Volume 20 Issue 7, July 2013

A shield of glycans coats the viral-envelope proteins of HIV. Recent work shows how broadly neutralizing antibodies can recognize this shield despite structural variation in these 'self' carbohydrate structures.

The anaphase-promoting complex/cyclosome (APC/C) is restricted by metazoan protein early mitotic inhibitor 1 (EMI1), a natural, potent inhibitor. New findings suggest that the multimodal inhibitory mechanisms of EMI1 control APC/C-dependent ubiquitylation.

SIMIBI-type NTP-binding proteins are an ancient subfamily of nucleotide-binding proteins that comprises both dimeric ATPases and GTPases (SIMIBI 'twins'). This Perspective focuses on a subset of SIMIBI proteins with a defined function in protein targeting and localization, and aims to define common mechanistic principles and differences for these proteins.

The proteasome 19S particle processes ubiquitinated substrates, unfolds the polypeptides and translocates them to the 20S core particle. Now cryo-EM analyses of the yeast proteasome in the presence of substrate show the 19S in an active conformation, with the AAA+ ring forming a wider central channel aligned with the 20S pore and the essential deubiquitinase Rpn11 positioned right above it.

Argonaute proteins are essential for miRNA function. Now a study in fly and mammalian systems shows that miRNA availability has an effect on the steady-state levels of Argonaute proteins. Argonaute is degraded by the ubiquitin-proteasome pathway, and increased miRNA levels lead to stabilization and accumulation of Argonaute.

Some broadly neutralizing antibodies to HIV-1 recognize glycan-dependent epitopes on gp120. Now X-ray crystallography and EM approaches, along with functional analyses, reveal how one particular antibody (PGT135) recognizes three glycan groups and can accommodate their conformational and chemical diversity.

Some broadly neutralizing antibodies against HIV-1 recognize glycopeptide epitopes in the gp120 V1–V2 region. Now X-ray crystallography and NMR approaches, together with functional analyses of two such antibodies (PG9 and PG16), reveal how glycan binding specificity is achieved. The authors also created a chimeric Fab that showed improved neutralization activity.

Argonaute (Ago) proteins that cleave target RNAs are known as slicer enzymes. Extensive mutagenesis studies combined with in vitro cleavage assays and structural modeling have now enabled catalytically inactive human Ago1 and Ago3 to be turned into slicer enzymes, providing insight into the molecular basis of human Ago1 and Ago3 inactivity.

What makes human Argonaute 2 (Ago2) slicing competent in contrast to AGO1, 3 and 4, which lack RNA cleavage activity, has remained unclear. Now, a molecular evolution approach, called DNA family shuffle (DFS), reveals features of AGO1, 3 and 4 that explain their RNA-slicing deficiency and suggests the juxtaposition and combined action of multiple disseminated protein modules.

APC/C is an E3 ligase complex of ~1.5 MDa that regulates cell division. APC/C^CDH1 is inhibited during interphase by EMI1. Now, NMR spectroscopy, electron microscopy and enzymology analyses are integrated, revealing that EMI1's 143-residue C-terminal domain binds distinct regions of APC/C^CDH1 to block the substrate-binding site and inhibit ubiquitin-chain elongation.

Double-strand-break repair by homologous recombination requires processing of the DNA ends by resection. In vitro and in vivo analysis of the Exo1 and Sgs1–Dna2 resection pathways in budding yeast demonstrates that both resection pathways are hindered by the presence of nucleosomes and require distinct chromatin remodeling events to navigate the chromatin structure.

Nascent polypeptides undergo various cotranslational maturation steps, including N-terminal enzymatic processing, chaperone-assisted folding and membrane targeting. Kinetic analyses now demonstrate that N-terminal processing enzymes have fast ribosome binding kinetics, and premature chaperone recruitment or folding negatively affects processing efficiency, thereby separating nascent chain processing from later events.

The presence of PHO84 sense and antisense transcripts in single cells is strongly anticorrelated, suggesting a switch-like regulation mechanism. Data from single-molecule fluorescent in situ hybridization experiments provide evidence that the exosome component Rrp6 does not degrade full-length PHO84 antisense transcripts but prevents antisense transcription elongation by favoring early termination.

The repressor–antirepressor complex AppA–PpsR is a transcriptional regulator that permits Rhodobacter to switch between photosynthesis and respiration in response to light and oxygen. A combination of crystallography, hydrogen/deuterium-exchange MS and binding assays shows how illumination reduces the affinity of the complex for DNA and provides insight into the mechanism of signal transduction by photoreceptors.

In mammalian spermatozoa, most of the genome is densely packaged by protamines, whereas only a very small percentage of nucleosomes is retained. Genome-wide nucleosome occupancy studies in mouse spermatozoa containing one percent residual histones now demonstrate that nucleosomes in sperm mainly localize to unmethylated CpG-rich sequences in a histone variant–specific manner and are differentially modified.

Glucocorticoid receptor (GR) binds as a dimer to DNA, with the canonical binding sequence featuring two half-sites, each bound by one monomer, separated by a 3-bp spacer. The specific bases contacted by GR are known to direct receptor activity. Now NMR and functional analyses show that nonspecific interactions, including with the spacer region, can also affect GR conformation and activity.

The yeast Sen1p helicase is involved in the transcription termination of most noncoding RNAs transcribed by RNA polymerase II. In vitro analyses now show that Sen1p is the key enzyme in the termination reaction and reveal important features of the mechanism, which resembles that of bacterial termination factor Rho.

The RECQL5 DNA helicase interacts with RNA polymerase II (Pol II) and inhibits transcription through an unknown mechanism. A series of structural and functional analyses now reveals that RECQL5 sterically blocks elongation while also competing with elongation factor TFIIS for binding to Pol II, suggesting a dual mode of transcriptional repression.

The 70-kDa heat-shock protein 70 (Hsp70) molecular chaperones have a nucleotide binding domain and a substrate binding domain that become allosterically coupled when Hsp70s bind ATP. The crystal structure of the prototypical Hsp70, Escherichia coli DnaK, in an ATP-bound state is now presented. The structure and functional analyses reveal the conformational changes and interdomain interactions that underlie allosteric coupling.

By integrating the expression profiles of long noncoding RNAs (lncRNAs) with clinical outcome and somatic copy-number alteration, the authors identified new lncRNAs that are associated with certain cancer subtypes and clinical prognoses. Experimental validation of the prostate cancer cell growth dependence of two new lncRNAs demonstrates the power of this approach for discovering disease-related lncRNAs.