A role for neuronal piRNAs in the epigenetic control of memory-related synaptic plasticity.

Small RNA-mediated gene regulation during development causes long-lasting changes in cellular phenotypes. To determine whether small RNAs of the adult brain can regulate memory storage, a process that requires stable and long-lasting changes in the functional state of neurons, we generated small RNA libraries from the Aplysia CNS. In these libraries, we discovered an unexpectedly abundant expression of a 28 nucleotide sized class of piRNAs in brain, which had been thought to be germline specific. These piRNAs have unique biogenesis patterns, predominant nuclear localization, and robust sensitivity to serotonin, a modulatory transmitter that is important for memory. We find that the Piwi/piRNA complex facilitates serotonin-dependent methylation of a conserved CpG island in the promoter of CREB2, the major inhibitory constraint of memory in Aplysia, leading to enhanced long-term synaptic facilitation. These findings provide a small RNA-mediated gene regulatory mechanism for establishing stable long-term changes in neurons for the persistence of memory.

Critical role of amyloid-like oligomers of Drosophila Orb2 in the persistence of memory.

A long-standing question in the study of long-term memory is how a memory trace persists for years when the proteins that initiated the process turn over and disappear within days. Previously, we postulated that self-sustaining amyloidogenic oligomers of cytoplasmic polyadenylation element-binding protein (CPEB) provide a mechanism for the maintenance of activity-dependent synaptic changes and, thus, the persistence of memory. Here, we found that the Drosophila CPEB Orb2 forms amyloid-like oligomers, and oligomers are enriched in the synaptic membrane fraction. Of the two protein isoforms of Orb2, the amyloid-like oligomer formation is dependent on the Orb2A form. A point mutation in the prion-like domain of Orb2A, which reduced amyloid-like oligomerization of Orb2, did not interfere with learning or memory persisting up to 24 hr. However the mutant flies failed to stabilize memory beyond 48 hr. These results support the idea that amyloid-like oligomers of neuronal CPEB are critical for the persistence of long-term memory.

Aplysia CPEB can form prion-like multimers in sensory neurons that contribute to long-term facilitation.

Prions are proteins that can assume at least two distinct conformational states, one of which is dominant and self-perpetuating. Previously we found that a translation regulator CPEB from Aplysia, ApCPEB, that stabilizes activity-dependent changes in synaptic efficacy can display prion-like properties in yeast. Here we find that, when exogenously expressed in sensory neurons, ApCPEB can form an amyloidogenic self-sustaining multimer, consistent with it being a prion-like protein. In addition, we find that conversion of both the exogenous and the endogenous ApCPEB to the multimeric state is enhanced by the neurotransmitter serotonin and that an antibody that recognizes preferentially the multimeric ApCPEB blocks persistence of synaptic facilitation. These results are consistent with the idea that ApCPEB can act as a self-sustaining prion-like protein in the nervous system and thereby might allow the activity-dependent change in synaptic efficacy to persist for long periods of time.

Essential role of coiled coils for aggregation and activity of Q/N-rich prions and PolyQ proteins.

The functional switch of glutamine/asparagine (Q/N)-rich prions and the neurotoxicity of polyQ-expanded proteins involve complex aggregation-prone structural transitions, commonly presumed to be forming ß sheets. By analyzing sequences of interaction partners of these proteins, we discovered a recurrent presence of coiled-coil domains both in the partners and in segments that flank or overlap Q/N-rich and polyQ domains. Since coiled coils can mediate protein interactions and multimerization, we studied their possible involvement in Q/N-rich and polyQ aggregations. Using circular dichroism and chemical crosslinking, we found that Q/N-rich and polyQ peptides form α-helical coiled coils in vitro and assemble into multimers. Using structure-guided mutagenesis, we found that coiled-coil domains modulate in vivo properties of two Q/N-rich prions and polyQ-expanded huntingtin. Mutations that disrupt coiled coils impair aggregation and activity, whereas mutations that enhance coiled-coil propensity promote aggregation. These findings support a coiled-coil model for the functional switch of Q/N-rich prions and for the pathogenesis of polyQ-expansion diseases.

The evolution of synaptic and cognitive capacity: Insights from the nervous system transcriptome of Aplysia.

The gastropod mollusk Aplysia is an important model for cellular and molecular neurobiological studies, particularly for investigations of molecular mechanisms of learning and memory. We developed an optimized assembly pipeline to generate an improved Aplysia nervous system transcriptome. This improved transcriptome enabled us to explore the evolution of cognitive capacity at the molecular level. Were there evolutionary expansions of neuronal genes between this relatively simple gastropod Aplysia (20,000 neurons) and Octopus (500 million neurons), the invertebrate with the most elaborate neuronal circuitry and greatest behavioral complexity? Are the tremendous advances in cognitive power in vertebrates explained by expansion of the synaptic proteome that resulted from multiple rounds of whole genome duplication in this clade? Overall, the complement of genes linked to neuronal function is similar between Octopus and Aplysia. As expected, a number of synaptic scaffold proteins have more isoforms in humans than in Aplysia or Octopus. However, several scaffold families present in mollusks and other protostomes are absent in vertebrates, including the Fifes, Lev10s, SOLs, and a NETO family. Thus, whereas vertebrates have more scaffold isoforms from select families, invertebrates have additional scaffold protein families not found in vertebrates. This analysis provides insights into the evolution of the synaptic proteome. Both synaptic proteins and synaptic plasticity evolved gradually, yet the last deuterostome-protostome common ancestor already possessed an elaborate suite of genes associated with synaptic function, and critical for synaptic plasticity.

Authentication of a lophotrochozoan adipokinetic hormone receptor in a Gastropod, Aplysia californica.

Gonadotropin-releasing hormone (GnRH) superfamily comprises multiple families of signaling peptides in both protostomes and deuterostomes. Among this superfamily, vertebrate GnRH stimulates reproduction, but other GnRH superfamily members elicit diverse pleiotropic effects. Within the GnRH superfamily members, adipokinetic hormone (AKH) and its receptor are well described in ecdysozoans but understudied in other lineages. To fill this knowledge gap, we deorphanized a putative receptor for a lophotrochozoan AKH in a gastropod mollusk, Aplysia californica, and named it Aplca-AKHR. Phylogenetic analysis revealed an orthologous relationship of Aplca-AKHR with ecdysozoan AKHRs and other putative lophotrochozoan AKHRs. Aplca-AKHR bound specifically to the previously identified Aplca-AKH with high affinity and activated the inositol phosphate pathway. Aplca-AKHR was expressed widely among central and peripheral tissues, but most prominently in several central ganglia and the heart. The expression of Aplca-AKHR was downregulated by a hyposaline challenge, consistent with a role in volume and fluid regulation previously described for its ligand, Aplca-AKH. In summary, this is the first pairing of a lophotrochozoan AKH with its cognate receptor. Expression data further support diverse central and peripheral roles, including volume and fluid control, of this ligand/receptor pair.

AI protein structure prediction-based modeling and mutagenesis of a protostome receptor and peptide ligands reveal key residues for their interaction.

The protostome leucokinin (LK) signaling system, including LK peptides and their G protein-coupled receptors, has been characterized in several species. Despite the progress, molecular mechanisms governing LK peptide-receptor interactions remain to be elucidated. Previously, we identified a precursor protein for Aplysia leucokinin-like peptides (ALKs) that contains the greatest number of amidated peptides among LK precursors in all species identified so far. Here, we identified the first ALK receptor from Aplysia, ALKR. We used cell-based IP1 activation assays to demonstrate that two ALK peptides with the most copies, ALK1 and ALK2, activated ALKR with high potencies. Other endogenous ALK-derived peptides bearing the FXXWX-amide motif also activated ALKR to various degrees. Our examination of cross-species activity of ALKs with the Anopheles LK receptor was consistent with a critical role for the FXXWX-amide motif in receptor activity. Furthermore, we showed, through alanine substitution of ALK1, the highly conserved phenylalanine (F), tryptophan (W), and C-terminal amidation were each essential for receptor activation. Finally, we used an artificial intelligence-based protein structure prediction server (Robetta) and Autodock Vina to predict the ligand-bound conformation of ALKR. Our model predicted several interactions (i.e., hydrophobic interactions, hydrogen bonds, and amide-pi stacking) between ALK peptides and ALKR, and several of our substitution and mutagenesis experiments were consistent with the predicted model. In conclusion, our results provide important information defining possible interactions between ALK peptides and their receptors. The workflow utilized here may be useful for studying other ligand-receptor interactions for a neuropeptide signaling system, particularly in protostomes.

Structural and functional analysis of a tandem repeat galacturonic acid-binding lectin from the sea hare Aplysia californica.

A d-galacturonic acid-specific lectin, named AcL, was purified from the sea hare Aplysia californica by galactose-agarose affinity chromatography. AcL has a molecular mass of 27.5 kDa determined by MALDI-TOF mass spectrometry. This lectin shows a good affinity for d-galacturonic acid and a lower affinity for galactosides: raffinose, melibiose, α and ß-lactose, and d-galactose. We determined the amino acid sequence of AcL by trypsin digestion and subsequent peptide analysis by mass spectrometry, resulting in a 238 amino acid protein with a theoretical molecular mass of 26.4 kDa. The difference between the theoretical and experimental values can be attributed to post-translational modifications. Thiol-disulfide quantification discerned five disulfide bonds and three free cysteines. The structure of Acl is mainly comprised of beta sheets, determined by circular dichroism, and predicted with AlphaFold. Theoretical models depict three nearly identical tandem domains consisting of two beta sheets each. From docking analysis, we identified AcL glycan-binding sites as multiple conserved motifs in each domain. Furthermore, phylogenetic analysis based on its structure and sequence showed that AcL and its closest homologues (GalULs) form a clear monophyletic group, distinct from other glycan-binding proteins with a jelly-roll fold: lectins of types F and H. GalULs possess four conserved sequence regions that distinguish them and are either ligand-binding motifs or stabilizing network hubs. We suggest that this new family should be referred to as GalUL or D-type, following the traditional naming of lectins; D standing for depilans, the epithet for the species (Aplysia depilans) from which a lectin of this family was first isolated and described.

Dynein-mediated microtubule translocation powering neurite outgrowth in chick and Aplysia neurons requires microtubule assembly.

Previously, we have shown that bulk microtubule (MT) movement correlates with neurite elongation, and blocking either dynein activity or MT assembly inhibits both processes. However, whether the contributions of MT dynamics and dynein activity to neurite elongation are separate or interdependent is unclear. Here, we investigated the underlying mechanism by testing the roles of dynein and MT assembly in neurite elongation of Aplysia and chick neurites using time-lapse imaging, fluorescent speckle microscopy, super-resolution imaging and biophysical analysis. Pharmacologically inhibiting either dynein activity or MT assembly reduced neurite elongation rates as well as bulk and individual MT anterograde translocation. Simultaneously suppressing both processes did not have additive effects, suggesting a shared mechanism of action. Single-molecule switching nanoscopy revealed that inhibition of MT assembly decreased the association of dynein with MTs. Finally, inhibiting MT assembly prevented the rise in tension induced by dynein inhibition. Taken together, our results suggest that MT assembly is required for dynein-driven MT translocation and neurite outgrowth.

Effective Synthesis and Antifouling Activity of Dolastatin 16 Derivatives.

Some derivatives of dolastatin 16, a depsipeptide natural product first obtained from the sea hare Dolabella auricularia, were synthesized through second-generation synthesis of two unusual amino acids, dolaphenvaline and dolamethylleuine. The second-generation synthesis enabled derivatizations such as functionalization of the aromatic ring in dolaphenvaline. The derivatives of fragments and whole structures were evaluated for antifouling activity against the cypris larvae of Amphibalanus amphitrite. Small fragments inhibited the settlement of the cypris larvae at potent to moderate concentrations (EC50 = 0.60-4.62 µg/mL), although dolastatin 16 with a substituent on the aromatic ring (24) was much less potent than dolastatin 16.

Divergent CPEB prion-like domains reveal different assembly mechanisms for a generic amyloid-like fold.

BACKGROUND: Amyloids are ordered, insoluble protein aggregates, characterized by a cross-ß sheet quaternary structure in which molecules in a ß-strand conformation are stacked along the filament axis via intermolecular interactions. While amyloids are typically associated with pathological conditions, functional amyloids have also been identified and are present in a wide variety of organisms ranging from bacteria to humans. The cytoplasmic polyadenylation element-binding (CPEB) prion-like protein is an mRNA-binding translation regulator, whose neuronal isoforms undergo activity-dependent aggregation, a process that has emerged as a plausible biochemical substrate for memory maintenance. CPEB aggregation is driven by prion-like domains (PLD) that are divergent in sequence across species, and it remains unknown whether such divergent PLDs follow a similar aggregating assembly pathway. Here, we describe the amyloid-like features of the neuronal Aplysia CPEB (ApCPEB) PLD and compare them to those of the Drosophila ortholog, Orb2 PLD. RESULTS: Using in vitro single-molecule and bulk biophysical methods, we find transient oligomers and mature amyloid-like filaments that suggest similarities in the late stages of the assembly pathway for both ApCPEB and Orb2 PLDs. However, while prior to aggregation the Orb2 PLD monomer remains mainly as a random coil in solution, ApCPEB PLD adopts a diversity of conformations comprising α-helical structures that evolve to coiled-coil species, indicating structural differences at the beginning of their amyloid assembly pathways. CONCLUSION: Our results indicate that divergent PLDs of CPEB proteins from different species retain the ability to form a generic amyloid-like fold through different assembly mechanisms.

Overexpression of ARHI increases the sensitivity of cervical cancer cells to paclitaxel through inducing apoptosis and autophagy.

Cervical cancer (CC) is a common malignant tumor of the female reproductive system. This study investigated the role of aplysia ras homolog I (ARHI) in resistance to CC in vitro and in patients' tissues. Hela cells were continuously treated with different concentrations of paclitaxel (1-10 nM) to construct paclitaxel-resistant cell model (Hela-TR). CC or CC-TR tissues were obtained from CC patients or CC patients who had developed paclitaxel resistance. The level of ARHI and multidrug resistance gene 1 (MDR1) in cells and tissues were detected by qRT-PCR and immunohistochemistry (IHC) staining. Cell viability, apoptosis and the number of colonies were assessed by MTT, flow cytometry and cell clone assay in Hela and Hela-TR cells after the ARHI plasmid or shARHI were transfected into cells. The autophagy and apoptosis signaling related proteins were analyzed by western blotting. The results revealed that the levels of ARHI mRNA and protein were down-regulated in CC tissues, and were further reduced in paclitaxel-resistant tissues and Hela cell model. High expression of ARHI inhibited the expression of MDR1 in Hela and Hela-TR cells. The cell viability and cell clone of Hela and Hela-TR cells were decreased by ARHI overexpression but increased by ARHI suppression. In addition, highly expressed ARHI promoted apoptosis and activated autophagy by increasing LC3-II/LC3-I through inactivating AKT/mTOR signaling pathway. In conclusion, overexpression of ARHI can increase the sensitivity of CC to paclitaxel through promoting apoptosis and autophagy in a AKT/mTOR inactivation dependent pathway.

Anterograde and retrograde signaling by an Aplysia neurotrophin forms a transsynaptic functional unit.

Whereas short-term synaptic plasticity is often either pre- or postsynaptic, intermediate- and long-term plasticity generally require coordinated pre- and postsynaptic mechanisms. Thus, the transition from presynaptic short-term facilitation (STF) to intermediate-term facilitation (ITF) induced by 5HT at Aplysia sensory-to-motor neuron synapses requires the recruitment of postsynaptic mechanisms and activation of protein synthesis in both neurons. In the companion paper to this report, we found that presynaptic autocrine signaling by an Aplysia neurotrophin (ApNT) forms a positive feedback loop that drives the synapses from STF to ITF. Here we report that ApNT also acts through both anterograde and retrograde signaling to form a transsynaptic positive feedback loop that orchestrates cellular functions in both the presynaptic and postsynaptic neurons during the induction of ITF. These two feedback loops activate protein synthesis in each synaptic compartment, which in both cases depends on signaling from the other synaptic compartment. These results suggest that the pre- and postsynaptic compartments act as one functional unit during the consolidation of learning-related facilitation induced by 5HT.

Halogenated Metabolites from the Diet of Aplysia dactylomela Rang.

Invertebrates are an important source of structurally-diverse and biologically-active halogenated metabolites. The sea hare Aplysia dactylomela Rang has long been known to possess halogenated metabolites of dietary origin that are used as a self-defense mechanism. The compounds from Aplysia dactylomela Rang are comprised mainly of terpenoids and small percentages of C-15 acetogenins, indoles, macrolides, sterols and alkaloids with potent cytotoxic, anti-microbial and anti-inflammatory properties. For decades the metabolites discovered have been investigated for their medical and pharmaceutical applications, so much so that the ecological role of the metabolites has been overlooked. The interaction between Aplysia dactylomela Rang and its diet that is comprised of seaweed can provide information into the distribution and diversity of the seaweed, the application of bioaccumulated secondary metabolites as part of its defense mechanism and the potential roles of these metabolites for adaptation in the marine environment. This paper compiles the diversity of halogenated secondary metabolites documented from Aplysia dactylomela Rang.

Binding of Sulfoxaflor to Aplysia californica-AChBP: Computational Insights from Multiscale Approaches.

Structural features and binding properties of sulfoxaflor (SFX) with Ac-AChBP, the surrogate of the insect nAChR ligand binding domain (LBD), are reported herein using various complementary molecular modeling approaches (QM, molecular docking, molecular dynamics, and QM/QM'). The different SFX stereoisomers show distinct behaviors in terms of binding and interactions with Ac-AChBP. Molecular docking and Molecular Dynamics (MD) simulations highlight the specific intermolecular contacts involved in the binding of the different SFX isomers and the relative contribution of the SFX functional groups. QM/QM' calculations provide further insights and a significant refinement of the geometric and energetic contributions of the various residues leading to a preference for the SS and RR stereoisomers. Notable differences in terms of binding interactions are pointed out for the four stereoisomers. The results point out the induced fit of the Ac-AChBP binding site according to the SFX stereoisomer. In this process, the water molecules-mediated contacts play a key role, their energetic contribution being among the most important for the various stereoisomers. In all cases, the interaction with Trp147 is the major binding component, through CH···π and π···π interactions. This study provides a rationale for the binding of SFX to insect nAChR, in particular with respect to the new class of sulfoximine-based insect nAChR competitive modulators, and points out the requirements of various levels of theory for an accurate description of ligand-receptor interactions.

Whole-transcriptome changes in gene expression accompany aging of sensory neurons in Aplysia californica.

BACKGROUND: Large-scale molecular changes occur during aging and have many downstream consequences on whole-organism function, such as motor function, learning, and memory. The marine mollusk Aplysia californica can be used to study transcriptional changes that occur with age in identified neurons of the brain, because its simplified nervous system allows for more direct correlations between molecular changes, physiological changes, and their phenotypic outcomes. Behavioral deficits in the tail-withdrawal reflex of aged animals have been correlated with reduced excitation in sensory neurons that control the reflex. RNASeq was used to investigate whole-transcriptome changes in tail-withdrawal sensory neurons of sexually mature and aged Aplysia to correlate transcriptional changes with reduced behavioral and physiological responses. RESULTS: Paired-end sequencing resulted in 210 million reads used for differential expression analysis. Aging significantly altered expression of 1202 transcripts in sensory neurons underlying the tail-withdrawal reflex, with an approximately equal number of these genes up- and down regulated with age. Despite overall bidirectionality of expression changes, > 80% of ion channel genes that were differentially expressed had decreased expression with age. In particular, several voltage-gated K+ and Ca2+ channels were down regulated. This marked decrease in ion channel expression may play an important role in previously observed declines in aged sensory neuron excitability. We also observed decreased expression of genes and pathways involved in learning and memory. Genes involved in the stress response showed increased expression in aged Aplysia neurons. CONCLUSIONS: Significantly altered expression of many genes between sexually mature and aged Aplysia suggests large molecular changes that may impact neuronal function. Decreased ion channel mRNA observed could mean fewer receptors present in aged neurons, resulting in reduced excitability of PVC sensory neurons, ultimately leading to reduced tail-withdrawal reflex observed in aged Aplysia. Significant changes in other genes and pathways, such as stress response and learning and memory, have previously been shown to occur with age in many vertebrate organisms. This suggests that some effects of aging are common across many animal phyla.

Single Synaptic Observation of Cholinergic Neurotransmission on Living Neurons: Concentration and Dynamics.

Acetylcholine, the first neurotransmitter identified more than a century ago, plays critical roles in human activities and health; however, its synaptic concentration dynamics have remained unknown. Here, we demonstrate the in situ simultaneous measurements of synaptic cholinergic transmitter concentration and release dynamics. We used nanoscale electroanalytical methods: nanoITIES electrode of 15 nm in radius and nanoresolved scanning electrochemical microscopy (SECM). Time-resolved in situ measurements unveiled information on synaptic acetylcholine concentration and release dynamics of living Aplysia neurons. The measuring technique enabled the quantitative sensing of acetylcholine with negligible interference of other ionic and redox-active species. We measured cholinergic transmitter concentrations very close to the synapse, with values as high as 2.4 mM. We observed diverse synaptic transmitter concentration dynamics consisting of singlet, doublet and multiplet events with a signal-to-noise ratio of 6 to 130. The unprecedented details about synaptic neurotransmission unveiled are instrumental for understanding brain communication and diseases in a way distinctive from extra-synaptic studies.

Functional validation of Ca2+-binding residues from the crystal structure of the BK ion channel.

BK channels are dually regulated by voltage and Ca2+, providing a cellular mechanism to couple electrical and chemical signalling. Intracellular Ca2+ concentration is sensed by a large cytoplasmic region in the channel known as "gating ring", which is formed by four tandems of regulator of conductance for K+ (RCK1 and RCK2) domains. The recent crystal structure of the full-length BK channel from Aplysia californica has provided new information about the residues involved in Ca2+ coordination at the high-affinity binding sites located in the RCK1 and RCK2 domains, as well as their cooperativity. Some of these residues have not been previously studied in the human BK channel. In this work we have investigated, through site directed mutagenesis and electrophysiology, the effects of these residues on channel activation by voltage and Ca2+. Our results demonstrate that the side chains of two non-conserved residues proposed to coordinate Ca2+ in the A. californica structure (G523 and E591) have no apparent functional role in the human BK Ca2+ sensing mechanism. Consistent with the crystal structure, our data indicate that in the human channel the conserved residue R514 participates in Ca2+ coordination in the RCK1 binding site. Additionally, this study provides functional evidence indicating that R514 also interacts with residues E902 and Y904 connected to the Ca2+ binding site in RCK2. Interestingly, it has been proposed that this interaction may constitute a structural correlate underlying the cooperative interactions between the two high-affinity Ca2+ binding sites regulating the Ca2+ dependent gating of the BK channel. This article is part of a Special Issue entitled: Beyond the Structure-Function Horizon of Membrane Proteins edited by Ute Hellmich, Rupak Doshi and Benjamin McIlwain.

Conformations and Binding Properties of Thiametoxam and Clothianidin Neonicotinoid Insecticides to Nicotinic Acetylcholine Receptors: The Contribution of σ-Hole Interactions.

The structural features and molecular-interaction properties of thiamethoxam (THA) and clothianidin (CLO) - two neonicotinoids - have been investigated through a combined approach based on a wide range of molecular modeling methods and X-ray-structure observations. Despite their close chemical structures, significant differences are emphasized by QM (DFT), docking, molecular dynamics, and QM/QM' calculations. Thus, for the first time, their propensity to interact through chalcogen-bond interactions is highlighted. The influence of the surroundings on this behavior is pointed out: in CLO, an intramolecular S⋅⋅⋅N chalcogen bond is shown to stabilize the structure in the solid state whereas the interaction leads to the preferred conformations in the isolated and continuum solvent models for both compounds. Interestingly, this interaction potential appears to be used for their binding to Ac-AChBP through intermolecular S⋅⋅⋅O chalcogen bonds with the hydroxyl group of Tyr195. The use of a suitable level of theory to describe properly these interactions is underlined, the classical methods being unsuited to highlight these interactions. The contribution of halogen bonding through the chlorine atom of the chlorothiazole ring in the binding of the two compounds is also underlined, both in the solid state and in the Ac-AChBP surroundings. However, the accommodation of the two insecticides in the binding site leads to the fact that a halogen-bond contribution is pointed out only for CLO.

Conformational investigation of the structure-activity relationship of GdFFD and its analogues on an achatin-like neuropeptide receptor of Aplysia californica involved in the feeding circuit.

Proteins and peptides in nature are almost exclusively made from l-amino acids, and this is even more absolute in the metazoan. With the advent of modern bioanalytical techniques, however, previously unappreciated roles for d-amino acids in biological processes have been revealed. Over 30 d-amino acid containing peptides (DAACPs) have been discovered in animals where at least one l-residue has been isomerized to the d-form via an enzyme-catalyzed process. In Aplysia californica, GdFFD and GdYFD (the lower-case letter "d" indicates a d-amino acid residue) modulate the feeding behavior by activating the Aplysia achatin-like neuropeptide receptor (apALNR). However, little is known about how the three-dimensional conformation of DAACPs influences activity at the receptor, and the role that d-residues play in these peptide conformations. Here, we use a combination of computational modeling, drift-tube ion-mobility mass spectrometry, and receptor activation assays to create a simple model that predicts bioactivities for a series of GdFFD analogs. Our results suggest that the active conformations of GdFFD and GdYFD are similar to their lowest energy conformations in solution. Our model helps connect the predicted structures of GdFFD analogs to their activities, and highlights a steric effect on peptide activity at position 1 on the GdFFD receptor apALNR. Overall, these methods allow us to understand ligand-receptor interactions in the absence of high-resolution structural data.