Clefts, grooves, and (small) pockets: the structure of the retinoblastoma tumor suppressor in complex with its cellular target E2F unveiled.

T umor suppressors are functional antagonists of oncogenes and their loss of function importantly contributes to human carcinogenesis. The sequence of the first human tumor suppressor, the retinoblastoma susceptibility gene product pRb, was published in 1986 (1). In this issue of PNAS, Xiao et al. (2) report the crystal structure of a pRb fragment in complex with a peptide derived from the E2F transcription factor, a key cellular target of pRb. An article by Lee et al. (3) recently published elsewhere reports similar findings. Thus, in less than four National Institutes of Health grant funding cycles since its initial discovery, we have arrived at a detailed structural understanding of how pRb inhibits cell growth, how this activity is regulated in normal cells, and how pRb is put out of action in tumor cells. Loss of pRb function is the ratelimiting step in the development of retinoblastoma, a rare childhood eye tumor. There is compelling evidence that pRb is at the center of a crucial cellular regulatory circuit that is perturbed in most human tumors (reviewed in ref. 4) (Fig. 1). Thus, the pRb pathway offers attractive targets for cancer therapy, and the delineation of the E2F pRb structure is good news and highly significant. One important function of pRb is to inhibit cellular DNA synthesis. In resting cells, pRb is hypophosphorylated and in complex with E2F, a group of heterodimeric transcription factors consisting of an E2F family member (E2F-1 to 6) and a dimerization partner (DP-1 or DP-2) (reviewed in ref. 5). E2F pRb complexes are transcriptional repressors and prevent cells from undergoing DNA replication (6). In preparation for DNA synthesis, pRb is phosphorylated by cyclin-dependent kinases (cdks) and E2F pRB complexes break apart. Free E2F acts as a transcriptional activator to drive DNA synthesis (reviewed in ref. 7) (Fig. 1). After cell division, pRb is dephosphorylated, binds up free E2F, and prevents cell cycle reentry. This delicately tuned regulatory circuit is almost universally subverted in tumors. Hyperactivity of cyclin D, cdk4, or cdk6, or inactivation of the cdk inhibitor p16INK4A each cause constitutive pRb phosphorylation. In cervical cancers that are caused by infections with high-risk human papillomaviruses (HPVs) the E7 oncoprotein binds and degrades pRb and disrupts E2F pRb complexes (reviewed in ref. 8) (Fig. 1). The adenovirus E1A protein, simian virus 40 large tumor antigen (SV40 TAg) and HPV E7 oncoproteins can each interact with pRb (9–11) and thwart the pRb regulatory circuit to accomplish expression of host cellular enzymes necessary for viral genome replication. These viral oncoproteins each contain an LXCXE domain, a small block of highly conserved amino acid residues that include the sequence leucine-X-cysteine-X-glutamate (X denotes any amino acid residue), which represents the core pRb-binding site and is necessary for induction of DNA synthesis and cellular transformation (reviewed in ref. 12). Whereas some cellular pRb interacting proteins also contain LXCXE domains, E2F proteins do not share significant sequence similarity to the viral oncoproteins and contain no LXCXE domains. Nevertheless, the viral oncoproteins and E2F transcription factors interact with similar pRb sequences, a central ‘‘pocket domain,’’ which consists of two densely packed subdomains that are connected by a flexible linker. Two additional cellular proteins p107 and p130 also contain pocket domains. Like pRb, they interact with E2F transcription factors and are targeted by viral oncoproteins. Despite these functional similarities, p107 and p130 are not frequently mutated in human tumors and may not be tumor suppressors. Proteins related to pRb and E2F have been discovered in many multicellular organisms including plants, suggesting that this regulatory principle is highly conserved (reviewed in refs. 13 and 14). Consistent with this notion, certain plant viruses encode replication proteins that interact with pRb (reviewed in ref. 15). Crystal structures of the pRB pocket domain in complex with LXCXE peptides of HPV E7 and SV40 TAg have been solved (16, 17). The LXCXE motif binds a conserved shallow groove within pRb pocket domain B. The E2F binding site is located on the opposite face of the pRb pocket within the central cleft formed by the interface of domains A and B and is 30 Å apart from the E7 binding site (2, 3) (Fig. 2). Similar to the LXCXE binding site it corresponds to a domain that is highly conserved in p107 and p130 and pRb-related proteins from different organisms. As insightful as the E2F pRb structure is it does not directly explicate a number of interesting questions. How does pRb phosphorylation regulate E2F binding? Are there, as suggested by other studies, additional E2F sequences that contribute to pRb binding? How does binding of E7 disrupt E2F pRb complexes? The authors have addressed each of these issues by using isothermal titration calorimetry, and these studies offer some additional interesting insights (2). Most of the cdk phosphorylation sites are within the C-terminal domain and are not included on the crystallized pRb fragment. It has been proposed that initial phosphorylation of the pRb C terminus may disrupt intramolecular interactions between the C terminus and the pocket. This renders the pocket accessible for phosphorylation, which in turn