Articles in 2011

RNF4 is an E3 ligase involved in ubiquitinating poly-SUMOylated proteins. The structure of the RNF4 dimer, along with modeling and functional analyses, now indicate that the dimer itself, rather than acting as a scaffold, plays a specific role in recognition by binding the E2~ubiquitin thioester and activating it for catalysis.

Resection of DNA 5′ ends is the initial step for repair of double-strand breaks via homologous recombination. DNA resection is controlled in a cell-cycle dependent manner, with yeast Cdk1 known to control Sae2, a nuclease that initiates resection. Now recruitment to DNA damage sites of Dna2, a nuclease responsible for extensive resection, is also shown to be controlled by Cdk1.

The pseudokinase JH2 domain of JAK2 is a negative regulator of JAK2 activity, but the mechanism for this is unclear. Now it is shown that JH2 is actually an active kinase that phosphorylates negative regulatory sites on JAK2, thus inhibiting JAK2 signaling.

Kinesin-1 is a motor protein that transports cargo unidirectionally along microtubules by alternatively advancing each of its two heads. Here single-molecule approaches show that kinesin-1 mutants in which both motor heads remain bound to the microtubule are able to step forward as well as backward, allowing the development of a five-state kinetic model that accounts quantitatively for both forward and backward stepping by kinesin-1.

The active EGFR kinase domain dimerizes through an asymmetrical interface, and it is even more active when an asymmetric dimer is formed. Negative-stain EM analysis of EGFR now shows that the kinase domains of a dimer can exist in three different states depending on the presence of ligand, mutations and inhibitors, whereas the receptor ectodomain remains in just one conformation.

The TAP-p15 heterodimer is involved in nuclear export of messenger RNAs. The crystal structure of TAP (RRM + LRR domains) bound to part of the export element found in simian type D retroviral RNAs, together with analysis of mutants in export assays, now provides insight into how this element is recognized and exploits the host nuclear transport system.

By examining the structure of phospholipase C-β, an autoinhibitory helix that interacts with the catalytic core of this enzyme is now identified. Disrupting this interaction enhances basal activity and decreases stimulation by Gα_q, supporting an allosteric mechanism for PLCβ activation through displacement of the autoinhibitory helix by Gαq.

The process by which mammalian L1 retrotransposons move around the genome is not entirely clear. Now structural work on L1ORF1 protein, an RNA-binding protein essential for retrotransposition, reveals a trimeric organization and considerable interdomain flexibility. The latter is shown by mutagenesis to be critical for retrotransposition.

Chromatin has been implicated in splicing regulation, but whether a causal relationship exists between pre-mRNA splicing and histone modification has been unclear. New genome-wide analyses now show that pre-mRNA splicing influences H3K36 trimethylation, and that splicing inhibition impairs the recruitment of H3K36 methyltransferase HYPB/Setd2, while splicing activation has the opposite effect.

Structural studies of complexin bound to a mimetic of a prefusion SNARE complex provide insight into how complexin 'clamps' SNARE complex assembly, thereby inhibiting membrane fusion. While the central helix of complexin is anchored to a SNARE complex, its accessory helix extends away and bridges to a second SNARE complex, generating a zigzag array incompatible with fusion.

Comparison of the prefusion and postfusion structures of complexin associated with SNAREs reveals a conformational switch in complexin, which is triggered by the zippering of three key Asp residues in the “switch” region of v-SNAREs into t-SNAREs. This causes complexin to move its accessory helix, thereby releasing the clamped state. This switch is triggered when the calcium sensor synaptotagmin binds calcium ions.

The SNARE-binding protein complexin (CPX) can both facilitate and inhibit membrane fusion, which has been puzzling. Using the Surface Forces Apparatus (SFA) system to obtain distance and interaction measurements of SNARE complexes assembling between bilayers demonstrates that CPX stabilizes an intermediate energetic state in which the SNAREpins are half-zippered, explaining activation and inhibition as the dual consequences of this single mechanism of action.

Rubisco plays a central role in photosynthetic carbon fixation. Form I Rubisco is composed of eight large (RbcL) and eight small (RbcS) subunits. The assembly of the RbcL octamer requires dedicated chaperone RbcX. Now the crystal structure of the RbcL core bound to RbcX reveals how the latter assists assembly of the octamer, and has implications for the role of RbcS.

The condensin complex contributes to the compaction of eukaryotic chromosomes during mitosis and meiosis. How condensin organizes and compacts chromatin is now investigated by the introduction of specific cleavage sites into yeast condensin subunits. The results indicate that condensin forms a ring-like structure that encircles DNA.

Plasmodium parasites use Duffy-binding proteins (DBPs) to invade erythrocyte precursor cells via binding to host protein DARC. Now this interaction is examined by structural and functional work on Plasmodium vivax DBP, revealing that it binds to DARC as a dimer and providing insights into host protective responses and immune evasion by the parasite.

WIF-1 inhibits Wnt signaling by binding Wnt ligands. Structural and biochemical analysis of WIF-1 shows the EGF-like domains wrapping back to contact the ligand-binding WD domain, which also binds a phospholipid near the interaction site for Wnt ligands. The tail of EGF-like domains also harbors a proteoglycan binding site, indicating that all domains of WIF-1 contribute to the regulation of Wnt signaling in vivo.

A previously uncharacterized member of the I-BAR subfamily of BAR-domain proteins, Pinkbar, is expressed in epithelial cells, where it stabilizes planar membrane sheets. Structural, biochemical and mutagenesis analyses suggest a molecular mechanism for this novel membrane-deforming activity.

Cdc13 forms a complex with Stn1-Ten1 that caps budding yeast telomeres; Cdc13 also recruits the telomerase complex. Indeed, Cdk1 phosphorylates Cdc13 and this favors the Cdc13- Est1p, at the expense of Cdc13 association with Stn1, which inhibits telomerase action at telomeres. Now a second, independent mechanism to control Cdc13-Stn1 interaction is revealed: sumoylation of Cdc13 promotes its interaction with Stn1 and prevents telomerase activity.

Previous models of γ-tubulin ring complex (γTuRC) assembly implied that γ-tubulin complex proteins (GCPs) 4–6 serve as a scaffold for arranging multiple γ-tubulin small complexes (γTuSCs) into large γTuRCs. The crystal structure of human GCP4 represents a prototype for all GCPs and can be precisely positioned within γTuSC, providing a new framework for understanding γTuRC formation.

Using purified chromatin rings, what defines a promoter is now explored. Exposure of purified templates to the RSC ATP-dependent remodeling complex leads to Translocation of promoter rather than open reading frame nucleosomes. Together with analysis of the effect of histone deacetylase on this process, this argues that nucleosomes define promoter-specificity.