Elucidating the crystal structure of the retinoblastoma amino-terminal domain (RBN) has provided insight into the way it contributes to RB function and tumor suppression.
The retinoblastoma tumour-suppressor protein (RB) has a key role in the regulation of cell proliferation, and loss of signalling through RB is common in tumour development. The central pocket and carboxy-terminal domains are involved in interactions that are important in the cell cycle but, although mutations in the amino-terminal domain (RBN) are found in familial retinoblastoma, the function of this region is not understood. Hassler et al. have now determined the crystal structure of RBN and suggest mechanisms by which RBN contributes to tumour suppression.
Crystallization of RBN revealed a globular structure consisting of a pair of tandem cyclin-like folds (lobe A and B) linked by a long helix (see figure). This architecture replicates the structure of the central pocket, which suggests evolution through duplication from an ancestral pocket-like fold. The authors identified four candidate functional surfaces within RBN that could be involved in protein–protein interactions: two clusters of conserved, surface-exposed residues; the hydrophobic projection region that appends to lobe B of the cyclin fold; and a 'cyclin wedge' in lobe B. In other proteins the cyclin wedge mediates high-affinity ligand interactions and the LxCxE motif that is common to many RB ligands docks in the RB pocket. Hassler et al. found that several of the mutations in RBN that are seen in retinoblastoma map to the cyclin wedge and the projection, implying that these surfaces are important in tumour suppression.
So, what protein interactions might involve the RBN domain? Some interactions that had previously been reported, such as with p84N5 and glutamate receptor-interacting protein 1, were confirmed. In addition, Hassler et al. found that RBN facilitates a homotypic interaction involving the RB pocket and a heterotypic interaction involving the E1A-like inhibitor of differentiation (EID1). EID1 is known to inhibit RB function and has previously been shown to interact with the RB pocket through its LxCxE motif. Targeted mutagenesis studies showed that EID1 interacts with RBN via an arginine-rich linker, which lies close to, and might form part of, the RBN cyclin wedge. This arginine-rich linker includes two cyclin-dependent kinase phosphorylation sites, and phosphorylation at these residues abrogated interaction with EID1. Peptide-array analysis confirmed that EID1 interacts with RBN through the arginine-rich linker and with the RB pocket, probably through the LxCxE-binding site. When EID1 binds the RB pocket, the pocket is displaced from RBN. The authors suggest that EID1 binding facilitates changing a closed conformation of RB, in which RBN interacts directly with the RB pocket, to an open conformation. This could allow the assembly of specific RB complexes and/or cause functional inactivation.
Elucidating the structure of RBN has provided insight into the mechanisms by which RBN contributes to RB function and tumour suppression.
Ezzie Hutchinson
References
Hassler, M. et al. Crystal structure of the retinoblastoma protein N domain provides insight into tumor suppression, ligand interaction, and holoprotein architecture. Mol. Cell28, 371–385 (2007) | Article | PubMed |
