Structural and Biochmical Studies of Nuclear Import Factor Karyopherin Beta-2
Cansizoglu, Ahmet Ertugrul
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Karyopherinbeta (Kapbeta ) proteins recognize nuclear localization and export signals (NLS/NES) and mediate the transport of macromolecules across the nuclear envelope, a process regulated by Ran GTPase through its nucleotide cycle. Diversity and few number of available signal sequences recognized by Kapbeta s have prevented prediction of new Kapbeta substrates. The structure of Kapbeta 2 (also known as Transportin) in complex with its most well studied substrate, the NLS of hnRNP A1 (M9NLS), elucidates the mechanism of substrate release by Ran GTPase. Analyses of the structure in conjunction with available NLS sequences reveal three general rules for NLS recognition by Kapbeta 2: NLSs are structurally disordered in substrates, they have overall basic character, and they carry an Nterminal hydrophobic or basic sequence motif followed by a C-terminal R/H/KX(2-5)PY consensus sequence. These rules successfully identify NLSs in seven previously known Kapbeta 2 substrates. These studies define and validate a new type of NLS, we term as PY-NLSs that could not be predicted by primary sequence analysis alone. After solving the structure of the basic-PY NLS (M9NLS) complex structure, I solved the structure of a basic-PY NLS substrate (hnRNP-M NLS) in complex with Kapbeta 2. Kapbeta 2 complexes carrying hydrophobic (M9NLS) and basic PY-NLSs converge in structure only at previously identified consensus motifs, which explains the ligand diversity. Using complementary biochemical experiments, we designed a chimeric Kapbeta 2 substrate, M9M, which acts as a pathway specific nuclear import inhibitor. To complete the Kapbeta 2 import cycle picture, I solved the structure of Kapbeta 2 in its unliganded form. The structure of the unliganded Kapbeta 2 together with structures of hydrophobic, basic PY-NLSs and Ran complexes provides understanding of conformational heterogeneity that accompanies ligand binding. The Kapbeta 2 superhelix is divided into three major segments. Two of them (HEAT repeats 9- 13 and 14-18), which constitute the substrate binding site, are rigid elements that rotate relative to each other about a flexible hinge. The third (HEAT repeats 1-8), which constitute the Ran binding site, exhibits conformational changes throughout its length. An analogous segmental architecture is also observed in Importinbeta suggesting that it is functionally significant and may be conserved in other import Karyopherins.