Articles in 2010

The anaphase-promoting complex/cyclosome (APC/C) is key to cell cycle regulation and is an E3 ubiquitin ligase. The overall shape of the substrate-free complex has previously been determined in various species. Budding yeast and human APC/Cs are now analyzed by EM structural and biochemical approaches to assess the substrate-binding site and elucidate the relative positioning of Doc1, a factor known to promote processive substrate ubiquitylation.

The K+ channels can display three distinct gating modes. The molecular basis for two of these modes (low open probability and flickery) are now examined by a combination of single-channel recording, crystallography and modeling of mutants in Glu71, revealing that changes in ion and water occupancy in and around the selectivity filter determine modal gating.

Prdm14 is expressed in embryonic stem (ES) cells and germ cells. It is now shown that this transcription factor safeguards the stem cell state by preventing development of mouse ES cells into extraembryonic endoderm cells by repressing loci related to differentiation along this developmental pathway. This suggests that Pdrm14 is part of the network of factors that maintains the ES cell state in mice.

Mitochondria go through cycles of fusion and fission. The yeast dynamin-related protein, Dnm1, localizes to sites of membrane fission and fusion. Hinshaw and coworkers solve the structure of Dnm1-lipid tubes by cryo-EM and compare it to dynamin. Upon GTP addition, the Dnm1-lipid tubes constrict by ~50 nm, reducing the underlying lipid membrane. This suggests that Dnm1 has the ability to impart a large contractile force during mitochondrial division.

The chaperone Hsp90 interacts with different cochaperones during its reaction cycle. Now a ternary complex of Hsp90 with ATPase inhibitor Sti1 and prolyl isomerase Cpr6 is identified as an intermediate by fluorescence resonance energy transfer (FRET) and analytical ultracentrifugation, with support from genetic data.

The human ether-a-go-go–related gene (hERG) channel regulates the heartbeat rhythm. The transition from the open to the inactivated state of human K⁺ channel ERG is analyzed by phi-value analysis, revealing a complex rearrangement of all domains (extracellular, transmembrane and cytoplasmic). The work thus reveals the sequence of conformational changes underlying selectivity filter gating, and may be relevant for other K⁺ channels.

CCT/TRiC is a eukaryotic multi-subunit chaperonin that promotes the correct folding of many cytosolic proteins, including tubulin, within its cavity. Now the crystal structure of CCT in its open state is solved to 5.5-Å resolution and, together with EM and biochemical analysis, allows the observation of a bound tubulin molecule interacting with CCT loops in the apical and equatorial domains.

Dmc1 is a Rad51 paralog with a central role in meiotic recombination. Both Rad51 and Dmc1 form filaments on single-stranded DNA that can promote strand exchange with similar efficiencies. Now, D loops formed with DMC1 are shown to be more resistant to dissociation by BLM or RAD54 than those formed with RAD51.

DNA methyltransferase-1 (DNMT1) is involved in CpG methylation, and its stability is known to be regulated by lysine methylation. Now it has been shown that methylation of DNMT1 and AKT1 phosphorylation of DNMT1 are mutually exclusive and that antagonism between these modifications affects DNMT1 turnover.

Chromosomal translocations can occur as a consequence of the repair of DNA double-strand breaks via the alternative nonhomologous end-joining pathway. Now the DNA end processing factor CtIP is shown to play a role during the formation of chromosomal translocations.

Mammalian protein CtIP processes DNA double-stranded breaks during homologous recombination and possibly for repair via alternative nonhomologous end-joining (alt-NHEJ). Now CtIP is shown to promote alt-NHEJ during class switch recombination in murine B cells.

The precise execution of biological processes depends on various different macromolecules, such as proteins, working together in a coordinated manner. The idea is that the mRNA level of essential genes encoding proteins in the same complex or pathway would be more correlated than transcripts of functionally unrelated genes. It turns out that this holds true for induced genes but not for transcripts of constitutive genes encoding essential subunits of multi-protein complexes. These transcripts are not correlated any more than functionally unrelated genes. The coordination of these functional complexes must occur post-transcriptionally, and likely post-translationally.

β2-microglobulin can form different forms of amyloid fibrils within joints of dialysis patients. Now the crystal structures of a β2m domain swapped dimer and of the steric zipper formed by a short peptide indicate how these fibrils might form.

Circadian rhythms rely on a molecular clock that effects specific gene expression. Recently it has become clear that there is a tight connection between circadian gene transcription and histone acetylation. Histone methyltransferase MLL1, and its product histone H3K4me3, are now shown to cycle at clock-responsive promoters. The former binds key circadian transcription factors and is required for rhythmic gene expression and chromatin modification.

Amyloid fibrils feature in many human diseases and in epigenetic memory, but understanding their molecular structure has been difficult. Now, through a combination of optical trapping and fluorescent imaging to examine amyloid fibrils of the yeast prion protein Sup35, the unexpected unfolding of individual subdomains has been detected, suggesting strong noncovalent interactions maintain the fibril even if individual monomers unfold.

Resection of DNA double strands is known to require a number of factors, but the exact roles each one plays in the process are still unclear. Now the resection events in yeast are reconstituted biochemically, showing that the MRX complex and Sae2 directly stimulate the resection of 5' strands by Exo1.

Dom34 and Hbs1 are involved in no-go decay (NGD) and nonfunctional 18S rRNA decay (18S NRD) pathways that eliminate RNAs causing translation stalling. Now structural work reveals the similarity of the Dom34–Hbs1 complex with elongation factor–tRNA and translation termination eRF1–eRF3 complexes. Mutagenesis analysis of Hbs1 shows that NGD and 18S NRD can be genetically uncoupled.

Reverse transcriptase is an essential enzyme for HIV replication. Single-molecule studies reveal for the first time the structural dynamics of the reverse transcription initiation complex in real time. Reverse transcriptase can bind its tRNA–vRNA substrate in two opposite orientations and flip between these two states.

Alterations in BRCA1 transcription can contribute to sporadic forms of breast cancer. Now the dynamics between transcriptional coactivators and co-repressors at the BRCA1 promoter reveal a central role for the metabolic sensor CtBP in the response to estrogen or cellular metabolic status

A number of antibodies against the membrane-proximal region of HIV-1 gp41 cannot neutralize the virus, despite their high affinity. Now binding studies along with a crystal structure indicate that these non-neutralizing antibodies recognize gp41 in a post-fusion conformation.