RESUMEN
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.
Asunto(s)
Proteína de Unión a CREB/química , Proteínas E7 de Papillomavirus/química , Secuencia de Aminoácidos , Animales , Proteína de Unión a CREB/genética , Transformación Celular Neoplásica , Secuencia Conservada , Interacciones Microbiota-Huesped , Humanos , Proteínas Intrínsecamente Desordenadas/química , Proteínas Intrínsecamente Desordenadas/genética , Ratones , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Proteínas E7 de Papillomavirus/genética , Unión Proteica , Conformación Proteica , Dominios y Motivos de Interacción de ProteínasRESUMEN
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.
Asunto(s)
Proteína p300 Asociada a E1A/metabolismo , Complejos Multiproteicos/metabolismo , Proteínas E7 de Papillomavirus/metabolismo , Proteína de Retinoblastoma/metabolismo , Secuencia de Aminoácidos , Unión Competitiva , Western Blotting , Línea Celular , Transformación Celular Neoplásica/genética , Proteína p300 Asociada a E1A/química , Fibroblastos/citología , Fibroblastos/metabolismo , Polarización de Fluorescencia , Humanos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Datos de Secuencia Molecular , Complejos Multiproteicos/química , Mutación , Proteínas E7 de Papillomavirus/química , Proteínas E7 de Papillomavirus/genética , Unión Proteica , Multimerización de Proteína , Estructura Terciaria de Proteína , Proteína de Retinoblastoma/química , Factores de Riesgo , Homología de Secuencia de AminoácidoRESUMEN
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.
