Volume 18 Issue 9, September 2011

One of the most versatile regulators of actin assembly, the WASP homology 2 (WH2) domain, reveals previously unknown facets by combining with a newly discovered actin-nucleating dimeric structure in the effector protein VopL from Vibrio parahaemolyticus.

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.

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.

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.

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.

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.

Toll-like receptors (TLRs) recognize pathogens and initiate innate immune responses. TLR4 associates with adaptor molecule MD-2 to recognize LPS, and this complex is regulated by a homologous complex, RP105–MD-1. The crystal structure of RP105–MD-1 reveals a unique organization, suggesting a mechanism for regulation of TLR4 response to LPS.

In the RNA world, there would have been ribozymes able to catalyze RNA replication. An artificial ribozyme able to catalyze RNA-templated RNA polymerization has been developed. Now biochemical work and crystal structures of this ribozyme's catalytic core trapped in a state prior to catalysis reveal the complex catalytic strategies it employs.

The contributions that tRNA-ribosome interactions make to the dynamics of translocation are now assessed using single-molecule FRET analysis. The analysis indicates that the flexibility of tRNA plays a key role in tuning the dynamics of pre-translocation complex during translocation.

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.

Pathogen proteins targeting the actin cytoskeleton often serve as model systems to understand their eukaryotic analogs. Structure-function studies of the bacterial actin nucleator VopL suggest that dimerization and pointed-end binding play crucial roles in VopL-mediated nucleation, by enabling the formation of a hexameric pointed end actin nucleus, and that eukaryotic actin nucleators may also function as dimers or higher oligomers.

VopL is a bacterial actin nucleation factor that induces actin stress fibers when injected by bacteria into eukaryotic host cells. Biochemical and structural analyses of VopL-mediated actin nucleation suggest a model in which contacts between the Wiskott-Aldrich homology 2 motifs and the C-terminal domain of VopL stabilize interstrand contacts between the initial actin monomers to create a filament nucleus.

The nature of small RNA species derived from gene termini and intron-exon junctions is now further examined. By sequencing transcripts between 12 and 100 nucleotides derived from cells depleted for RNA decay factors as well as those associated with Argonaute proteins, insights into how these RNAs are produced is provided. Moreover, new small RNAs are identified.