CRM1 is an export receptor for leucine-rich nuclear export signals. Support Center Support Center. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. We apologize for the inadvertent omission of an explanation of the error used in the original Figure 4. Selenomethionine hRio2 NES peptide was also generated and anomalous data were collected to confirm correct placement of its methionine, and unambiguously confirm the direction of the NES polypeptide chain Figure 1—figure supplement 1. Prior to use, Ran nucleotides were exchanged into Ran by adding nucleotide and MgCl 2 to final concentrations of 1 mM and 5 mM, respectively, and then incubating at room temperature for 30 min.
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We revised the fitting procedure as described in the Methods and recalculated the correlation coefficients presented in Figure 2-figure supplement 2. Annual Review of Biochemistry. D Quantification of C and the cem1 stoichiometry of the complex.
Are their densities sitting on top of large and equal constant densities? Each CRM1 cargo contains multiple sequences that match NES consensus patterns but most of these sequences are not functional export signals.
A step-wise increase in imidazole concentration eluted proteins from the resin.
The streptavidin-labeled RRE sample adds 60 kD of mass, also outside the envelope, and the cryo-EM averages bring out features of the RNA not visible by negative stain. CRM1 is also a promising cancer drug target, and a small molecule inhibitor of CRM1 named Selinexor is currently in more than 40 clinical trials for a variety of cancers clinicaltrials.
This fitting suggested that the species-specific residues may form a protein—protein interface between the Crm1 monomers, and indeed, we discovered that this d interface formed a crystal contact between human Crm1 monomers in the crystal structure bound to Snurportin1 Dong et al. A solution to limited genomic capacity: There is some concern about complex dissociation, and indeed we observe a minor population of free Crm1 monomers.
Other issues for the authors’ consideration: Data were indexed, integrated, and scaled using HKL Minor et al. Copyright and License information Disclaimer.
Structural determinants of nuclear export signal orientation in binding to exportin CRM1
Prompted by this comment, we have re-evaluated fitting the Hill model to the binding data and used an alternative binding model in the text and Figure 2d. We, therefore, only interpreted the volume with no symmetry applied. The authors should address the following questions and comments in the form of revision. Proteins were expressed in E. Journal of Structural Biology. Nonetheless, the accompanying crystal structure paper provides some structural rationale to reconcile the stoichiometry of a Rev hexamer docking to a Crm1 dimer.
Support Center Support Center. Structure of importin-beta bound to the IBB domain of importin-alpha.
For each class average of untilted images, we calculated a random conical tilt RCT reconstruction from tilted particles with angular parameters from the in plane rotation of class averages and from the fx angle determined during particle picking.
The selective pressures that fixed the dimer interface in the simian primate lineage are unknown, but its evolution has certainly provided an advantage for HIV replication. Fitted binding curves are overlaid onto data points with error bars representing the mean and standard deviation of triplicate titrations.
The required shift of the backbone to put the plus and minus side chains in ex same position is more likely for linear motifs than for extensive interfaces between two folded proteins, since the latter are constrained by many additional crj1 outside of the helix-binding groove.
Copyright and License information Disclaimer. The Rev protein then binds to a group of host proteins called the Crm1 export complex to send the viral RNA molecules to the cytoplasm. Seminars in Cancer Biology.
The export receptor Crm1 forms a dimer to promote nuclear export of HIV RNA
The washed RNA pellet was resuspended in buffer A. This conclusion came from comparing the location of extra densities from tagged versions of Rev or the RRE, but given the caveats above, we have eliminated the latter half of the sentence. CRM1 recognizes its protein cargos through 8—15 residue long nuclear export signals NESs in the proteins la Cour et al. Reviewers have the opportunity to discuss the decision before the letter is sent see review process. Negative-stain electron microscopy 2.
We searched for sequences that match class 1-R dc patterns in the Dbase data set, which compiled NES-containing CRM1 cargos from previously published literature Xu et al.
Current Opinion in Structural Biology.