RESUMEN
Toll-like receptor signaling requires interactions of the Toll/IL-1 receptor (TIR) domains of the receptor and adapter proteins. Using the mammalian protein-protein interaction trap strategy, homology modeling, and site-directed mutagenesis, we identify the interaction surfaces in the TLR4 TIR domain for the TLR4-TLR4, TLR4-MyD88 adapter-like (MAL), and TLR4-TRIF-related adapter molecule (TRAM) interaction. Two binding sites are equally important for TLR4 dimerization and adapter recruitment. In a model based on the crystal structure of the dimeric TLR10 TIR domain, the first binding site mediates TLR4-TLR4 TIR-TIR interaction. Upon dimerization, two identical second binding sites of the TLR4 TIR domain are juxtaposed and form an extended binding platform for both MAL and TRAM. In our mammalian protein-protein interaction trap assay, MAL and TRAM compete for binding to this platform. Our data suggest that adapter binding can stabilize the TLR4 TIR dimerization.
Asunto(s)
Modelos Moleculares , Multimerización de Proteína/fisiología , Receptor Toll-Like 4/química , Animales , Humanos , Ratones , Mutagénesis Sitio-Dirigida , Factor 88 de Diferenciación Mieloide/química , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/metabolismo , Mapeo Peptídico , Estructura Terciaria de Proteína , Homología Estructural de Proteína , Receptor Toll-Like 10/química , Receptor Toll-Like 10/genética , Receptor Toll-Like 10/metabolismo , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/metabolismoRESUMEN
Toll-like receptors (TLRs) are pattern recognition receptors that recognize pathogens based on distinct molecular signatures. The human (h)TLR1, 2, 6 and 10 belong to the hTLR1 subfamilies, which are localized in the extracellular regions and activated in response to diverse ligand molecules. Due to the unavailability of the hTLR10 crystal structure, the understanding of its homo and heterodimerization with hTLR2 and hTLR1 and the ligand responsible for its activation is limited. To improve our understanding of the TLR10 receptor-ligand interaction, we used homology modeling to construct a three dimensional (3D) structure of hTLR10 and refined the model through molecular dynamics (MD) simulations. We utilized the optimized structures for the molecular docking in order to identify the potential site of interactions between the homo and heterodimer (hTLR10/2 and hTLR10/1). The docked complexes were then used for interaction with ligands (Pam(3)CSK(4) and PamCysPamSK(4)) using MOE-Dock and ASEDock. Our docking studies have shown the binding orientations of hTLR10 heterodimer to be similar with other TLR2 family members. However, the binding orientation of hTLR10 homodimer is different from the heterodimer due to the presence of negative charged surfaces at the LRR11-14, thereby providing a specific cavity for ligand binding. Moreover, the multiple protein-ligand docking approach revealed that Pam(3)CSK(4) might be the ligand for the hTLR10/2 complex and PamCysPamSK(4,) a di-acylated peptide, might activate hTLR10/1 hetero and hTLR10 homodimer. Therefore, the current modeled complexes can be a useful tool for further experimental studies on TLR biology.
Asunto(s)
Transducción de Señal , Receptor Toll-Like 10/química , Receptor Toll-Like 10/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Dimerización , Humanos , Ligandos , Lipopéptidos/química , Lipopéptidos/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Estabilidad Proteica , Estructura Terciaria de Proteína , Alineación de Secuencia , Receptor Toll-Like 1/química , Receptor Toll-Like 1/genética , Receptor Toll-Like 1/metabolismo , Receptor Toll-Like 10/genética , Receptor Toll-Like 2/química , Receptor Toll-Like 2/genética , Receptor Toll-Like 2/metabolismoRESUMEN
TLRs are central receptors of the innate immune system that drive host inflammation and adaptive immune responses in response to invading microbes. Among human TLRs, TLR10 is the only family member without a defined agonist or function. Phylogenetic analysis reveals that TLR10 is most related to TLR1 and TLR6, both of which mediate immune responses to a variety of microbial and fungal components in cooperation with TLR2. The generation and analysis of chimeric receptors containing the extracellular recognition domain of TLR10 and the intracellular signaling domain of TLR1, revealed that TLR10 senses triacylated lipopeptides and a wide variety of other microbial-derived agonists shared by TLR1, but not TLR6. TLR10 requires TLR2 for innate immune recognition, and these receptors colocalize in the phagosome and physically interact in an agonist-dependent fashion. Computational modeling and mutational analysis of TLR10 showed preservation of the essential TLR2 dimer interface and lipopeptide-binding channel found in TLR1. Coimmunoprecipitation experiments indicate that, similar to TLR2/1, TLR2/10 complexes recruit the proximal adaptor MyD88 to the activated receptor complex. However, TLR10, alone or in cooperation with TLR2, fails to activate typical TLR-induced signaling, including NF-kappaB-, IL-8-, or IFN-beta-driven reporters. We conclude that human TLR10 cooperates with TLR2 in the sensing of microbes and fungi but possesses a signaling function distinct from that of other TLR2 subfamily members.
Asunto(s)
Inmunidad Innata , Modelos Inmunológicos , Transducción de Señal/inmunología , Receptor Toll-Like 10/fisiología , Receptor Toll-Like 1/fisiología , Secuencia de Aminoácidos , Animales , Línea Celular , Línea Celular Tumoral , Espacio Extracelular/química , Espacio Extracelular/genética , Espacio Extracelular/inmunología , Humanos , Inmunidad Innata/genética , Lipopéptidos/síntesis química , Lipopéptidos/metabolismo , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Multimerización de Proteína/genética , Multimerización de Proteína/inmunología , Estructura Terciaria de Proteína/genética , Seudogenes/inmunología , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal/genética , Receptor Toll-Like 1/agonistas , Receptor Toll-Like 1/química , Receptor Toll-Like 1/deficiencia , Receptor Toll-Like 10/agonistas , Receptor Toll-Like 10/química , Receptor Toll-Like 10/deficiencia , Receptor Toll-Like 2/química , Receptor Toll-Like 2/genética , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 2/fisiologíaRESUMEN
The Toll/interleukin-1 receptor (TIR) domain is a highly conserved signaling domain found in the intracellular regions of Toll-like receptors (TLRs), in interleukin-1 receptors, and in several cytoplasmic adaptor proteins. TIR domains mediate receptor signal transduction through recruitment of adaptor proteins and play critical roles in the innate immune response and inflammation. This work presents the 2.2A crystal structure of the TIR domain of human TLR10, revealing a symmetric dimer in the asymmetric unit. The dimer interaction surface contains residues from the BB-loop, DD-loop, and alphaC-helix, which have previously been identified as important structural motifs for signaling in homologous TLR receptors. The interaction surface is extensive, containing a central hydrophobic patch surrounded by polar residues. The BB-loop forms a tight interaction, where a range of consecutive residues binds in a pocket formed by the reciprocal BB-loop and alphaC-helix. This pocket appears to be well suited for binding peptide substrates, which is consistent with the notion that peptides and peptide mimetics of the BB-loop are inhibitors for TLR signaling. The TLR10 structure is in good agreement with available biochemical data on TLR receptors and is likely to provide a good model for the physiological dimer.
Asunto(s)
Citoplasma/química , Transducción de Señal , Receptor Toll-Like 10/química , Secuencia Conservada , Cristalografía por Rayos X , Dimerización , Humanos , Modelos Moleculares , Mutación/genética , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
The mammalian Toll-like receptors (TLRs) play an important role in the recognition of invading pathogens and the modulation of innate immune responses. The primary objective of this study was to characterize single nucleotide polymorphisms (SNPs) and insertion-deletion polymorphisms (indels) within bovine TLRs 1, 5, and 10, thereby facilitating future TLR signaling and association studies relevant to bovine innate immunity. Comparative sequence analysis for 10 bovine breeds derived from Bos taurus and Bos indicus revealed 98 polymorphisms (92 SNPs and 6 indels), with at least 14 nonsynonymous SNPs located within predicted TLR domains considered to be of functional significance. Of the 98 polymorphisms detected, 94 are reported here for the first time. Notably, 2 nonsynonymous SNPs were determined to modulate the prediction of a novel leucine-rich repeat (LRR) domain within B. indicusTLR5. Prediction and comparison of TLR protein domain architectures for multiple species revealed seven conserved regions of LRR patterning associated with the three genes investigated.