Research articles

The contribution of hydrogen bonding to the thermodynamics of protein folding is not well understood. The strength of hydrogen bonds is now found to depend on the polarity of their microenvironment, being stronger in non-polar surroundings. Thus, the burial or solvent exposure of a few hydrogen bonds near the surface of a protein can significantly stabilize or destabilize its native state.

Telomeres alternate between telomerase-extendable and telomerase-unextendable states. Now this switch is reconstituted in vitro, using DNA templates and purified telomeric proteins from yeast. The molecular chaperone Hsp82 is shown to have a role in this switch by modulating the DNA binding activity of Cdc13.

ESCRT-III proteins play important roles in multivesicular body (MVB) formation, cytokinesis, and enveloped virus budding. The structure of Ist1, which also functions in cytokinesis and MVB sorting, reveals that it, too, is an ESCRT-III family member and suggests that this protein family uses a common mode of autoinhibition.

The Brr2 ATPase is a large DExD/H-box helicase required for key snRNA-remodeling steps during the splicing reaction. The structure of part of Brr2, in conjunction with modeling and functional analysis, indicates that it probably resembles the Hel308 DNA helicase and may share a similar helicase mechanism.

Nonsense-mediated decay is a surveillance pathway that removes transcripts containing a premature stop codon. UPF3 is unusual among the trans-acting factors in the pathway because there are two distinct homologs, UPF3A and UPF3B. Given that patients with reduced UPF3B contain upregulated levels of UPF3A, a regulatory interplay between the two factors is uncovered, where competition for UPF2 binding destabilizes the unbound factor.

The nuclear pore complex mediates nucleocytoplasmic transport and consists of an assembly of multiple copies of ∼30 different proteins called nucleoporins. Kampmann and Blobel describe the structure and flexibility of the heptameric Nup84 complex by single-particle, negative-stain EM. They find that the arrangement of β-propeller and α-solenoid folds within the heptamer resembles that of the clathrin triskelion, which has been proposed to share a common evolutionary origin with the heptameric complex.

Covalent histone modifications can affect the structure of chromatin. Expression of underlying monomethylated histone H3K27 is associated with chromocenters in Arabidopsis, but its presence is unaffected by mutations in the expected methyltransferases. Data now indicate that this modification is catalyzed by Arabidopsis ATRX5 and ATXR6 and is required for silencing, but in a pathway independent of that involving DNA methyltransferases.

The box H/ACA pseudouridine synthase complex guides modification of small nucleolar and Cajal body ribonucleoproteins (sno/scaRNAs), which are essential for maturation of the ribosome and spliceosome. The structure of a functional H/ACA complex containing L7Ae, Nop1 and Cbf5 proteins bound to the substrate and guide RNAs and with a catalytically rearranged substrate in the active site is now presented.

Protein kinase C epsilon (PKCε) priming involves phosphorylation of the kinase domain at 3 different sites. Whether these phosphorylation events were autocatalytic was unclear. Now Parker and colleagues use different PKCε mutants and inhibitors to demonstrate that the occupancy of the nucleotide binding pocket, and not catalytic activity, promote priming of PKCε.

The signaling adaptor TRAF6 is a ubiquitin E3 ligase whose activity can lead to activation of NF-κB and MAPK pathways. New data based on the structure of TRAF6 in complex with the ubiquitin E2 Ubc13 suggest that other TRAFs do not interact with Ubc13 and that oligomerization of TRAF6 is needed for downstream signal transduction.

The amino-terminal domain (ATD) of ionotropic glutamate receptors (iGluRs) mediates their assembly into AMPA-, kainate- and NMDA-sensitive subtypes. The crystal structure of the ATD from the kainate receptor GluR6 reveals a dimeric organization and likely determinants for subtype-selective assembly.

piRNAs have been implicated in transposon silencing. Tudor domain–containing protein-1 (Tdrd1) is now shown to interact with the mouse Piwi ortholog Mili and be part of a complex that contains Mili-associated piRNAs. Interaction occurs through the N terminus of Mili, which is dimethylated, a modification that promotes Tdrd1 interaction. The Tdrd1 mutant shares phenotypes with the Mili mutant but shows a strong effect on the nature of the small RNA pool associated with Mili.

Pupylation is the bacterial equivalent of ubiquitin conjugation, and it involves C-terminal Pup conjugation to lysines to target proteins for proteasomal degradation. This modification reaction has now been reconstituted in vitro using enzymes from the pathogen Mycobacterium tuberculosis. The Pup deamidase (Dop) of this pathway has been defined, and PafA has been shown to conjugate deamidated Pup to substrates.

Topoisomerases alter DNA topology, an essential activity in all organisms. Bacterial type II topoisomerases are targets for antimicrobials such as quinolones, whose binding mode was unclear. Now the crystal structures of pneumococcal topoisomerase IV in a DNA cleavage complex bound to moxifloxacin or clinafloxacin provide insight into how these drugs work and how bacteria can acquire resistance.

A large-scale screen for changes in alternative splice forms in cancer now reveals that almost half of the active alternative splicing events are shifted in breast and ovarian cancer tissues. In addition, many of these changes occur near consensus binding sequences for FOX2 binding sites. This correlates with changes in Fox2 expression or splicing in tissues assessed, and FOX2 depletion results in similar shifts in splice isoforms.

Sertraline (Zoloft) and fluoxetine (Prozac) are selective serotonin-reuptake inhibitors (SSRIs) that are widely prescribed to treat depression. They bind to the presynaptic plasma membrane serotonin transporter (SERT) and inhibit serotonin uptake. Both these drugs possess halogen atoms, but the structural basis for the specificity of SERT for these inhibitors was not known. Zhou et al. now report the crystal structure of LeuT, a bacterial SERT homolog in complex with three different SSRIs. The halogen atoms of all three bind within exactly the same pocket of LeuT, and mutations within this pocket in SERT markedly reduce the transporter's affinity for SSRIs but not for tricyclic antidepressants.

Turnover of mRNA has mainly been studied in the budding yeast, Saccharomyces cerevisiae, and is thought to be initiated by deadenylation. Now Rissland and Norbury reveal that additional, parallel decay pathways are at work in the fission yeast, Schizosaccharomyces pombe. They find that mRNA decapping is frequently independent of deadenylation and that Cid1-dependent uridylation of polyadenylated mRNAs seems to stimulate decapping as part of a novel mRNA turnover pathway. As human cells contain Cid1 orthologs, uridylation may form the basis of a widespread, conserved mechanism of mRNA decay.

The enzyme activation-induced deaminase (AID) promotes antibody diversification after B-cell activation, by causing mutagenic lesions on DNA. Hence, AID's actions must be tightly controlled. AID is found mainly in the cytosolic compartment and contains a known nuclear export sequence. Now a structural nuclear localization sequence and a cytosolic-retention determinant are identified in AID and found to have a role in localization and function.

Gram-negative bacteria use type III secretion systems (T3SSs) to pass virulence factors into host cells, making them potential therapeutic targets to combat bacterial infection. A new EM study of the needle complex from the Shigella T3SS reveals 12-fold symmetry throughout and suggests interactions important for self-assembly and complex stability.

The type III secretion system (T3SS) of pathogenic bacteria is composed of a series of rings in the inner and outer bacterial membranes. Crystallographic studies of EscJ and PrgH, proteins that comprise the two inner membrane rings of the T3SS, suggest that a conserved structural motif serves as a platform for ring assembly. Additional docking and modeling studies reveal details of the T3SS architecture and assembly.