RESUMEN
During eukaryotic transcription elongation, RNA polymerase II (RNAP2) is regulated by a chorus of factors. Here, we identified a common binary interaction module consisting of TFIIS N-terminal domains (TNDs) and natively unstructured TND-interacting motifs (TIMs). This module was conserved among the elongation machinery and linked complexes including transcription factor TFIIS, Mediator, super elongation complex, elongin, IWS1, SPT6, PP1-PNUTS phosphatase, H3K36me3 readers, and other factors. Using nuclear magnetic resonance, live-cell microscopy, and mass spectrometry, we revealed the structural basis for these interactions and found that TND-TIM sequences were necessary and sufficient to induce strong and specific colocalization in the crowded nuclear environment. Disruption of a single TIM in IWS1 induced robust changes in gene expression and RNAP2 elongation dynamics, which underscores the functional importance of TND-TIM surfaces for transcription elongation.
Asunto(s)
Proteínas Intrínsecamente Desordenadas/química , ARN Polimerasa II/metabolismo , Proteínas de Unión al ARN/química , Elongación de la Transcripción Genética , Factores de Transcripción/química , Factores de Elongación Transcripcional/química , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Línea Celular Tumoral , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Expresión Génica , Humanos , Proteínas Intrínsecamente Desordenadas/metabolismo , Modelos Moleculares , Mutación , Unión Proteica , Dominios Proteicos , Dominios y Motivos de Interacción de Proteínas/genética , Mapas de Interacción de Proteínas , ARN Polimerasa II/química , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Factores de Elongación Transcripcional/metabolismoRESUMEN
Dimerization of many eukaryotic transcription regulatory factors is critical for their function. Regulatory role of an epigenetic reader lens epithelium-derived growth factor/p75 (LEDGF/p75) requires at least two copies of this protein to overcome the nucleosome-induced barrier to transcription elongation. Moreover, various LEDGF/p75 binding partners are enriched for dimeric features, further underscoring the functional regulatory role of LEDGF/p75 dimerization. Here, we dissected the minimal dimerization region in the C-terminal part of LEDGF/p75 and, using paramagnetic NMR spectroscopy, identified the key molecular contacts that helped to refine the solution structure of the dimer. The LEDGF/p75 dimeric assembly is stabilized by domain swapping within the integrase binding domain and additional electrostatic "stapling" of the negatively charged α helix formed in the intrinsically disordered C-terminal region. We validated the dimerization mechanism using structure-inspired dimerization defective LEDGF/p75 variants and chemical crosslinking coupled to mass spectrometry. We also show how dimerization might affect the LEDGF/p75 interactome.
Asunto(s)
Péptidos y Proteínas de Señalización Intercelular/química , Multimerización de Proteína , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Dominios Proteicos , Electricidad EstáticaRESUMEN
Accurate prediction of protein-ligand binding affinities is essential for hit-to-lead optimization and virtual screening. The reliability of scoring functions can be improved by including quantum effects. Here, we demonstrate the ranking power of the semiempirical quantum mechanics (SQM)/implicit solvent (COSMO) scoring function by using a challenging set of 10 inhibitors binding to carbonic anhydraseâ II through Zn2+ in the active site. This new dataset consists of the high-resolution (1.1-1.4â Å) crystal structures and experimentally determined inhibitory constant (Ki ) values. It allows for evaluation of the common approximations, such as representing the solvent implicitly or by using a single target conformation combined with a set of ligand docking poses. SQM/COSMO attained a good correlation of R2 of 0.56-0.77 with the experimental inhibitory activities, benefiting from careful handling of both noncovalent interactions (e.g. charge transfer) and solvation. This proof-of-concept study of SQM/COSMO ranking for metalloprotein-ligand systems demonstrates its potential for hit-to-lead applications.
Asunto(s)
Anhidrasa Carbónica II/metabolismo , Inhibidores de Anhidrasa Carbónica/metabolismo , Sulfonamidas/metabolismo , Anhidrasa Carbónica II/química , Inhibidores de Anhidrasa Carbónica/química , Diseño de Fármacos , Ligandos , Modelos Químicos , Simulación del Acoplamiento Molecular , Unión Proteica , Teoría Cuántica , Sulfonamidas/químicaRESUMEN
Lens epithelium-derived growth factor (LEDGF/p75) is an epigenetic reader and attractive therapeutic target involved in HIV integration and the development of mixed lineage leukaemia (MLL1) fusion-driven leukaemia. Besides HIV integrase and the MLL1-menin complex, LEDGF/p75 interacts with various cellular proteins via its integrase binding domain (IBD). Here we present structural characterization of IBD interactions with transcriptional repressor JPO2 and domesticated transposase PogZ, and show that the PogZ interaction is nearly identical to the interaction of LEDGF/p75 with MLL1. The interaction with the IBD is maintained by an intrinsically disordered IBD-binding motif (IBM) common to all known cellular partners of LEDGF/p75. In addition, based on IBM conservation, we identify and validate IWS1 as a novel LEDGF/p75 interaction partner. Our results also reveal how HIV integrase efficiently displaces cellular binding partners from LEDGF/p75. Finally, the similar binding modes of LEDGF/p75 interaction partners represent a new challenge for the development of selective interaction inhibitors.
Asunto(s)
Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas/metabolismo , Proteínas Represoras/metabolismo , Transposasas/metabolismo , Secuencia de Aminoácidos , Secuencia de Consenso , Dimerización , Escherichia coli , Integrasa de VIH/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Lentivirus/enzimología , Datos de Secuencia Molecular , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Estructura Terciaria de Proteína , Proteínas de Unión al ARN , Factores de TranscripciónRESUMEN
The monoclonal antibodies 1696 and F11.2.32 strongly inhibit the activity of wild-type HIV-1 protease (PR) by binding to epitopes at the enzyme N-terminus (residues 1-6) and flap residues 36-46, respectively. Here we demonstrate that these antibodies are also potent inhibitors of PR variants resistant to active-site inhibitors used as anti-AIDS drugs. Our in vitro experiments revealed that the inhibitory potency of single-chain fragments (scFv) of these antibodies is not significantly affected by the presence of mutations in PR; inhibition constants for drug-resistant protease variants are 5-11 nM and 13-169 nM for scFv1696 and for scFvF11.2.32, respectively. Tethered dimer of HIV-1 PR variant proved to be a model protease variant resistant to dissociative inhibition by 1696, and, strikingly, it also displayed resistance to inhibition by F11.2.32 suggesting that dimer dissociation also plays a role in the inhibitory action of F11.2.32.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Farmacorresistencia Viral/genética , Variación Genética , Proteasa del VIH/efectos de los fármacos , VIH-1/enzimología , Fragmentos de Inmunoglobulinas/farmacología , Proteínas Recombinantes/farmacología , Anticuerpos Monoclonales/inmunología , Terapia Antirretroviral Altamente Activa , Dimerización , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/virología , Proteasa del VIH/genética , Proteasa del VIH/inmunología , Inhibidores de la Proteasa del VIH/farmacología , VIH-1/efectos de los fármacos , VIH-1/genética , Humanos , Fragmentos de Inmunoglobulinas/inmunología , Modelos Moleculares , Mutación , Proteínas Recombinantes/inmunologíaRESUMEN
Specific antibodies interfere with the function of human tumor-associated carbonic anhydrase IX (CA IX), and show potential as tools for anticancer interventions. In this work, a correlation between structural elements and thermodynamic parameters of the association of antibody fragment Fab M75 to a peptide corresponding to its epitope in the proteoglycan-like domain of CA IX, is presented. Comparisons of the crystal structures of free Fab M75 and its complex with the epitope peptide reveal major readjustments of CDR-H1 and CDR-H3. In contrast, the overall conformations and positions of CDR-H2 and CDR-L2 remain unaltered, and their positively charged residues may thus present a fixed frame for epitope recognition. Adoption of the altered CDR-H3 conformation in the structure of the complex is accompanied by an apparent local stabilization. Analysis of domain mobility with translation-libration-screw (TLS) method shows that librations of the entire heavy chain variable domain (V(H)) decrease and reorient in the complex, which correlates well with participation of the heavy chain in ligand binding. Isothermal titration microcalorimetry (ITC) experiments revealed a highly unfavorable entropy term, which can be attributed mainly to the decrease in the degrees of freedom of the system, the loss of conformational freedom of peptide and partially to a local stabilization of CDR-H3. Moreover, it was observed that one proton is transferred from the environment to the protein-ligand complex upon binding. Molecular dynamics simulations followed by molecular mechanics/generalized Born surface area (MM-GBSA) calculations of the ligand (epitope peptide) binding energy yielded energy values that were in agreement with the ITC measurements and indicated that the charged residues play crucial role in the epitope binding. Theoretical arguments presented in this work indicate that two adjacent arginine residues (ArgH50 and ArgH52) are responsible for the observed proton transfer.
Asunto(s)
Anticuerpos Monoclonales/química , Antígenos de Neoplasias/química , Sitios de Unión de Anticuerpos , Anhidrasas Carbónicas/química , Simulación por Computador , Epítopos/química , Fragmentos Fab de Inmunoglobulinas/química , Secuencia de Aminoácidos , Antígenos de Neoplasias/inmunología , Calorimetría , Anhidrasa Carbónica IX , Anhidrasas Carbónicas/inmunología , Línea Celular Tumoral , Cristalografía por Rayos X , Epítopos/inmunología , Humanos , Isoenzimas/química , Isoenzimas/inmunología , Datos de Secuencia Molecular , TermodinámicaRESUMEN
Depending on the excess of ligand used for complex formation, the HIV-1 protease complexed with a novel phenylnorstatine inhibitor forms crystals of either hexagonal (P6(1)) or orthorhombic (P2(1)2(1)2(1)) symmetry. The orthorhombic form shows an unusual complexity of crystal packing: in addition to one inhibitor molecule that is bound to the enzyme active site, the second inhibitor molecule is bound as an outer ligand at the protein interface. Binding of the outer ligand apparently increases the crystal-quality parameters so that the diffraction data allow solution of the structure of the complex at 1.03 A, the best resolution reported to date. The outer ligand interacts with all four surrounding HIV-1 protease molecules and has a bent conformation owing to its accommodation in the intermolecular space. The parameters of the solved structures of the orthorhombic and hexagonal forms are compared.
Asunto(s)
Inhibidores de la Proteasa del VIH/química , Inhibidores de la Proteasa del VIH/metabolismo , Proteasa del VIH/química , Proteasa del VIH/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Ligandos , Modelos Moleculares , Conformación Proteica , Sensibilidad y EspecificidadRESUMEN
The X-ray structure of a complex of HIV-1 protease (PR) with a phenylnorstatine inhibitor Z-Pns-Phe-Glu-Glu-NH(2) has been determined at 1.03 A, the highest resolution so far reported for any HIV PR complex. The inhibitor shows subnanomolar K(i) values for both the wild-type PR and the variant representing one of the most common mutations linked to resistance development. The structure comprising the phenylnorstatine moiety of (2R,3S)-chirality displays a unique pattern of hydrogen bonding to the two catalytic aspartate residues. This high resolution makes it possible to assess the donor and acceptor relations of this hydrogen bonding and to indicate a proton shared by the two catalytic residues. A structural mechanism for the unimpaired inhibition of the protease Val82Ala mutant is also suggested, based on energy calculations and analyses.
Asunto(s)
Inhibidores de la Proteasa del VIH/química , Proteasa del VIH/química , Fenilbutiratos/química , Sitios de Unión , Cristalografía por Rayos X , Farmacorresistencia Viral/genética , Proteasa del VIH/genética , Modelos Moleculares , Mutación , Unión Proteica , Estereoisomerismo , TermodinámicaRESUMEN
BACKGROUND: Uraemia and haemodialysis treatment are associated with microinflammation and oxidative as well as carbonyl stress, which result in enhanced formation of glycoxidation products. Although both glycoxidation and inflammation can contribute to severe vascular and cardiovascular complications, the role that these pathogenic mechanisms play in the complex response of the whole organism remains to be elucidated. METHODS: We performed a cross-sectional study in 34 clinically stable chronic haemodialysis patients and in 14 healthy controls while determining serum concentrations of pentosidine, fluorescent advanced glycation end-products (AGEs), advanced oxidation protein products (AOPPs) and acute phase reactants. We further assessed the relationship between these glycoxidation products and parameters of inflammation. RESULTS: Glycoxidation products as well as certain acute phase reactants were elevated in haemodialysis patients. There were significant correlations between AOPPs and inflammatory parameters such as orosomucoid (0.39, P < 0.05), fibrinogen (0.49, P < 0.05) and pregnancy-associated protein A (PAPP-A; 0.46, P < 0.05), but no correlations between pentosidine or fluorescent AGEs and any of the inflammatory parameters. CONCLUSION: Oxidative damage showed a closer relationship to inflammation than advanced glycation (glycoxidation). AOPPs may represent a superior acute biochemical marker, whereas AGEs may better describe chronic long-lasting damage.