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Structure of RCC1 chromatin factor bound to the nucleosome core particle.
Makde RD
,
England JR
,
Yennawar HP
,
Tan S
.
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The small GTPase Ran enzyme regulates critical eukaryotic cellular functions including nuclear transport and mitosis through the creation of a RanGTP gradient around the chromosomes. This concentration gradient is created by the chromatin-bound RCC1 (regulator of chromosome condensation) protein, which recruits Ran to nucleosomes and activates Ran's nucleotide exchange activity. Although RCC1 has been shown to bind directly with the nucleosome, the molecular details of this interaction were not known. Here we determine the crystal structure of a complex of Drosophila RCC1 and the nucleosome core particle at 2.9 Å resolution, providing an atomic view of how a chromatin protein interacts with the histone and DNA components of the nucleosome. Our structure also suggests that the Widom 601 DNA positioning sequence present in the nucleosomes forms a 145-base-pair nucleosome core particle, not the expected canonical 147-base-pair particle.
Figure 2. Interactions of RCC1, LANA peptide and H4 peptide with the nucleosome histone dimer acidic patch. (a) RCC1-histone interactions in ribbon representation. RCC1 employs side chains in its switchback loop to bind to the histone dimer. Hydrogen bonds are depicted as yellow dotted lines. (b) RCC1 recognizes acidic histone H2A/H2B dimer surface. APBS49-calculated electrostatics (â3 to +20 kT) were mapped onto histone surfaces using same view as in (a). (c) LANA-histone interactions in LANA-nucleosome crystal structure (PDB id 1ZLA). Key side chains of the LANA peptide (blue) are shown. (d) Histone H4-histone crystal contacts in nucleosome core particle (NCP) crystal structure (PDB id 1KX5).
Figure 3. RCC1-nucleosomal DNA interactions. RCC1âs N-terminal tail approaches the DNA minor groove at superhelical location (SHL) 6.5, while its DNA-binding and adjacent loop binds across the major groove at SHL 6. Hydrogen bonds (<3.2 Ã ) are shown as yellow dotted lines, while potential hydrogen bonds are shown in blue.
Figure 4. The Widom 601 sequence forms a 145 bp nucleosome core particle. (a) Superposition of 601 nucleosome core particle DNA (in blue) and human αsatellite nucleosome core particle DNA (peach) in simplified cartoon representation. The major differences between the two DNA structures at SHL +5 and â5 are highlighted in red and green respectively. (b) and (c) Superpositions of the two DNA structures at SHL +5 and â5 respectively. Numbers shown are the base positions from the nucleosome dyad. (d) Alignment of DNA present in nucleosome core particle crystal structures shows 5 bp major and minor grooves blocks. The 5 or 6 bp major groove blocks at SHL ±2 and ±5 accommodates differences in the DNA structures (red arrows). DNA dyad indicated by black arrow. Human α-satellite sequence alignments based on Fig.1 of Ong et al13.
Figure 5. Model for Ran-RCC1-nucleosome core particle complex assuming no conformational changes. Ran (light blue) was modeled by aligning the RCC1 component of the Ran-RCC1 complex crystal structure (PDB id 1I2M) with one of the RCC1 subunits in the RCC1-nucleosome core particle structure. The flexible C-terminus of Ran is shown in white. The GDP nucleotide (magenta) was modeled using the nucleotide present in the RanGDP structure (PDB id 3GJ0). If Ran interacts with histones in the Ran-RCC1-nucleosome complex, a conformational change in either RCC1 or Ran presumably must occur.
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