The E7 oncoprotein from human Papillomavirus (HPV) mediates cell transformation in part by binding to the human pRb tumor suppressor protein and E2F transcription factors, resulting in the dissociation of pRb from E2F transcription factors and the premature cell progression into the S-phase of the cell cycle. This activity is mediated by the LXCXE motif and the CR3 zinc binding domain of the E7 protein. In this study we report the x-ray crystal structure of the CR3 region of HPV E7 and a structure-based mutational analysis to investigate its mode of pRb and E2F binding and E2F displacement from pRb. The structure reveals a novel zinc-bound E7-CR3 obligate homodimer that contains two surface patches of sequence conservation. Mutation of residues within these patches reveals that one patch is required for pRb binding, whereas the other is required for E2F binding. We also show that both E7-mediated interactions are required to disrupt pRb⅐E2F complexes. Based on these studies we present a mechanistic model for how E7 displaces E2F from pRb. Because the CR3 region of HPV E7 has no detectable homology to other human proteins, the structure-function studies presented here provide an avenue for developing small molecule compounds that inhibit HPV-E7-mediated cell transformation.Human papillomaviruses (HPVs) 2 are the causative agents for more than 90% of cervical cancers (1). More than 200 HPV genotypes have been identified based on genomic differences (2), and these genotypes fall into low risk (for example types 1A, 6B, and 11) and high risk (for example, types 5, 8, 16, 18, 31, and 33) forms that are correlated with benign and malignant lesions, respectively (3) (supplemental Fig. 1). Host cell transformation by HPV is mediated by two viral oncogenes, E6 and E7 (4 -6). E6 proteins contain four CXXC motifs that presumably form two zinc binding regions (3, 7-9), and E6 proteins of high risk type 16 and 18 cooperate with the host cellular factor E6-AP (E6-associated protein) to target the tumor suppressor p53 to ubiquitin-mediated degradation (10, 11).E7 proteins resemble adenovirus E1A and SV40 large T antigen both in primary sequences and in transactivation and transformation properties. Based on amino acid sequence homology within E7 proteins, they can be separated into three conserved regions denoted in an analogous fashion to adenovirus E1A as CR1, CR2, and CR3 (12). The CR2 and CR3 regions of HPV E7 share sequence homology with the corresponding regions of adenovirus E1A and SV40 large T antigen, including a strictly conserved LXCXE motif that mediates high affinity binding to pRb (13,14). The CR3 region of E7 contains two CXXC motifs that are separated by 29 or 30 residues, forming a novel zinc binding domain (3), which is also present in two copies in the primary sequence of E6 (15). The HPV E7 CR3 region has been shown to mediate protein dimerization (15-17) and to mediate direct interaction with several E7-interacting proteins. In particular, the E7 CR3 region contacts the C-terminal region of pRb (18), ...
The retinoblastoma tumor suppressor protein (pRb) regulates the cell cycle, facilitates differentiation, and restrains apoptosis. Furthermore, dysfunctional pRb is thought to be involved in the development of most human malignancies. Many of the functions of pRb are mediated by its regulation of the E2F transcription factors. To understand the structural basis for this regulation, we have determined the crystal structure of a fragment of E2F in complex with the pocket domain of the tumor suppressor protein.The pRb pocket, comprising the A and B cyclin-like domains, is the major focus of tumourigenic mutations in the protein. The fragment of E2F used in our structural studies, residues 409 -426 of E2F-1, represents the core of the pRb-binding region of the transcription factor. The structure shows that E2F binds at the interface of the A and B domains of the pocket making extensive interactions with conserved residues from both. We show by solution studies that a second site, probably contained within the ''marked box'' region of E2F, is responsible for additional interactions with the pRb pocket but is insufficient for complex formation on its own. In addition, we show that the interaction of the core binding fragment of E2F with pRb is inhibited by phosphorylation of the tumor suppressor protein by CDK2͞cyclin D͞E. Finally, our data reveal that the tight binding of the human papillomavirus E7 oncoprotein to pRb prevents subsequent interactions with the marked box region of E2F but not with its core binding region.
The human p300/CBP-associating factor, PCAF, mediates transcriptional activation through its ability to acetylate nucleosomal histone substrates as well as transcriptional activators such as p53. We have determined the 2.3 Å crystal structure of the histone acetyltransferase (HAT) domain of PCAF bound to coenzyme A. The structure reveals a central protein core associated with coenzyme A binding and a pronounced cleft that sits over the protein core and is flanked on opposite sides by the N-and C-terminal protein segments. A correlation of the structure with the extensive mutagenesis data for PCAF and the homologous yeast GCN5 protein implicates the cleft and the N-and C-terminal protein segments as playing an important role in histone substrate binding, and a glutamate residue in the protein core as playing an essential catalytic role. A structural comparison with the coenzyme-bound forms of the related N-acetyltransferases, HAT1 (yeast histone acetyltransferase 1) and SmAAT (Serratia marcescens aminoglycoside 3-N-acetyltransferase), suggests the mode of substrate binding and catalysis by these enzymes and establishes a paradigm for understanding the structure-function relationships of other enzymes that acetylate histones and transcriptional regulators to promote activated transcription.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.