosphatase Mu (MAM) domain . XM_0021Hydra oligactis using SSH. Among these genes, we identified 17 that encoded TSR domain proteins. Of the cDNA clones obtained in the present study, 25.3% were derived from genes encoding TSR domain proteins. Our WISH analyses confirmed that eight of the 17 genes we identified were exclusively expressed in the hypostome, five were expressed in the other regions as well as in the hypostome, and three were expressed only in the tentacles  [Hydra vulgaris (HyBra2) [Hydra by WISH.As suggested by the results of WISH analyses, our SSH procedure may effectively concentrate cDNA clones for head-specific genes. Notably, multiple cDNA clones for a (Wnts)  and Hydr(HyBra2) . FurtherThe TSR domain, which consists of ~60 amino acid residues, was initially detected in the thrombospondin-1 (THBS1) protein in human platelets. THBS1 is a multifunctional matrix glycoprotein that plays roles in platelet aggregation, angiogenesis, and tumorigenesis . TSR domHydra, we identified 185 TSR domain proteins in the NCBI Protein database (http://www.ncbi.nlm.nih.gov/protein). Thus, the TSR domain proteins identified in the present study account for a small proportion (9.2%) of all Hydra TSR domain proteins.TSR domain proteins have been identified in various species from protozoa to vertebrates. In The TSR domain proteins in the present study were tentatively classified into five groups according to the domain combinations. In order to determine whether these domain combinations are evolutionary conserved, we used the Conserved Domain Architecture Retrieval Tool (CDART)  at the NHydra magnipapillata, the genome of which has been sequenced and analyzed. Thus, we cannot rule out the possibility that the bona fide domain structures of these TSR domain proteins are different from those shown here. Moreover, lack of the information about the genomic and cDNA sequences of Hydra oligactis did not allow us to directly determine the domain structures of the TSR domain proteins in Hydra oligactis. Further studies to determine full-length cDNA sequences for the subtracted clones encoding the TSR domain proteins are required.Most of the domain structures of TSR domain proteins shown here are predicted from the genomic and cDNA sequences of Hydra plays important roles in establishing and maintaining the head organizer [The TSR domain has been identified within a number of proteins including R-spondin, F-spondin, SCO-spondin, Semaphorin-5A (Sema5A), UNC-5, complement factors, the TRAP proteins of plasmodium, and the ADAMTS family , 29. Therganizer , 9, someHydra.Several TSR domain proteins, such as Sema5A, F-spondin, SCO-spondin, TSP-1 and UNC-5, are known to be involved in axonal guidance and growth in the vertebrate nervous system , 29. TheHydra may be in defense against pathogens. The most frequently cloned gene, Tsrp2 (AM233513), encodes a rhamnospondin (Rsp)-like protein [Hydractinia symbiolongicarpus (Phylum Cnidaria) and are predicted to encode multi-domain secretory proteins containing a rhamnose-binding lectin domain and eight TSR domains [Hydra, the Tsrp2 gene may act in a similar manner to Rsp.Another possible function of the TSR domain proteins in  protein . Rsp gen domains . The Rsp domains . In HydrS1 FigExpression patterns of the 5 genes in A group (A-E), the 4 genes in D group (F-I) and the 2 genes in E group (J and K). Accession numbers were labeled under each panel. The domain structures of the genes were shown on the top of photo panels for A group, and the right side for D and E group.(PDF)Click here for additional data file.S1 TableThis table is continued from (DOCX)Click here for additional data file.


Binding the C-terminus of heat shock protein 90 (Hsp 90) to the tetratricopeptide repeat (TPR) domain of cyclophilin 40 (Cyp40) allosterically changes the dynamics of the cyclophilin-active site and reduces peptidyl-prolyl isomerase (PPIase) activity. Cyclophilin 40 (Cyp40) comprises an N-terminal cyclophilin domain with peptidyl-prolyl isomerase (PPIase) activity and a C-terminal tetratricopeptide repeat (TPR) domain that binds to the C-terminal–EEVD sequence common to both heat shock protein 70 (Hsp70) and Hsp90. We show in the present study that binding of peptides containing the MEEVD motif reduces the PPIase activity by ∼30%. CD and fluorescence assays show that the TPR domain is less stable than the cyclophilin domain and is stabilized by peptide binding. Isothermal titration calorimetry (ITC) shows that the affinity for the–MEEVD peptide is temperature sensitive in the physiological temperature range. Results from these biophysical studies fit with the MD simulations of the apo and holo (peptide-bound) structures which show a significant reduction in root mean square (RMS) fluctuation in both TPR and cyclophilin domains when–MEEVD is bound. The MD simulations of the apo-protein also highlight strong anti-correlated motions between residues around the PPIase-active site and a band of residues running across four of the seven helices in the TPR domain. Peptide binding leads to a distortion in the shape of the active site and a significant reduction in these strongly anti-correlated motions, providing an explanation for the allosteric effect of ligand binding and loss of PPIase activity. Together the experimental and MD results suggest that on heat shock, dissociation of Cyp40 from complexes mediated by the TPR domain leads to an increased pool of free Cyp40 capable of acting as an isomerase/chaperone in conditions of cellular stress. There are 20 cyclophilin paralogues in the human genome which are found in diverse cellular compartments and form components of multiple cell signalling pathways and steroid receptor complexes  essentially as described for CypA by Wear et al. [Cyp40  was compared for the unliganded (apo) form and in the presence of 250 μM XXXXXMEEVD peptide (holo) at 10°C with the substrate Suc-A-A-P-F-p-NA (Sigma); 50 mM Hepes, pH 8; 100 mM sodium chloride; 1 mM DTT determined as previously described . using t−1; data pitch 0.1 nm; response time 1 s between 185 and 285 nm in a 0.1 cm path-length quartz cuvette between 10°C and 60°C at 10°C intervals (JASCO-810 spectrometer). The protein was exchanged into 10 mM sodium phosphate, pH 8; 150 mM sodium fluoride prior to analysis . Spectra were corrected by subtracting a buffer baseline recorded at each temperature, spectra were recorded in triplicate. Secondary structure was estimated using the Dichroweb CD secondary structure analysis server using the methods CONTIN, SELCON3 and CDSSTR and data set SP175; mean results are presented with S.E.M [The far UV CD spectrum of 0.75 μM Cyp40 was recorded at 20 nm minth S.E.M –34.Steady-state fluorescent emission was recorded for 1 μM Cyp40 in the apo form and after the addition of 250 μM SRMEEVD (Hsp90 peptide) between 15°C and 45°C at 5°C intervals in 50 mM Hepes, pH 8; 100 mM sodium chloride; 1 mM DTT . Tryptophan was selectively excited at 295 nm (5 nm band pass excitation and emission) and emission spectra recorded from 310 to 400 nm, 1 nm interval; integration time 1 s; scans were repeated in triplicate, mean spectra are presented. Protein scans were blanked by subtracting a spectrum of a similar solution without protein at each temperature.Thermal denaturation fluorescence (TDF) melting curves were recorded for 2 μM Cyp40 in the apo form and in the presence of 4 μM CsA and 250 μM SRMEEVD peptide alone or together; 50 mM Hepes, pH8; 100 mM sodium chloride; 1 mM DTT. Fluorescence intensity was measured in relative fluorescence units (RFU) using excitation/emission wavelengths of 485 nm/575 nm between 25°C and 70°C, interval 0.5°C, 30 s hold at each temperature as previously described  . Data weIsothermal titration calorimetry (ITC) experiments were carried out to determine the affinity of Cyp40 for Hsp90 peptide over the temperature range 10°C–30°C at 5°C intervals . Cyp40 was exchanged into the experimental buffer; 50 mM Hepes, pH 8; 100 mM sodium chloride; 1 mM DTT  immediately before the experiment. SRMEEVD (500 μM) was titrated into 7.7 μM Cyp40 in 15 × 2.5 μl aliquots. Data were analysed using non-linear regression in the MicroCal ORIGIN software package assuming a 1:1 binding model.All the MD simulations were carried out in explicit solvent (water) using NAMD 2.9 with CHARMM27 force field . All simKd values of ∼5 μM  and at the concentration used in this assay an estimated over 98% of Cyp40 would be bound [be bound . The simThe change in secondary structure as a function of increasing temperature was monitored using CD spectroscopy A. The pe239 forms an interface between the hairpin loop of helix 1 and 2 and helix 3; it makes hydrophobic contacts with Phe234, Gln237, Lys285 and Met286 and shows little solvent exposure. The crystal structure of bCyp40 has high B-factors at the hinge between at the opposite ends of helix 2 and 3 . Movement, perpendicular to the plane of the indole ring, at this hinge would have a direct effect on the environment of Trp239 and lead to exposure of the face of the indole group to solvent and the increased possibility of rotation about Cβ γ. Trp289 forms contacts between helices 3 and 4 and helix 5 and Trp317 in buried in the groove formed by helices 4, 5 and 6.Human Cyp40 is unusual in the cyclophilin family in that there are no tryptophan residues in the PPIase domain or linker region. There are three tryptophan residues in the TPR domain A. These Typically solvent-exposed tryptophan residues fluoresce at a longer wavelength and have a smaller quantum yield than buried residues ; thus flAddition of Hsp90 peptide causes an enhancement in fluorescence at all temperatures and is consistent with the tryptophan side chains being in a less solvent-exposed environment, though the red shift observed on melting is similar to the unliganded protein B and 4C.Tm=42°C (mid-point melting temperature) but starts to melt at 30°C (solid line). Addition of Hsp90 peptide -MEEVD delays onset of melting by ∼5°C, however Tm only increases by ∼1°C (dashed line). Comparison of the shapes of these two melting curves suggests that the TPR domain and PPIase domain do not start to melt at the same temperature. Addition of Hsp90 peptide stabilizes the TPR domain and influences the shape of the melting curve at temperatures below those where the cyclophilin domain is significantly destabilized. Addition of the tight-binding CsA ligand (PPIase domain) raises the Tm by 4°C upto 46°C. The shape of the melting curve for addition of the MEEVD peptide along with CsA again suggests a delay in melting of the TPR domain but this has little effect on the final Tm of the protein which remains at 46°C.The increase in fluorescence measured in the TDF assay results from a dye probe binding to exposed hydrophobic residues as the protein melts and unfolds. The melting curves shown in Kd for Cyp40 and the MEEVD peptide is ∼5 μM which rises to a value of ∼25 μM over which was measured at the highest attainable temperature of 30°C. The graph of changes in ΔH and ΔS with temperature shows a point of inflection between 25°C and 30°C suggesting, in line with data from CD and fluorescence, this corresponds with the onset of melting of the TPR  using the X-ray structure 1IHG.pdb as the starting model. A second MD simulation was carried out on holo-Cyp40 with the MEEVD peptide docked in the binding groove of the TPR domain of Cyp40. The X-ray structure of the E3 ligase CHIP (PDB code 2C2L) has a peptide with an MEEVD motif bound to a TPR domain that is structurally homologous to the TPR domain of Cyp40 . A model of holo Cyp40 (bound to the peptide MEEVD) was generated by overlaying the two structures. The RMS fit for 90 corresponding atoms from the two structures is 1.1 Å. MD simulations were calculated for 100 ns using this docked structure as the starting model for holo Cyp40.Ri)2> are related to the temperature factor (Bi) by:Average fluctuations  for each residue were calculated from the 100 ns simulations. The mean square displacement values for each atom < is found to be slightly more flexible with B values of 32.6 and 56.2 Å2 for the apo and holo forms respectively. It is clear that binding of the MEEVD peptide to the TPR domain has a significant effect on reducing fluctuations on both the TPR and the cyclophilin domains.The calculated B-factors determined from the holo and apo simulations are shown in 2 and for the TPR domain (residues 213–360), the average B-value is 12.5 Å2. When these values are converted into corresponding B-values at 310 K  the B values are 33.8 and 38.8 Å2 respectively. The average crystallographic B-factors measured at 100 K were taken from X-ray structure 1IHG. For the backbone atoms of the cyclophilin domain (residues 2–186), the average B-value is 10.9 ÅRi and ΔRj of ith and jth (α-carbon) atoms can be assessed by calculating the projection of the fluctuation of one atom on the other [calculated as the magnitude of the dot product (ΔRi · ΔRj) at every ps) and averaged over the full 100 ns trajectory .Here, the dot in the numerator denotes the scalar product and the angular brackets are time averages. With this normalization, Cij values are normalized between +1 and–1 and there are only six regions of the molecule that are involved in significant anti-correlated motions with Cij values <–0.75  is shown in An analysis of the 100 ns MD trajectories of the holo (MMEVD-bound) Cyp40 complex showed a very significant reduction in the anti-correlated motions between the cyclophilin and the TPR domains. A comparison of the correlation matrices of holo and apo structures (both contoured at 80 and His141 (and R75-L42) at one end of the cleft while closing the gap between Gly92 and Ala123 at the other end. The distance between the middle residues (Gln83 and His146) remains constant. These changes are consistent with a scissor-like motion  lie on either side of a binding cleft . The paie motion  that disPrevious studies on TPR domains have proposed that plasticity in the TPR domain enable it to adopt a range of conformations with different protein partners ,41. The The allosteric effect of peptide binding to reduce PPIase activity of Cyp40 Isomerases by ∼30% was unexpected, but can now be rationalized in terms of protein dynamics. The PPIase mechanism of CypA has been well studied and there is good evidence for a coupling between dynamics and substrate turn-over. NMR relaxation experiments have established that millisecond motions within the PPIase domain of CypA during catalysis occur with a frequency comparable to substrate turnover ,42–44. MKd is 2.9 μM . This suggests that the biological function may require ready dissociation of the Cyp40–Hsp90 complex. The strong temperature dependence of the interaction between Hsp90 and Cyp40 described in the present study is in the physiological temperature range. Upon heat shock, the affinity of Cyp40 for MEEVD is significantly reduced and equilibrium between bound and unbound Cyp40 is shifted towards the more enzymatically-active unbound form of the enzyme. A reasonable hypothesis is that enhanced isomerase activity may be able to rescue partially folded proteins from unfavourable interactions under conditions of heat or possibly other stress conditions.The measured dissociation constants between Cyp40 and Hsp90 are relatively weak; for full length Hsp90 at 20°C, the


The permeability barrier of nuclear pore complexes (NPCs) controls bulk nucleocytoplasmic exchange. It consists of nucleoporin domains rich in phenylalanine-glycine motifs (FG domains). As a bottom-up nanoscale model for the permeability barrier, we have used planar films produced with three different end-grafted FG domains, and quantitatively analyzed the binding of two different nuclear transport receptors (NTRs), NTF2 and Importin β, together with the concomitant film thickness changes. NTR binding caused only moderate changes in film thickness; the binding isotherms showed negative cooperativity and could all be mapped onto a single master curve. This universal NTR binding behavior – a key element for the transport selectivity of the NPC – was quantitatively reproduced by a physical model that treats FG domains as regular, flexible polymers, and NTRs as spherical colloids with a homogeneous surface, ignoring the detailed arrangement of interaction sites along FG domains and on the NTR surface.DOI:http://dx.doi.org/10.7554/eLife.14119.001 The cells of animals, plants and other eukaryotic organisms contain a compartment called the nucleus that contains most of the cell's genetic material. Proteins and other molecules – collectively known as cargos – can enter and exit the nucleus via tiny channels in the membrane that surrounds and protects it. Receptor proteins – called nuclear transport receptors – bind to potential cargos and shuttle them through the channels.This selective transport process relies on the nuclear transport receptors being attracted to flexible, spaghetti-like proteins that are anchored to the walls on the inside of each channel. However, because of their flexible and disordered nature, these so-called FG domains are difficult to study, and the details of the transport process are poorly understood.Zahn, Osmanović et al. decided to study how the FG domains behave and what happens when they interact with nuclear transport receptors by using ultrathin films made of just the FG domains. This is a good model system because the films are easier to study than the whole channels, but are likely to retain the essential properties of the real barrier formed in the nuclear envelope. Zahn, Osmanović et al. compared the binding of two nuclear transport receptors of different sizes, taken from humans and yeast, to FG domain films made from one of three different FG domains. The experiments showed that the different nuclear transport receptors bind to the different FG domains in very similar ways.Zahn, Osmanović et al. then used a computational model that essentially represented the FG domains as sticky spaghetti and the nuclear transport receptors as perfectly round meatballs. This sticky-spaghetti-with-meatballs model reproduced the experimental data, implying that the exact chemical make-up and structure of the molecules may not be critical for controlling the transport of cargo across the nuclear envelope. Future studies will test whether the generic physical features of nuclear transport receptors and FG domains can indeed explain how the cargo molecules pass through the nuclear envelope.DOI:http://dx.doi.org/10.7554/eLife.14119.002 In eukaryotic organisms, the nuclear envelope separates the nucleoplasm from the cytoplasm and encloses most of the genetic material in the cell. The ordered course of gene expression requires selective transport through this double membrane. This function is provided by nuclear pore complexes (NPCs), large membrane-spanning protein complexes that perforate the nuclear envelope . AlthougThe permeability barrier in the 30 to 50 nm diameter central transport channel of NPCs is formed by domains of NPC proteins (nucleoporins) that are rich in phenylalanine-glycine motifs  and that are grafted to the channel walls at a high density . The FG Because of the low degree of order in the FG domain meshwork and its spatial confinement within the NPC, it has been difficult to address these questions using traditional biochemical assays and structure determination methods. Alternatively, computational models can provide valuable insights into collective behavior of FG domains, but are affected by the size and complexity of the NPC, and in particular by the experimental uncertainty on protein distributions and interactions (see Homo sapiens (NTF2) and Importin β from Saccharomyces cerevisiae (Impβ), to plane-grafted FG domain films that each are generated from one of three different FG domains: the FG domain of Nsp1 from S. cerevisiae (that has FxFG and just FG motifs), a glycosylated FG domain of Nup98 from Xenopus tropicalis  and an artificial, regular repeat with exclusively FSFG motifs (reg-FSFG). The two transport receptors differ in size  and in the number and distribution of binding sites for FG domains. Two identical sites are located between the subunits of NTF2  computational modeling for a quantitative interpretation of the experimental results in terms of FG domain morphology and intermolecular interactions. Specifically, we produce films of end-grafted purified FG domains that are similar to the protein meshwork in NPCs, both in their thickness and in their FG motif density ; 2013. W of NTF2 , whereas of NTF2  and mole of NTF2 . Recent  of NTF2 , suggest of NTF2 , as well of NTF2  , and effective film thicknesses, d, respectively.Selected FG domains, i.e., Nsp1, Nup98-glyco and reg-FSFG, were purified  and anch2 , by varying the FG domain solution concentration and incubation time. This range covers and extends around the estimated grafting density in a yeast NPC that is thought to be 5.2 to 6.9 pmol/cm2 ; this estimate is based on the assumption of a cylindrical channel of 35–40 nm in diameter and 30–35 nm in length (2 for Nup98-glyco) over which the FG domain films maintain a homogeneous appearance, as previously verified by atomic force microscopy  remained largely unchanged with FG domain type and grafting density. This common shape prompted a more detailed analysis, including the use of phenomenological models , as expected from the low-concentration limit of a Langmuir isotherm. This indicates that – at low concentrations – individual NTR molecules bind to the FG domain film independently. In this concentration range, the ratio ΓNTR,eq / cNTR= PC × d was constant, with partition coefficients PC between 103 and 105 , with ΓNTR,max the maximum areal density of bound NTRs and K0.5 the concentration for half-maximum binding) failed to faithfully describe the data (dashed line). This is in line with earlier observations (solid line), i.e., K0.5 for the different FG domain types and grafting densities  versus normalized NTR concentrations (cNTR/K0.5), with ΓNTR,max and K0.5 determined from the Hill fit, to show that this reduces all data to a single master curve  . The Hilα+cNTRα) . This naensities , confirmill fits . The unier curve . The agrer curve  and thatcNTR = 10 μM, the films contained at least 10 and 50 FG motifs per bound NTF2 dimers and per bound Impβ, respectively  following NTR binding were generally moderate. At NTF2 solution concentrations up to 1 μM, the thickness remained virtually unchanged for Nup98-glyco and reg-FSFG, and decreased marginally (by up to 7%) for Nsp1. At higher concentrations, the thickness gradually increased, by between 5 and 35% at 10 μM compared to the pristine FG domain film, depending on the FG domain type and grafting density. For Impβ binding to Nsp1, there was a moderate and gradual thickness increase up to 25% at 10 μM. In all cases, the increase in film thickness was smaller than or comparable with the dimensions of the NTRs. These findings are in clear disagreement with the film collapse by more than 50%, reported by Lim et al. on nanoscale islands of FG domain assemblies  should match the effective thickness measured by QCM-D most accurately. Reassuringly, however, the results were only weakly influenced by the precise definition of film thickness that was used in the computational model, i.e., density thresholds of 1% or 10% instead of 5% typically altered the estimates for εpp by less than 10%. From the comparison of computation and experiment, it was clear that εpp for Nup98-glyco exceeded that of Nsp1, in good agreement with our previous study  shows computational data for parameter sets of εpp and εpc that best match the experimental results for the different FG domain and NTR types, where εpp was determined by the film thickness measurement in the absence of NTRs (see pc) over several orders of magnitude in bound NTR and in NTR concentration, the agreement with the experimental data is remarkably good. With εpp estimated from the thickness in the absence of NTR , the performance of the model was further validated via the film thickness as a function of NTR concentration in solution. There is good agreement between the experimental data and the computational results (bottom row). pc varying less than ~20% between the different FG domains and NTRs. Taken together, the measured binding of NTRs to FG domain films was accurately modeled by our simplified description of the relevant interactions in terms of the two key parameters εpp and εpc, where we treat all amino acids in the FG domain chains identically and the NTR surfaces as homogeneous.Next, we analyzed the binding isotherms. For a given cohesiveness parameter εsotherms  and 5B. NTRs see . Taking e of NTR  and εpc sotherms , top rowpc to a rough estimate for the binding energy of an NTR in the FG domain film, one may assume the NTR colloid to be surrounded by polymer beads at the maximum polymer packing in our calculations (~20%), yielding at most ~26 and ~53 polymer beads in contact with the colloidal surface, for the NTF2- and Impβ-mimicking colloids, respectively. Hence for NTF2, the corresponding binding energy is pc; and εpc between 0.3 and 0.4 kBT implies a binding energy of kBT per NTF2 homodimer. Similarly, εpc ~ 0.4 kBT implies a binding energy of kBT per Impβ. Hence our results would correspond to a few kBT binding energy per FG-binding site on the NTRs, in reasonable agreement with the millimolar affinities per FG motif observed with Impβ  for cNTR = 10 μM, i.e., in the physiological range of total NTR concentrations. They reveal that, given parameter settings that best match the experimental system  demonstrates that a relatively small change in the FG domain cohesiveness can have a dramatic effect on the NTR distribution. For example, with the NTF2•reg-FSFG interaction maintained at εpc = 0.4 kBT, an increase in inter-FG domain attraction by 33%, from εpp = 0.03 kBT to 0.04 kBT, essentially impaired NTF2 accumulation inside the film, and the NTF2 was enriched instead at the film-solution interface – that is to say, on the film rather than in the film. A similar trend is observed for all other combinations of NTRs and FG domains tested, albeit to a lesser extent for the least cohesive FG domains (Nsp1) with the smaller NTR (NTF2).pc by only 25% compared to the best match for the respective NTRs and FG domains, there is a reduction in colloid binding by more than one order of magnitude. Such a dramatic effect is remarkable: For comparison, a reduction of less than 25% would be expected for simple Langmuir-type one-to-one binding. Collectively, these results suggest that the native system is tuned to operate within a rather narrow parameter space in εpc and εpp that facilitates the strong enrichment of NTRs within the FG domain film, whereas similarly sized proteins with weaker binding strength are effectively excluded.The model also indicated that a minor reduction in the colloid•FG domain interaction strength drastically reduces binding , 3 and 5pp, and defines the compactness of the FG domain phase  and flexible peptide chains, as follows from an extrapolation of the computational data , and th (~25 nm ). This wfinement .On exposing the FG domain films to NTF2 and Impβ, we find a remarkable quantitative similarity  in the btop row). In our computational model, these effects are included, and the amount of NTR binding follows from collective low-affinity interactions – the balance between the overall cohesiveness of the FG domains (εpp) and the smeared-out, average NTR•FG domain interactions (εpc).Our analysis suggests that NTR binding is largely determined by generic effects such as overall binding energy, crowding and excluded-volume interactions, and by the entropic costs of NTR absorption that, in turn, reduce the conformational freedom of the grafted and flexible FG domains. Such effects may well be expected since, at physiological NTR concentrations, the absorption of NTRs roughly doubles the total density of the films , top rowtop rows). The binding energy is balanced by excluded-volume interactions and polymer cohesiveness and entropy, i.e., by generic physical effects. At equilibrium, this balance is such that, in spite of the high binding energy between NTRs and FG domains, NTRs can exchange between the NPC and the nucleus/cytoplasm at minimal cost, thus facilitating cargo uptake and release.In this context, it is important to emphasize that enrichment of NTRs in an FG domain phase – in our model films but also in the NPC – vastly differs from NTR•FG motif binding under dilute conditions. This can be illustrated, for example, by comparing the enrichment of Nsp1 films for NTF2 and Impβ: the higher total Impβ•Nsp1 binding energy (integrated over the Impβ surface) does not translate into a higher enrichment than for NTF2, even in the limit of low NTR concentrations , which results in NTRs preferentially binding on top of (and not into) the FG domain assembly  system. For the entire NPC, such rigorous testing of computational models is presently complicated by experimental uncertainties in the locations of different FG domains inside the NPC, by the difficulties in accurately validating interaction parameters for the ensemble of FG domains in the NPC, and by the predicted bistable behavior of polymers grafted in nanopore geometries  changes in interaction parameters can result in qualitatively different behaviors.10 tag; Nup98-glyco (58.3 kDa), amino acids 1 to 485 of Nup98 with ~30 O-GlcNAc modified S and T residues per chain , an artificially designed regular FSFG domain with 16 repetitions of the sequence STPAFSFGASNNNSTNNGT and an N-terminal His14-TEV tag; reg-SSSG (32.2 kDa), a polypeptide identical to reg-FSFG but with phenylalanines replaced by serines. All FG domains were purified as described earlier ; and Impβ from S. cerevisiae (95.2 kDa). NTF2, the W7A mutant of NTF2 and Impβ were expressed and purified as previously described (We used the following FG domains: Nsp1 (64.1 kDa), amino acids 2 to 601 of Nsp1 with a C-terminal Hiser chain  and an N earlier  and storescribed  and storescribed .The formation of FG domain films and the binding of NTRs to FG domain films were simultaneously followed by SE and QCM-D on the same surface and in a liquid environment  Richter. To thisif and ΔiD) were acquired for six overtones  with a time resolution better than 1 s. Prior to each measurement, the walls of the cuvette were passivated by incubation with a buffer solution containing 10 mg/mL of bovine serum albumin  for 30 min. The cuvette was rinsed with buffer, ultrapure water and blow-dried with nitrogen. For the measurement, the cuvette was filled with ~2 mL working buffer, continuously stirred and held at a temperature of 23°C. Samples were injected directly into the buffer-filled cuvette at desired concentrations. To remove samples, the cuvette content was diluted by repeated addition of excess buffer and removal of excess liquid until the concentration of soluble sample, estimated from the dilution rate, was below 10 ng/mL.In the SE measurements, ellipsometric angles (Δ and ψ) were acquired over a wavelength range of λ = 380 to 1000 nm at 70° angle of incidence and about 5 s time resolution. In the QCM-D measurements, frequency and dissipation shifts    into the buffer-filled SE/QCM-D fluid cell. SLB formation was monitored by SE and QCM-D and only SLBs of good quality  were used for further measurements.For measurements with NTF2 and Nsp1, we used His tag capturing QCM-D sensors . These sensors are coated with a thin layer of poly(ethylene glycol) (PEG) that exposes Cus (SLBs) . For meaeviously ; 2013. Bris-NTA) . SLBs weWe formed FG domain films by injecting the FG domains directly into the SE/QCM-D cuvette equipped with a functionalized sensor. FG domain film formation was monitored, and FG domain concentration (up to 2.9 µM) and incubation time (up to 90 min) modulated to obtain FG domain films of desired grafting density . NTRs weG’) and a loss modulus (G”) that depend on frequency in the form of a power law. The film density was fixed based on the areal mass density  and partial specific volume of proteins, and the density of water. The semi-infinite bulk solution was assumed to be a Newtonian fluid with the density and viscosity of water.We determined the thickness of FG domain films by fitting the QCM-D data to a continuum viscoelastic model, as described in detail previously . BrieflyThe interface between the FG domain film and the bulk solution is not ideally sharp, and the definition of film thickness thus not trivial. The viscoelastic model neglects the fuzzy interface and assumes a homogeneous film. However, because the acoustic contrast in polymer materials is generally high , the ac. The spedSE and a wavelength-dependent refractive index n(λ). Protein surface densities Γ where obtained through de Fejter’s equation (dSEΔn / (MW × dn/dc), where Δn is the difference in refractive index between the FG domain film and the buffer solution (assumed to be wavelength independent) and MW the protein molecular mass. We used dn/dc = 0.18 cm3/g as refractive index increment for our protein films , to quantify protein surface densities. The fitting methods for QSX335 and QSX340 sensor substrates are described in detail in refs.  and Eis, respectequation , i.e., Γin films . The resthe film .2, i.e., 3.3 polymers per 100 nm2, to approximately match the FG domain grafting density in the NPC and the majority of the experimental data sets presented here. It was kept identical for the different FG domains to facilitate comparison and cross-validation of the computational results. For significantly lower grafting densities, lateral heterogeneity became increasingly important – as also observed experimentally , thus reducing the system to two dimensions for computational efficiency. For consistency with our previously developed algorithms, zero-density boundary conditions were imposed for both polymers and colloids at the side walls of the cylinder. At the cylinder top and bottom, zero-density boundary conditions were used for the polymers and periodic boundary conditions for the colloids.r weighting factor in cylindrical coordinates). The results were verified for robustness against the exact threshold setting , and found to typically correspond to the thickness that includes Local  film thicknesses were defined via iso-density profiles corresponding to a defined fraction  of the maximal density within the film. For comparison to the experimental data, these thicknesses were averaged across the cylinder  of the cylinder. To test for boundary-related artifacts in the computational results, areal colloid densities were also computed as averages across the central part (of 50 nm diameter) of the cylinder. Within the range of parameters relevant for comparison with experiment, the results agreed to within less than a factor of two.Polymer and colloid density profiles were determined from respective packing fractions versus the axis that is normal to the grafting surface. Analysis of a profile was performed on the average of profiles for different radial positions in the cylinder. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Vivek Malhotra as the Senior Editor.Thank you for submitting your article "A minimal physical model describing the interaction of nuclear transport receptors with FG nucleoporin domain assemblies" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.We would caution using the current title, since the theory on which DFT is based is far from trivial, and just because they can model based on this "complex model" with very few parameters a few experimental observables (binding isotherms etc.), I would not be certain that this is the "minimalistic" model. Other computational models have also been able to capture certain experimental results .Summary:eLife. However, there are several issues that remain unanswered that will have to be addressed to make the paper suitable for publication. The essential revisions required follow.The work is of high technical quality and it is interesting that the computational model, which in their previous work was used to describe AFM data on physiological NPCs, can capture experiments with different NTR-FG systems. Overall, the data in Essential revisions:1) The Hill analysis yields Hill coefficients that are less than unity and this is thought to imply negative cooperativity. It is worth emphasizing that a Hill coefficient that is less than unity is necessary but not sufficient to invoke negative cooperativity. Further, the modeling does not seem to provide a rationalization for the proposed negative cooperativity.2) As for the model itself, it is unclear if the beads are intended to represent FG motifs or collections of residues that incorporate multiple FG motifs. This is relevant because the question is if the energy scale for polymer-polymer interactions includes only FG motifs. If yes, then what is the basis for ignoring interactions that do not involve FG motifs?3) The choice of the polymer model needs further rationalization. In particular, is it possible to obtain equivalent fits with a polymer model that belongs to a completely different universality class such as a self-avoiding random walk for example? A suitable set of reference models needs to be constructed, as should a suitable random prior to understand why one should put faith in the model.4) The manuscript ends rather abruptly as far as pushing the model is concerned. Although the matching of experimental data is nice, the predictive insights that emerge from the model remain unclear. This is important because one would like to know specific predictions in order to design ways to test the merits of the model.5) Muthukumar has developed an elegant family of theories for macromolecular translocation and affords several analytical routes to make specific predictions through analysis of the simulation results. We would caution using the current title, since the theory on which DFT is based is far from trivial, and just because they can model based on this "complex model" with very few parameters a few experimental observables (binding isotherms etc.), I would not be certain that this is the "minimalistic" model. Other computational models have also been able to capture certain experimental results . Our model is minimal in the way it approaches nucleoporins as homogeneous polymers and transport receptors as spherical colloids. We consider this minimal in the scientific context relevant for the NPC. However, the notion of “minimal” or “minimalistic” clearly depends on the reader’s perspective. We have therefore followed the reviewers’ recommendation and have removed “minimal” from the title, as well as at several occurrences in the manuscript.Moreover, we feel that our model does considerably more than describing “a few experimental observables”. The model describes quantitatively the overall binding isotherms for different nucleoporins and nuclear transport receptors, together with also accounting for the overall resulting change in morphology  of precisely defined and characterised FG domain assemblies. Moreover, it does so over several orders of magnitude of receptor concentration. We believe that this represents a considerable advance towards understanding the transport selectivity of the nuclear pore complex (NPC).In addition, we have made more explicit that other computational models  have also been able to capture certain experimental results, in the section “Towards a minimal physical model for the NPC”.Essential revisions:1) The Hill analysis yields Hill coefficients that are less than unity and this is thought to imply negative cooperativity. It is worth emphasizing that a Hill coefficient that is less than unity is necessary but not sufficient to invoke negative cooperativity. Further, the modeling does not seem to provide a rationalization for the proposed negative cooperativity. We thank the reviewers for this question regarding the negative cooperativity and its implications for the interactions involved in transport through NPCs. We followed the standard textbook definitions when describing the binding as negatively cooperative when the Hill coefficients are less than unity. For example, Voet D & Voet JG, Biochemistry, 4th ed. , specifies: “The quantity n, the Hill constant, increases with the degree of cooperativity of a reaction and thereby provides a convenient, although simplistic, characterization of a ligand-binding reaction. […] If n < 1, the reaction is termed negatively cooperative”. Because of the rather simplistic nature of this definition, in the text we explicitly describe the meaning of “negatively cooperative” in the context of our work and say “that the free energy of binding per NTR decreases with an increasing amount of bound NTR in the film, and that this decrease is more pronounced than would be expected for the one-to-one binding of NTRs to independent and uncorrelated binding sites ”. We included this more precise definition in the section “Universal aspects and negative cooperativity in NTR binding to FG domains”.We have also clarified the rationalisation of the negative cooperativity in the same section , where we discuss the generic nature of the negative cooperativity and attribute it to generic polymer behaviour of the FG domains. Specifically, we infer that, beside the binding energy (that is included in simple Langmuir behaviour), NTR binding is affected by “crowding and excluded-volume interactions, and by the entropic costs of NTR absorption that, in turn, reduce the conformational freedom of the grafted and flexible FG domains”.2) As for the model itself, it is unclear if the beads are intended to represent FG motifs or collections of residues that incorporate multiple FG motifs. This is relevant because the question is if the energy scale for polymer-polymer interactions includes only FG motifs. If yes, then what is the basis for ignoring interactions that do not involve FG motifs? We are grateful for this suggestion and have now expanded the text in the section “Computational approach and cohesiveness of the FG domain films” to clarify the nature of the beads.Specifically, the beads are not intended to represent specific FG motifs nor necessarily collections of residues that incorporate multiple FG motifs.pp for the pair-potential between two beads, in addition to the excluded-volume interactions, which are treated separately . The parameter εpp thus effectively includes interactions between FG motifs but also interactions with other parts of the FG domain.The model considers the polymers as homogeneous, ignoring the exact distribution of FG motifs in the FG domains. Polymer-polymer interactions are thus considered smeared out along the polymer. They are included in one effective, overall attractive phenomenological interaction parameter ε3) The choice of the polymer model needs further rationalization. In particular, is it possible to obtain equivalent fits with a polymer model that belongs to a completely different universality class such as a self-avoiding random walk for example? A suitable set of reference models needs to be constructed, as should a suitable random prior to understand why one should put faith in the model. The choice of the polymer model used in this manuscript is based on our earlier work (pp = 0 (just hard-sphere interactions). However, this model does not provide a good match to the experimental data as can be inferred from the revised section “FG domain films are significantly compacted compared to non-cohesive polymer brushes”.4) The manuscript ends rather abruptly as far as pushing the model is concerned. Although the matching of experimental data is nice, the predictive insights that emerge from the model remain unclear. This is important because one would like to know specific predictions in order to design ways to test the merits of the model. We are grateful for this suggestion and have expanded the text to outline more fully the implications of the modelling for nucleocytoplasmic transport. In our view, one of the most exciting perspectives is to predict and to test transport selectivity through the NPC, along the lines we discuss in the final paragraph of the section “Towards a minimal physical model for the NPC”. We have expanded our original discussion here and make more explicit our prediction that this transport selectivity may be modelled and understood by taking into account the flexible nature and cohesion of FG domains and crowding alone, so that one can circumvent the complications inherent in including precise details of much of the underlying chemistry and sub-molecular structure.Currently a rigorous testing of this prediction is complicated, until more data becomes available, by experimental uncertainties in, for example, the locations of different FG domains inside the NPC, the difficulties in accurately validating interaction parameters for the ensemble of FG domains in the NPC, together with the predicted bistable behaviour of polymers grafted in nanopore geometries  Muthukumar has developed an elegant family of theories for macromolecular translocation and affords several analytical routes to make specific predictions through analysis of the simulation results.Muthukumar’s pioneering work  refers to the uptake and translocation of flexible polymers  in solid-state and α-haemolysin nanopores and, although it represents a crucial advance in this area, it refers to a quite different type of transport to that seen with NPCs. Thus, transport through NPCs generally concerns the translocation of folded, globular proteins, and so does the uptake of transport receptors in our FG domain assemblies and consequently is on a different scale to the passage of flexible polymers through very narrow pores. Consequently we feel that Muthukumar’s work is not strictly relevant to the system that is being investigated in our manuscript, but would be delighted to follow more specific guidance by the reviewers on this topic.