Biomedical subjects

Luis R Comolli

Publications and source records attributed to Luis R Comolli.

6 recordsLinked to original sources

PubMed2006

Novel crystal form of the ColE1 Rom protein.

The RNA I modulator protein (Rom) acts as a co-regulator of ColE1 plasmid copy number by binding to RNA kissing hairpins and stabilizing their interaction. The structure of Rom has been determined in a new crystal form from X-ray diffraction data to 2.5 A resolution. In this structure, a dimer of the 57-amino-acid protein is found in the asymmetric unit. Each subunit consists almost entirely of two antiparallel alpha-helices joined by a short hairpin bend. The dimer contains a non-crystallographic twofold axis and forms a highly regular four-alpha-helical bundle. The structural packing in this novel crystal form is different from previously known Rom structures. The asymmetric unit contains one dimer, giving a crystal volume per protein weight (V(M)) of 1.83 A(3) Da(-1) and a low solvent content of 30%. Strong packing interactions and low solvation are characteristic of the structure. The Rom protein was cocrystallized with the Tar-Tar* kissing hairpin RNA. Although the electron-density maps do not show bound RNA, altered conformations in the side chains of Rom that are known to be involved in RNA binding have been identified. These results provide additional information about Rom protein conformational flexibility and suggest that the presence of a highly charged polymer such as RNA can promote tight packing of an RNA-binding protein, even when the RNA itself is not observed in the crystal.

Bacterial Proteins↗

PubMed2006

Oligomerization states of the association domain and the holoenyzme of Ca2+/CaM kinase II.

Ca2+/calmodulin activated protein kinase II (CaMKII) is an oligomeric protein kinase with a unique holoenyzme architecture. The subunits of CaMKII are bound together into the holoenzyme by the association domain, a C-terminal region of approximately 140 residues in the CaMKII polypeptide. Single particle analyses of electron micrographs have suggested previously that the holoenyzme forms a dodecamer that contains two stacked 6-fold symmetric rings. In contrast, a recent crystal structure of the isolated association domain of mouse CaMKIIalpha has revealed a tetradecameric assembly with two stacked 7-fold symmetric rings. In this study, we have determined the crystal structure of the Caenorhabditis elegans CaMKII association domain and it too forms a tetradecamer. We also show by electron microscopy that in its fully assembled form the CaMKII holoenzyme is a dodecamer but without the kinase domains, either from expression of the isolated association domain in bacteria or following their removal by proteolysis, the association domains form a tetradecamer. We speculate that the holoenzyme is held in its 6-fold symmetric state by the interactions of the N-terminal approximately 1-335 residues and that the removal of this region allows the association domain to convert into a more stable 7-fold symmetric form.

Animals↗

PubMed2005

Dose tolerance at helium and nitrogen temperatures for whole cell electron tomography.

Electron tomography is currently the only method that allows the direct three-dimensional visualization of macromolecules in an unperturbed cellular context. In principle, tomography should enable the identification and localization of the major macromolecular complexes within intact bacteria, embedded in amorphous ice. In an effort to optimize conditions for recording data that would bring us close to the theoretical limits, we present here a comparison of the dose tolerance of Caulobacter crescentus cells embedded in amorphous ice at liquid helium versus liquid nitrogen temperature. The inner and outer cell membranes, and the periodic structure of the S-layer of this Gram-negative bacterium provide ideal features to monitor changes in contrast and order as a function of dose. The loss of order in the S-layer occurs at comparable doses at helium and nitrogen temperatures. Macroscopic bubbling within the cell and the plastic support develops at both temperatures, but more slowly at helium temperature. The texture of the bubbles is finer in initial stages at helium temperature, giving an impression of contrast reversal in some parts of the specimen. Bubbles evolve differently in different organelles, presumably a consequence of their different chemical composition and mechanical properties. Finally, the amorphous ice "flows" at helium temperature, causing changes in the relative positions of markers within the specimen and distorting the cells. We conclude that for cryo-electron tomography of whole cells liquid nitrogen temperature provides better overall data quality.

Animals↗

PubMed2005

Distinct constrictive processes, separated in time and space, divide caulobacter inner and outer membranes.

Cryoelectron microscope tomography (cryoEM) and a fluorescence loss in photobleaching (FLIP) assay were used to characterize progression of the terminal stages of Caulobacter crescentus cell division. Tomographic cryoEM images of the cell division site show separate constrictive processes closing first the inner membrane (IM) and then the outer membrane (OM) in a manner distinctly different from that of septum-forming bacteria. FLIP experiments had previously shown cytoplasmic compartmentalization (when cytoplasmic proteins can no longer diffuse between the two nascent progeny cell compartments) occurring 18 min before daughter cell separation in a 135-min cell cycle so the two constrictive processes are separated in both time and space. In the very latest stages of both IM and OM constriction, short membrane tether structures are observed. The smallest observed pre-fission tethers were 60 nm in diameter for both the inner and outer membranes. Here, we also used FLIP experiments to show that both membrane-bound and periplasmic fluorescent proteins diffuse freely through the FtsZ ring during most of the constriction procession.

Bacterial Outer Membrane Proteins↗

PubMed2002

A molecular switch underlies a human telomerase disease.

Telomerase is a ribonucleoprotein (RNP) required for maintenance of telomeres. Although up-regulated telomerase activity is closely linked to the cellular immortality characteristic of late stage carcinogenesis, recently, mutations in the telomerase RNA gene in humans have been associated with dyskeratosis congenita and aplastic anemia, both typified by impaired haemopoietic function. These mutations include base changes in a highly conserved putative telomerase RNA pseudoknot. Here, by using in vitro telomerase assays, NMR, and UV absorbance melting analyses of model oligonucleotides designed to form a "trans-pseudoknot," we describe functional, structural, and energetic properties of this structure. We demonstrate that the pseudoknot domain exists in two alternative states of nearly equal stability in solution: one is the previously proposed pseudoknot formed by pairing P3 with the loop domain of P2b, and the other is a structured P2b loop alone. We show that the two-base mutation (GC1078 --> AG) present in one gene copy in a family with dyskeratosis congenita abrogates telomerase activity. This mutation hyperstabilizes the P2b intraloop structure, blocking pseudoknot formation. Conversely, when the P3 pseudoknot pairing is hyperstabilized by deleting a conserved bulge in P3, telomerase activity also decreases. We propose that the P2bP3 pseudoknot domain acts as a molecular switch, and interconversion between its two states is important for telomerase function. Phylogenetic covariation in the P2b and P3 sequences of 35 species provides a compelling set of "natural" compensatory base pairing changes supporting the existence of the crucial molecular switch.

Anemia, Aplastic↗

PubMed2002

NMR structure of the 3' stem-loop from human U4 snRNA.

The NMR structure of the 3' stem-loop (3'SL) from human U4 snRNA was determined to gain insight into the structural basis for conservation of this stem-loop sequence from vertebrates. 3'SL sequences from human, rat, mouse and chicken U4 snRNA each consist of a 7 bp stem capped by a UACG tetraloop. No high resolution structure has previously been reported for a UACG tetraloop. The UACG tetraloop portion of the 3'SL was especially well defined by the NMR data, with a total of 92 NOE-derived restraints (about 15 per residue), including 48 inter-residue restraints (about 8 per residue) for the tetraloop and closing C-G base pair. Distance restraints were derived from NOESY spectra using MARDIGRAS with random error analysis. Refinement of the 20mer RNA hairpin structure was carried out using the programs DYANA and miniCarlo. In the UACG tetraloop, U and G formed a base pair stabilized by two hydrogen bonds, one between the 2'-hydroxyl proton of U and carbonyl oxygen of G, another between the imino proton of G and carbonyl oxygen O2 of U. In addition, the amino group of C formed a hydrogen bond with the phosphate oxygen of A. G adopted a syn orientation about the glycosidic bond, while the sugar puckers of A and C were either C2'-endo or flexible. The conformation of the UACG tetraloop was, overall, similar to that previously reported for UUCG tetraloops, another member of the UNCG class of tetraloops. The presence of an A, rather than a U, at the variable position, however, presents a distinct surface for interaction of the 3'SL tetraloop with either RNA or protein residues that may stabilize interactions important for active spliceosome formation. Such tertiary interactions may explain the conservation of the UACG tetraloop motif in 3'SL sequences from U4 snRNA in vertebrates.

Conserved Sequence↗