Characterization of the High-Affinity Fuzzy Complex between the Disordered Domain of the E7 Oncoprotein from High-Risk HPV and the TAZ2 Domain of CBP.

The intrinsically disordered N-terminal region of the E7 protein from high-risk human papillomavirus (HPV) strains is responsible for oncogenic transformation of host cells through its interaction with a number of cellular factors, including the TAZ2 domain of the transcriptional coactivator CREB-binding protein. Using a variety of spectroscopic and biochemical tools, we find that despite its nanomolar affinity, the HPV16 E7 complex with TAZ2 is disordered and highly dynamic. The disordered domain of HPV16 E7 protein does not adopt a single conformation on the surface of TAZ2 but engages promiscuously with its target through multiple interactions involving two conserved motifs, termed CR1 and CR2, that occupy an extensive binding surface on TAZ2. The fuzzy nature of the complex is a reflection of the promiscuous binding repertoire of viral proteins, which must efficiently dysregulate host cell processes by binding to a variety of host factors in the cellular environment.

NMR analysis of the structure, dynamics, and unique oligomerization properties of the chemokine CCL27.

Chemokines have two essential interactions in vivo, with G protein-coupled receptors, which activate intracellular signaling pathways, and with glycosaminoglycans (GAGs), which are involved in cell surface localization and transport. Although it has been shown that chemokines bind and activate their respective G protein-coupled receptors as monomers, many chemokines oligomerize upon GAG binding, and the ability to oligomerize and bind GAGs is required for in vivo function. In this study, we investigated the structure, dynamics, and oligomerization behavior of cutaneous T-cell-attracting chemokine (CTACK, also known as CCL27) by NMR. (15)N relaxation and translational self-diffusion rates indicate that CCL27 oligomerizes, but in contrast to many other chemokines that form relatively discrete oligomers, CCL27 transitions between monomer, dimer, and tetramer species over a relatively narrow concentration range. A three-dimensional structure determination was pursued under conditions where CCL27 is primarily dimeric, revealing the standard motif for a chemokine monomer. Analysis of chemical shift perturbations of (1)H-(15)N HSQC spectra, relaxation-dispersion experiments, and filtered nuclear Overhauser effects suggest that CCL27 does not adopt a discrete CXC or CC dimer motif. Instead, CCL27 has uncommon oligomerization behavior, where several equilibria involving relatively low affinity interactions between different interfaces seem to be simultaneously at work. However, interaction with heparin avidly promotes oligomerization under conditions where CCL27 is monomeric by itself. We hypothesize that the plasticity in the oligomerization state may enable CCL27 to adopt different oligomeric structures, depending on the nature of the GAG binding partner, thereby providing a mechanism for increased diversity and specificity in GAG-binding and GAG-related functions.

Characterization of the chemokine CXCL11-heparin interaction suggests two different affinities for glycosaminoglycans.

Chemokines orchestrate the migration of leukocytes in the context of homeostasis and inflammation. In addition to interactions of chemokines with receptors on migrating cells, these processes require interactions of chemokines with glycosaminoglycans (GAGs) for cell surface localization. Most chemokines are basic proteins with Arg/Lys/His residue clusters functioning as recognition epitopes for GAGs. In this study we characterized the GAG-binding epitopes of the chemokine I-TAC/CXCL11. Four separate clusters of basic residues were mutated to alanine and tested for their ability to bind to GAGs in vitro and to activate the receptor, CXCR3. Mutation of a set of basic residues in the C-terminal helix (the 50s cluster, (57)KSKQAR(62)) along with Lys(17), significantly impaired heparin binding in vitro, identifying these residues as components of the dominant epitope. However, this GAG mutant retained nearly wild type receptor binding affinity, and its ability to induce cell migration in vitro was only mildly perturbed. Nevertheless, the mutant was unable to induce cell migration in vivo, establishing a requirement of CXCL11 for GAG binding for in vivo function. These studies also led to some interesting findings. First, CXCL11 exhibits conformational heterogeneity, as evidenced by the doubling of peaks in its HSQC spectra. Second, it exhibits more than one affinity state for both heparin and CXCR3, which may be related to its structural plasticity. Finally, although the binding affinities of chemokines for GAGs are typically weaker than interactions with receptors, the high affinity GAG binding state of CXCL11 is comparable with typical receptor binding affinities, suggesting some unique properties of this chemokine.

Verification of a designed intramolecular hydrogen bond in a drug scaffold by nuclear magnetic resonance spectroscopy.

2D 1H-15N HMBC NMR acquired at natural abundance and DMSO titration monitored by 1D 1H NMR verified the existence of an intramolecular hydrogen bond that was designed to mimic the pyrimidinone ring of a class of kinase inhibitors. A scalar coupling across the hydrogen bond was detected in organic and aqueous solvent, suggesting a simple and general approach for testing the propensity of intramolecular hydrogen bonds to stabilize pseudo-rings in drug scaffolds.

The high-risk HPV16 E7 oncoprotein mediates interaction between the transcriptional coactivator CBP and the retinoblastoma protein pRb.

The oncoprotein E7 from human papillomavirus (HPV) strains that confer high cancer risk mediates cell transformation by deregulating host cellular processes and activating viral gene expression through recruitment of cellular proteins such as the retinoblastoma protein (pRb) and the cyclic-AMP response element binding binding protein (CBP) and its paralog p300. Here we show that the intrinsically disordered N-terminal region of E7 from high-risk HPV16 binds the TAZ2 domain of CBP with greater affinity than E7 from low-risk HPV6b. HPV E7 and the tumor suppressor p53 compete for binding to TAZ2. The TAZ2 binding site in E7 overlaps the LxCxE motif that is crucial for interaction with pRb. While TAZ2 and pRb compete for binding to a monomeric E7 polypeptide, the full-length E7 dimer mediates an interaction between TAZ2 and pRb by promoting formation of a ternary complex. Cell-based assays show that expression of full-length HPV16 E7 promotes increased pRb acetylation and that this response depends both on the presence of CBP/p300 and on the ability of E7 to form a dimer. These observations suggest a model for the oncogenic effect of high-risk HPV16 E7. The disordered region of one E7 molecule in the homodimer interacts with the pocket domain of pRb, while the same region of the other E7 molecule binds the TAZ2 domain of CBP/p300. Through its ability to dimerize, E7 recruits CBP/p300 and pRb into a ternary complex, bringing the histone acetyltransferase domain of CBP/p300 into proximity to pRb and promoting acetylation, leading to disruption of cell cycle control.

Chapter 2. Homo- and hetero-oligomerization of chemokines.

Chemokines function in cell migration by binding and activating seven transmembrane G protein-coupled receptors (GPCRs) on leukocytes and many other diverse cell types. The extracellular binding event stabilizes specific conformations of the receptor that trigger cascades of intracellular signaling pathways involved in cell movement and activation (Baggiolini, 1998; Baggiolini et al., 1997; Charo and Ransohoff, 2006; Hartley et al., 2003; Kunkel and Butcher, 2002; Loetscher and Clark-Lewis, 2001). Although the current consensus is that monomeric forms of chemokines are necessary for receptor binding to induce cell migration, oligomeric states of chemokines may be associated with other complex functional roles such as regulation, haptotactic gradient formation, protection from proteolysis, and signaling related to processes distinct from migration. Accordingly, diverse biophysical methods have been used to identify and characterize the details of these quaternary interactions. This chapter aims to summarize these methods and to provide guidelines for their application in future studies.

Automated microflow NMR: routine analysis of five-microliter samples.

A microflow CapNMR probe double-tuned for 1H and 13C was installed on a 400-MHz NMR spectrometer and interfaced to an automated liquid handler. Individual samples dissolved in DMSO-d6 are submitted for NMR analysis in vials containing as little as 10 microL of sample. Sets of samples are submitted in a low-volume 384-well plate. Of the 10 microL of sample per well, as with vials, 5 microL is injected into the microflow NMR probe for analysis. For quality control of chemical libraries, 1D NMR spectra are acquired under full automation from 384-well plates on as many as 130 compounds within 24 h using 128 scans per spectrum and a sample-to-sample cycle time of approximately 11 min. Because of the low volume requirements and high mass sensitivity of the microflow NMR system, 30 nmol of a typical small molecule is sufficient to obtain high-quality, well-resolved, 1D proton or 2D COSY NMR spectra in approximately 6 or 20 min of data acquisition time per experiment, respectively. Implementation of pulse programs with automated solvent peak identification and suppression allow for reliable data collection, even for samples submitted in fully protonated DMSO. The automated microflow NMR system is controlled and monitored using web-based software.