A bacterial tungsten-containing aldehyde oxidoreductase forms an enzymatic decorated protein nanowire.

Aldehyde oxidoreductases (AORs) are tungsten enzymes catalyzing the oxidation of many different aldehydes to the corresponding carboxylic acids. In contrast to other known AORs, the enzyme from the denitrifying betaproteobacterium Aromatoleum aromaticum (AORAa) consists of three different subunits (AorABC) and uses nicotinamide adenine dinucleotide (NAD) as an electron acceptor. Here, we reveal that the enzyme forms filaments of repeating AorAB protomers that are capped by a single NAD-binding AorC subunit, based on solving its structure via cryo-electron microscopy. The polyferredoxin-like subunit AorA oligomerizes to an electron-conducting nanowire that is decorated with enzymatically active and W-cofactor (W-co) containing AorB subunits. Our structure further reveals the binding mode of the native substrate benzoate in the AorB active site. This, together with quantum mechanics:molecular mechanics (QM:MM)-based modeling for the coordination of the W-co, enables formulation of a hypothetical catalytic mechanism that paves the way to further engineering for applications in synthetic biology and biotechnology.

InsP6 binding to PIKK kinases revealed by the cryo-EM structure of an SMG1-SMG8-SMG9 complex.

We report the 3.45-Å resolution cryo-EM structure of human SMG1-SMG8-SMG9, a phosphatidylinositol-3-kinase (PI(3)K)-related protein kinase (PIKK) complex central to messenger RNA surveillance. Structural and MS analyses reveal the presence of inositol hexaphosphate (InsP6) in the SMG1 kinase. We show that the InsP6-binding site is conserved in mammalian target of rapamycin (mTOR) and potentially other PIKK members, and that it is required for optimal in vitro phosphorylation of both SMG1 and mTOR substrates.

Molecular Basis for poly(A) RNP Architecture and Recognition by the Pan2-Pan3 Deadenylase.

The stability of eukaryotic mRNAs is dependent on a ribonucleoprotein (RNP) complex of poly(A)-binding proteins (PABPC1/Pab1) organized on the poly(A) tail. This poly(A) RNP not only protects mRNAs from premature degradation but also stimulates the Pan2-Pan3 deadenylase complex to catalyze the first step of poly(A) tail shortening. We reconstituted this process in vitro using recombinant proteins and show that Pan2-Pan3 associates with and degrades poly(A) RNPs containing two or more Pab1 molecules. The cryo-EM structure of Pan2-Pan3 in complex with a poly(A) RNP composed of 90 adenosines and three Pab1 protomers shows how the oligomerization interfaces of Pab1 are recognized by conserved features of the deadenylase and thread the poly(A) RNA substrate into the nuclease active site. The structure reveals the basis for the periodic repeating architecture at the 3' end of cytoplasmic mRNAs. This illustrates mechanistically how RNA-bound Pab1 oligomers act as rulers for poly(A) tail length over the mRNAs' lifetime.

Structure of the nuclear exosome captured on a maturing preribosome.

The RNA exosome complex processes and degrades a wide range of transcripts, including ribosomal RNAs (rRNAs). We used cryo-electron microscopy to visualize the yeast nuclear exosome holocomplex captured on a precursor large ribosomal subunit (pre-60S) during 7S-to-5.8S rRNA processing. The cofactors of the nuclear exosome are sandwiched between the ribonuclease core complex (Exo-10) and the remodeled "foot" structure of the pre-60S particle, which harbors the 5.8S rRNA precursor. The exosome-associated helicase Mtr4 recognizes the preribosomal substrate by docking to specific sites on the 25S rRNA, captures the 3' extension of the 5.8S rRNA, and channels it toward Exo-10. The structure elucidates how the exosome forms a structural and functional unit together with its massive pre-60S substrate to process rRNA during ribosome maturation.

Reconstitution of the complete pathway of ITS2 processing at the pre-ribosome.

Removal of internal transcribed spacer 2 (ITS2) from pre-ribosomal RNA is essential to make functional ribosomes. This complicated processing reaction begins with a single endonucleolytic cleavage followed by exonucleolytic trimming at both new cleavage sites to generate mature 5.8S and 25S rRNA. We reconstituted the 7S→5.8S processing branch within ITS2 using purified exosome and its nuclear cofactors. We find that both Rrp44's ribonuclease activities are required for initial RNA shortening followed by hand over to the exonuclease Rrp6. During the in vitro reaction, ITS2-associated factors dissociate and the underlying 'foot' structure of the pre-60S particle is dismantled. 7S pre-rRNA processing is independent of 5S RNP rotation, but 26S→25S trimming is a precondition for subsequent 7S→5.8S processing. To complete the in vitro assay, we reconstituted the entire cycle of ITS2 removal with a total of 18 purified factors, catalysed by the integrated activities of the two participating RNA-processing machines, the Las1 complex and nuclear exosome.

Effect of lipid head groups on double-layered two-dimensional crystals formed by aquaporin-0.

Aquaporin-0 (AQP0) is a lens-specific water channel that also forms membrane junctions. Reconstitution of AQP0 with dimyristoyl phosphatidylcholine (DMPC) and E. coli polar lipids (EPL) yielded well-ordered, double-layered two-dimensional (2D) crystals that allowed electron crystallographic structure determination of the AQP0-mediated membrane junction. The interacting tetramers in the two crystalline layers are exactly in register, resulting in crystals with p422 symmetry. The high-resolution density maps also allowed modeling of the annular lipids surrounding the tetramers. Comparison of the DMPC and EPL bilayers suggested that the lipid head groups do not play an important role in the interaction of annular lipids with AQP0. We now reconstituted AQP0 with the anionic lipid dimyristoyl phosphatidylglycerol (DMPG), which yielded a mixture of 2D crystals with different symmetries. The different crystal symmetries result from shifts between the two crystalline layers, suggesting that the negatively charged PG head group destabilizes the interaction between the extracellular AQP0 surfaces. Reconstitution of AQP0 with dimyristoyl phosphatidylserine (DMPS), another anionic lipid, yielded crystals that had the usual p422 symmetry, but the crystals showed a pH-dependent tendency to stack through their cytoplasmic surfaces. Finally, AQP0 failed to reconstitute into membranes that were composed of more than 40% dimyristoyl phosphatidic acid (DMPA). Hence, although DMPG, DMPS, and DMPA are all negatively charged lipids, they have very different effects on AQP0 2D crystals, illustrating the importance of the specific lipid head group chemistry beyond its mere charge.

Molecular architecture of the HerA-NurA DNA double-strand break resection complex.

DNA double-strand breaks can be repaired by homologous recombination, during which the DNA ends are long-range resected by helicase-nuclease systems to generate 3' single strand tails. In archaea, this requires the Mre11-Rad50 complex and the ATP-dependent helicase-nuclease complex HerA-NurA. We report the cryo-EM structure of Sulfolobus solfataricus HerA-NurA at 7.4Å resolution and present the pseudo-atomic model of the complex. HerA forms an ASCE hexamer that tightly interacts with a NurA dimer, with each NurA protomer binding three adjacent HerA HAS domains. Entry to NurA's nuclease active sites requires dsDNA to pass through a 23Å wide channel in the HerA hexamer. The structure suggests that HerA is a dsDNA translocase that feeds DNA into the NurA nuclease sites.

Fast and accurate reference-free alignment of subtomograms.

In cryoelectron tomography alignment and averaging of subtomograms, each dnepicting the same macromolecule, improves the resolution compared to the individual subtomogram. Major challenges of subtomogram alignment are noise enhancement due to overfitting, the bias of an initial reference in the iterative alignment process, and the computational cost of processing increasingly large amounts of data. Here, we propose an efficient and accurate alignment algorithm via a generalized convolution theorem, which allows computation of a constrained correlation function using spherical harmonics. This formulation increases computational speed of rotational matching dramatically compared to rotation search in Cartesian space without sacrificing accuracy in contrast to other spherical harmonic based approaches. Using this sampling method, a reference-free alignment procedure is proposed to tackle reference bias and overfitting, which also includes contrast transfer function correction by Wiener filtering. Application of the method to simulated data allowed us to obtain resolutions near the ground truth. For two experimental datasets, ribosomes from yeast lysate and purified 20S proteasomes, we achieved reconstructions of approximately 20Å and 16Å, respectively. The software is ready-to-use and made public to the community.

Structure and catalytic mechanism of a cyclic dipeptide prenyltransferase with broad substrate promiscuity.

Fungal indole prenyltransferases (PTs) typically act on specific substrates, and they are able to prenylate their target compounds with remarkably high regio- and stereoselectivity. Similar to several indole PTs characterized to date, the cyclic dipeptide N-prenyltransferase (CdpNPT) is able to prenylate a range of diverse substrates, thus exhibiting an unusually broad substrate promiscuity. To define the structural basis for this promiscuity, we have determined crystal structures of unliganded CdpNPT and of a ternary complex of CdpNPT bound to (S)-benzodiazepinedione and thiolodiphosphate. Analysis of the structures reveals a limited number of specific interactions with (S)-benzodiazepinedione, which projects into a largely hydrophobic surface. This surface can also accommodate other substrates, explaining the ability of the enzyme to prenylate a range of compounds. The location of the bound substrates suggests a likely reaction mechanism for the conversion of (S)-benzodiazepinedione. Structure-guided mutagenesis experiments confirm that, in addition to (S)-benzodiazepinedione, CdpNPT can also act on (R)-benzodiazepinedione and several cyclic dipeptides, albeit with relaxed specificity. Finally, nuclear magnetic resonance spectroscopy demonstrates that CdpNPT is a C-3 reverse PT that catalyzes the formation of C-3ß prenylated indolines from diketopiperazines of tryptophan-containing cyclic dipeptides.