Project description:The human papillomavirus (HPV) capsid consists of 360 copies of the major capsid protein, L1, arranged as 72 pentamers on a T=7 icosahedral lattice, with substoichiometric amounts of the minor capsid protein, L2. In order to understand the arrangement of L2 within the HPV virion, we have defined and biochemically characterized a domain of L2 that interacts with L1 pentamers. We utilized an in vivo binding assay involving the coexpression of recombinant HPV type 11 (HPV11) L1 and HPV11 glutathione S-transferase (GST) L2 fusion proteins in Escherichia coli. In this system, L1 forms pentamers, GST=L2 associates with these pentamers, and L1+L2 complexes are subsequently isolated by using the GST tag on L2. The stoichiometry of L1:L2 in purified L1+L2 complexes was 5:1, indicating that a single molecule of L2 interacts with an L1 pentamer. Coexpression of HPV11 L1 with deletion mutants of HPV11 L2 defined an L1-binding domain contained within amino acids 396 to 439 near the carboxy terminus of L2. L2 proteins from eight different human and animal papillomavirus serotypes were tested for their ability to interact with HPV11 L1. This analysis targeted a hydrophobic region within the L1-binding domain of L2 as critical for L1 binding. Introduction of negative charges into this hydrophobic region by site-directed mutagenesis disrupted L1 binding. L1-L2 interactions were not significantly disrupted by treatment with high salt concentrations (2 M NaCl), weak detergents, and urea concentrations of up to 2 M, further indicating that L1 binding by this domain is mediated by strong hydrophobic interactions. L1+L2 protein complexes were able to form virus-like particles in vitro at pH 5.2 and also at pH 6.8, a pH that is nonpermissive for assembly of L1 protein alone. Thus, L1/L2 interactions are primarily hydrophobic, encompass a relatively short stretch of amino acids, and have significant effects upon in vitro assembly.
Project description:Cisplatin is a widely used anti-tumor agent for the treatment of testicular and ovarian cancers. Carboplatin is used extensively for small cell, non small cell lung cancer and ovarian cancer. Oxaliplatin has recently been approved in the United States (US) for treatment of colorectal cancer. A large portion (in the range of 65% to 98%) of cisplatin in the blood plasma was bound to protein within a day after intravenous administration. The binding of cisplatin and other analogues to proteins and enzymes is generally believed to be the cause of several severe side effects such as ototoxicity and nephrotoxicity. The interactions between platinum based chemotherapy drugs and proteins is proposed to play important roles in both drug activity and toxicity. Therefore, a better understanding of the molecular mechanism of platinum-protein interactions may have an impact on optimization of strategies for treatment. The objective is to develop novel approaches and techniques to provide detailed mechanistic, kinetic and high-resolution structural information on the binding of platinum analogues to blood proteins, and to improve treatment efficacy and reduce side effects.
Project description:Myeloid differentiation 88 (MyD88) is the key signaling adapter of Toll-like and interleukin-1 receptors. Recurrent lymphoma-associated mutations, particularly Leu265Pro (L265P), within the MyD88 Toll/interleukin-1 receptor (TIR) domain sustain lymphoma cell survival due to constitutive nuclear factor ?B signaling. We found that mutated TIR domains displayed an intrinsic propensity for augmented oligomerization and spontaneous formation of cytosolic Myddosome aggregates in lymphoma cell lines, mimicking the effect of dimerized TIR domains. Blocking of MyD88 oligomerization induced apoptosis. The L265P TIR domain can recruit the endogenous wild-type MyD88 for oligomer formation and hyperactivity. Molecular dynamics simulations and analysis of additional mutations suggest that constitutive activity is caused by allosteric oligomerization.
Project description:N-terminal signal peptides mediate the interaction of native proteins with the translocon complex of the endoplasmic reticulum membrane and are cleaved off during early protein biogenesis. The corticotropin-releasing factor receptor type 2a (CRF(2(a))R) possesses an N-terminal pseudo signal peptide, which represents a so far unique domain within the large protein family of G protein-coupled receptors (GPCRs). In contrast to a conventional signal peptide, the pseudo signal peptide remains uncleaved and consequently forms a hydrophobic extension at the N terminus of the receptor. The functional consequence of the presence of the pseudo signal peptide is not understood. Here, we have analyzed the significance of this domain for receptor dimerization/oligomerization in detail. To this end, we took the CRF(2(a))R and the homologous corticotropin-releasing factor receptor type 1 (CRF(1)R) possessing a conventional cleaved signal peptide and conducted signal peptide exchange experiments. Using single cell and single molecule imaging methods (fluorescence resonance energy transfer and fluorescence cross-correlation spectroscopy, respectively) as well as biochemical experiments, we obtained two novel findings; we could show that (i) the CRF(2(a))R is expressed exclusively as a monomer, and (ii) the presence of the pseudo signal peptide prevents its oligomerization. Thus, we have identified a novel functional domain within the GPCR protein family, which plays a role in receptor oligomerization and which may be useful to study the functional significance of this process in general.
Project description:Interaction of TLR9 with ligands activates NF-κB, leading to proinflammatory cytokine production. Excessive TLR activation is a pathogenic factor for inflammatory diseases. This study has examined cell-permeating decoy peptides (CPDPs) derived from the TLR9 Toll/IL-1R resistance (TIR) domain. CPDP 9R34, which included AB loop, β-strand B, and N-terminal BB loop residues, inhibited TLR9 signaling most potently. CPDPs derived from α-helices C, D, and E (i.e., 9R6, 9R9, and 9R11) also inhibited TLR9-induced cytokines but were less potent than 9R34. 9R34 did not inhibit TLR2/1, TLR4, or TLR7 signaling. The N-terminal deletion modification of 9R34, 9R34-ΔN, inhibited TLR9 as potently as the full length 9R34. Binding of 9R34-ΔN to TIR domains was studied using cell-based Förster resonance energy transfer/fluorescence lifetime imaging approach. Cy3-labeled 9R34-ΔN dose-dependently decreased fluorescence lifetime of TLR9 TIR-Cerulean (Cer) fusion protein. Cy3-9R34-ΔN also bound TIRAP TIR, albeit with a lesser affinity, but not MyD88 TIR, whereas CPDP from the opposite TIR surface, 9R11, bound both adapters and TLR9. i.p. administration of 9R34-ΔN suppressed oligonucleotide-induced systemic cytokines and lethality in mice. This study identifies a potent, TLR9-specific CPDP that targets both receptor dimerization and adapter recruitment. Location of TIR segments that represent inhibitory CPDPs suggests that TIR domains of TLRs and TLR adapters interact through structurally homologous surfaces within primary receptor complex, leading to formation of a double-stranded, filamentous structure. In the presence of TIRAP and MyD88, primary complex can elongate bidirectionally, from two opposite ends, whereas in TIRAP-deficient cells, elongation is unidirectional, only through the αE side.
Project description:GPCRs have been shown to form oligomers, which generate distinctive signaling outcomes. However, the structural nature of the oligomerization process remains uncertain. We have characterized oligomeric configurations of the adenosine A2a receptor (A2aR) by combining large-scale molecular dynamics simulations with Markov state models. These oligomeric structures may also serve as templates for studying oligomerization of other class A GPCRs. Our simulation data revealed that receptor activation results in enhanced oligomerization, more diverse oligomer populations, and a more connected oligomerization network. The active state conformation of the A2aR shifts protein-protein association interfaces to those involving intracellular loop ICL3 and transmembrane helix TM6. Binding of PIP2 to A2aR stabilizes protein-protein interactions via PIP2-mediated association interfaces. These results indicate that A2aR oligomerization is responsive to the local membrane lipid environment. This, in turn, suggests a modulatory effect on A2aR whereby a given oligomerization profile favors the dynamic formation of specific supramolecular signaling complexes.
Project description:Toll/Interleukin-1 receptor (TIR) domains are cytoplasmic domain that mediates receptor signalling. These domains are present in proteins like Toll-like receptors (TLR), its signaling adaptors and Interleukins, that form a major part of the immune system. These TIR domain containing signaling adaptors binds to the TLRs and interacts with their TIR domains for downstream signaling. We have examined the evolutionary divergence across the tree of life of two of these TIR domain containing adaptor molecules (TICAM) i.e., TIR domain-containing adapter-inducing interferon-β (TRIF/TICAM1) and TIR domain containing adaptor molecule2 (TRAM/TICAM2), by using computational approaches. We studied their orthologs, domain architecture, conserved motifs, and amino acid variations. Our study also adds a timeframe to infer the duplication of TICAM protein from Leptocardii and later divergence into TICAM1/TRIF and TICAM2/TRAM. More evidence of TRIF proteins was seen, but the absence of conserved co-existing domains such as TRIF-NTD, TIR, and RHIM domains in distant relatives hints on diversification and adaptation to different biological functions. TRAM was lost in Actinopteri and has conserved domain architecture of TIR across species except in Aves. An additional isoform of TRAM, TAG (TRAM adaptor with the GOLD domain), could be identified in species in the Mesozoic era. Finally, the Hypothesis based Likelihood ratio test was applied to look for selection pressure amongst orthologues of TRIF and TRAM to search for positively selected sites. These residues were mostly seen in the non-structural region of the proteins. Overall, this study unravels evolutionary information on the adaptors TRAM and TRIF and how well they had duplicated to perform diverse functions by changes in their domain architecture across lineages.
Project description:Plant nucleotide-binding leucine-rich repeat (NLR) immune receptors with an N-terminal Toll/interleukin-1 receptor (TIR) domain mediate recognition of strain-specific pathogen effectors, typically via their C-terminal ligand-sensing domains1. Effector binding enables TIR-encoded enzymatic activities that are required for TIR-NLR (TNL)-mediated immunity2,3. Many truncated TNL proteins lack effector-sensing domains but retain similar enzymatic and immune activities4,5. The mechanism underlying the activation of these TIR domain proteins remain unclear. Here we show that binding of the TIR substrates NAD+ and ATP induces phase separation of TIR domain proteins in vitro. A similar condensation occurs with a TIR domain protein expressed via its native promoter in response to pathogen inoculation in planta. The formation of TIR condensates is mediated by conserved self-association interfaces and a predicted intrinsically disordered loop region of TIRs. Mutations that disrupt TIR condensates impair the cell death activity of TIR domain proteins. Our data reveal phase separation as a mechanism for the activation of TIR domain proteins and provide insight into substrate-induced autonomous activation of TIR signalling to confer plant immunity.
Project description:Whether thymocytes adopt an ?? or a ?? T cell fate in the thymus is determined at the ? selection checkpoint by the relatively weak or strong signals that are delivered by either the pre-T cell receptor (preTCR) or the ?? TCR, respectively. Signal initiation at the ? selection checkpoint is thought to be independent of ligand engagement of these receptors. Some reports have suggested that receptor oligomerization, which is thought to be mediated by either the immunoglobulin (Ig)-like domain of the preTCR? (pT?) chain or the variable domain of TCR?, is a unifying mechanism that initiates signaling in early CD4(-)CD8(-) double-negative (DN) thymocyte progenitors. Here, we demonstrate that the extracellular regions of pT? and TCR? that are implicated in mediating receptor oligomerization were not required for signal initiation from the preTCR or TCR??. Indeed, a truncated TCR?? that lacked all of its extracellular Ig-like domains still formed a signaling-competent TCR that drove cells through the ? selection checkpoint. These observations suggest that signal initiation in DN thymocytes is simply a consequence of the surface-pairing of TCR chains, with signal strength being a function of the abundances of surface TCRs. Thus, processes that regulate the surface abundances of TCR complexes in DN cells, such as oligomerization-induced endocytosis, would be predicted to have a major influence in determining whether cells adopt an ?? versus ?? T cell fate.