HEPN Domain Research Articles

Structural insight into the Csx1-Crn2 fusion self-limiting ribonuclease of type III CRISPR system.

In the type III CRISPR system, cyclic oligoadenylate (cOA) molecules act as second messengers, activating various promiscuous ancillary nucleases that indiscriminately degrade host and viral DNA/RNA. Conversely, ring nucleases, by specifically cleaving cOA molecules, function as off-switches to protect host cells from dormancy or death, and allow viruses to counteract immune responses. The fusion protein Csx1-Crn2, combining host ribonuclease with viral ring nuclease, represents a unique self-limiting ribonuclease family. Here, we describe the structures of Csx1-Crn2 from the organism of Marinitoga sp., in both its full-length and truncated forms, as well as in complex with cA4. We show that Csx1-Crn2 operates as a homo-tetramer, a configuration crucial for preserving the structural integrity of the HEPN domain and ensuring effective ssRNA cleavage. The binding of cA4 to the CARF domain triggers significant conformational changes across the CARF, HTH, and into the HEPN domains, leading the two R-X4-6-H motifs to form a composite catalytic site. Intriguingly, an acetate ion was found to bind at this composite site by mimicking the scissile phosphate. Further molecular docking analysis reveals that the HEPN domain can accommodate a single ssRNA molecule involving both R-X4-6-H motifs, underscoring the importance of HEPN domain dimerization for its activation.

CoCoNuTs are a diverse subclass of Type IV restriction systems predicted to target RNA

A comprehensive census of McrBC systems, among the most common forms of prokaryotic Type IV restriction systems, followed by phylogenetic analysis, reveals their enormous abundance in diverse prokaryotes and a plethora of genomic associations. We focus on a previously uncharacterized branch, which we denote coiled-coil nuclease tandems (CoCoNuTs) for their salient features: the presence of extensive coiled-coil structures and tandem nucleases. The CoCoNuTs alone show extraordinary variety, with three distinct types and multiple subtypes. All CoCoNuTs contain domains predicted to interact with translation system components, such as OB-folds resembling the SmpB protein that binds bacterial transfer-messenger RNA (tmRNA), YTH-like domains that might recognize methylated tmRNA, tRNA, or rRNA, and RNA-binding Hsp70 chaperone homologs, along with RNases, such as HEPN domains, all suggesting that the CoCoNuTs target RNA. Many CoCoNuTs might additionally target DNA, via McrC nuclease homologs. Additional restriction systems, such as Type I RM, BREX, and Druantia Type III, are frequently encoded in the same predicted superoperons. In many of these superoperons, CoCoNuTs are likely regulated by cyclic nucleotides, possibly, RNA fragments with cyclic termini, that bind associated CARF (CRISPR-Associated Rossmann Fold) domains. We hypothesize that the CoCoNuTs, together with the ancillary restriction factors, employ an echeloned defense strategy analogous to that of Type III CRISPR-Cas systems, in which an immune response eliminating virus DNA and/or RNA is launched first, but then, if it fails, an abortive infection response leading to PCD/dormancy via host RNA cleavage takes over.

CoCoNuTs are a diverse subclass of Type IV restriction systems predicted to target RNA.

A comprehensive census of McrBC systems, among the most common forms of prokaryotic Type IV restriction systems, followed by phylogenetic analysis, reveals their enormous abundance in diverse prokaryotes and a plethora of genomic associations. We focus on a previously uncharacterized branch, which we denote coiled-coil nuclease tandems (CoCoNuTs) for their salient features: the presence of extensive coiled-coil structures and tandem nucleases. The CoCoNuTs alone show extraordinary variety, with three distinct types and multiple subtypes. All CoCoNuTs contain domains predicted to interact with translation system components, such as OB-folds resembling the SmpB protein that binds bacterial transfer-messenger RNA (tmRNA), YTH-like domains that might recognize methylated tmRNA, tRNA, or rRNA, and RNA-binding Hsp70 chaperone homologs, along with RNases, such as HEPN domains, all suggesting that the CoCoNuTs target RNA. Many CoCoNuTs might additionally target DNA, via McrC nuclease homologs. Additional restriction systems, such as Type I RM, BREX, and Druantia Type III, are frequently encoded in the same predicted superoperons. In many of these superoperons, CoCoNuTs are likely regulated by cyclic nucleotides, possibly, RNA fragments with cyclic termini, that bind associated CARF (CRISPR-Associated Rossmann Fold) domains. We hypothesize that the CoCoNuTs, together with the ancillary restriction factors, employ an echeloned defense strategy analogous to that of Type III CRISPR-Cas systems, in which an immune response eliminating virus DNA and/or RNA is launched first, but then, if it fails, an abortive infection response leading to PCD/dormancy via host RNA cleavage takes over.

Molecular mechanism for target RNA recognition and cleavage of Cas13h.

RNA-targeting type VI CRISPR-Cas effectors are widely used in RNA applications. Cas13h is a recently identified subtype of Cas13 ribonuclease, with strong RNA cleavage activity and robust in vivo RNA knockdown efficiency. However, little is known regarding its biochemical properties and working mechanisms. Biochemical characterization of Cas13h1 indicated that it lacks in vitro pre-crRNA processing activity and adopts a central seed. The cleavage activity of Cas13h1 is enhanced by a R(G/A) 5'-PFS, and inhibited by tag:anti-tag RNA pairing. We determined the structures of Cas13h1-crRNA binary complex at 3.1 Å and Cas13h1-crRNA-target RNA ternary complex at 3.0 Å. The ternary complex adopts an elongated architecture, and encodes a nucleotide-binding pocket within Helical-2 domain to recognize the guanosine at the 5'-end of the target RNA. Base pairing between crRNA guide and target RNA disrupts Cas13h1-guide interactions, leading to dramatic movement of HEPN domains. Upon target RNA engagement, Cas13h1 adopts a complicated activation mechanism, including separation of HEPN catalytic residues and destabilization of the active site loop and NTD domain, to get activated. Collectively, these insights expand our understanding into Cas13 effectors.

Molecular mechanism of allosteric activation of the CRISPR ribonuclease Csm6 by cyclic tetra-adenylate

Type III CRISPR systems are innate immune systems found in bacteria and archaea, which produce cyclic oligoadenylate (cOA) second messengers in response to viral infections. In these systems, Csm6 proteins serve as ancillary nucleases that degrade single-stranded RNA (ssRNA) upon activation by cOA. In addition, Csm6 proteins also possess cOA-degrading activity as an intrinsic off-switch to avoid degradation of host RNA and DNA that would eventually lead to cell dormancy or cell death. Here, we present the crystal structures of Thermus thermophilus (Tt) Csm6 alone, and in complex with cyclic tetra-adenylate (cA4) in both pre- and post-cleavage states. These structures establish the molecular basis of the long-range allosteric activation of TtCsm6 ribonuclease by cA4. cA4 binding induces significant conformational changes, including closure of the CARF domain, dimerization of the HTH domain, and reorganization of the R-X4-6-H motif within the HEPN domain. The cleavage of cA4 by the CARF domain restores each domain to a conformation similar to its apo state. Furthermore, we have identified hyperactive TtCsm6 variants that exhibit sustained cA4-activated RNase activity, showing great promise for their applications in genome editing and diagnostics.

A high-quality, haplotype-phased genome reconstruction reveals unexpected haplotype diversity in a pearl oyster.

Homologous chromosomes in the diploid genome are thought to contain equivalent genetic information, but this common concept has not been fully verified in animal genomes with high heterozygosity. Here we report a near-complete, haplotype-phased, genome assembly of the pearl oyster, Pinctada fucata, using hi-fidelity (HiFi) long reads and chromosome conformation capture data. This assembly includes 14 pairs of long scaffolds (>38 Mb) corresponding to chromosomes (2n = 28). The accuracy of the assembly, as measured by an analysis of k-mers, is estimated to be 99.99997%. Moreover, the haplotypes contain 95.2% and 95.9%, respectively, complete and single-copy BUSCO genes, demonstrating the high quality of the assembly. Transposons comprise 53.3% of the assembly and are a major contributor to structural variations. Despite overall collinearity between haplotypes, one of the chromosomal scaffolds contains megabase-scale non-syntenic regions, which necessarily have never been detected and resolved in conventional haplotype-merged assemblies. These regions encode expanded gene families of NACHT, DZIP3/hRUL138-like HEPN, and immunoglobulin domains, multiplying the immunity gene repertoire, which we hypothesize is important for the innate immune capability of pearl oysters. The pearl oyster genome provides insight into remarkable haplotype diversity in animals.

Structural basis for self-cleavage prevention by tag:anti-tag pairing complementarity in type VI Cas13 CRISPR systems

Bacteria and archaea apply CRISPR-Cas surveillance complexes to defend against foreign invaders. These invading genetic elements are captured and integrated into the CRISPR array as spacer elements, guiding sequence-specific DNA/RNA targeting and cleavage. Recently, invivo studies have shown that target RNAs with extended complementarity with repeat sequences flanking the target element (tag:anti-tag pairing) can dramatically reduce RNA cleavage by the type VI-A Cas13a system. Here, we report the cryo-EM structure of Leptotrichia shahii LshCas13acrRNA in complex with target RNA harboring tag:anti-tag pairing complementarity, with the observed conformational changes providing a molecular explanation for inactivation of the composite HEPN domain cleavage activity. These structural insights, together with invitro biochemical and invivo cell-based assays on key mutants, define the molecular principles underlying Cas13a's capacity to target and discriminate between self and non-self RNA targets. Our studies illuminate approaches to regulate Cas13a's cleavage activity, thereby influencing Cas13a-mediated biotechnological applications.

Type III-A CRISPR-associated protein Csm6 degrades cyclic hexa-adenylate activator using both CARF and HEPN domains.

The type III CRISPR–Cas systems provide immunity against invading nucleic acids through the coordinated transcription-dependent DNA targeting and cyclic adenylate (cAn)-activated RNA degradation. Here, we show that both these pathways contribute to the Streptococcus thermophilus (St) type III-A CRISPR–Cas immunity. HPLC-MS analysis revealed that in the heterologous Escherichia coli host the StCsm effector complex predominantly produces cA5 and cA6. cA6 acts as a signaling molecule that binds to the CARF domain of StCsm6 to activate non-specific RNA degradation by the HEPN domain. By dissecting StCsm6 domains we demonstrate that both CARF and HEPN domains act as ring nucleases that degrade cAns to switch signaling off. CARF ring nuclease converts cA6 to linear A6>p and to the final A3>p product. HEPN domain, which typically degrades RNA, also shows ring nuclease activity and indiscriminately degrades cA6 or other cAns down to A>p. We propose that concerted action of both ring nucleases enables self-regulation of the RNase activity in the HEPN domain and eliminates all cAn secondary messengers in the cell when viral infection is combated by a coordinated action of Csm effector and the cA6-activated Csm6 ribonuclease.

Activation and self-inactivation mechanisms of the cyclic oligoadenylate-dependent CRISPR ribonuclease Csm6

Bacterial and archaeal CRISPR-Cas systems provide RNA-guided immunity against genetic invaders such as bacteriophages and plasmids. Upon target RNA recognition, type III CRISPR-Cas systems produce cyclic-oligoadenylate second messengers that activate downstream effectors, including Csm6 ribonucleases, via their CARF domains. Here, we show that Enteroccocus italicus Csm6 (EiCsm6) degrades its cognate cyclic hexa-AMP (cA6) activator, and report the crystal structure of EiCsm6 bound to a cA6 mimic. Our structural, biochemical, and in vivo functional assays reveal how cA6 recognition by the CARF domain activates the Csm6 HEPN domains for collateral RNA degradation, and how CARF domain-mediated cA6 cleavage provides an intrinsic off-switch to limit Csm6 activity in the absence of ring nucleases. These mechanisms facilitate rapid invader clearance and ensure termination of CRISPR interference to limit self-toxicity.

Regulation of the RNA and DNA nuclease activities required for Pyrococcus furiosus Type III-B CRISPR-Cas immunity.

Type III CRISPR–Cas prokaryotic immune systems provide anti-viral and anti-plasmid immunity via a dual mechanism of RNA and DNA destruction. Upon target RNA interaction, Type III crRNP effector complexes become activated to cleave both target RNA (via Cas7) and target DNA (via Cas10). Moreover, trans-acting endoribonucleases, Csx1 or Csm6, can promote the Type III immune response by destroying both invader and host RNAs. Here, we characterize how the RNase and DNase activities associated with Type III-B immunity in Pyrococcus furiosus (Pfu) are regulated by target RNA features and second messenger signaling events. In vivo mutational analyses reveal that either the DNase activity of Cas10 or the RNase activity of Csx1 can effectively direct successful anti-plasmid immunity. Biochemical analyses confirmed that the Cas10 Palm domains convert ATP into cyclic oligoadenylate (cOA) compounds that activate the ribonuclease activity of Pfu Csx1. Furthermore, we show that the HEPN domain of the adenosine-specific endoribonuclease, Pfu Csx1, degrades cOA signaling molecules to provide an auto-inhibitory off-switch of Csx1 activation. Activation of both the DNase and cOA generation activities require target RNA binding and recognition of distinct target RNA 3′ protospacer flanking sequences. Our results highlight the complex regulatory mechanisms controlling Type III CRISPR immunity.

CRISPR-Cas III-A Csm6 CARF Domain Is a Ring Nuclease Triggering Stepwise cA4 Cleavage with ApA>p Formation Terminating RNase Activity.

Type III-A CRISPR-Cas surveillance complexes containing multi-subunit Csm effector, guide, and target RNAs exhibit multiple activities, including formation of cyclic-oligoadenylates (cAn) from ATP and subsequent cAn-mediated cleavage of single-strand RNA(ssRNA) by the trans-acting Csm6 RNase. Our structure-function studies have focused on Thermococcus onnurineus Csm6 to deduce mechanistic insights into how cA4 binding to the Csm6 CARF domain triggers the RNase activity of the Csm6 HEPN domain and what factors contribute to regulation of RNA cleavage activity. We demonstrate thatthe Csm6 CARF domain is a ring nuclease, whereby bound cA4 is stepwise cleaved initially to ApApApA>p and subsequently to ApA>p in its CARF domain-binding pocket, with such cleavage bursts using a timer mechanism to regulate the RNase activity of the Csm6 HEPN domain. In addition, we establish T.onnurineus Csm6 as an adenosine-specific RNase and identify a histidine in thecA4 CARF-binding pocket involved in autoinhibitory regulation of RNase activity.

Two HEPN domains dictate CRISPR RNA maturation and target cleavage in Cas13d

Cas13d, the type VI-D CRISPR-Cas effector, is an RNA-guided ribonuclease that has been repurposed to edit RNA in a programmable manner. Here we report the detailed structural and functional analysis of the uncultured Ruminococcus sp. Cas13d (UrCas13d)-crRNA complex. Two hydrated Mg2+ ions aid in stabilizing the conformation of the crRNA repeat region. Sequestration of divalent metal ions does not alter pre-crRNA processing, but abolishes target cleavage by UrCas13d. Notably, the pre-crRNA processing is executed by the HEPN-2 domain. Furthermore, both the structure and sequence of the nucleotides U(-8)-C(-1) within the repeat region are indispensable for target cleavage, and are specifically recognized by UrCas13d. Moreover, correct base pairings within two separate spacer regions (an internal and a 3′-end region) are essential for target cleavage. These findings provide a framework for the development of Cas13d into a tool for a wide range of applications.

Structures of ubiquitin-like (Ubl) and Hsp90-like domains of sacsin provide insight into pathological mutations

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a neurodegenerative disease that is caused by mutations in the SACS gene. The product of this gene is a very large 520-kDa cytoplasmic protein, sacsin, with a ubiquitin-like (Ubl) domain at the N terminus followed by three large sacsin internal repeat (SIRPT) supradomains and C-terminal J and HEPN domains. The SIRPTs are predicted to contain Hsp90-like domains, suggesting a potential chaperone activity. In this work, we report the structures of the Hsp90-like Sr1 domain of SIRPT1 and the N-terminal Ubl domain determined at 1.55- and 2.1-Å resolutions, respectively. The Ubl domain crystallized as a swapped dimer that could be relevant in the context of full-length protein. The Sr1 domain displays the Bergerat protein fold with a characteristic nucleotide-binding pocket, although it binds nucleotides with very low affinity. The Sr1 structure reveals that ARSACS-causing missense mutations (R272H, R272C, and T201K) disrupt protein folding, most likely leading to sacsin degradation. This work lends structural support to the view of sacsin as a molecular chaperone and provides a framework for future studies of this protein.

The ribonuclease activity of Csm6 is required for anti-plasmid immunity by Type III-A CRISPR-Cas systems

ABSTRACTCRISPR-Cas systems provide prokaryotes with RNA-based adaptive immunity against viruses and plasmids. A unique feature of Type III CRISPR-Cas systems is that they selectively target transcriptionally-active invader DNA, and can cleave both the expressed RNA transcripts and source DNA. The Type III-A effector crRNP (CRISPR RNA-Cas protein complex), which contains Cas proteins Csm1-5, recognizes and degrades invader RNA and DNA in a crRNA-guided, manner. Interestingly, Type III-A systems also employ Csm6, an HEPN family ribonuclease that does not stably associate with the Type III-A effector crRNP, but nevertheless contributes to defense via mechanistic details that are still being determined. Here, we investigated the mechanism of action of Csm6 in Type III-A CRISPR-Cas systems from Lactococcus lactis, Staphylococcus epidermidis, and Streptococcus thermophilus expressed in Escherichia coli. We found that L. lactis and S. epidermidis Csm6 cleave RNA specifically after purines in vitro, similar to the activity reported for S. thermophilus Csm6. Moreover, L. lactis Csm6 functions as a divalent metal-independent, single strand-specific endoribonuclease that depends on the conserved HEPN domain. In vivo, we show that deletion of csm6 or expression of an RNase-defective form of Csm6 disrupts crRNA-dependent loss of plasmid DNA in all three systems expressed in E. coli. Mutations in the Csm1 palm domain, which are known to deactivate Csm6 ribonuclease activity, also prevent plasmid loss in the three systems. The results indicate that Csm6 ribonuclease activity rather than Csm1-mediated DNase activity effects anti-plasmid immunity by the three Type III-A systems investigated.

Complementary and alternative medicine beliefs and complementary medicine use in Australians with type 2 diabetes: The Fremantle Diabetes Study Phase II

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated (Cas) system is an adaptive immune system in bacteria and archaea that resists exogenous invasion through nucleic acid-mediated cleavage. In the type III-A system, the Csm complex contains five effectors and a CRISPR RNA, which edits both single stranded RNA and double stranded DNA. It has recently been demonstrated that cyclic oligoadenylates (cOAs), which are synthesized by the Csm complex, act as second messengers that bind and activate Csm6. Here, we report the crystal structures of Staphylococcus epidermidis Csm3 (SeCsm3) and an N-terminally truncated Csm6 (SeCsm6ΔN) at 2.26 and 2.0 Å, respectively. The structure of SeCsm3 highly resembled previously reported Csm3 structures from other species; however, it provided novel observations allowing further enzyme characterization. The homodimeric SeCsm6ΔN folds into a compact structure. The dimerization of the HEPN domain leads to the formation of the ribonuclease active site, which is consistent with the reported Csm6 structures. Altogether, our studies provide a structural view of the ribonuclease activity mediated by Csm3 and Csm6 of the type III-A CRISPR-Cas system.

Structural insights into the CRISPR-Cas-associated ribonuclease activity of Staphylococcus epidermidis Csm3 and Csm6

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated (Cas) system is an adaptive immune system in bacteria and archaea that resists exogenous invasion through nucleic acid-mediated cleavage. In the type III-A system, the Csm complex contains five effectors and a CRISPR RNA, which edits both single stranded RNA and double stranded DNA. It has recently been demonstrated that cyclic oligoadenylates (cOAs), which are synthesized by the Csm complex, act as second messengers that bind and activate Csm6. Here, we report the crystal structures of Staphylococcus epidermidis Csm3 (SeCsm3) and an N-terminally truncated Csm6 (SeCsm6ΔN) at 2.26 and 2.0 Å, respectively. The structure of SeCsm3 highly resembled previously reported Csm3 structures from other species; however, it provided novel observations allowing further enzyme characterization. The homodimeric SeCsm6ΔN folds into a compact structure. The dimerization of the HEPN domain leads to the formation of the ribonuclease active site, which is consistent with the reported Csm6 structures. Altogether, our studies provide a structural view of the ribonuclease activity mediated by Csm3 and Csm6 of the type III-A CRISPR-Cas system.

Two Distant Catalytic Sites Are Responsible for C2c2 RNase Activities

C2c2, the effector of type VI CRISPR-Cas systems, has two RNase activities-one for cutting its RNA target and the other for processing the CRISPR RNA (crRNA). Here, we report the structures of Leptotrichia shahii C2c2 in its crRNA-free and crRNA-bound states. While C2c2 has a bilobed structure reminiscent of all other Class 2 effectors, it also exhibits different structural characteristics. It contains the REC lobe with a Helical-1 domain and the NUC lobe with two HEPN domains. The two RNase catalytic pockets responsible for cleaving pre-crRNA and target RNA are independently located on Helical-1 and HEPN domains, respectively. crRNA binding induces significant conformational changes that are likely to stabilize crRNA binding and facilitate target RNA recognition. These structures provide important insights into the molecular mechanism of dual RNase activities of C2c2 and establish a framework for its future engineering as a RNA editing tool.

Structural basis for the endoribonuclease activity of the type III-A CRISPR-associated protein Csm6.

Prokaryotic CRISPR–Cas systems provide an RNA-guided mechanism for genome defense against mobile genetic elements such as viruses and plasmids. In type III-A CRISPR–Cas systems, the RNA-guided multisubunit Csm effector complex targets both single-stranded RNAs and double-stranded DNAs. In addition to the Csm complex, efficient anti-plasmid immunity mediated by type III-A systems also requires the CRISPR-associated protein Csm6. Here we report the crystal structure of Csm6 from Thermus thermophilus and show that the protein is a ssRNA-specific endoribonuclease. The structure reveals a dimeric architecture generated by interactions involving the N-terminal CARF and C-terminal HEPN domains. HEPN domain dimerization leads to the formation of a composite ribonuclease active site. Consistently, mutations of invariant active site residues impair catalytic activity in vitro. We further show that the ribonuclease activity of Csm6 is conserved across orthologs, suggesting that it plays an important functional role in CRISPR–Cas systems. The dimer interface of the CARF domains features a conserved electropositive pocket that may function as a ligand-binding site for allosteric control of ribonuclease activity. Altogether, our work suggests that Csm6 proteins provide an auxiliary RNA-targeting interference mechanism in type III-A CRISPR–Cas systems that operates in conjunction with the RNA- and DNA-targeting endonuclease activities of the Csm effector complex.

New Insights into the RNA-Binding and E3 Ubiquitin Ligase Activities of Roquins.

Roquins are a family of highly conserved RNA-binding proteins that also contain a RING-type E3 ubiquitin ligase domain. They repress constitutive decay elements containing mRNAs and play a critical role in RNA homeostasis and immunological self-tolerance. Here we present the crystal structures of the RNA-binding region of Roquin paralog RC3H2 in both apo- and RNA-bound forms. The RNA-binding region has a bipartite architecture composed of ROQ and HEPN domains, and can bind to stem-loop and double-stranded RNAs simultaneously. The two domains undergo a large orientation change to accommodate RNA duplex binding. We profiled E2 ubiquitin-conjugating enzymes that pair with Roquins and found that RC3H1 and RC3H2 interact with two sets of overlapping but not identical E2 enzymes to drive the assembly of polyubiquitin chains of different linkages. Crystal structures, small-angle X-ray scattering, and E2 profiling revealed that while the two paralogs are highly homologous, RC3H2 and RC3H1 are different in their structures and functions. We also demonstrated that RNA duplex binding to RC3H2 cross-talks with its E3 ubiquitin ligase function using an in vitro auto-ubiquitination assay.

High-Throughput Screening for Ligands of the HEPN Domain of Sacsin.

Sacsin is a large protein implicated in the neurodevelopmental and neurodegenerative disease autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS), which features the loss of Purkinje neurons in the cerebellum. Although the domain architecture of sacsin suggests that it is a neuronal chaperone assisting in protein quality control, the precise function of sacsin remains elusive. Using fluorescence polarization (FP) assays, we confirmed that the HEPN domain of sacsin binds to nucleotides with low micromolar affinities. FP competition assays with a variety of nucleotides and nucleotide analogs revealed that the binding is primarily mediated by the phosphate groups of nucleotides. A high-throughput screen subsequently identified novel small molecule ligands of HEPN, providing new chemical probes for cell culture studies and drug development. Together, the results are consistent with the HEPN domain contributing to the functional activity of sacsin by binding to nucleotides or other multiply charged anionic compounds in neurons.