Jianlin Lei

Structure of the Nav1.4-β1 Complex from Electric Eel

Voltage-gated sodium (Nav) channels initiate and propagate action potentials. Here, we present the cryo-EM structure of EeNav1.4, the Nav channel from electric eel, in complex with the β1 subunit at 4.0xa0Å resolution. The immunoglobulin domain of β1 docks onto the extracellular L5I and L6IV loops of EeNav1.4 via extensive polar interactions, and the single transmembrane helix interacts with the third voltage-sensing domain (VSDIII). The VSDs exhibit up conformations, while the intracellular gate of the pore domain is kept open by a digitonin-like molecule. Structural comparison with closed NavPaS shows that the outward transfer of gating charges is coupled to the iris-like pore domain dilation through intricate force transmissions involving multiple channel segments. The IFM fast inactivation motif on the III-IV linker is plugged into the corner enclosed by the outer S4-S5 and inner S6 segments in repeats III and IV, suggesting a potential allosteric blocking mechanism for fast inactivation.

An Atomic Structure of the Human Spliceosome

Mechanistic understanding of pre-mRNA splicing requires detailed structural information on various states of the spliceosome. Here we report the cryo electron microscopy (cryo-EM) structure of the human spliceosome just before exon ligation (the C∗ complex) at an average resolution of 3.76xa0Å. The splicing factor Prp17 stabilizes the active site conformation. The step II factor Slu7 adopts an extended conformation, binds Prp8 and Cwc22, and is poised for selection of the 3-splice site. Remarkably, the intron lariat traverses through a positively charged central channel of RBM22; this unusual organization suggests mechanisms of intron recruitment, confinement, and release. The protein PRKRIP1 forms a 100-Å α helix linking the distant U2 snRNP to the catalytic center. A 35-residue fragment of the ATPase/helicase Prp22 latches onto Prp8, and the quaternary exon junction complex (EJC) recognizes upstream 5-exon sequences and associates with Cwc22 and the GTPase Snu114. These structural features reveal important mechanistic insights into exon ligation.

Structure of the Human Lipid Exporter ABCA1.

ABCA1, an ATP-binding cassette (ABC) subfamily A exporter, mediates the cellular efflux of phospholipids and cholesterol to the extracellular acceptor apolipoprotein A-I (apoA-I) for generation of nascent high-density lipoprotein (HDL). Mutations of human ABCA1 are associated with Tangier disease and familial HDL deficiency. Here, we report the cryo-EM structure of human ABCA1 with nominal resolutions of 4.1xa0Å for the overall structure and 3.9xa0Å for the massive extracellular domain. The nucleotide-binding domains (NBDs) display a nucleotide-free state, while the two transmembrane domains (TMDs) contact each other through a narrow interface in the intracellular leaflet of the membrane. In addition to TMDs and NBDs, two extracellular domains of ABCA1 enclose an elongated hydrophobic tunnel. Structural mapping of dozens of disease-related mutations allows potential interpretation of their diverse pathogenic mechanisms. Structural-based analysis suggests a plausible lateral access mechanism for ABCA1-mediated lipid export that may be distinct from the conventional alternating-access paradigm.

Structure of an Intron Lariat Spliceosome from Saccharomyces cerevisiae

The disassembly of the intron lariat spliceosome (ILS) marks the end of a splicing cycle. Here we report a cryoelectron microscopy structure of the ILS complex from Saccharomyces cerevisiae at an average resolution of 3.5xa0Å. The intron lariat remains bound in the spliceosome whereas the ligated exon is already dissociated. The step II splicing factors Prp17 and Prp18, along with Cwc21 and Cwc22 that stabilize the 5 exon binding to loop I of U5 small nuclear RNA (snRNA), have been released from the active site assembly. The DEAH family ATPase/helicase Prp43 binds Syf1 at the periphery of the spliceosome, with its RNA-binding site close to the 3 end of U6 snRNA. The C-terminal domain of Ntr1/Spp382 associates with the GTPase Snu114, and Ntr2 is anchored to Prp8 while interacting with the superhelical domain of Ntr1. These structural features suggest a plausible mechanism for the disassembly of the ILS complex.

Structure of the Post-catalytic Spliceosome from Saccharomyces cerevisiae

Removal of an intron from a pre-mRNA by the spliceosome results in the ligation of two exons in the post-catalytic spliceosome (known as the P complex). Here, we present a cryo-EM structure of the Pxa0complex from Saccharomyces cerevisiae at an average resolution of 3.6xa0Å. The ligated exon is held in the active site through RNA-RNA contacts. Three bases at the 3 end of the 5 exon remain anchored to loop I of U5 small nuclear RNA, and the conserved AG nucleotides of the 3-splice site (3SS) are specifically recognized by the invariant adenine of the branch point sequence, the guanine base at the 5xa0end of the 5SS, and an adenine base of U6 snRNA. The 3SS is stabilized through an interaction with thexa01585-loop of Prp8. The P complex structure provides a view on splice junction formation critical for understanding the complete splicing cycle.

Structure of a human catalytic step I spliceosome

Structure of the human spliceosome Catalyzed by the spliceosome, precursor mRNA splicing proceeds in two steps: branching and exon ligation. Transition from the C (catalytic post-branching spliceosome) to the C* (catalytic pre-exon ligation spliceosome) complex is driven by the adenosine triphosphatase/helicase Prp16. Zhan et al. report the cryo-electron microscopy structure of the human C complex, showing that two step I splicing factors stabilize the active site and link it to Prp16. Science, this issue p. 537 The cryo–electron microscopy structure of the human C complex spliceosome reveals mechanistic insights into ribonucleoprotein remodeling. Splicing by the spliceosome involves branching and exon ligation. The branching reaction leads to the formation of the catalytic step I spliceosome (C complex). Here we report the cryo–electron microscopy structure of the human C complex at an average resolution of 4.1 angstroms. Compared with the Saccharomyces cerevisiae C complex, the human complex contains 11 additional proteins. The step I splicing factors CCDC49 and CCDC94 (Cwc25 and Yju2 in S. cerevisiae, respectively) closely interact with the DEAH-family adenosine triphosphatase/helicase Prp16 and bridge the gap between Prp16 and the active-site RNA elements. These features, together with structural comparison of the human C and C* complexes, provide mechanistic insights into ribonucleoprotein remodeling and allow the proposition of a working mechanism for the C-to-C* transition.

Structures of the fully assembled Saccharomyces cerevisiae spliceosome before activation

Structural basis for spliceosome assembly The spliceosome removes noncoding sequences from precursor RNA and ligates coding sequences into useful mRNA. The pre-spliceosome (A complex) associates with a small nuclear ribonucleoprotein (snRNP) complex called U4/U6.U5 tri-snRNP to form the pre-B complex, which is converted into the precatalytic B complex. Bai et al. solved the cryo–electron microscopy structures of the pre-B and B complexes isolated from yeast. These structures show the U1 and U2 snRNPs and allow modeling of the A complex to reveal the early steps of spliceosome assembly and activation. Science, this issue p. 1423 Cryo–electron microscopy structures of the pre-B and B complexes reveal the mechanism of assembly and activation for the yeast spliceosome. The precatalytic spliceosome (B complex) is preceded by the pre-B complex. Here we report the cryo–electron microscopy structures of the Saccharomyces cerevisiae pre-B and B complexes at average resolutions of 3.3 to 4.6 and 3.9 angstroms, respectively. In the pre-B complex, the duplex between the 5′ splice site (5′SS) and U1 small nuclear RNA (snRNA) is recognized by Yhc1, Luc7, and the Sm ring. In the B complex, U1 small nuclear ribonucleoprotein is dissociated, the 5′-exon–5′SS sequences are translocated near U6 snRNA, and three B-specific proteins may orient the precursor messenger RNA. In both complexes, U6 snRNA is anchored to loop I of U5 snRNA, and the duplex between the branch point sequence and U2 snRNA is recognized by the SF3b complex. Structural analysis reveals the mechanism of assembly and activation for the yeast spliceosome.

Cryo-EM structure of the polycystic kidney disease-like channel PKD2L1

PKD2L1, also termed TRPP3 from the TRPP subfamily (polycystic TRP channels), is involved in the sour sensation and other pH-dependent processes. PKD2L1 is believed to be a nonselective cation channel that can be regulated by voltage, protons, and calcium. Despite its considerable importance, the molecular mechanisms underlying PKD2L1 regulations are largely unknown. Here, we determine the PKD2L1 atomic structure at 3.38u2009Å resolution by cryo-electron microscopy, whereby side chains of nearly all residues are assigned. Unlike its ortholog PKD2, the pore helix (PH) and transmembrane segment 6 (S6) of PKD2L1, which are involved in upper and lower-gate opening, adopt an open conformation. Structural comparisons of PKD2L1 with a PKD2-based homologous model indicate that the pore domain dilation is coupled to conformational changes of voltage-sensing domains (VSDs) via a series of π–π interactions, suggesting a potential PKD2L1 gating mechanism.Polycystic kidney disease 2-like 1 protein (PKD2L1) is a voltage-dependent calcium-dependent nonselective ion channel involved in sour taste perception and regulation of pH-dependent action potential of spinal cord neurons. Here the authors present the 3.4u2009Å cryo-EM structure of PKD2L1 in the open state and propose a model for the gating mechanism.

Structural basis for the modulation of voltage-gated sodium channels by animal toxins

Structures of voltage-gated sodium channels In “excitable” cells, like neurons and muscle cells, a difference in electrical potential is used to transmit signals across the cell membrane. This difference is regulated by opening or closing ion channels in the cell membrane. For example, mutations in human voltage-gated sodium (Nav) channels are associated with disorders such as chronic pain, epilepsy, and cardiac arrhythmia. Pan et al. report the high-resolution structure of a human Nav channel, and Shen et al. report the structures of an insect Nav channel bound to the toxins that cause pufferfish and shellfish poisoning in humans. Together, the structures give insight into the molecular basis of sodium ion permeation and provide a path toward structure-based drug discovery. Science, this issue p. eaau2486, p. eaau2596 Structures provide insight into how voltage-gated sodium channels function and how they can be inhibited. INTRODUCTION Almost all venoms contain toxins that modulate the activity of voltage-gated sodium (Nav) channels in order to incapacitate prey or predators. The single-chain eukaryotic Nav channels comprise four homologous repeats. The central pore domain is constituted by the carboxyl-terminal segments from all four repeats, and each repeat also has a voltage-sensing domain (VSD). Toxins are broadly divided into two categories—pore blockers that physically occlude the channel pore and gating modifiers that alter channel gating by interfering with the VSDs. Whereas small-molecule neurotoxins such as tetrodotoxin (TTX) and saxitoxin (STX) function as pore blockers, most peptidic Nav channel toxins are gating modifiers that trap the channel in a particular stage of the gating cycle through interactions with one or more VSDs. In neither case is the structural basis of channel modulation fully understood. RATIONALE Dc1a is a peptidic gating modifier toxin (GMT) from venom of the desert bush spider Diguetia canities that specifically binds to VSDII of insect Nav channels to promote channel opening. We showed through biochemical analysis that Dc1a interacts with NavPaS, a Nav channel from the American cockroach Periplaneta americana, for which a cryo–electron microscopy (cryo-EM) structure was recently determined at 3.8-Å resolution. We therefore sought to solve the structure of the complex between NavPaS and Dc1a. As Dc1a occupies a distinctly different channel binding site to pore blockers, we also attempted to supplement the complex with TTX or STX to obtain structures of the ternary complexes. RESULTS The cryo-EM structure of NavPaS-Dc1a was determined to an overall resolution of 2.8 Å in the presence of 300 mM NaCl, whereas those of NavPaS-Dc1a-TTX and NavPaS-Dc1a-STX were resolved at 2.6 Å and 3.2 Å, respectively, in the presence of 150 mM NaCl. VSDII constitutes the primary docking site for Dc1a, which undergoes considerable structural rearrangement upon binding to the channel. The toxin inserts into the cleft between VSDII and the pore region, making intimate contacts with both domains. The network of intermolecular interactions seen in the cryo-EM structure was validated through examination of the effect of toxin and channel mutations using the orthologous NavBg channel from the German cockroach Blattella germanica. Four residues, Asp/Glu/Lys/Ala (DEKA), at a corresponding locus in the selectivity filter (SF) of each repeat confer Na+ selectivity. A Na+ ion was observed in the same position in the structures of NavPaS-Dc1a and NavPaS-Dc1a-TTX, coordinated by the Asp and Glu residues in the DEKA motif of the SF, and an invariant Glu on the P2 helix in repeat II, a helix in the entryway to the SF on the extracellular side. Both TTX and STX form extensive electrostatic interactions with residues in the outer electronegative ring that attracts cations into the SF and Asp and Glu in the DEKA motif, completely blocking access of Na+ ions to the SF. CONCLUSION The structure of the NavPaS-Dc1a complex suggests that the network of interactions between Nav channels and GMTs is more complex than previously anticipated. Therefore, caution has to be applied when using isolated Nav channel VSDs for drug discovery or for understanding the molecular basis of GMT action. The current structures elucidate the molecular basis for the insect selectivity of Dc1a and the subtype-specific binding of TTX or STX to Nav channels. Unambiguous structural elucidation of the bound TTX and STX, whose molecular weights are both around 300 Da, showcases the power of cryo-EM and its potential for structure-aided drug discovery. Structural basis for specific binding of GMT Dc1a and guanidinium pore blockers TTX and STX by NavPaS. (A) Dc1a inserts into the extracellular cavity between VSDII and the pore elements of repeat III. (B) Molecular mechanism for pore blockade by TTX and STX. Top: The carboxylate groups of Asp (D) and Glu (E) residues in the DEKA motif and an invariant Glu on P2II together constitute a potential Na+ binding site (designated the DEE site). Bottom: TTX and STX block access of Na+ to the DEE site from the extracellular side. A semitransparent presentation of the electrostatic surface potential of the entrance to the SF viewed from the extracellular side is shown. CTD, C-terminal domain; R, Arg; L, Leu; Y, Tyr; K, Lys. Animal toxins that modulate the activity of voltage-gated sodium (Nav) channels are broadly divided into two categories—pore blockers and gating modifiers. The pore blockers tetrodotoxin (TTX) and saxitoxin (STX) are responsible for puffer fish and shellfish poisoning in humans, respectively. Here, we present structures of the insect Nav channel NavPaS bound to a gating modifier toxin Dc1a at 2.8 angstrom-resolution and in the presence of TTX or STX at 2.6-Å and 3.2-Å resolution, respectively. Dc1a inserts into the cleft between VSDII and the pore of NavPaS, making key contacts with both domains. The structures with bound TTX or STX reveal the molecular details for the specific blockade of Na+ access to the selectivity filter from the extracellular side by these guanidinium toxins. The structures shed light on structure-based development of Nav channel drugs.

Structures of the human pre-catalytic spliceosome and its precursor spliceosome

The pre-catalytic spliceosome (B complex) is preceded by its precursor spliceosome (pre-B complex) and followed by the activated spliceosome (Bact complex). The pre-B-to-B and B-to-Bact transitions are driven by the ATPase/helicases Prp28 and Brr2, respectively. In this study, we report the cryo-electron microscopy structures of the human pre-B complex and the human B complex at an average resolution of 5.7 and 3.8u2009Å, respectively. In the pre-B complex, U1 and U2 small nuclear ribonucleoproteins (snRNPs) associate with two edges of the tetrahedron-shaped U4/U6.U5 tri-snRNP. The pre-mRNA is yet to be recognized by U5 or U6 small nuclear RNA (snRNA), and loop I of U5 snRNA remains unengaged. In the B complex, U1 snRNP and Prp28 are dissociated, the 5’-exon is anchored to loop I of U5 snRNA, and the 5′-splice site is recognized by U6 snRNA through duplex formation. In sharp contrast to S. cerevisiae, most components of U2 snRNP and tri-snRNP, exemplified by Brr2, undergo pronounced rearrangements in the human pre-B-to-B transition. Structural analysis reveals mechanistic insights into the assembly and activation of the human spliceosome.