PURPOSE:Protein-altering gene variants in SEPSECS disrupt the biosynthesis of selenoproteins, leading to a spectrum of neurological diseases. METHODS:We studied 27 individuals with biallelic SEPSECS variants, identifying 13 unreported gene variants. To better understand and diagnose the disorder, broad biochemical correlation of neurological symptoms, focused metabolomics, and structural and in vitro activity analyses were deployed. RESULTS:Our results suggest three general clinical courses: (i) severe early-onset with cerebellar or cerebral atrophy, (ii) milder early-onset with gradual deterioration, and (iii) late-onset, mild disease. SEPSECS variants primarily affect the brain. In only one individual out of eight, thyroid hormone measurements suggested a defect of T4 to T3 conversion. Accompanied increase in glutathione and sulfur metabolites in plasma indicates elevated oxidative stress. Variants mapping to conserved N- and C-termini and catalytic site elicit SEPSECS misfolding, aggregation, thermal instability, and loss of function, that could ultimately lead to ferroptosis of neurons and perhaps oligodendrocytes. For differential diagnosis and monitoring therapeutic attempts, we recommend measuring levels of plasma selenium, glutathione and sulfur metabolites, GPX activity, and SELENOP. Given the pontine involvement in less than half of the cases, we suggest renaming the syndrome from PCH2D to SEPSECS-related neurodevelopmental disorder. CONCLUSION:Our study expands the understanding of SEPSECS-related neurodevelopmental disorders, highlighting the need for updated diagnostic criteria and potential treatment strategies.

The efficiency and accuracy of CRISPR-Cas9 targeting varies considerably across genomic targets and remains a persistent issue for using this system in cells. Studies have shown that the use of 5' truncated single guide RNAs (sgRNAs) can reduce the rate of unwanted off-target recognition while still maintaining on-target specificity. However, it is not well-understood how reducing target complementarity enhances specificity or how truncation past 15 nucleotides (nts) prevents full Cas9 activation without compromising on-target binding. Here, we use biochemistry and cryogenic electron microscopy to investigate Cas9 structure and activity when bound to a 14-nt sgRNA. Our structures reveal that the shortened path of the displaced non-target strand (NTS) sterically occludes docking of the HNH L1 linker and prevents proper positioning of the nuclease domains. We show that cleavage inhibition can be alleviated by either artificially melting the protospacer adjacent motif (PAM)-distal duplex or providing a supercoiled substrate. Even though Cas9 forms a stable complex with its target, we find that plasmid cleavage is ∼1000-fold slower with a 14-nt sgRNA than with a full-length 20-nt sgRNA. Our results provide a structural basis for Cas9 target binding with 5' truncated sgRNAs and underline the importance of PAM-distal NTS availability in promoting Cas9 activation.

Selenophosphate synthetase (SEPHS) plays an essential role in selenium metabolism. Two mammalian SEPHS paralogues, SEPHS1 and SEPHS2, share high sequence identity and structural homology with SEPHS. Here, we report nine individuals from eight families with developmental delay, growth and feeding problems, hypotonia, and dysmorphic features, all with heterozygous missense variants in SEPHS1. Eight of these individuals had a recurrent variant at amino acid position 371 of SEPHS1 (p.Arg371Trp, p.Arg371Gln, and p.Arg371Gly); seven of these variants were known to be de novo. Structural modeling and biochemical assays were used to understand the effect of these variants on SEPHS1 function. We found that a variant at residue Trp352 results in local structural changes of the C-terminal region of SEPHS1 that decrease the overall thermal stability of the enzyme. In contrast, variants of a solvent-exposed residue Arg371 do not impact enzyme stability and folding but could modulate direct protein-protein interactions of SEPSH1 with cellular factors in promoting cell proliferation and development. In neuronal SH-SY5Y cells, we assessed the impact of SEPHS1 variants on cell proliferation and ROS production and investigated the mRNA expression levels of genes encoding stress-related selenoproteins. Our findings provided evidence that the identified SEPHS1 variants enhance cell proliferation by modulating ROS homeostasis. Our study supports the hypothesis that SEPHS1 plays a critical role during human development and provides a basis for further investigation into the molecular mechanisms employed by SEPHS1. Furthermore, our data suggest that variants in SEPHS1 are associated with a neurodevelopmental disorder.

The evolution of the genetic code to incorporate selenocysteine (Sec) enabled the development of a selenoproteome in all domains of life. O-phosphoseryl-tRNASec selenium transferase (SepSecS) catalyzes the terminal reaction of Sec synthesis on tRNASec in archaea and eukaryotes. Despite harboring four equivalent active sites, human SEPSECS binds no more than two tRNASec molecules. Though, the basis for this asymmetry remains poorly understood. In humans, an acidic, C-terminal, α-helical extension precludes additional tRNA-binding events in two of the enzyme monomers, stabilizing the SEPSECS•tRNASec complex. However, the existence of a helix exclusively in vertebrates raised questions about the evolution of the tRNA-binding mechanism in SEPSECS and the origin of its C-terminal extension. Herein, using a comparative structural and phylogenetic analysis, we show that the tRNA-binding motifs in SEPSECS are poorly conserved across species. Consequently, in contrast to mammalian SEPSECS, the archaeal ortholog cannot bind unacylated tRNASec and requires an aminoacyl group. Moreover, the C-terminal α-helix 16 is a mammalian innovation, and its absence causes aggregation of the SEPSECS•tRNASec complex at low tRNA concentrations. Altogether, we propose SEPSECS evolved a tRNASec binding mechanism as a crucial functional and structural feature, allowing for additional levels of regulation of Sec and selenoprotein synthesis.

O-Phosphoseryl-tRNASec selenium transferase (SepSecS) catalyzes the terminal step of selenocysteine (Sec) synthesis in archaea and eukaryotes. How the Sec synthetic machinery recognizes and discriminates tRNASec from the tRNA pool is essential to the integrity of the selenoproteome. Previously, we suggested that SepSecS adopts a competent conformation that is pre-ordered for catalysis. Herein, using high-resolution X-ray crystallography, we visualized tRNA-dependent conformational changes in human SepSecS that may be a prerequisite for achieving catalytic competency. We show that tRNASec binding organizes the active sites of the catalytic protomer, while stabilizing the N- and C-termini of the non-catalytic protomer. Binding of large anions to the catalytic groove may further optimize the catalytic site for substrate binding and catalysis. Our biochemical and mutational analyses demonstrate that productive SepSecS•tRNASec complex formation is enthalpically driven and primarily governed by electrostatic interactions between the acceptor-, TΨC-, and variable arms of tRNASec and helices α1 and α14 of SepSecS. The detailed visualization of the tRNA-dependent activation of SepSecS provides a structural basis for a revised model of the terminal reaction of Sec formation in archaea and eukaryotes.

The elongation of eukaryotic selenoproteins relies on a poorly understood process of interpreting in-frame UGA stop codons as selenocysteine (Sec). We used cryo-electron microscopy to visualize Sec UGA recoding in mammals. A complex between the noncoding Sec-insertion sequence (SECIS), SECIS-binding protein 2 (SBP2), and 40S ribosomal subunit enables Sec-specific elongation factor eEFSec to deliver Sec. eEFSec and SBP2 do not interact directly but rather deploy their carboxyl-terminal domains to engage with the opposite ends of the SECIS. By using its Lys-rich and carboxyl-terminal segments, the ribosomal protein eS31 simultaneously interacts with Sec-specific transfer RNA (tRNASec) and SBP2, which further stabilizes the assembly. eEFSec is indiscriminate toward l-serine and facilitates its misincorporation at Sec UGA codons. Our results support a fundamentally distinct mechanism of Sec UGA recoding in eukaryotes from that in bacteria.

The 'Yale years' were formative in many ways and consequently had left a deep imprint on my career.Perhaps the most remarkable feature of Tom's modus operandi was the he encouraged scientific freedom and independence of people who worked with him.Collaborative projects were often forged, and Tom would not be aware of such endeavors until after first scientific fruits were born.Herein, I will describe how one such collaboration that had been initiated while I was in Tom's laboratory led me to a path of studying a set of intriguing basic scientific questions to this day [1][2][3][4][5][6][7][8].The overall goal of these studies is to understand at the structural level how humans synthesize the amino acid selenocysteine and how is that amino acid incorporated into nascent selenoproteins.

The RNA-guided Cas9 endonuclease from Streptococcus pyogenes is a single-turnover enzyme that displays a stable product state after double-stranded-DNA cleavage. Here, we present cryo-EM structures of precatalytic, postcatalytic and product states of the active Cas9–sgRNA–DNA complex in the presence of Mg2+. In the precatalytic state, Cas9 adopts the ‘checkpoint’ conformation with the HNH nuclease domain positioned far away from the DNA. Transition to the postcatalytic state involves a dramatic ~34-Å swing of the HNH domain and disorder of the REC2 recognition domain. The postcatalytic state captures the cleaved substrate bound to the catalytically competent HNH active site. In the product state, the HNH domain is disordered, REC2 returns to the precatalytic conformation, and additional interactions of REC3 and RuvC with nucleic acids are formed. The coupled domain motions and interactions between the enzyme and the RNA-DNA hybrid provide new insights into the mechanism of genome editing by Cas9. Cryo-EM structures of the active Cas9–sgRNA–DNA complex in the presence of Mg2+ capture Cas9 in the pre- and postcatalytic states as well as in the product-bound state, and reveal coupled domain motions and interactions between the enzyme and nucleic acids.

X-ray crystallography remains a dominant approach in high-resolution structural studies.However, because of the nature of the crystallization process and stringent requirements for homogeneity of crystalline matter to be analyzed by X-rays, the method provides a somewhat 'static' representation of a macromolecule under study.In addition, in the absence of any prior structural information and phase estimates, X-ray crystallography alone cannot provide even simplest descriptors about the target molecule structure and may fail to fully inform about structural changes in solution.Here, I will present a summary of our projects that studied enzymes responsible for accurate synthesis of human selenoproteins [1][2][3].In all instances, X-ray crystallography and SAXS coupled to size-exclusion chromatography were pivotal for obtaining critical insights and successful completion of studies.Our results highlight the power of a synchronous application of X-ray crystallography and SAXS.

Background: Selenium, an essential dietary micronutrient, is incorporated into proteins as the amino acid selenocysteine (Sec) in response to in-frame UGA codons. Complex machinery ensures accurate recoding of Sec codons in higher organisms. A specialized elongation factor eEFSec is central to the process. Scope of review: Selenoprotein synthesis relies on selenocysteinyl-tRNA(Sec) (Sec-tRNA(Sec)), selenocysteine inserting sequence (SECIS) and other selenoprotein mRNA elements, an in-trans SECIS binding protein 2 (SBP2) protein factor, and eEFSec. The exact mechanisms of discrete steps of the Sec UGA recoding are not well understood. However, recent studies on mammalian model systems have revealed the first insights into these mechanisms. Herein, we summarize the current knowledge about the structure and role of mammalian eEFSec. Major conclusions: eEFSec folds into a chalice-like structure resembling that of the archaeal and bacterial orthologues SelB and the initiation protein factor IF2/eIF5B. The three N-terminal domains harbor major functional sites and adopt an EF-Tu-like fold. The C-terminal domain 4 binds to Sec-tRNA(Sec) and SBP2, senses distinct binding domains, and modulates the GTPase activity. Remarkably, GTP hydrolysis does not induce a canonical conformational change in eEFSec, but instead promotes a slight ratchet of domains 1 and 2 and a lever-like movement of domain 4, which may be critical for the release of Sec-tRNA(Sec) on the ribosome. General significance: Based on current findings, a non-canonical mechanism for elongation of selenoprotein synthesis at the Sec UGA codon is proposed. Although incomplete, our understanding of this fundamental biological process is significantly improved, and it is being harnessed for biomedical and synthetic biology initiatives. This article is part of a Special Issue entitled "Selenium research" in celebration of 200 years of selenium discovery, edited by Dr. Elias Arner and Dr. Regina Brigelius-Flohe.

Selenium is an essential micronutrient for human health. The biologically active form of selenium in all domains of life is the 21 st amino acid, selenocysteine (Sec). The incorporation of Sec into proteins is unique compared to the standard amino acids as Sec is encoded by a UGA stop codon and directly synthesized from a serine precursor on its cognate tRNA, tRNA Sec . Although not well understood, Sec biosynthesis requires three unique enzymes in archaea and eukaryotes. The enzyme selenocysteine synthase (SepSecS) is a tetrameric enzyme that catalyzes the terminal reaction of Sec synthesis in which a phosphoseryl intermediate is converted into the selenocysteinyl moiety while attached to tRNA Sec . Our previous studies have demonstrated that human SepSecS, despite containing four equivalent tRNA‐binding sites and active‐site grooves, only binds one or two substrate molecules at a given time. Recent structural data have provided a potential explanation for this restriction on the stoichiometry of the SepSecS catalytic complex. High‐resolution electron density maps of the SepSecS‐tRNA Sec complex have revealed a previously unobserved α‐helix positioned in the potential tRNA Sec binding site of the noncatalytic subunits of the tetramer. This helix directly interacts with residues that are critical for tRNA Sec recognition and binding. Thus, the presence of this α‐helix precludes binding of an additional tRNA Sec molecule. This helix is disordered in the structure of the SepSecS holoenzyme, suggesting that in the tRNA Sec ‐free state all four tRNA Sec binding sites are equally available for substrate binding. Our data suggest a model of allosteric regulation of SepSecS substrate binding in which binding of a single tRNA Sec molecule leads to a conformational change in the tetrameric enzyme that prevents binding at two of the four potential tRNA Sec binding sites. Support or Funding Information This work was supported by a grant from the National Institute of General Medical Sciences of the National Institutes of Health R01 GM097042.

Aminoacyl-tRNA synthetases (AaRSs) are ubiquitously expressed enzymes that ensure accurate translation of the genetic information into functional proteins. These enzymes also execute a variety of non-canonical functions that are significant for regulation of diverse cellular processes and that reside outside the realm of protein synthesis. Associations between faults in AaRS-mediated processes and human diseases have been long recognized. Most recent research findings strongly argue that 10 cytosolic and 14 mitochondrial AaRSs are implicated in some form of pathology of the human nervous system. The advent of modern whole-exome sequencing makes it all but certain that similar associations between the remaining 15 ARS genes and neurologic illnesses will be defined in future. It is not surprising that an intense scientific debate about the role of translational machinery, in general, and AaRSs, in particular, in the development and maintenance of the healthy human neural cell types and the brain is sparked. Herein, we summarize the current knowledge about causative links between mutations in human AaRSs and diseases of the nervous system and briefly discuss future directions.

Seryl-tRNA synthetase (SerRS) attaches L-serine to the cognate serine tRNA (tRNASer) and the noncognate selenocysteine tRNA (tRNASec). The latter activity initiates the anabolic cycle of selenocysteine (Sec), proper decoding of an in-frame Sec UGA codon, and synthesis of selenoproteins across all domains of life. While the accuracy of SerRS is important for overall proteome integrity, it is its substrate promiscuity that is vital for the integrity of the selenoproteome. This raises a question as to what elements in the two tRNA species, harboring different anticodon sequences and adopting distinct folds, facilitate aminoacylation by a common aminoacyl-tRNA synthetase. We sought to answer this question by analyzing the ability of human cytosolic SerRS to bind and act on tRNASer, tRNASec, and 10 mutant and chimeric constructs in which elements of tRNASer were transposed onto tRNASec We show that human SerRS only subtly prefers tRNASer to tRNASec, and that discrimination occurs at the level of the serylation reaction. Surprisingly, the tRNA mutants predicted to adopt either the 7/5 or 8/5 fold are poor SerRS substrates. In contrast, shortening of the acceptor arm of tRNASec by a single base pair yields an improved SerRS substrate that adopts an 8/4 fold. We suggest that an optimal tertiary arrangement of structural elements within tRNASec and tRNASer dictate their utility for serylation. We also speculate that the extended acceptor-TΨC arm of tRNASec evolved as a compromise for productive binding to SerRS while remaining the major recognition element for other enzymes involved in Sec and selenoprotein synthesis.

The human genome contains 25 genes coding for selenocysteine-containing proteins (selenoproteins). These proteins are involved in a variety of functions, most notably redox homeostasis. Selenoprotein enzymes with known functions are designated according to these functions: TXNRD1, TXNRD2, and TXNRD3 (thioredoxin reductases), GPX1, GPX2, GPX3, GPX4, and GPX6 (glutathione peroxidases), DIO1, DIO2, and DIO3 (iodothyronine deiodinases), MSRB1 (methionine sulfoxide reductase B1), and SEPHS2 (selenophosphate synthetase 2). Selenoproteins without known functions have traditionally been denoted by SEL or SEP symbols. However, these symbols are sometimes ambiguous and conflict with the approved nomenclature for several other genes. Therefore, there is a need to implement a rational and coherent nomenclature system for selenoprotein-encoding genes. Our solution is to use the root symbol SELENO followed by a letter. This nomenclature applies to SELENOF (selenoprotein F, the 15-kDa selenoprotein, SEP15), SELENOH (selenoprotein H, SELH, C11orf31), SELENOI (selenoprotein I, SELI, EPT1), SELENOK (selenoprotein K, SELK), SELENOM (selenoprotein M, SELM), SELENON (selenoprotein N, SEPN1, SELN), SELENOO (selenoprotein O, SELO), SELENOP (selenoprotein P, SeP, SEPP1, SELP), SELENOS (selenoprotein S, SELS, SEPS1, VIMP), SELENOT (selenoprotein T, SELT), SELENOV (selenoprotein V, SELV), and SELENOW (selenoprotein W, SELW, SEPW1). This system, approved by the HUGO Gene Nomenclature Committee, also resolves conflicting, missing, and ambiguous designations for selenoprotein genes and is applicable to selenoproteins across vertebrates.

Cytosolic glutaminyl-tRNA synthetase (GlnRS) is the singular enzyme responsible for translation of glutamine codons. Compound heterozygous mutations in GlnRS cause severe brain disorders by a poorly understood mechanism. Herein, we present crystal structures of the wild type and two pathological mutants of human GlnRS, which reveal, for the first time, the domain organization of the intact enzyme and the structure of the functionally important N-terminal domain (NTD). Pathological mutations mapping in the NTD alter the domain structure, and decrease catalytic activity and stability of GlnRS, whereas missense mutations in the catalytic domain induce misfolding of the enzyme. Our results suggest that the reduced catalytic efficiency and a propensity of GlnRS mutants to misfold trigger the disease development. This report broadens the spectrum of brain pathologies elicited by protein misfolding and provides a paradigm for understanding the role of mutations in aminoacyl-tRNA synthetases in neurological diseases.

Selenocysteine synthase (SepSecS) catalyzes the terminal reaction of selenocysteine, and is vital for human selenoproteome integrity. Autosomal recessive inheritance of mutations in SepSecS–Ala239Thr, Thr325Ser, Tyr334Cys and Tyr429*–induced severe, early-onset, neurological disorders in distinct human populations. Although harboring different mutant alleles, patients presented remarkably similar phenotypes typified by cerebellar and cerebral atrophy, seizures, irritability, ataxia, and extreme spasticity. However, it has remained unclear how these genetic alterations affected the structure of SepSecS and subsequently elicited the development of a neurological pathology. Herein, our biophysical and structural characterization demonstrates that, with the exception of Tyr429*, pathogenic mutations decrease protein stability and trigger protein misfolding. We propose that the reduced stability and increased propensity towards misfolding are the main causes for the loss of SepSecS activity in afflicted patients, and that these factors contribute to disease progression. We also suggest that misfolding of enzymes regulating protein synthesis should be considered in the diagnosis and study of childhood neurological disorders.

Selenocysteine is the only proteinogenic amino acid encoded by a recoded in-frame UGA codon that does not operate as the canonical opal stop codon. A specialized translation elongation factor, eEFSec in eukaryotes and SelB in prokaryotes, promotes selenocysteine incorporation into selenoproteins by a still poorly understood mechanism. Our structural and biochemical results reveal that four domains of human eEFSec fold into a chalice-like structure that has similar binding affinities for GDP, GTP and other guanine nucleotides. Surprisingly, unlike in eEF1A and EF-Tu, the guanine nucleotide exchange does not cause a major conformational change in domain 1 of eEFSec, but instead induces a swing of domain 4. We propose that eEFSec employs a non-canonical mechanism involving the distinct C-terminal domain 4 for the release of the selenocysteinyl-tRNA during decoding on the ribosome.