A Trigger Residue for Transmembrane Signaling in the Escherichia coli Serine Chemoreceptor.

UNLABELLED: The transmembrane Tsr protein of Escherichia coli mediates chemotactic responses to environmental serine gradients. Serine binds to the periplasmic domain of the homodimeric Tsr molecule, promoting a small inward displacement of one transmembrane helix (TM2). TM2 piston displacements, in turn, modulate the structural stability of the Tsr-HAMP domain on the cytoplasmic side of the membrane to control the autophosphorylation activity of the signaling CheA kinase bound to the membrane-distal cytoplasmic tip of Tsr. A five-residue control cable segment connects TM2 to the AS1 helix of HAMP and transmits stimulus and sensory adaptation signals between them. To explore the possible role of control cable helicity in transmembrane signaling by Tsr, we characterized the signaling properties of mutant receptors with various control cable alterations. An all-alanine control cable shifted Tsr output toward the kinase-on state, whereas an all-glycine control cable prevented Tsr from reaching either a fully on or fully off output state. Restoration of the native isoleucine (I214) in these synthetic control cables largely alleviated their signaling defects. Single amino acid replacements at Tsr-I214 shifted output toward the kinase-off (L, N, H, and R) or kinase-on (A and G) states, whereas other control cable residues tolerated most amino acid replacements with little change in signaling behavior. These findings indicate that changes in control cable helicity might mediate transitions between the kinase-on and kinase-off states during transmembrane signaling by chemoreceptors. Moreover, the Tsr-I214 side chain plays a key role, possibly through interaction with the membrane interfacial environment, in triggering signaling changes in response to TM2 piston displacements. IMPORTANCE: The Tsr protein of E. coli mediates chemotactic responses to environmental serine gradients. Stimulus signals from the Tsr periplasmic sensing domain reach its cytoplasmic kinase control domain through piston displacements of a membrane-spanning helix and an adjoining five-residue control cable segment. We characterized the signaling properties of Tsr variants to elucidate the transmembrane signaling role of the control cable, an element present in many microbial sensory proteins. Both the kinase-on and kinase-off output states of Tsr depended on control cable helicity, but only one residue, I214, was critical for triggering responses to attractant inputs. These findings suggest that signal transmission in Tsr involves modulation of control cable helicity through interaction of the I214 side chain with the cytoplasmic membrane.

HAMP domain structural determinants for signalling and sensory adaptation in Tsr, the Escherichia coli serine chemoreceptor.

HAMP domains mediate input-output transactions in many bacterial signalling proteins. To clarify the mechanistic logic of HAMP signalling, we constructed Tsr-HAMP deletion derivatives and characterized their steady-state signal outputs and sensory adaptation properties with flagellar rotation and receptor methylation assays. Tsr molecules lacking the entire HAMP domain or just the HAMP-AS2 helix generated clockwise output signals, confirming that kinase activation is the default output state of the chemoreceptor signalling domain and that attractant stimuli shift HAMP to an overriding kinase-off signalling state to elicit counter-clockwise flagellar responses. Receptors with deletions of the AS1 helices, which free the AS2 helices from bundle-packing constraints, exhibited kinase-off signalling behaviour that depended on three C-terminal hydrophobic residues of AS2. We conclude that AS2/AS2' packing interactions alone can play an important role in controlling output kinase activity. Neither kinase-on nor kinase-off HAMP deletion outputs responded to sensory adaptation control, implying that out-of-range conformations or bundle-packing stabilities of their methylation helices prevent substrate recognition by the adaptation enzymes. These observations support the previously proposed biphasic, dynamic-bundle mechanism of HAMP signalling and additionally show that the structural interplay of helix-packing interactions between HAMP and the adjoining methylation helices is critical for sensory adaptation control of receptor output.

The mobility of two kinase domains in the Escherichia coli chemoreceptor array varies with signalling state.

Motile bacteria sense their physical and chemical environment through highly cooperative, ordered arrays of chemoreceptors. These signalling complexes phosphorylate a response regulator which in turn governs flagellar motor reversals, driving cells towards favourable environments. The structural changes that translate chemoeffector binding into the appropriate kinase output are not known. Here, we apply high-resolution electron cryotomography to visualize mutant chemoreceptor signalling arrays in well-defined kinase activity states. The arrays were well ordered in all signalling states, with no discernible differences in receptor conformation at 2-3 nm resolution. Differences were observed, however, in a keel-like density that we identify here as CheA kinase domains P1 and P2, the phosphorylation site domain and the binding domain for response regulator target proteins. Mutant receptor arrays with high kinase activities all exhibited small keels and high proteolysis susceptibility, indicative of mobile P1 and P2 domains. In contrast, arrays in kinase-off signalling states exhibited a range of keel sizes. These findings confirm that chemoreceptor arrays do not undergo large structural changes during signalling, and suggest instead that kinase activity is modulated at least in part by changes in the mobility of key domains.

Biphasic control logic of HAMP domain signalling in the Escherichia coli serine chemoreceptor.

HAMP domains mediate input-output communication in many bacterial signalling proteins. To explore the dynamic bundle model of HAMP signalling (Zhou et al., Mol. Microbiol. 73: 801, 2009), we characterized the signal outputs of 118 HAMP missense mutants of the serine chemoreceptor, Tsr, by flagellar rotation patterns. Receptors with proline or charged amino acid replacements at critical hydrophobic packing residues in the AS1 and AS2 HAMP helices had locked kinase-off outputs, indicating that drastic destabilization of the Tsr-HAMP bundle prevents kinase activation, both in the absence and presence of the sensory adaptation enzymes, CheB and CheR. Attractant-mimic lesions that enhance the structural stability of the HAMP bundle also suppressed kinase activity, demonstrating that Tsr-HAMP has two kinase-off output states at opposite extremes of its stability range. HAMP mutants with locked-on kinase outputs appeared to have intermediate bundle stabilities, implying a biphasic relationship between HAMP stability and kinase activity. Some Tsr-HAMP mutant receptors exhibited reversed output responses to CheB and CheR action that are readily explained by a biphasic control logic. The findings of this study provide strong support for a three-state dynamic bundle model of HAMP signalling in Tsr, and possibly in other bacterial transducers as well.

Mutational analysis of the control cable that mediates transmembrane signaling in the Escherichia coli serine chemoreceptor.

During transmembrane signaling by Escherichia coli Tsr, changes in ligand occupancy in the periplasmic serine-binding domain promote asymmetric motions in a four-helix transmembrane bundle. Piston displacements of the signaling TM2 helix in turn modulate the HAMP bundle on the cytoplasmic side of the membrane to control receptor output signals to the flagellar motors. A five-residue control cable joins TM2 to the HAMP AS1 helix and mediates conformational interactions between them. To explore control cable structural features important for signal transmission, we constructed and characterized all possible single amino acid replacements at the Tsr control cable residues. Only a few lesions abolished Tsr function, indicating that the chemical nature and size of the control cable side chains are not individually critical for signal control. Charged replacements at I214 mimicked the signaling consequences of attractant or repellent stimuli, most likely through aberrant structural interactions of the mutant side chains with the membrane interfacial environment. Prolines at residues 214 to 217 also caused signaling defects, suggesting that the control cable has helical character. However, proline did not disrupt function at G213, the first control cable residue, which might serve as a structural transition between the TM2 and AS1 helix registers. Hydrophobic amino acids at S217, the last control cable residue, produced attractant-mimic effects, most likely by contributing to packing interactions within the HAMP bundle. These results suggest a helix extension mechanism of Tsr transmembrane signaling in which TM2 piston motions influence HAMP stability by modulating the helicity of the control cable segment.

Mutational analyses of HAMP helices suggest a dynamic bundle model of input-output signalling in chemoreceptors.

To test the gearbox model of HAMP signalling in the Escherichia coli serine receptor, Tsr, we generated a series of amino acid replacements at each residue of the AS1 and AS2 helices. The residues most critical for Tsr function defined hydrophobic packing faces consistent with a four-helix bundle. Suppression patterns of helix lesions conformed to the predicted packing layers in the bundle. Although the properties and patterns of most AS1 and AS2 lesions were consistent with both proposed gearbox structures, some mutational features specifically indicate the functional importance of an x-da bundle over an alternative a-d bundle. These genetic data suggest that HAMP signalling could simply involve changes in the stability of its x-da bundle. We propose that Tsr HAMP controls output signals by modulating destabilizing phase clashes between the AS2 helices and the adjoining kinase control helices. Our model further proposes that chemoeffectors regulate HAMP bundle stability through a control cable connection between the transmembrane segments and AS1 helices. Attractant stimuli, which cause inward piston displacements in chemoreceptors, should reduce cable tension, thereby stabilizing the HAMP bundle. This study shows how transmembrane signalling and HAMP input-output control could occur without the helix rotations central to the gearbox model.

Complete structure of the chemosensory array core signalling unit in an E. coli minicell strain.

Motile bacteria sense chemical gradients with transmembrane receptors organised in supramolecular signalling arrays. Understanding stimulus detection and transmission at the molecular level requires precise structural characterisation of the array building block known as a core signalling unit. Here we introduce an Escherichia coli strain that forms small minicells possessing extended and highly ordered chemosensory arrays. We use cryo-electron tomography and subtomogram averaging to provide a three-dimensional map of a complete core signalling unit, with visible densities corresponding to the HAMP and periplasmic domains. This map, combined with previously determined high resolution structures and molecular dynamics simulations, yields a molecular model of the transmembrane core signalling unit and enables spatial localisation of its individual domains. Our work thus offers a solid structural basis for the interpretation of a wide range of existing data and the design of further experiments to elucidate signalling mechanisms within the core signalling unit and larger array.

THE EFFECT OF AN ANTI-GRAVITY TREADMILL ON RUNNING CADENCE.

BACKGROUND: Running cadence, or step rate, is often measured in running gait analysis and manipulated in gait retraining. A lower body positive pressure treadmill, or anti-gravity treadmill, allows users to walk/run in a reduced gravity environment. PURPOSE: The primary purpose of this study was to determine how natural running cadence is affected by running on an anti-gravity treadmill compared to a standard treadmill in a healthy, active population. The secondary purpose was to determine if natural and increased cadence is affected by amount of body weight support. STUDY DESIGN: Cross-sectional study (convenience sample). METHODS: Thirty participants were recruited to run on an anti-gravity treadmill (AlterG Anti-Gravity TreadmillTM M320) at their pre-determined, self-selected, comfortable treadmill speed. Cadence was recorded at nine randomized bodyweight conditions, ranging from 100% of body weight to 20% of body weight, in 10% increments. An additional nine participants were recruited to try to replicate their natural, standard treadmill cadence, as well as increase it by 5% and 10%, while on an anti-gravity treadmill with the same randomized body weight conditions. RESULTS: Thirty participants, 19 females and 11 males, mean age 27.3 years (range, 22-45), completed Part 1 of the study protocol, while nine additional participants (2 females and 7 males) with a mean age of 29.6 years old (range, 25-40 years) completed Part 2 of the protocol. There was a significant effect of natural running cadence on the anti-gravity treadmill at reduced body weight percentages (p<.01). Post-hoc t-tests revealed that every 10% bodyweight interval was significantly lower than the previous 10% interval (p<.01) on the anti-gravity treadmill, with cadence decreases ranging from 1.5%-3.5% between intervals. Seven of the nine (77.8%) participants in Part 2 were able to replicate and increase their cadence at all body weight levels on the anti-gravity treadmill. CONCLUSIONS: Decreasing bodyweight level on an anti-gravity treadmill yields a significant and linear decrease in running cadence when performed at a self-selected, moderate intensity pace. Further, the vast majority of participants were able to successfully replicate and increase cadence at all levels of bodyweight percentage.

In Situ Conformational Changes of the Escherichia coli Serine Chemoreceptor in Different Signaling States.

Tsr, the serine chemoreceptor in Escherichia coli, transduces signals from a periplasmic ligand-binding site to its cytoplasmic tip, where it controls the activity of the CheA kinase. To function, Tsr forms trimers of homodimers (TODs), which associate in vivo with the CheA kinase and CheW coupling protein. Together, these proteins assemble into extended hexagonal arrays. Here, we use cryo-electron tomography and molecular dynamics simulation to study Tsr in the context of a near-native array, characterizing its signaling-related conformational changes at both the individual dimer and the trimer level. In particular, we show that individual Tsr dimers within a trimer exhibit asymmetric flexibilities that are a function of the signaling state, highlighting the effect of their different protein interactions at the receptor tips. We further reveal that the dimer compactness of the Tsr trimer changes between signaling states, transitioning at the glycine hinge from a compact conformation in the kinase-OFF state to an expanded conformation in the kinase-ON state. Hence, our results support a crucial role for the glycine hinge: to allow the receptor flexibility necessary to achieve different signaling states while also maintaining structural constraints imposed by the membrane and extended array architecture.IMPORTANCE In Escherichia coli, membrane-bound chemoreceptors, the histidine kinase CheA, and coupling protein CheW form highly ordered chemosensory arrays. In core signaling complexes, chemoreceptor trimers of dimers undergo conformational changes, induced by ligand binding and sensory adaptation, which regulate kinase activation. Here, we characterize by cryo-electron tomography the kinase-ON and kinase-OFF conformations of the E. coli serine receptor in its native array context. We found distinctive structural differences between the members of a receptor trimer, which contact different partners in the signaling unit, and structural differences between the ON and OFF signaling complexes. Our results provide new insights into the signaling mechanism of chemoreceptor arrays and suggest an important functional role for a previously postulated flexible region and glycine hinge in the receptor molecule.

Mutational analysis of the connector segment in the HAMP domain of Tsr, the Escherichia coli serine chemoreceptor.

HAMP domains are approximately 50-residue motifs, found in many bacterial signaling proteins, that consist of two amphiphilic helices joined by a nonhelical connector segment. The HAMP domain of Tsr, the serine chemoreceptor of Escherichia coli, receives transmembrane input signals from the periplasmic serine binding domain and in turn modulates output signals from the Tsr kinase control domain to elicit chemotactic responses. We created random amino acid replacements at each of the 14 connector residues of Tsr-HAMP to identify those that are critical for Tsr function. In all, we surveyed 179 connector missense mutants and identified three critical residues (G235, L237, and I241) at which most replacements destroyed Tsr function and another important residue (G245) at which most replacements impaired Tsr function. The region surrounding G245 tolerated 1-residue deletions and insertions of up to 10 glycines, suggesting a role as a relatively nonspecific, flexible linker. The critical connector residues are consistent with a structural model of the Tsr-HAMP domain based on the solution structure of an isolated thermophile HAMP domain (M. Hulko, F. Berndt, M. Gruber, J. U. Linder, V. Truffault, A. Schultz, J. Martin, J. E. Schultz, A. N. Lupas, and M. Coles, Cell 126:929-940, 2006) in which G235 defines a critical turn at the C terminus of the first helix and L237 and I241 pack against the helices, perhaps to stabilize alternative HAMP signaling conformations. Most I241 lesions locked Tsr signal output in the kinase-on mode, implying that this residue is responsible mainly for stabilizing the kinase-off signaling state. In contrast, lesions at L237 resulted in a variety of aberrant output patterns, suggesting a role in toggling output between signaling states.

All-Codon Mutagenesis for Structure-Function Studies of Chemotaxis Signaling Proteins.

The technique of all-codon mutagenesis can generate mutants that represent all possible amino acid replacements at any particular residue in a protein. It is thus a powerful tool to probe structure-function relationships in proteins of interest. In this chapter, we describe how we used all-codon mutagenesis to obtain mutants of the Escherichia coli serine receptor Tsr with amino acid replacements at residue F373, a functionally important site in this protein. We provide general protocols for mutagenesis of a target codon in a plasmid-borne gene and for the selection and screening of the resultant mutants. These techniques should be adaptable for the study of a variety of bacterial proteins.

Phenotypic suppression methods for analyzing intra- and inter-molecular signaling interactions of chemoreceptors.

The receptors that mediate chemotactic behaviors in E. coli and other motile bacteria and archaea are exquisite molecular machines. They detect minute concentration changes in the organism's chemical environment, integrate multiple stimulus inputs, and generate a highly amplified output signal that modulates the cell's locomotor pattern. Genetic dissection and suppression analyses have played an important role in elucidating the molecular mechanisms that underlie chemoreceptor signaling. This chapter discusses three examples of phenotypic suppression analyses of receptor signaling defects. (i) Balancing suppression can occur in mutant receptors that have biased output signals and involves second-site mutations that create an offsetting bias change. Such suppressors can arise in many parts of the receptor and need not involve directly interacting parts of the molecule. (ii) Conformational suppression within a mutant receptor molecule occurs through a mutation that directly compensates for the initial structural defect. This form of suppression should be highly dependent on the nature of the structural alterations caused by the original mutation and its suppressor, but in practice may be difficult to distinguish from balancing suppression without high-resolution structural information about the mutant and pseudorevertant proteins. (iii) Conformational suppression between receptor molecules involves correction of a functional defect in one receptor by a mutational change in a heterologous receptor with which it normally interacts. The suppression patterns exhibit allele-specificity with respect to the compensatory residue positions and amino acid side chains, a hallmark of stereospecific protein-protein interactions.

Collaborative signaling by bacterial chemoreceptors.

Motile bacteria seek optimal living habitats by following gradients of attractant and repellent chemicals in their environment. The signaling machinery for these chemotactic behaviors, although assembled from just a few protein components, has extraordinary information-processing capabilities. Escherichia coli, the best-studied model, employs a networked cluster of transmembrane receptors to detect minute chemical stimuli, to integrate multiple and conflicting inputs, and to generate an amplified output signal that controls the cell's flagellar motors. Signal gain arises through cooperative action of chemoreceptors of different types. The signaling-teams within a receptor cluster may be built from trimers of receptor dimers that communicate through shared connections to their partner signaling proteins.

Evidence for a Helix-Clutch Mechanism of Transmembrane Signaling in a Bacterial Chemoreceptor.

The Escherichia coli Tsr protein contains a periplasmic serine-binding domain that transmits ligand occupancy information to a cytoplasmic kinase-control domain to regulate the cell's flagellar motors. The Tsr input and output domains communicate through conformational changes transmitted through a transmembrane helix (TM2), a five-residue control cable helix at the membrane-cytoplasm interface, and a four-helix HAMP bundle. Changes in serine occupancy are known to promote TM2 piston displacements in one subunit of the Tsr homodimer. We explored how such piston motions might be relayed through the control cable to reach the input AS1 helix of HAMP by constructing and characterizing mutant receptors that had one-residue insertions or deletions in the TM2-control cable segment of Tsr. TM2 deletions caused kinase-off output shifts; TM2 insertions caused kinase-on shifts. In contrast, control cable deletions caused kinase-on output, whereas insertions at the TM2-control cable junction caused kinase-off output. These findings rule out direct mechanical transmission of TM2 conformational changes to HAMP. Instead, we suggest that the Tsr control cable transmits input signals to HAMP by modulating the intensity of structural clashes between out-of-register TM2 and AS1 helices. Inward displacement of TM2 might alter the sidechain environment of control cable residues at the membrane core-headgroup interface, causing a break in the control cable helix to attenuate the register mismatch and enhance HAMP packing stability, leading to a kinase-off output response. This helix-clutch model offers a new perspective on the mechanism of transmembrane signaling in chemoreceptors.

Chemotactic Signaling by Single-Chain Chemoreceptors.

Bacterial chemoreceptors of the methyl-accepting chemotaxis protein (MCP) family operate in commingled clusters that enable cells to detect and track environmental chemical gradients with high sensitivity and precision. MCP homodimers of different detection specificities form mixed trimers of dimers that facilitate inter-receptor communication in core signaling complexes, which in turn assemble into a large signaling network. The two subunits of each homodimeric receptor molecule occupy different locations in the core complexes. One subunit participates in trimer-stabilizing interactions at the trimer axis, the other lies on the periphery of the trimer, where it can interact with two cytoplasmic proteins: CheA, a signaling autokinase, and CheW, which couples CheA activity to receptor control. As a possible tool for independently manipulating receptor subunits in these two structural environments, we constructed and characterized fused genes for the E. coli serine chemoreceptor Tsr that encoded single-chain receptor molecules in which the C-terminus of the first Tsr subunit was covalently connected to the N-terminus of the second with a polypeptide linker. We showed with soft agar assays and with a FRET-based in vivo CheA kinase assay that single-chain Tsr~Tsr molecules could promote serine sensing and chemotaxis responses. The length of the connection between the joined subunits was critical. Linkers nine residues or shorter locked the receptor in a kinase-on state, most likely by distorting the native structure of the receptor HAMP domain. Linkers 22 or more residues in length permitted near-normal Tsr function. Few single-chain molecules were found as monomer-sized proteolytic fragments in cells, indicating that covalently joined receptor subunits were responsible for mediating the signaling responses we observed. However, cysteine-directed crosslinking, spoiling by dominant-negative Tsr subunits, and rearrangement of ligand-binding site lesions revealed subunit swapping interactions that will need to be taken into account in experimental applications of single-chain chemoreceptors.

A phenylalanine rotameric switch for signal-state control in bacterial chemoreceptors.

Bacterial chemoreceptors are widely used as a model system for elucidating the molecular mechanisms of transmembrane signalling and have provided a detailed understanding of how ligand binding by the receptor modulates the activity of its associated kinase CheA. However, the mechanisms by which conformational signals move between signalling elements within a receptor dimer and how they control kinase activity remain unknown. Here, using long molecular dynamics simulations, we show that the kinase-activating cytoplasmic tip of the chemoreceptor fluctuates between two stable conformations in a signal-dependent manner. A highly conserved residue, Phe396, appears to serve as the conformational switch, because flipping of the stacked aromatic rings of an interacting F396-F396' pair in the receptor homodimer takes place concomitantly with the signal-related conformational changes. We suggest that interacting aromatic residues, which are common stabilizers of protein tertiary structure, might serve as rotameric molecular switches in other biological processes as well.

Conformational suppression of inter-receptor signaling defects.

Motile bacteria follow gradients of attractant and repellent chemicals with high sensitivity. Their chemoreceptors are physically clustered, which may enable them to function as a cooperative array. Although native chemoreceptor molecules are typically transmembrane homodimers, they appear to associate through their cytoplasmic tips to form trimers of dimers, which may be an important architectural element in the assembly and operation of receptor clusters. The five receptors of Escherichia coli that mediate most of its chemotactic and aerotactic behaviors have identical trimer contact residues and have been shown by in vivo crosslinking methods to form mixed trimers of dimers. Mutations at the trimer contact sites of Tsr, the serine chemoreceptor, invariably abrogate Tsr function, but some of those lesions (designated Tsr*) are epistatic and block the function of heterologous chemoreceptors. We isolated and characterized mutations (designated Tar()) in the aspartate chemoreceptor that restored function to Tsr* receptors. The suppressors arose at or near the Tar trimer contact sites and acted in an allele-specific fashion on Tsr* partners. Alone, many Tar() receptors were unable to mediate chemotactic responses to aspartate, but all formed clusters with varying efficiencies. Most of those Tar() receptors were epistatic to WT Tsr, but some regained Tar function in combination with a suppressible Tsr* partner. Tar()-Tsr* suppression most likely occurs through compensatory changes in the conformation or dynamics of a mixed receptor signaling complex, presumably based on trimer-of-dimer interactions. These collaborative teams may be responsible for the high-gain signaling properties of bacterial chemoreceptors.

Collaborative signaling by mixed chemoreceptor teams in Escherichia coli.

Chemoreceptors of the methyl-accepting chemotaxis protein family form clusters, typically at the cell pole(s), in both Bacteria and Archaea. To elucidate the architecture and signaling role of receptor clusters, we investigated interactions between the serine (Tsr) and aspartate (Tar) chemoreceptors in Escherichia coli by constructing Tsr mutations at the six hydrophobic and five polar residues implicated in "trimer of dimers" formation. Tsr mutants with proline replacements could not mediate serine chemotaxis, receptor clustering, or clockwise flagellar rotation. Alanine and tryptophan mutants, although also nonchemotactic, formed receptor clusters, and some produced clockwise flagellar rotation, indicating receptor-coupled activation of the signaling CheA kinase. The alanine and tryptophan mutants evidently assemble defective receptor complexes that cannot modulate CheA activity in response to serine stimuli. In cells containing wild-type Tar receptors, tryptophan replacements in Tsr interfered with Tar function, whereas four Tsr mutants with alanine replacements regained Tsr function. These epistatic and rescuable phenotypes imply interactions between Tsr and Tar dimers in higher-order signaling teams. The bulky side chain in tryptophan mutants may prevent stimulus-induced conformational changes in the team, whereas the small side chain in alanine mutants may permit signaling control when teamed with functional receptor molecules. Direct physical interactions between Tsr and Tar molecules were observed by in vivo chemical crosslinking. Wild-type Tsr crosslinked to Tar, whereas a clustering-defective proline replacement mutant did not. These findings indicate that bacterial chemoreceptor clusters are comprised of signaling teams, seemingly based on trimers of dimers, that can contain different receptor types acting collaboratively.