Single-molecule imaging with cell-derived nanovesicles reveals early binding dynamics at a cyclic nucleotide-gated ion channel

Ligand binding to membrane proteins is critical for many biological signaling processes. However, individual binding events are rarely directly observed, and their asynchronous dynamics are occluded in ensemble-averaged measures. For membrane proteins, single-molecule approaches that resolve these dynamics are challenged by dysfunction in non-native lipid environments, lack of access to intracellular sites, and costly sample preparation. Here, we introduce an approach combining cell-derived nanovesicles, microfluidics, and single-molecule fluorescence colocalization microscopy to track individual binding events at a cyclic nucleotide-gated TAX-4 ion channel critical for sensory transduction. Our observations reveal dynamics of both nucleotide binding and a subsequent conformational change likely preceding pore opening. Kinetic modeling suggests that binding of the second ligand is either independent of the first ligand or exhibits up to ~10-fold positive binding cooperativity. This approach is broadly applicable to studies of binding dynamics for proteins with extracellular or intracellular domains in native cell membrane.

Modulation of Toll-like receptor 1 intracellular domain structure and activity by Zn2+ ions

Toll-like receptors (TLRs) play an important role in the innate immune response. While a lot is known about the structures of their extracellular parts, many questions are still left unanswered, when the structural basis of TLR activation is analyzed for the TLR intracellular domains. Here we report the structure and dynamics of TLR1 toll-interleukin like (TIR) cytoplasmic domain in crystal and in solution. We found that the TLR1-TIR domain is capable of specific binding of Zn with nanomolar affinity. Interactions with Zn are mediated by cysteine residues 667 and 686 and C667 is essential for the Zn binding. Potential structures of the TLR1-TIR/Zn complex were predicted in silico. Using the functional assays for the heterodimeric TLR1/2 receptor, we found that both Zn addition and Zn depletion affect the activity of TLR1, and C667A mutation disrupts the receptor activity. Analysis of C667 position in the TLR1 structure and possible effects of C667A mutation, suggests that zinc-binding ability of TLR1-TIR domain is critical for the receptor activation.

Topography and motion of the acid-sensing ion channel intracellular domains

Acid-sensing ion channels (ASICs) are trimeric cation-selective channels activated by decreases in extracellular pH. The intracellular N and C terminal tails of ASIC1 influence channel gating, trafficking, and signaling in ischemic cell death. Despite several x-ray and cryo-EM structures of the extracellular and transmembrane segments of ASIC1, these important intracellular tails remain unresolved. Here we describe the coarse topography of the chicken ASIC1 intracellular domains determined by FRET, measured using either fluorescent lifetime imaging or patch clamp fluorometry. We find the C terminal tail projects into the cytosol by approximately 35 Å and that the N and C tail from the same subunits are closer than adjacent subunits. Using pH-insensitive fluorescent proteins, we fail to detect any relative movement between the N and C tails upon extracellular acidification but do observe axial motions of the membrane proximal segments towards the plasma membrane. Taken together, our study furnishes a coarse topographic map of the ASIC intracellular domains while providing directionality and context to intracellular conformational changes induced by extracellular acidification.

Single-molecule imaging with cell-derived nanovesicles reveals early binding dynamics at a cyclic nucleotide-gated ion channel

Ligand binding to membrane proteins is critical for many biological signaling processes. However, individual binding events are rarely directly observed, and their asynchronous dynamics are occluded in ensemble-averaged measures. For membrane proteins, single-molecule approaches that resolve these dynamics are challenged by dysfunction in nonnative lipid environments, lack of access to intracellular sites, and costly sample preparation. Here, we introduce an approach combining cell-derived nanovesicles, microfluidics, and single-molecule fluorescence colocalization microscopy to track individual binding events at a cyclic nucleotide-gated TAX-4 ion channel critical for sensory transduction. Our observations reveal dynamics of both nucleotide binding and a subsequent conformational change likely preceding pore opening. We further show that binding of the second ligand in the tetrameric channel is less cooperative than previously estimated from ensemble-averaged binding measures. This approach is broadly applicable to studies of binding dynamics for proteins with extracellular or intracellular domains in native cell membrane.

Chimeric human opsins as optogenetic light sensitisers

Human opsin-based photopigments have great potential as light-sensitisers, but their requirement for phototransduction cascade-specific second messenger proteins may restrict their functionality in non-native cell types. In this study, eight chimeric human opsins were generated consisting of a backbone of either a rhodopsin (RHO) or long-wavelength-sensitive (LWS) opsin and intracellular domains from Gq/11-coupled human melanopsin. Rhodopsin/melanopsin chimeric opsins coupled to both Gi and Gq/11 pathways. Greater substitution of the intracellular surface with corresponding melanopsin domains generally showed greater Gq/11 activity with a decrease in Gi activation. Unlike melanopsin, rhodopsin and rhodopsin/melanopsin chimeras were dependent upon exogenous chromophore to function. By contrast, wild type LWS opsin and LWS/melanopsin chimeras showed only weak Gi activation in response to light, whilst Gq/11 pathway activation was not detected. Immunocytochemistry demonstrated that chimeric opsins with more intracellular domains of melanopsin were less likely to be trafficked to the plasma membrane. This study demonstrates the importance of Gα coupling efficiency to the speed of cellular responses and created human opsins with a unique combination of properties to expand the range of customised optogenetic biotools for basic research and translational therapies.

New Structural Perspectives in G Protein-Coupled Receptor-Mediated Src Family Kinase Activation

Src family kinases (SFKs) are key regulators of cell proliferation, differentiation, and survival. The expression of these non-receptor tyrosine kinases is strongly correlated with cancer development and tumor progression. Thus, this family of proteins serves as an attractive drug target. The activation of SFKs can occur via multiple signaling pathways, yet many of them are poorly understood. Here, we summarize the current knowledge on G protein-coupled receptor (GPCR)-mediated regulation of SFKs, which is of considerable interest because GPCRs are among the most widely used pharmaceutical targets. This type of activation can occur through a direct interaction between the two proteins or be allosterically regulated by arrestins and G proteins. We postulate that a rearrangement of binding motifs within the active conformation of arrestin-3 mediates Src regulation by comparison of available crystal structures. Therefore, we hypothesize a potentially different activation mechanism compared to arrestin-2. Furthermore, we discuss the probable direct regulation of SFK by GPCRs and investigate the intracellular domains of exemplary GPCRs with conserved polyproline binding motifs that might serve as scaffolding domains to allow such a direct interaction. Large intracellular domains in GPCRs are often understudied and, in general, not much is known of their contribution to different signaling pathways. The suggested direct interaction between a GPCR and a SFK could allow for a potential immediate allosteric regulation of SFKs by GPCRs and thereby unravel a novel mechanism of SFK signaling. This overview will help to identify new GPCR–SFK interactions, which could serve to explain biological functions or be used to modulate downstream effectors.

An Inside Job: Molecular Determinants for Postsynaptic Localization of Nicotinic Acetylcholine Receptors

Nicotinic acetylcholine receptors (nAChRs) mediate fast synaptic transmission at neuromuscular and autonomic ganglionic synapses in the peripheral nervous system. The postsynaptic localization of muscle ((α1)2β1γδ) and neuronal ((α3β4)2β4) nicotinic receptors at these synapses is mediated by interactions between the nAChR intracellular domains and cytoplasmic scaffolding proteins. Recent high resolution structures and functional studies provide new insights into the molecular determinants that mediate these interactions. Surprisingly, they reveal that the muscle nAChR binds 1–3 rapsyn scaffolding molecules, which dimerize and thereby form an interconnected lattice between receptors. Moreover, rapsyn binds two distinct sites on the nAChR subunit cytoplasmic loops; the MA-helix on one or more subunits and a motif specific to the β subunit. Binding at the latter site is regulated by agrin-induced phosphorylation of βY390, and increases the stoichiometry of rapsyn/AChR complexes. Similarly, the neuronal nAChR may be localized at ganglionic synapses by phosphorylation-dependent interactions with 14-3-3 adaptor proteins which bind specific motifs in each of the α3 subunit cytoplasmic loops. Thus, postsynaptic localization of nAChRs is mediated by regulated interactions with multiple scaffolding molecules, and the stoichiometry of these complexes likely helps regulate the number, density, and stability of receptors at the synapse.

Enhancing and inhibitory motifs have coevolved to regulate CD4 activity

SUMMARYCD4+ T cells use T cell receptor (TCR)-CD3 complexes, and CD4, to respond to peptide antigens within MHCII molecules (pMHCII). We report here that, through ∼435 million years of evolution in jawed vertebrates, purifying selection has shaped motifs in the extracellular, transmembrane, and intracellular domains of eutherian CD4 that both enhance pMHCII responses and are coevolving with residues in an intracellular motif that inhibits pMHCII responses. Importantly, while CD4 interactions with the Src kinase, Lck, are classically viewed as the key determinant of CD4’s contribution to pMHCII responses, we found that without the inhibitory motif CD4-Lck interactions are not necessary for robust responses to pMHCII. In summary, motifs that mediate events on the outside and inside of CD4+ T cells coevolved to finetune the relay of pMHCII-specific information across the membrane. These results have implications for the evolution and function of complex transmembrane receptors and for biomimetic engineering.

Smoothened transduces hedgehog signals via activity-dependent sequestration of PKA catalytic subunits

The Hedgehog (Hh) pathway is essential for organ development, homeostasis, and regeneration. Dysfunction of this cascade drives several cancers. To control expression of pathway target genes, the G protein–coupled receptor (GPCR) Smoothened (SMO) activates glioma-associated (GLI) transcription factors via an unknown mechanism. Here, we show that, rather than conforming to traditional GPCR signaling paradigms, SMO activates GLI by binding and sequestering protein kinase A (PKA) catalytic subunits at the membrane. This sequestration, triggered by GPCR kinase (GRK)-mediated phosphorylation of SMO intracellular domains, prevents PKA from phosphorylating soluble substrates, releasing GLI from PKA-mediated inhibition. Our work provides a mechanism directly linking Hh signal transduction at the membrane to GLI transcription in the nucleus. This process is more fundamentally similar between species than prevailing hypotheses suggest. The mechanism described here may apply broadly to other GPCR- and PKA-containing cascades in diverse areas of biology.

A pathogen-encoded signaling receptor mediating host-like interactions through intrinsic disorder

The translocated intimin receptor (Tir) is a central effector of Attaching and Effacing (A/E) pathogens responsible for worldwide foodborne disease cases. Upon delivery into host cells, Tir acts as a cell-signaling receptor, rewiring host cellular processes to assist infection. We found that this bacterial-encoded transmembrane protein comprises highly disordered intracellular domains bearing host-like motifs that bind host proteins. This unexpected trait was found prevalent in several other effectors secreted by A/E bacteria. We assessed Tir’s intrinsic disorder by an integrative structural biophysics approach, unveiling that its intracellular side comprises a partially structured N-terminal dimer (N-Tir) and a disordered C-terminal tail (C-Tir). NMR analysis revealed that C-Tir has pre-existing transient structures at phosphorylation sites, including host-like immunoreceptor tyrosine-based inhibitory motifs (ITIMs). These ITIM-like sequences were found to bind lipid bilayers as previously observed for host T-cell receptor cytoplasmic disordered domains. C-Tir’s membrane affinity is residue-specific and modulated by lipid composition, suggesting a regulation layer based on membrane composition. Using NMR, we also observed that the disordered C-Tir displays multisite tyrosine-phosphorylation sites that mediate promiscuous binding to the C-terminal SH2 domain of host SHP-1 in dynamic equilibrium. Together, these novel insights provide an updated picture of Tir’s structural features and highlight Tir-mediated mimicry of host disordered membrane receptors as a molecular strategy for host cell subversion.SummaryTir is a cellular receptor secreted by life-threatening pathogens. Upon delivery into host cells, Tir inserts the host plasma membrane providing a means for these extracellular pathogens to control host intracellular processes. To prevent pathogens from relying on Tir, it is essential to understand its intracellular mechanics. This paper provides a coherent picture of the intracellular side of Tir, highlighting its ability to copycat the interactions of disordered intracellular domains of host immune receptors. This copycatting allows the bacterial pathogens to modulate critical host processes, allowing infection to spread further without triggering the immune system response. This work proposes that other bacterial secreted pathogenic proteins exploit intrinsic disorder to hijack human cells, suggesting a widespread host subversion mechanism.