Structure of the human Papillomavirus E7 oncoprotein and its mechanism for inactivation of the retinoblastoma tumor suppressor.

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 pRb.E2F 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.

Fluorescence-based peptide labeling and fractionation strategies for analysis of cysteine-containing peptides.

This study demonstrates that 1,5-I-AEDANS (5-({2-[(iodoacetyl)amino]ethyl}amino)naphthalene-1-sulfonic acid) can be used as a versatile fluorescence-based peptide quantification tool and provides readily interpretable tandem mass spectra for de novo peptide sequencing. Two AEDANS-cysteinyl-peptide fractionation strategies were evaluated. One AEDANS-cysteinyl-peptide fractionation strategy employs immobilized metal affinity chromatography (IMAC) to recover AEDANS-labeled peptides and reduce the complexity of peptide mixtures. In an alternate solid-phase approach, 1,5-I-AEDANS was coupled to an o-nitrobenzyl-based photocleavable resin to produce a resin that can label and isolate thiols and cysteine-containing peptides with a modified-AEDANS label (mAEDANS: 5-((4-amino-4-oxobutanoyl){2-[(iodoacetyl)amino]ethyl}amino)naphthalene-1-sulfonic acid). This fractionation protocol enriches cysteine-containing peptides more specifically than the IMAC strategy. Using micro-LC-ESI-MS with an on-line fluorescence detector and a Q-TOF mass spectrometer, we generated fluorescence-based elution profiles and corresponding positive ion mass spectra of AEDANS-labeled peptides. This study demonstrates that AEDANS-peptides produce positive ion ESI-MS mass spectra with detection limits comparable to those of the unlabeled peptide. Collision-induced dissociation (CID) of fluorescent AEDANS-peptides revealed readily interpretable product ion spectra with the label intact. Similar to the AEDANS-labeled peptide, an mAEDANS-labeled thiol is fluorescent and CID of a mAEDANS-labeled peptide also reveals an interpretable product ion spectrum with the label intact.

Structural basis for histone and phosphohistone binding by the GCN5 histone acetyltransferase.

Distinct posttranslational modifications on histones occur in specific patterns to mediate certain chromosomal events. For example, on histone H3, phosphorylation at Ser10 can enhance GCN5-mediated Lys14 acetylation to promote transcription. To gain insight into the mechanism underlying this synergism, we determined the structure of Tetrahymena GCN5 (tGCN5) and coenzyme A (CoA) bound to unmodified and Ser10-phosphorylated 19 residue histone H3 peptides (H3p19 and H3p19Pi, respectively). The tGCN5/CoA/H3p19 structure reveals that a 12 amino acid core sequence mediates extensive contacts with the protein, providing the structural basis for substrate specificity by the GCN5/PCAF family of histone acetyltransferases. Comparison with the tGCN5/CoA/H3p19Pi structure reveals that phospho-Ser10 and Thr11 mediate significant histone-protein interactions, and nucleate additional interactions distal to the phosphorylation site. Functional studies show that histone H3 Thr11 is necessary for optimal transcription at yGcn5-dependent promoters requiring Ser10 phosphorylation. Together, these studies reveal how one histone modification can modulate another to affect distinct transcriptional signals.

Structure and autoregulation of the yeast Hst2 homolog of Sir2.

Yeast Hst2 (yHst2) is a member of the silencing information regulator 2 (Sir2) family of NAD(+)-dependent protein deacetylases that are implicated in transcriptional silencing, DNA repair, genome stability and longevity. The X-ray crystal structure of the full-length yHst2 protein reveals a central catalytic core domain fold that is characteristic of the other Sir2 homologs, and C- and N-terminal extensions that interact with the NAD(+) and acetyl-lysine substrate-binding sites, respectively, suggesting an autoregulatory function for these domains. Moreover, the N-terminal extension mediates formation of a homotrimer within the crystal lattice. Enzymatic and sedimentation equilibrium studies using deletion constructs of yHst2 support the involvement of the N- and C-terminal yHst2 regions and trimer formation in catalysis by yHst2. Together, these studies indicate that the sequence-divergent N- and C-terminal regions of the eukaryotic Sir2 proteins may have a particularly important role in their distinct substrate-binding properties, biological activities or both.

Crystal structure of the retinoblastoma tumor suppressor protein bound to E2F and the molecular basis of its regulation.

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 CDK2cyclin DE. 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.