Volume 17 Issue 3, March 2010

Synaptotagmins and SNAREs are known to couple Ca²⁺ sensing to membrane fusion during Ca²⁺-triggered exocytosis, but unraveling the mechanism of this coupling has proven extremely difficult. Two studies in this issue now provide crucial insights into the nature of synaptotagmin-SNARE interactions and reveal unsuspected similarities between synaptotagmins and viral fusion proteins.

The CaMKII complex can enter a state of Ca²⁺-independent activity in response to high frequency Ca²⁺ pulses. Using functional and structural analyses, it is now shown that activation is cooperative and the basis of this is the intersubunit capture of the regulatory subunit of one kinase module by its neighbor.

By controlling interaction with RNA polymerase II, the Head module portion of Mediator plays a critical role in transcriptional regulation. A study now characterizes different conformations of the Head and also its interaction with polymerase and the TATA-binding protein.

Munc13 primes synaptic vesicles for exocytosis. Studies now reveal that the central C2B domain of Munc13 contains a structurally unusual Ca²⁺ binding site that confers specific phospholipid-binding properties to Munc13 and is essential for regulating exocytosis upon trains of action potentials.

The tuberactinomycin antibiotic family is one of the most effective against multi-drug resistant M. tuberculosis. The structures of two members of this family, viomycin and capreomycin, bound to the ribosome now indicate that they act by stabilizing the A site tRNA in a pre-translocation state and may suggest further avenues for drug development.

The crystal structure of FANCL, the catalytic subunit of the Fanconi Anemia core complex, reveals an unexpected domain architecture. The molecule comprises an N-terminal E2-like fold, a novel double-RWD domain, which is found to be responsible for substrate binding, and a C-terminal RING domain that is predicted to facilitate E2 binding.

H2AX phosphorylation is an early response to DNA damage and is mediated by the ATM/ATR kinases. By examining the genome-wide location of γ-H2A in wild type and mutant S. cerevisiae strains, loci that tend to engage ATR (Mec1) and ATM (Tel1) are identified as a route to mapping fragile sites in this genome.

SF2/ASF is a key splicing factor and is abnormally overexpressed in some tumors. By examining the post-transcriptional regulation of this factor, it is now found that SF2/ASF downregulates its own expression through multiple mechanisms.

Previous studies argued that nascent polypeptide chains can form secondary structure in the ribosome exit tunnel despite spatial constraints. Using single-particle cryo-EM reconstructions of eukaryotic ribosomes carrying nascent chains with high helical propensity, density consistent with helix formation is now observed in the exit tunnel as are interactions with tunnel proteins.

Single-molecule FRET studies have resulted in an experimentally derived model of a synaptotagmin–SNARE complex. In this complex of SNARE with synaptotagmin 1, the arrangement of the Ca²⁺-binding loops is similar to that of the structure of SNARE-induced Ca²⁺-bound synaptotagmin 3. This suggests a common molecular mechanism by which the synaptotagmin–SNARE interaction plays a role in Ca²⁺-triggered vesicle fusion.

The interaction between synaptotagmin and SNAREs was characterized by a combination of single-molecule FRET and crystallography. The arrangement of the two Ca²⁺-binding loops of synaptotagmin 3 within SNARE-induced Ca²⁺-bound synaptotagmin 3 matches that of SNARE-bound synaptotagmin 1, suggesting a common molecular mechanism by which the synaptotagmin–SNARE interaction plays a role in Ca²⁺-triggered vesicle fusion.

ABC transporters move substrates across the membrane. The substrate is often delivered by a binding protein. Functional analysis of the bacterial BtuCD-F system now reveals a distinct mechanism for substrate delivery different from other ABC transporters, whereby the binding protein associates with the transporter in the absence of substrate, and substrate or ATP binding destabilize the complex.

T-cadherin is a non-classical GPI-anchored cadherin. The crystal structures of T-cadherins from different organisms now reveal that the EC1-EC2 regions can form dimers via an interface near the EC1-EC2 calcium binding sites, different from the EC1 strand swapping seen in classical cadherin homophilic interactions.

Classical cadherins form two types of dimers: a strand swap version and an “X dimer” that lacks strand exchange between monomers. Strand swapping mutants are now found to form the X-dimer structure. Together with further experiments, this supports the idea that the X-dimer is an intermediate configuration that promotes strand swapped dimer formation.

Most intracellular membrane fusion reactions in eukaryotes are mediated by SNARE proteins present in both fusing membranes. It has been unclear how many SNARE complexes are needed for fusion. FRET studies now show that liposomes with a single SNARE molecule are able to fuse with other liposomes or with purified synaptic vesicles.

Upon formation of DNA double-strand breaks, many protein phosphorylation events are involved in activating cellular responses and DNA repair processes. Now a phosphatase complex containing PP4C and regulatory subunit PP4R2 is shown to dephosphorylate RPA2, and this is necessary to allow efficient DNA repair via homologous recombination.

The crystal structure of the peripheral stalk of the A-type ATPase/synthase (A-ATPase) from Thermus thermophilus reveals a heterodimeric right-handed coiled coil, a protein fold never observed before. Fitting of the stalk structure into the EM density of intact A-ATPase provides the most complete composite model so far.

Aromatic amines can form covalent DNA adducts, in which the damaged base can cause mutations in the vicinity of the lesion. A mechanism for such semi-targeted mutagenesis is now proposed, based on structural and functional data on Dpo4: the bulky lesion-bypass polymerase interaction leads to a conformation of Dpo4 that stabilizes misaligned intermediates.