FUS Phase Separation Is Modulated by a Molecular Chaperone and Methylation of Arginine Cation-π Interactions.

Reversible phase separation underpins the role of FUS in ribonucleoprotein granules and other membrane-free organelles and is, in part, driven by the intrinsically disordered low-complexity (LC) domain of FUS. Here, we report that cooperative cation-π interactions between tyrosines in the LC domain and arginines in structured C-terminal domains also contribute to phase separation. These interactions are modulated by post-translational arginine methylation, wherein arginine hypomethylation strongly promotes phase separation and gelation. Indeed, significant hypomethylation, which occurs in FUS-associated frontotemporal lobar degeneration (FTLD), induces FUS condensation into stable intermolecular ß-sheet-rich hydrogels that disrupt RNP granule function and impair new protein synthesis in neuron terminals. We show that transportin acts as a physiological molecular chaperone of FUS in neuron terminals, reducing phase separation and gelation of methylated and hypomethylated FUS and rescuing protein synthesis. These results demonstrate how FUS condensation is physiologically regulated and how perturbations in these mechanisms can lead to disease.

A Molecular Grammar Governing the Driving Forces for Phase Separation of Prion-like RNA Binding Proteins.

Proteins such as FUS phase separate to form liquid-like condensates that can harden into less dynamic structures. However, how these properties emerge from the collective interactions of many amino acids remains largely unknown. Here, we use extensive mutagenesis to identify a sequence-encoded molecular grammar underlying the driving forces of phase separation of proteins in the FUS family and test aspects of this grammar in cells. Phase separation is primarily governed by multivalent interactions among tyrosine residues from prion-like domains and arginine residues from RNA-binding domains, which are modulated by negatively charged residues. Glycine residues enhance the fluidity, whereas glutamine and serine residues promote hardening. We develop a model to show that the measured saturation concentrations of phase separation are inversely proportional to the product of the numbers of arginine and tyrosine residues. These results suggest it is possible to predict phase-separation properties based on amino acid sequences.

A self-healing multispectral transparent adhesive peptide glass.

Despite its disordered liquid-like structure, glass exhibits solid-like mechanical properties1. The formation of glassy material occurs by vitrification, preventing crystallization and promoting an amorphous structure2. Glass is fundamental in diverse fields of materials science, owing to its unique optical, chemical and mechanical properties as well as durability, versatility and environmental sustainability3. However, engineering a glassy material without compromising its properties is challenging4-6. Here we report the discovery of a supramolecular amorphous glass formed by the spontaneous self-organization of the short aromatic tripeptide YYY initiated by non-covalent cross-linking with structural water7,8. This system uniquely combines often contradictory sets of properties; it is highly rigid yet can undergo complete self-healing at room temperature. Moreover, the supramolecular glass is an extremely strong adhesive yet it is transparent in a wide spectral range from visible to mid-infrared. This exceptional set of characteristics is observed in a simple bioorganic peptide glass composed of natural amino acids, presenting a multi-functional material that could be highly advantageous for various applications in science and engineering.

The intrinsic substrate specificity of the human tyrosine kinome.

Phosphorylation of proteins on tyrosine (Tyr) residues evolved in metazoan organisms as a mechanism of coordinating tissue growth1. Multicellular eukaryotes typically have more than 50 distinct protein Tyr kinases that catalyse the phosphorylation of thousands of Tyr residues throughout the proteome1-3. How a given Tyr kinase can phosphorylate a specific subset of proteins at unique Tyr sites is only partially understood4-7. Here we used combinatorial peptide arrays to profile the substrate sequence specificity of all human Tyr kinases. Globally, the Tyr kinases demonstrate considerable diversity in optimal patterns of residues surrounding the site of phosphorylation, revealing the functional organization of the human Tyr kinome by substrate motif preference. Using this information, Tyr kinases that are most compatible with phosphorylating any Tyr site can be identified. Analysis of mass spectrometry phosphoproteomic datasets using this compendium of kinase specificities accurately identifies specific Tyr kinases that are dysregulated in cells after stimulation with growth factors, treatment with anti-cancer drugs or expression of oncogenic variants. Furthermore, the topology of known Tyr signalling networks naturally emerged from a comparison of the sequence specificities of the Tyr kinases and the SH2 phosphotyrosine (pTyr)-binding domains. Finally we show that the intrinsic substrate specificity of Tyr kinases has remained fundamentally unchanged from worms to humans, suggesting that the fidelity between Tyr kinases and their protein substrate sequences has been maintained across hundreds of millions of years of evolution.

Legionella effector LnaB is a phosphoryl-AMPylase that impairs phosphosignalling

AMPylation is a post-translational modification in which AMP is added to the amino acid side chains of proteins1,2. Here we show that, with ATP as the ligand and actin as the host activator, the effector protein LnaB of Legionella pneumophila exhibits AMPylase activity towards the phosphoryl group of phosphoribose on PRR42-Ub that is generated by the SidE family of effectors, and deubiquitinases DupA and DupB in an E1- and E2-independent ubiquitination process3-7. The product of LnaB is further hydrolysed by an ADP-ribosylhydrolase, MavL, to Ub, thereby preventing the accumulation of PRR42-Ub and ADPRR42-Ub and protecting canonical ubiquitination in host cells. LnaB represents a large family of AMPylases that adopt a common structural fold, distinct from those of the previously known AMPylases, and LnaB homologues are found in more than 20 species of bacterial pathogens. Moreover, LnaB also exhibits robust phosphoryl AMPylase activity towards phosphorylated residues and produces unique ADPylation modifications in proteins. During infection, LnaB AMPylates the conserved phosphorylated tyrosine residues in the activation loop of the Src family of kinases8,9, which dampens downstream phosphorylation signalling in the host. Structural studies reveal the actin-dependent activation and catalytic mechanisms of the LnaB family of AMPylases. This study identifies, to our knowledge, an unprecedented molecular regulation mechanism in bacterial pathogenesis and protein phosphorylation.

Covalent targeted radioligands potentiate radionuclide therapy.

Targeted radionuclide therapy, in which radiopharmaceuticals deliver potent radionuclides to tumours for localized irradiation, has addressed unmet clinical needs and improved outcomes for patients with cancer1-4. A therapeutic radiopharmaceutical must achieve both sustainable tumour targeting and fast clearance from healthy tissue, which remains a major challenge5,6. A targeted ligation strategy that selectively fixes the radiopharmaceutical to the target protein in the tumour would be an ideal solution. Here we installed a sulfur (VI) fluoride exchange (SuFEx) chemistry-based linker on radiopharmaceuticals to prevent excessively fast tumour clearance. When the engineered radiopharmaceutical binds to the tumour-specific protein, the system undergoes a binding-to-ligation transition and readily conjugates to the tyrosine residues through the 'click' SuFEx reaction. The application of this strategy to a fibroblast activation protein (FAP) inhibitor (FAPI) triggered more than 80% covalent binding to the protein and almost no dissociation for six days. In mice, SuFEx-engineered FAPI showed 257% greater tumour uptake than did the original FAPI, and increased tumour retention by 13-fold. The uptake in healthy tissues was rapidly cleared. In a pilot imaging study, this strategy identified more tumour lesions in patients with cancer than did other methods. SuFEx-engineered FAPI also successfully achieved targeted ß- and α-radionuclide therapy, causing nearly complete tumour regression in mice. Another SuFEx-engineered radioligand that targets prostate-specific membrane antigen (PSMA) also showed enhanced therapeutic efficacy. Considering the broad scope of proteins that can potentially be ligated to SuFEx warheads, it might be possible to adapt this strategy to other cancer targets.

Molecular mechanism of choline and ethanolamine transport in humans.

Human feline leukaemia virus subgroup C receptor-related proteins 1 and 2 (FLVCR1 and FLVCR2) are members of the major facilitator superfamily1. Their dysfunction is linked to several clinical disorders, including PCARP, HSAN and Fowler syndrome2-7. Earlier studies concluded that FLVCR1 may function as a haem exporter8-12, whereas FLVCR2 was suggested to act as a haem importer13, yet conclusive biochemical and detailed molecular evidence remained elusive for the function of both transporters14-16. Here, we show that FLVCR1 and FLVCR2 facilitate the transport of choline and ethanolamine across the plasma membrane, using a concentration-driven substrate translocation process. Through structural and computational analyses, we have identified distinct conformational states of FLVCRs and unravelled the coordination chemistry underlying their substrate interactions. Fully conserved tryptophan and tyrosine residues form the binding pocket of both transporters and confer selectivity for choline and ethanolamine through cation-π interactions. Our findings clarify the mechanisms of choline and ethanolamine transport by FLVCR1 and FLVCR2, enhance our comprehension of disease-associated mutations that interfere with these vital processes and shed light on the conformational dynamics of these major facilitator superfamily proteins during the transport cycle.

Tyrosine - a structural glue for hierarchical protein assembly.

Protein self-assembly, guided by the interplay of sequence- and environment-dependent liquid-liquid phase separation (LLPS), constitutes a fundamental process in the assembly of numerous intrinsically disordered proteins. Heuristic examination of these proteins has underscored the role of tyrosine residues, evident in their conservation and pivotal involvement in initiating LLPS and subsequent liquid-solid phase transitions (LSPT). The development of tyrosine-templated constructs, designed to mimic their natural counterparts, emerges as a promising strategy for creating adaptive, self-assembling systems with diverse applications. This review explores the central role of tyrosine in orchestrating protein self-assembly, delving into key interactions and examining its potential in innovative applications, including responsive biomaterials and bioengineering.

Visualizing protein breathing motions associated with aromatic ring flipping.

Aromatic residues cluster in the core of folded proteins, where they stabilize the structure through multiple interactions. Nuclear magnetic resonance (NMR) studies in the 1970s showed that aromatic side chains can undergo ring flips-that is, 180° rotations-despite their role in maintaining the protein fold1-3. It was suggested that large-scale 'breathing' motions of the surrounding protein environment would be necessary to accommodate these ring flipping events1. However, the structural details of these motions have remained unclear. Here we uncover the structural rearrangements that accompany ring flipping of a buried tyrosine residue in an SH3 domain. Using NMR, we show that the tyrosine side chain flips to a low-populated, minor state and, through a proteome-wide sequence analysis, we design mutants that stabilize this state, which allows us to capture its high-resolution structure by X-ray crystallography. A void volume is generated around the tyrosine ring during the structural transition between the major and minor state, and this allows fast flipping to take place. Our results provide structural insights into the protein breathing motions that are associated with ring flipping. More generally, our study has implications for protein design and structure prediction by showing how the local protein environment influences amino acid side chain conformations and vice versa.

MARK4 controls ischaemic heart failure through microtubule detyrosination.

Myocardial infarction is a major cause of premature death in adults. Compromised cardiac function after myocardial infarction leads to chronic heart failure with systemic health complications and a high mortality rate1. Effective therapeutic strategies are needed to improve the recovery of cardiac function after myocardial infarction. More specifically, there is a major unmet need for a new class of drugs that can improve cardiomyocyte contractility, because inotropic therapies that are currently available have been associated with high morbidity and mortality in patients with systolic heart failure2,3 or have shown a very modest reduction of risk of heart failure4. Microtubule detyrosination is emerging as an important mechanism for the regulation of cardiomyocyte contractility5. Here we show that deficiency of microtubule-affinity regulating kinase 4 (MARK4) substantially limits the reduction in the left ventricular ejection fraction after acute myocardial infarction in mice, without affecting infarct size or cardiac remodelling. Mechanistically, we provide evidence that MARK4 regulates cardiomyocyte contractility by promoting phosphorylation of microtubule-associated protein 4 (MAP4), which facilitates the access of vasohibin 2 (VASH2)-a tubulin carboxypeptidase-to microtubules for the detyrosination of α-tubulin. Our results show how the detyrosination of microtubules in cardiomyocytes is finely tuned by MARK4 to regulate cardiac inotropy, and identify MARK4 as a promising therapeutic target for improving cardiac function after myocardial infarction.

The ß-subunit of tryptophan synthase is a latent tyrosine synthase.

Aromatic amino acids and their derivatives are diverse primary and secondary metabolites with critical roles in protein synthesis, cell structure and integrity, defense and signaling. All de novo aromatic amino acid production relies on a set of ancient and highly conserved chemistries. Here we introduce a new enzymatic transformation for L-tyrosine synthesis by demonstrating that the ß-subunit of tryptophan synthase-which natively couples indole and L-serine to form L-tryptophan-can act as a latent 'tyrosine synthase'. A single substitution of a near-universally conserved catalytic residue unlocks activity toward simple phenol analogs and yields exclusive para carbon-carbon bond formation to furnish L-tyrosines. Structural and mechanistic studies show how a new active-site water molecule orients phenols for a nonnative mechanism of alkylation, with additional directed evolution resulting in a net >30,000-fold rate enhancement. This new biocatalyst can be used to efficiently prepare valuable L-tyrosine analogs at gram scales and provides the missing chemistry for a conceptually different pathway to L-tyrosine.

The structure of human thyroglobulin.

Thyroglobulin (TG) is the protein precursor of thyroid hormones, which are essential for growth, development and the control of metabolism in vertebrates1,2. Hormone synthesis from TG occurs in the thyroid gland via the iodination and coupling of pairs of tyrosines, and is completed by TG proteolysis3. Tyrosine proximity within TG is thought to enable the coupling reaction but hormonogenic tyrosines have not been clearly identified, and the lack of a three-dimensional structure of TG has prevented mechanistic understanding4. Here we present the structure of full-length human thyroglobulin at a resolution of approximately 3.5 Å, determined by cryo-electron microscopy. We identified all of the hormonogenic tyrosine pairs in the structure, and verified them using site-directed mutagenesis and in vitro hormone-production assays using human TG expressed in HEK293T cells. Our analysis revealed that the proximity, flexibility and solvent exposure of the tyrosines are the key characteristics of hormonogenic sites. We transferred the reaction sites from TG to an engineered tyrosine donor-acceptor pair in the unrelated bacterial maltose-binding protein (MBP), which yielded hormone production with an efficiency comparable to that of TG. Our study provides a framework to further understand the production and regulation of thyroid hormones.

KIR signaling is regulated by electrostatic interaction of its cytosolic tail with the plasma membrane despite being neutral polyampholyte.

Many receptors signal upon phosphorylation of tyrosine-based motifs in their cytosolic tail, with intrinsic disorder as a common feature. Studies on CD3ζ and CD3ε tails, which are disordered and polybasic, suggested regulation of phosphorylation through accessibility of tyrosines, governed by electrostatic interactions with membrane anionic lipids. We noticed characteristics of intrinsic disorder and previously unappreciated features in tyrosine-based motif-bearing cytosolic tails of many, especially, inhibitory receptors. They are neutral or acidic polyampholytes, with acidic and basic residues linearly segregated. To explore roles of these electrostatic features, we studied inhibitory killer-cell immunoglobulin-like receptor (KIR). Its cytosolic tail is a disordered neutrally charged polyampholyte, wherein juxtamembrane and membrane distal stretches are basic, and the intervening stretch is acidic. Despite lacking net charge, it interacted electrostatically with the plasma membrane. The juxtamembrane stretch was crucial for overall binding, which sequestered tyrosines in the lipid bilayer and restrained their constitutive phosphorylation. Human leukocyte antigen-C ligand binding to KIR released its tail from the plasma membrane to initiate signaling. Tail release occurred independently of KIR polymerization, clustering, or tyrosine phosphorylation, but required acidic residues of the acidic stretch. Tail interaction with the plasma membrane dictated signaling strength of KIR. These results revealed an electrostatic protein-lipid interaction that is unusual in being governed by segregated clusters of acidic and basic residues in polyampholytic disordered region of protein. In contrast to previously known, segregated distribution of oppositely charged residues made both binding and unbinding modules inherent to receptor tail, which could make the interaction an independent signaling switch.

The detyrosination/re-tyrosination cycle of tubulin and its role and dysfunction in neurons and cardiomyocytes.

Among the variety of post-translational modifications to which microtubules are subjected, the detyrosination/re-tyrosination cycle is specific to tubulin. It is conserved by evolution and characterized by the enzymatic removal and re-addition of a gene-encoded tyrosine residue at the C-terminus of α-tubulin. Detyrosinated tubulin can be further converted to Δ2-tubulin by the removal of an additional C-terminal glutamate residue. Detyrosinated and Δ2-tubulin are carried by stable microtubules whereas tyrosinated microtubules are present on dynamic polymers. The cycle regulates trafficking of many cargo transporting molecular motors and is linked to the microtubule dynamics via regulation of microtubule interactions with specific cellular effectors such as kinesin-13. Here, we give an historical overview of the general features discovered for the cycle. We highlight the recent progress toward structure and functioning of the enzymes that keep the levels of tyrosinated and detyrosinated tubulin in cells, the long-known tubulin tyrosine ligase and the recently discovered vasohibin-SVBP complexes. We further describe how the cycle controls microtubule functions in healthy neurons and cardiomyocytes and how deregulations of the cycle are involved in dysfunctions of these highly differentiated cells, leading to neurodegeneration and heart failure in humans.

Structure, dynamics, and redox reactivity of an all-purpose flavodoxin.

The flavodoxin of Rhodopseudomonas palustris CGA009 (Rp9Fld) supplies highly reducing equivalents to crucial enzymes such as hydrogenase, especially when the organism is iron-restricted. By acquiring those electrons from photodriven electron flow via the bifurcating electron transfer flavoprotein, Rp9Fld provides solar power to vital metabolic processes. To understand Rp9Fld's ability to work with diverse partners, we solved its crystal structure. We observed the canonical flavodoxin (Fld) fold and features common to other long-chain Flds but not all the surface loops thought to recognize partner proteins. Moreover, some of the loops display alternative structures and dynamics. To advance studies of protein-protein associations and conformational consequences, we assigned the 19F NMR signals of all five tyrosines (Tyrs). Our electrochemical measurements show that incorporation of 3-19F-Tyr in place of Tyr has only a modest effect on Rp9Fld's redox properties even though Tyrs flank the flavin on both sides. Meanwhile, the 19F probes demonstrate the expected paramagnetic effect, with signals from nearby Tyrs becoming broadened beyond detection when the flavin semiquinone is formed. However, the temperature dependencies of chemical shifts and linewidths reveal dynamics affecting loops close to the flavin and regions that bind to partners in a variety of systems. These coincide with patterns of amino acid type conservation but not retention of specific residues, arguing against detailed specificity with respect to partners. We propose that the loops surrounding the flavin adopt altered conformations upon binding to partners and may even participate actively in electron transfer.

Tyrosine-modifying glycosylation by Yersinia effectors.

Mono-O-glycosylation of target proteins by bacterial toxins or effector proteins is a well-known mechanism by which bacteria interfere with essential functions of host cells. The respective glycosyltransferases are important virulence factors such as the Clostridioides difficile toxins A and B. Here, we describe two glycosyltransferases of Yersinia species that have a high sequence identity: YeGT from the zoonotic pathogen Yersinia enterocolitica and YkGT from the murine pathogen Yersinia kristensenii. We show that both modify Rho family proteins by attachment of GlcNAc at tyrosine residues (Tyr-34 in RhoA). Notably, the enzymes differed in their target protein specificity. While YeGT modified RhoA, B, and C, YkGT possessed a broader substrate spectrum and glycosylated not only Rho but also Rac and Cdc42 subfamily proteins. Mutagenesis studies indicated that residue 177 is important for this broader target spectrum. We determined the crystal structure of YeGT shortened by 16 residues N terminally (sYeGT) in the ligand-free state and bound to UDP, the product of substrate hydrolysis. The structure assigns sYeGT to the GT-A family. It shares high structural similarity to glycosyltransferase domains from toxins. We also demonstrated that the 16 most N-terminal residues of YeGT and YkGT are important for the mediated translocation into the host cell using the pore-forming protective antigen of anthrax toxin. Mediated introduction into HeLa cells or ectopic expression of YeGT and YkGT caused morphological changes and redistribution of the actin cytoskeleton. The data suggest that YeGT and YkGT are likely bacterial effectors belonging to the family of tyrosine glycosylating bacterial glycosyltransferases.

PECAM-1 supports leukocyte diapedesis by tension-dependent dephosphorylation of VE-cadherin.

Leukocyte extravasation is an essential step during the immune response and requires the destabilization of endothelial junctions. We have shown previously that this process depends in vivo on the dephosphorylation of VE-cadherin-Y731. Here, we reveal the underlying mechanism. Leukocyte-induced stimulation of PECAM-1 triggers dissociation of the phosphatase SHP2 which then directly targets VE-cadherin-Y731. The binding site of PECAM-1 for SHP2 is needed for VE-cadherin dephosphorylation and subsequent endocytosis. Importantly, the contribution of PECAM-1 to leukocyte diapedesis in vitro and in vivo was strictly dependent on the presence of Y731 of VE-cadherin. In addition to SHP2, dephosphorylation of Y731 required Ca2+ -signaling, non-muscle myosin II activation, and endothelial cell tension. Since we found that ß-catenin/plakoglobin mask VE-cadherin-Y731 and leukocyte docking to endothelial cells exert force on the VE-cadherin-catenin complex, we propose that leukocytes destabilize junctions by PECAM-1-SHP2-triggered dephosphorylation of VE-cadherin-Y731 which becomes accessible by actomyosin-mediated mechanical force exerted on the VE-cadherin-catenin complex.

Structural basis for the distinct roles of non-conserved Pro116 and conserved Tyr124 of BCH domain of yeast p50RhoGAP.

p50RhoGAP is a key protein that interacts with and downregulates the small GTPase RhoA. p50RhoGAP is a multifunctional protein containing the BNIP-2 and Cdc42GAP Homology (BCH) domain that facilitates protein-protein interactions and lipid binding and the GAP domain that regulates active RhoA population. We recently solved the structure of the BCH domain from yeast p50RhoGAP (YBCH) and showed that it maintains the adjacent GAP domain in an auto-inhibited state through the ß5 strand. Our previous WT YBCH structure shows that a unique kink at position 116 thought to be made by a proline residue between alpha helices α6 and α7 is essential for the formation of intertwined dimer from asymmetric monomers. Here we sought to establish the role and impact of this Pro116. However, the kink persists in the structure of P116A mutant YBCH domain, suggesting that the scaffold is not dictated by the proline residue at this position. We further identified Tyr124 (or Tyr188 in HBCH) as a conserved residue in the crucial ß5 strand. Extending to the human ortholog, when substituted to acidic residues, Tyr188D or Tyr188E, we observed an increase in RhoA binding and self-dimerization, indicative of a loss of inhibition of the GAP domain by the BCH domain. These results point to distinct roles and impact of the non-conserved and conserved amino acid positions in regulating the structural and functional complexity of the BCH domain.

Kinetic model for reversible radical transfer in ribonucleotide reductase.

The enzyme ribonucleotide reductase (RNR), which catalyzes the reduction of ribonucleotides to deoxynucleotides, is vital for DNA synthesis, replication, and repair in all living organisms. Its mechanism requires long-range radical translocation over ∼32 Šthrough two protein subunits and the intervening aqueous interface. Herein, a kinetic model is designed to describe reversible radical transfer in Escherichia coli RNR. This model is based on experimentally studied photoRNR systems that allow the photochemical injection of a radical at a specific tyrosine residue, Y356, using a photosensitizer. The radical then transfers across the interface to another tyrosine residue, Y731, and continues until it reaches a cysteine residue, C439, which is primed for catalysis. This kinetic model includes radical injection, an off-pathway sink, radical transfer between pairs of residues along the pathway, and the conformational flipping motion of Y731 at the interface. Most of the input rate constants for this kinetic model are obtained from previous experimental measurements and quantum mechanical/molecular mechanical free-energy simulations. Ranges for the rate constants corresponding to radical transfer across the interface are determined by fitting to the experimentally measured Y356 radical decay times in photoRNR systems. This kinetic model illuminates the time evolution of radical transport along the tyrosine and cysteine residues following radical injection. Further analysis identifies the individual rate constants that may be tuned to alter the timescale and probability of the injected radical reaching C439. The insights gained from this kinetic model are relevant to biochemical understanding and protein-engineering efforts with potential pharmacological implications.

A family of unusual immunoglobulin superfamily genes in an invertebrate histocompatibility complex.

Most colonial marine invertebrates are capable of allorecognition, the ability to distinguish between themselves and conspecifics. One long-standing question is whether invertebrate allorecognition genes are homologous to vertebrate histocompatibility genes. In the cnidarian Hydractinia symbiolongicarpus, allorecognition is controlled by at least two genes, Allorecognition 1 (Alr1) and Allorecognition 2 (Alr2), which encode highly polymorphic cell-surface proteins that serve as markers of self. Here, we show that Alr1 and Alr2 are part of a family of 41 Alr genes, all of which reside in a single genomic interval called the Allorecognition Complex (ARC). Using sensitive homology searches and highly accurate structural predictions, we demonstrate that the Alr proteins are members of the immunoglobulin superfamily (IgSF) with V-set and I-set Ig domains unlike any previously identified in animals. Specifically, their primary amino acid sequences lack many of the motifs considered diagnostic for V-set and I-set domains, yet they adopt secondary and tertiary structures nearly identical to canonical Ig domains. Thus, the V-set domain, which played a central role in the evolution of vertebrate adaptive immunity, was present in the last common ancestor of cnidarians and bilaterians. Unexpectedly, several Alr proteins also have immunoreceptor tyrosine-based activation motifs and immunoreceptor tyrosine-based inhibitory motifs in their cytoplasmic tails, suggesting they could participate in pathways homologous to those that regulate immunity in humans and flies. This work expands our definition of the IgSF with the addition of a family of unusual members, several of which play a role in invertebrate histocompatibility.