Divalent Metal Ions Tune the Self-Splicing Reaction of the Yeast Mitochondrial Group II Intron Sc.ai5γ

Group II introns are large ribozymes, consisting of six functionally distinct domains that assemble in the presence of Mg2+ to the active structure catalyzing a variety of reactions. The first step of intron splicing is well characterized by a Michaelis–Menten-type cleavage reaction using a two-piece group II intron: the substrate RNA, the 5′-exon covalently linked to domains 1, 2, and 3, is cleaved upon addition of domain 5 acting as a catalyst. Here we investigate the effect of Ca2+, Mn2+, Ni2+, Zn2+, Cd2+, Pb2+, and [Co(NH3)6]3+ on the first step of splicing of the Saccharomyces cerevisiae mitochondrial group II intron Sc.ai5γ. We find that this group II intron is very sensitive to the presence of divalent metal ions other than Mg2+. For example, the presence of only 5% Ca2+ relative to Mg2+ results in a decrease in the maximal turnover rate k cat by 50%. Ca2+ thereby has a twofold effect: this metal ion interferes initially with folding, but then also competes directly with Mg2+ in the folded state, the latter being indicative of at least one specific Ca2+ binding pocket interfering directly with catalysis. Similar results are obtained with Mn2+, Cd2+, and [Co(NH3)6]3+. Ni2+ is a much more powerful inhibitor and the presence of either Zn2+ or Pb2+ leads to rapid degradation of the RNA. These results show a surprising sensitivity of such a large multidomain RNA on trace amounts of cations other than Mg2+ and raises the question of biological relevance at least in the case of Ca2+

Structure Determination of Catalytic RNAs and Investigations of Their Metal Ion-Binding Properties

Naturally occurring RNA molecules exhibit many unexpected and fascinating properties in living cells such as protein synthesis and transport, regulation of metabolic functions, and catalytic cleavage reactions. To understand this functional diversity, a detailed knowledge of RNA structure and metal ion-binding properties is crucial. In our research group, we address these problems by combining various biochemical, analytical and spectroscopic techniques. A large part of our work is devoted to the structure determination of catalytic RNA molecules, i.e. ribozymes, by NMR. Based on the three-dimensional structure, further experiments are carried out to understand in detail the effects of different metal ions on the local and global structure, as well as catalysis itself

Specific phosphorothioate substitution within domain 6 of a group II intron ribozyme leads to changes in local structure and metal ion binding

Group II introns are large self-splicing ribozymes that require high amounts of monovalent and divalent metal ions for folding and catalysis under in vitro conditions. Domain 6 of these ribozymes contains a highly conserved adenosine whose 2′-OH acts as a nucleophile during self-cleavage via the branching pathway. We have previously suggested a divalent metal ion that binds to the major groove at the GU wobble pair above the branch-A in a minimal, but active branch domain construct (D6–27) from the yeast mitochondrial intron Sc.ai5γ. Here we characterize metal ion binding to the phosphate oxygens at the branch site. In vitro transcription yielded a D6–27 construct where all R P oxygens of the uridine phosphate groups are replaced by sulfur (α-thio-D6–27). We determined its NMR structure, the second RNA-only structure containing thiophosphate groups. [31P] resonances were assigned and chemical shift changes monitored upon titration with Cd2+. In addition, the two uridines flanking the branch-point, U19 and U21 were specifically thioated by chemical synthesis (thio-U19-D6–27 and thio-U19/U21-D6–27), enabling us to study Cd2+ binding at the R P-, as well as the S P- position of the corresponding phosphate oxygens. Our studies reveal that both non-bridging phosphate oxygens of U19 are involved in metal ion coordination, whereas only the major groove phosphate oxygen of U21 is influenced. Together with NOE data of a hexaamminecobalt(III) titration, this suggests a single metal ion binding site at the GU wobble pair above the branch point in the major groove of D6 of this group II intron ribozyme

A dynamically interacting flexible loop assists oligomerisation of the Caenorhabditis elegans centriolar protein SAS-6

Abstract Centrioles are conserved organelles fundamental for the organisation of microtubules in animal cells. Oligomerisation of the spindle assembly abnormal protein 6 (SAS-6) is an essential step in the centriole assembly process and may act as trigger for the formation of these organelles. SAS-6 oligomerisation is driven by two independent interfaces, comprising an extended coiled coil and a dimeric N-terminal globular domain. However, how SAS-6 oligomerisation is controlled remains unclear. Here, we show that in the Caenorhabditis elegans SAS-6, a segment of the N-terminal globular domain, unresolved in crystallographic structures, comprises a flexible loop that assists SAS-6 oligomerisation. Atomistic molecular dynamics simulations and nuclear magnetic resonance experiments suggest that transient interactions of this loop across the N-terminal dimerisation interface stabilise the SAS-6 oligomer. We discuss the possibilities presented by such flexible SAS-6 segments for the control of centriole formation

Structural analysis of the G-Box domain of the microcephaly protein CPAP suggests a role in centriole architecture

Centrioles are evolutionarily conserved eukaryotic organelles composed of a protein scaffold surrounded by sets of microtubules organized with a 9-fold radial symmetry. CPAP, a centriolar protein essential for microtubule recruitment, features a C-terminal domain of unknown structure, the G-box. A missense mutation in the G-box reduces affinity for the centriolar shuttling protein STIL and causes primary microcephaly. Here, we characterize the molecular architecture of CPAP and determine the G-box structure alone and in complex with a STIL fragment. The G-box comprises a single elongated β sheet capable of forming supramolecular assemblies. Structural and biophysical studies highlight the conserved nature of the CPAP-STIL complex. We propose that CPAP acts as a horizontal “strut” that joins the centriolar scaffold with microtubules, whereas G-box domains form perpendicular connections

Structural Basis of the 9-Fold Symmetry of Centrioles

The centriole, and the related basal body, is an ancient organelle characterized by a universal 9-fold radial symmetry and is critical for generating cilia, flagella, and centrosomes. The mechanisms directing centriole formation are incompletely understood and represent a fundamental open question in biology. Here, we demonstrate that the centriolar protein SAS-6 forms rod-shaped homodimers that interact through their N-terminal domains to form oligomers. We establish that such oligomerization is essential for centriole formation in C. elegans and human cells. We further generate a structural model of the related protein Bld12p from C. reinhardtii, in which nine homodimers assemble into a ring from which nine coiled-coil rods radiate outward. Moreover, we demonstrate that recombinant Bld12p self-assembles into structures akin to the central hub of the cartwheel, which serves as a scaffold for centriole formation. Overall, our findings establish a structural basis for the universal 9-fold symmetry of centrioles

A Plasmodium falciparum PHIST protein binds the virulence factor PfEMP1 and comigrates to knobs on the host cell surface

Uniquely among malaria parasites, Plasmodium falciparum-infected erythrocytes (iRBCs) develop membrane protrusions, known as knobs, where the parasite adhesion receptor P. falciparum erythrocyte membrane protein 1 (PfEMP1) clusters. Knob formation and the associated iRBC adherence to host endothelium are directly linked to the severity of malaria and are functional manifestations of protein export from the parasite to the iRBC. A family of exported proteins featuring Plasmodium helical interspersed subtelomeric (PHIST) domains has attracted attention, with members being implicated in host-parasite protein interactions and differentially regulated in severe disease and among parasite isolates. Here, we show that PHIST member PFE1605w binds the PfEMP1 intracellular segment directly with Kd = 5 ± 0.6 μM, comigrates with PfEMP1 during export, and locates in knobs. PHIST variants that do not locate in knobs (MAL8P1.4) or bind PfEMP1 30 times more weakly (PFI1780w) used as controls did not display the same pattern. We resolved the first crystallographic structure of a PHIST protein and derived a partial model of the PHIST-PfEMP1 interaction from nuclear magnetic resonance. We propose that PFE1605w reinforces the PfEMP1-cytoskeletal connection in knobs and discuss the possible role of PHIST proteins as interaction hubs in the parasite exportome.-Oberli, A., Slater, L. M., Cutts, E., Brand, F., Mundwiler-Pachlatko, E., Rusch, S., Masik, M. F. G., Erat, M. C., Beck, H.-P., Vakonakis, I. A Plasmodium falciparum PHIST protein binds the virulence factor PfEMP1 and comigrates to knobs on the host cell surface

Accurate analysis of Mg2+ binding to RNA: From classical methods to a novel iterative calculation procedure

Mg2+ acts as a catalytic cofactor in many ribozymes and specifically bound divalent metal ions have been implicated in the stabilization of structural motifs that are essential for RNA folding. The accurate calculation of intrinsic affinity constants of M2+ to specific binding sites in nucleic acids is therefore of high importance. Methods classically applied to determine the affinity constants of metal ions to RNAs are summarized in the first part of this review, e.g. hydrolytic cleavage experiments, equilibrium dialysis, and spectroscopic techniques like EPR and NMR. However, the fact that several binding sites of similar affinities are often present in a single RNA molecule is mostly neglected. The most immediate consequence of several binding sites is that less than the total amount of M2+ is available to bind to a particular binding site at a given total concentration. We have recently introduced a new iterative procedure that tackles this problem and have developed a rapid calculation tool (ISTAR) that is available from the authors. Here, we explain this procedure in detail under different assumptions and illustrate how the intrinsic affinity constants for Mg2+ to a short RNA hairpin, a minimal domain 6 from the group II intron Sc.ai5 gamma, change. We use ISTAR to calculate intrinsic affinities and to validate a particular binding stoichiometry by judging the quality of the fit to the experimental data for a given model. This is important since weak coordination sites exhibiting similar binding affinities, and being thus in direct competition to each other, are a characteristic feature of nucleic acids. With ISTAR these binding affinities can be calculated more accurately within minutes and we can gain a better understanding of these crucial metal ion-nucleic acid interactions