Archives

  • 2018-07
  • 2019-08
  • 2019-09
  • 2019-10
  • 2019-11
  • 2019-12
  • 2020-01
  • 2020-02
  • 2020-03
  • 2020-04
  • 2020-05
  • 2020-06
  • 2020-07
  • 2020-08
  • 2020-09
  • 2020-10
  • 2020-11
  • 2020-12
  • 2021-01
  • 2021-02
  • 2021-03
  • 2021-04
  • 2021-05
  • Vancomycin hydrochloride This review focuses on the atomic b

    2021-04-30

    This review focuses on the atomic basis of CRM1-mediated nuclear export. There are now 27 crystal structures of CRM1 in the Protein Data Bank (PDB) (www.rcsb.org) [64]. Collectively, this large body of work explains various aspects of CRM1 function. Here we summarize the structure–function studies that explain CRM1-cargo recognition, release and inhibition.
    CRM1 and the Ran cycle CRM1-mediated nuclear export requires the action of the small GTPase Ran. A RanGTP-RanGDP gradient is maintained across the nuclear envelope through compartmentalization of Ran regulators. Ran is predominantly in the GTP state in the nucleus because of efficient nucleotide exchange by its guanidine nucleotide exchange factor RCC1, which is tethered to Vancomycin hydrochloride through interactions with histones H2A and H2B (Fig. 1) [65], [66], [67]. In contrast, cytoplasmic Ran is predominantly in the GDP state because the GTPase-activating protein RanGAP1 that catalyzes hydrolysis of RanGTP to RanGDP is located in the cytoplasm or at the cytoplasmic fibrils of the NPC (Fig. 1) [68], [69], [70]. Binary interactions of CRM1 with either RanGTP or export cargos are very weak, but CRM1 binds both ligands cooperatively to form the CRM1-cargo-RanGTP export complex (Fig. 1) [71], [72]. The loading process is further facilitated by the Ran binding protein RanBP3 through a still unknown mechanism [73], [74]. The CRM1-cargo-RanGTP export complex binds various nucleoporins in the NPC including Nup98 on the nucleoplasmic side, Nup214-Nup88 on the cytoplasmic side of the NPC and various FG repeat-containing nucleoporins [21], [26], [27], [28], [29], [30]. In the cytoplasm, the CRM1-cargo-RanGTP complex encounters RanBP1 and Nup358 (also known as RanBP2), which facilitate cargo release and interactions of RanGTP with RanGAP1 (Fig. 1) [75], [76], [77], [78], [79]. Finally, RanGAP1 catalyzes hydrolysis of RanGTP to RanGDP to end the nuclear export process and CRM1 is then recycled back to the nucleus for additional rounds of export (Fig. 1) [68].
    A summary of CRM1 structures Many crystal structures of CRM1 have been published in the last five years. 27 CRM1 structures are now available in the PDB [64]. CRM1 from several organisms (human, mouse, fungi Chaetomium thermophilum and Saccharomyces cerevisiae) were used in these studies but CRM1 architecture appears conserved across these homologs. Structures of unliganded CRM1, a CRM1-cargo intermediate, CRM1-cargo-Ran export complexes, the post-release yeast CRM1-Ran-RanBP1 complex and several CRM1-inhibitor complexes, all provide snapshots of CRM1 in many steps of the nuclear export cycle and inform on various aspects of CRM1 function. We now have insights into the chemical basis of why unliganded CRM1 is inactive, how CRM1 recognizes NESs in the nucleus and releases them in the cytoplasm, and how various small molecule inhibitors block CRM1-mediated nuclear export.
    Conclusion and perspectives The first crystal structure of a small fragment of CRM1 was solved in 2004, the first structures of full length CRM were reported in 2009 and structural knowledge of CRM1-mediated nuclear export has grown tremendously since then [72]. It is now clear that CRM1 is a ring-shaped karyopherin, which uses its outer convex surface to bind protein cargos. CRM1-NES recognition is achieved through anchoring of key hydrophobic residues of the NES into the hydrophobic NES binding groove on the CRM1 convex surface, and variation in NES sequences is accommodated through variation of secondary structures of the bound NESs. The CRM1 protein adopts multiple conformations, including an inactive unliganded state with a closed NES groove and an active state with the groove open and NES-bound. These two conformations are stabilized by two different conformations of the CRM1 H9 loop, which are in turn controlled by different conformations of the CRM1 C-terminal extension (the H21B helix and C-terminal tail). Similarly, RanBP1, which accelerates cargo release when bound to RanGTP and CRM1, also stabilizes the inactive groove-closed state of CRM1 through stabilization of the inactive configuration of the CRM1 H9 loop. Finally, thus far, inhibition of CRM1 nuclear export by small molecule compounds has been achieved through direct conjugation of the inhibitors to the reactive cysteine residue in the NES groove of CRM1. However, the mechanisms of inhibition are not the same for all CRM1 inhibitors. LMB is first covalently conjugated to CRM1 and then hydrolyzed by the protein. LMB Vancomycin hydrochloride hydrolysis stabilizes the covalent CRM1-LMB interaction causing the inhibitor to be irreversibly bound to CRM1. The persistent shutdown of CRM1-mediated nuclear export is expected to be highly deleterious. KPT-SINEs, on the other hand, are not hydrolyzed by CRM1 and conjugate to CRM1 in a slowly reversible manner, which is potentially favorable for tolerance of the drugs.