Marco Marcia

Visualizing Group II Intron Catalysis through the Stages of Splicing.

Group II introns are self-splicing ribozymes that share a reaction mechanism and a common ancestor with the eukaryotic spliceosome, thereby providing a model system for understanding the chemistry of pre-mRNA splicing. Here we report 14 crystal structures of a group II intron at different stages of catalysis. We provide a detailed mechanism for the first step of splicing, we describe a reversible conformational change between the first and the second steps of splicing, and we present the ligand-free intron structure after splicing in an active state that corresponds to the retrotransposable form of the intron. During each reaction, the reactants are aligned and activated by a heteronuclear four-metal-ion center that contains a metal cluster and obligate monovalent cations, and they adopt a structural arrangement similar to that of protein endonucleases. Based on our data, we propose a model for the splicing cycle and show that it is applicable to the eukaryotic spliceosome.

The structure of Aquifex aeolicus sulfide:quinone oxidoreductase, a basis to understand sulfide detoxification and respiration

Sulfide:quinone oxidoreductase (SQR) is a flavoprotein with homologues in all domains of life except plants. It plays a physiological role both in sulfide detoxification and in energy transduction. We isolated the protein from native membranes of the hyperthermophilic bacterium Aquifex aeolicus, and we determined its X-ray structure in the “as-purified,” substrate-bound, and inhibitor-bound forms at resolutions of 2.3, 2.0, and 2.9 Å, respectively. The structure is composed of 2 Rossmann domains and 1 attachment domain, with an overall monomeric architecture typical of disulfide oxidoreductase flavoproteins. A. aeolicus SQR is a surprisingly trimeric, periplasmic integral monotopic membrane protein that inserts about 12 Å into the lipidic bilayer through an amphipathic helix–turn–helix tripodal motif. The quinone is located in a channel that extends from the si side of the FAD to the membrane. The quinone ring is sandwiched between the conserved amino acids Phe-385 and Ile-346, and it is possibly protonated upon reduction via Glu-318 and/or neighboring water molecules. Sulfide polymerization occurs on the re side of FAD, where the invariant Cys-156 and Cys-347 appear to be covalently bound to polysulfur fragments. The structure suggests that FAD is covalently linked to the polypeptide in an unusual way, via a disulfide bridge between the 8-methyl group and Cys-124. The applicability of this disulfide bridge for transferring electrons from sulfide to FAD, 2 mechanisms for sulfide polymerization and channeling of the substrate, S2−, and of the product, Sn, in and out of the active site are discussed.

A new structure-based classification of sulfide:quinone oxidoreductases.

Sulfide:quinone oxidoreductases (SQR) are ubiquitous membrane‐bound flavoproteins involved in sulfide detoxification, in sulfide‐dependent energy conservation processes and potenatially in the homeostasis of the neurotransmitter sulfide. The first 2 structures of SQRs from the bacterium Aquifex aeolicus (Marcia et al., Proc Natl Acad Sci USA 2009; 106:9625–9630) and the archaeon Acidianus ambivalens (Brito et al., Biochemistry 2009; 48:5613–5622) were determined recently by X‐ray crystallography revealing unexpected differences in the active sites and in flavin adenine dinucleotide binding. Besides the reciprocal differences, they show a different conformation of the active site compared with another sulfide oxidizing enzyme, the flavocytochrome c:sulfide dehydrogenase (FCSD) from Allochromatium vinosum (protein data bank id: 1FCD). In addition to the new structural data, the number of available SQR‐like protein sequences is continuously increasing (Pham et al., Microbiology 2008; 154:3112–3121) and the SQR activity of new members of this protein family was recently proven too (Chan et al., J Bacteriol 2009; 191:1026–1034). In the light of the new data, here we revisit the previously proposed contradictory SQR classification and we define new structure‐based sequence fingerprints that support a subdivision of the SQR family into six groups. Our report summarizes the state‐of‐art knowledge about SQRs and highlights the questions that still remain unanswered. Despite two decades of work already done on these enzymes, new and most exciting discoveries can be expected in the future. Proteins 2010.

Now on display: a gallery of group II intron structures at different stages of catalysis

Group II introns are mobile genetic elements that self-splice and retrotranspose into DNA and RNA. They are considered evolutionary ancestors of the spliceosome, the ribonucleoprotein complex essential for pre-mRNA processing in higher eukaryotes. Over a 20-year period, group II introns have been characterized first genetically, then biochemically, and finally by means of X-ray crystallography. To date, 17 crystal structures of a group II intron are available, representing five different stages of the splicing cycle. This review provides a framework for classifying and understanding these new structures in the context of the splicing cycle. Structural and functional implications for the spliceosome are also discussed.

Ion Binding and Internal Hydration in the Multidrug Resistance Secondary Active Transporter NorM Investigated by Molecular Dynamics Simulations

Recently, a 3.65 Å resolution structure of the transporter NorM from the multidrug and toxic compound extrusion family has been determined in the outward-facing conformation. This antiporter uses electrochemical gradients to drive substrate export of a large class of antibiotic and toxic compounds in exchange for small monovalent cations (H(+) and Na(+)), but the molecular details of this mechanism are still largely unknown. Here we report all-atom molecular dynamics simulations of NorM, with and without the bound Na(+) cation and at different ion concentrations. Spontaneous binding of Na(+) is observed in several independent simulations with transient ion binding to D36 being necessary to reach the final binding site for which two competitive binding modes occur. Finally, the simulations indicate that the extracellular vestibule of the transporter invariably loses its characteristic V shape indicated by the crystallographic data, and it is reduced to a narrow permeation pathway lined by polar residues that can act as a specific pore for the transport of small cations. This event, together with the available structures of evolutionarily related transporters of the major facilitator superfamily (MFS), suggests that differences in the hydrophobic content of the extracellular vestibule may be characteristic of multidrug resistance transporters in contrast to substrate-selective members of the MFS.

Principles of ion recognition in RNA: insights from the group II intron structures

Metal ions promote both RNA folding and catalysis, thus being essential in stabilizing the structure and determining the function of large RNA molecules, including group II introns. The latter are self-splicing metalloribozymes, containing a heteronuclear four-metal-ion center within the active site. In addition to these catalytic ions, group II introns bind many other structural ions, including delocalized ions that bind the RNA diffusively and well-ordered ions that bind the RNA tightly with high occupancy. The latter ions, which can be studied by biophysical methods, have not yet been analyzed systematically. Here, we compare crystal structures of the group IIC intron from Oceanobacillus iheyensis and classify numerous site-bound ions, which are primarily localized in the intron core and near long-range tertiary contacts. Certain ion-binding sites resemble motifs observed in known RNA structures, while others are idiosyncratic to the group II intron. Particularly interesting are (1) ions proximal to the active site, which may participate in splicing together with the catalytic four-metal-ion center, (2) organic ions that bind regions predicted to interact with intron-encoded proteins, and (3) unusual monovalent ions bound to GU wobble pairs, GA mismatches, the S-turn, the tetraloop-receptor, and the T-loop. Our analysis extends the general principles by which ions participate in RNA structural organization and it will aid in the determination and interpretation of future RNA structures.

Characterizing a monotopic membrane enzyme. Biochemical, enzymatic and crystallization studies on Aquifex aeolicus sulfide:quinone oxidoreductase

Monotopic membrane proteins are membrane proteins that interact with only one leaflet of the lipid bilayer and do not possess transmembrane spanning segments. They are endowed with important physiological functions but until now only few of them have been studied. Here we present a detailed biochemical, enzymatic and crystallographic characterization of the monotopic membrane protein sulfide:quinone oxidoreductase. Sulfide:quinone oxidoreductase is a ubiquitous enzyme involved in sulfide detoxification, in sulfide-dependent respiration and photosynthesis, and in heavy metal tolerance. It may also play a crucial role in mammals, including humans, because sulfide acts as a neurotransmitter in these organisms. We isolated and purified sulfide:quinone oxidoreductase from the native membranes of the hyperthermophilic bacterium Aquifex aeolicus. We studied the pure and solubilized enzyme by denaturing and non-denaturing polyacrylamide electrophoresis, size-exclusion chromatography, cross-linking, analytical ultracentrifugation, visible and ultraviolet spectroscopy, mass spectrometry and electron microscopy. Additionally, we report the characterization of its enzymatic activity before and after crystallization. Finally, we discuss the crystallization of sulfide:quinone oxidoreductase in respect to its membrane topology and we propose a classification of monotopic membrane protein crystal lattices. Our data support and complement an earlier description of the three-dimensional structure of A. aeolicus sulfide:quinone oxidoreductase (M. Marcia, U. Ermler, G. Peng, H. Michel, Proc Natl Acad Sci USA, 106 (2009) 9625-9630) and may serve as a reference for further studies on monotopic membrane proteins.

Solving nucleic acid structures by molecular replacement: examples from group II intron studies

Strategies for phasing nucleic acid structures by molecular replacement, using both experimental and de novo designed models, are discussed.

Role of the N-terminal signal peptide in the membrane insertion of Aquifex aeolicus F1F0 ATP synthase c-subunit.

Rotary ATPases are membrane protein complexes that couple ATP hydrolysis to ion translocation across the membrane. Overall, they are evolutionarily well conserved, but the N‐terminal segments of their rotary subunits (c‐subunits) possess different lengths and levels of hydrophobicity across species. By analyzing the N‐terminal variability, we distinguish four phylogenetic groups of c‐subunits (groups 1–4). We characterize a member of group 2, the c‐subunit from Aquifex aeolicus F1F0 ATP synthase, both in native cells and in a heterologous expression system. We demonstrate that its N‐terminal segment forms a signal peptide with signal recognition particle (SRP) recognition features and is obligatorily required for membrane insertion. Based on our study and on previous characterizations of c‐subunits from other organisms, we propose that c‐subunits follow different membrane insertion pathways.

Production of fully assembled and active Aquifex aeolicus F1FO ATP synthase in Escherichia coli

BACKGROUND F1FO ATP synthases catalyze the synthesis of ATP from ADP and inorganic phosphate driven by ion motive forces across the membrane. A number of ATP synthases have been characterized to date. The one from the hyperthermophilic bacterium Aquifex aeolicus presents unique features, i.e. a putative heterodimeric stalk. To complement previous work on the native form of this enzyme, we produced it heterologously in Escherichia coli. METHODS We designed an artificial operon combining the nine genes of A. aeolicus ATP synthase, which are split into four clusters in the A. aeolicus genome. We expressed the genes and purified the enzyme complex by affinity and size-exclusion chromatography. We characterized the complex by native gel electrophoresis, Western blot, and mass spectrometry. We studied its activity by enzymatic assays and we visualized its structure by single-particle electron microscopy. RESULTS We show that the heterologously produced complex has the same enzymatic activity and the same structure as the native ATP synthase complex extracted from A. aeolicus cells. We used our expression system to confirm that A. aeolicus ATP synthase possesses a heterodimeric peripheral stalk unique among non-photosynthetic bacterial F1FO ATP synthases. CONCLUSIONS Our system now allows performing previously impossible structural and functional studies on A. aeolicus F1FO ATP synthase. GENERAL SIGNIFICANCE More broadly, our work provides a valuable platform to characterize many other membrane protein complexes with complicated stoichiometry, i.e. other respiratory complexes, the nuclear pore complex, or transporter systems.