Dynamics and Driving Forces of Macromolecular Complexes

The ribosome, a two-subunit macromolecular machine, deciphers the genetic code and catalyzes peptide bond formation. Dynamic rotational movement between ribosomal subunits is likely required for efficient and accurate protein synthesis, but direct observation of intersubunit dynamics has been obscured by the repetitive, multistep nature of translation. Here, we report a collection of single-molecule fluorescence resonance energy transfer assays that reveal a ribosomal intersubunit conformational cycle in real time during initiation and the first round of elongation. After subunit joining and delivery of correct aminoacyl-tRNA to the ribosome, peptide bond formation results in a rapid conformational change, consistent with the counterclockwise rotation of the 30S subunit with respect to the 50S subunit implied by prior structural and biochemical studies. Subsequent binding of elongation factor G and GTP hydrolysis results in a clockwise rotation of the 30S subunit relative to the 50S subunit, preparing the ribosome for the next round of tRNA selection and peptide bond formation. The ribosome thus harnesses the free energy of irreversible peptidyl transfer and GTP hydrolysis to surmount activation barriers to large-scale conformational changes during translation. Intersubunit rotation is likely a requirement for the concerted movement of tRNA and mRNA substrates during translocation.

SecA, the ATPase of Sec translocase, mediates the post-translational translocation of preprotein through the protein-conducting channel SecYEG in the bacterial inner membrane. Here we report the structures of Escherichia coli Sec intermediates during preprotein translocation as visualized by electron microscopy to probe the oligomeric states of SecA during this process. We found that the translocase holoenzyme is symmetrically assembled by SecA and SecYEG on proteoliposomes, whereas the translocation intermediate 31 (I(31)) becomes asymmetric because of the presence of preprotein. Moreover, SecA is a dimer in these two translocation complexes. This work also shows surface topological changes in the components of translocation intermediates by immunogold labeling. The channel entry for preprotein translocation was found at the center of the I(31) structures. Our results indicate that the presence of preprotein introduces asymmetry into translocation intermediates, while SecA remains dimeric during the translocation process.

To the Editor: In their appealing study published in a recent issue of Stroke , Bolay et al1 extend their previous observations2 that relatively mild ischemia causes long-term dysfunction of synaptic transmission and now suggest that a likely mechanism for this phenomenon is persistent inhibition of presynaptic signaling and neurotransmitter release. In their convincing experiments, the authors demonstrate that 1-hour middle cerebral artery occlusion causes selective inhibition of synapsin-I phosphorylation in the ischemic penumbra of the rat cortex and propose this as a key reason for the suppression of vesicular neurotransmitter release. Interestingly, the postsynaptic elements of neuronal networks seem to remain intact and continue to respond to exogenously applied glutamate. These findings, together with previous data on ischemia-induced inactivation of Ca2+/calmodulin-dependent protein kinase II3 and protein kinase A,4 create a new picture of how relatively mild ischemia or transient ischemic attack may cause long-term impairment of brain function despite survival of neuronal cells. I agree with the authors that phosphorylation defects likely contribute to the long-lasting suppression of synaptic transmission. However, I would like to attract the attention of the authors and readers to an additional possible mechanism of …

Single particle electron cryomicroscopy (cryo-EM) has recently made significant progress in high-resolution structure determination of macromolecular complexes due to improvements in electron microscopic instrumentation and computational image analysis. However, cryo-EM structures can be highly non-uniform in local resolution and all structures available to date have been limited to resolutions above 3Å. Here we present the cryo-EM structure of the 70S ribosome from Escherichia coli in complex with elongation factor Tu, aminoacyl-tRNA and the antibiotic kirromycin at 2.65-2.9Å resolution using spherical aberration (Cs)-corrected cryo-EM. Overall, the cryo-EM reconstruction at 2.9Å resolution is comparable to the best-resolved X-ray structure of the E. coli 70S ribosome (2.8Å), but provides more detailed information (2.65Å) at the functionally important ribosomal core. The cryo-EM map elucidates for the first time the structure of all 35 rRNA modifications in the bacterial ribosome, explaining their roles in fine-tuning ribosome structure and function and modulating the action of antibiotics. We also obtained atomic models for flexible parts of the ribosome such as ribosomal proteins L9 and L31. The refined cryo-EM-based model presents the currently most complete high-resolution structure of the E. coli ribosome, which demonstrates the power of cryo-EM in structure determination of large and dynamic macromolecular complexes.

Dietary Polyunsaturated Fat that Is Low in (n-3) and High in (n-6) Fatty Acids Alters the SNARE Protein Complex and Nitrosylation in Rat Hippocampus ,

During protein synthesis, tRNAs move from the ribosome's aminoacyl to peptidyl to exit sites. Here we investigate conformational motions during spontaneous translocation, using molecular dynamics simulations of 13 intermediate-translocation-state models obtained by combining Escherichia coli ribosome crystal structures with cryo-EM data. Resolving fast transitions between states, we find that tRNA motions govern the transition rates within the pre- and post-translocation states. Intersubunit rotations and L1-stalk motion exhibit fast intrinsic submicrosecond dynamics. The L1 stalk drives the tRNA from the peptidyl site and links intersubunit rotation to translocation. Displacement of tRNAs is controlled by 'sliding' and 'stepping' mechanisms involving conserved L16, L5 and L1 residues, thus ensuring binding to the ribosome despite large-scale tRNA movement. Our results complement structural data with a time axis, intrinsic transition rates and molecular forces, revealing correlated functional motions inaccessible by other means.

Functional localization of acetylcholinesterase (AChE) in vertebrate muscle and brain depends on interaction of the tryptophan amphiphilic tetramerization (WAT) sequence, at the C-terminus of its major splice variant (T), with a proline-rich attachment domain (PRAD), of the anchoring proteins, collagenous (ColQ) and proline-rich membrane anchor. The crystal structure of the WAT/PRAD complex reveals a novel supercoil structure in which four parallel WAT chains form a left-handed superhelix around an antiparallel left-handed PRAD helix resembling polyproline II. The WAT coiled coils possess a WWW motif making repetitive hydrophobic stacking and hydrogen-bond interactions with the PRAD. The WAT chains are related by an approximately 4-fold screw axis around the PRAD. Each WAT makes similar but unique interactions, consistent with an asymmetric pattern of disulfide linkages between the AChE tetramer subunits and ColQ. The P59Q mutation in ColQ, which causes congenital endplate AChE deficiency, and is located within the PRAD, disrupts crucial WAT-WAT and WAT-PRAD interactions. A model is proposed for the synaptic AChE(T) tetramer.

The major function of the ribosome is its ability to catalyze formation of peptide bonds, and it is carried out by the ribosomal peptidyltransferase. Recent evidence suggests that the catalyst of peptide bond formation is the 23S rRNA of the large ribosomal subunit. We have developed an in vitro system for the determination of peptidyltransferase activity in yeast ribosomes. Using this system, a kinetic analysis of a model reaction for peptidyltransferase is described with Ac-Phe-tRNA as the peptidyl donor and puromycin as the acceptor. The Ac-Phe-tRNA-poly(U)-80S ribosome complex (complex C) was isolated and then reacted with excess puromycin to give Ac-Phe-puromycin. This reaction (puromycin reaction) followed first-order kinetics. At saturating concentrations of puromycin, the first-order rate constant (k(3)) is identical to the catalytic rate constant (k(cat)) of peptidyltransferase. This k(cat) from wild-type yeast strains was equal to 2.18 min(-1) at 30 degrees C. We now present for the first time kinetic evidence that yeast ribosomes lacking a particular protein of the 60S subunit may possess significantly altered peptide bond-forming ability. The k(cat) of peptidyltransferase from mutants lacking ribosomal protein L24 was decreased 3-fold to 0.69 min(-1), whereas the k(cat) from mutants lacking L39 was slightly increased to 3.05 min(-1) and that from mutants lacking both proteins was 1.07 min(-1). These results suggest that the presence of ribosomal proteins L24 and, to a lesser extent, L39 is required for exhibition of the normal catalytic activity of the ribosome. Finally, the L24 or L39 mutants did not affect the rate or the extent of the translocation phase of protein synthesis. However, the absence of L24 caused increased resistance to cycloheximide, a translocation inhibitor. Translocation of Ac-Phe-tRNA from the A- to P-site was inhibited by 50% at 1.4 microM cycloheximide for the L24 mutant compared to 0.7 microM for the wild type.

The quantum physics of synaptic communication via the SNARE protein complex

Base-edited cynomolgus monkeys mimic core symptoms of STXBP1 encephalopathy

During neurotransmitter release, SVs (synaptic vesicles) fuse at the active zone and are recovered predominantly via clathrin-mediated endocytosis at the presynaptic compartment surrounding the site of release, referred to as the periactive zone. Exo- and endo-cytosis in synapses are tightly temporarily and spatially coupled to sustain synaptic transmission. The molecular mechanisms linking these two cellular events, which take place in separate compartments of the nerve terminal, remain largely enigmatic. Several lines of evidence indicate that multiple factors may be involved in exocytic-endocytic coupling including SV integral membrane proteins, SV membrane lipids and the membrane-associated actin cytoskeleton. A number of recent studies also indicate that multimodular adaptor proteins shuttling between the active and periactive zones aid the dynamic assembly of macromolecular protein complexes that execute the exo- and endo-cytic limbs of the SV cycle. Here, we discuss recent evidence implicating the multidomain scaffolding and adaptor protein ITSN1 (intersectin 1) as a central regulator of SV cycling.

Serine-proteinase-catalyzed peptide splicing was demonstrated in analogues of the trypsin inhibitor SFTI-1: both single peptides and two-peptide chains (C- and N-terminal peptide chains linked by a disulfide bridge). In the second series, peptide splicing with catalytic amount of proteinase was observed only when formation of acyl-enzyme intermediate was preceded by hydrolysis of the substrate Lys-Ser peptide bond. Here we demonstrate that with an equimolar amount of the proteinase, splicing occurs in all the two-peptide-chain analogues. This conclusion was supported by high resolution crystal structures of selected analogues in complex with trypsin. We showed that the process followed a direct transpeptidation mechanism. Thus, the acyl-enzyme intermediate was formed and was immediately used for a new peptide bond formation; products associated with the hydrolysis of the acyl-enzyme were not observed. The peptide splicing was sequence- not structure-specific.

This review summarizes our current understanding of translation in prokaryotes, focusing on the mechanistic and structural aspects of each phase of translation: initiation, elongation, termination, and ribosome recycling. The assembly of the initiation complex provides multiple checkpoints for messenger RNA (mRNA) and start-site selection. Correct codon-anticodon interaction during the decoding phase of elongation results in major conformational changes of the small ribosomal subunit and shapes the reaction pathway of guanosine triphosphate (GTP) hydrolysis. The ribosome orchestrates proton transfer during peptide bond formation, but requires the help of elongation factor P (EF-P) when two or more consecutive Pro residues are to be incorporated. Understanding the choreography of transfer RNA (tRNA) and mRNA movements during translocation helps to place the available structures of translocation intermediates onto the time axis of the reaction pathway. The nascent protein begins to fold cotranslationally, in the constrained space of the polypeptide exit tunnel of the ribosome. When a stop codon is reached at the end of the coding sequence, the ribosome, assisted by termination factors, hydrolyzes the ester bond of the peptidyl-tRNA, thereby releasing the nascent protein. Following termination, the ribosome is dissociated into subunits and recycled into another round of initiation. At each step of translation, the ribosome undergoes dynamic fluctuations between different conformation states. The aim of this article is to show the link between ribosome structure, dynamics, and function.

Ectoine is a solute compatible with the physiologies of both prokaryotic and eukaryotic cells and is widely synthesized by bacteria as an osmotic stress protectant. Because it preserves functional attributes of proteins and macromolecular complexes, it is considered a chemical chaperone and has found numerous practical applications. However, the mechanism of its biosynthesis is incompletely understood. The second step in ectoine biosynthesis is catalyzed by l-2,4-diaminobutyrate acetyltransferase (EctA; EC 2.3.1.178), which transfers the acetyl group from acetyl-CoA to EctB-formed l-2,4-diaminobutyrate (DAB), yielding N-γ-acetyl-l-2,4-diaminobutyrate (N-γ-ADABA), the substrate of ectoine synthase (EctC). Here, we report the biochemical and structural characterization of the EctA enzyme from the thermotolerant bacterium Paenibacillus lautus (Pl). We found that (Pl)EctA forms a homodimer whose enzyme activity is highly regiospecific by producing N-γ-ADABA but not the ectoine catabolic intermediate N-α-acetyl-l-2,4-diaminobutyric acid. High-resolution crystal structures of (Pl)EctA (at 1.2-2.2 Å resolution) (i) for its apo-form, (ii) in complex with CoA, (iii) in complex with DAB, (iv) in complex with both CoA and DAB, and (v) in the presence of the product N-γ-ADABA were obtained. To pinpoint residues involved in DAB binding, we probed the structure-function relationship of (Pl)EctA by site-directed mutagenesis. Phylogenomics shows that EctA-type proteins from both Bacteria and Archaea are evolutionarily highly conserved, including catalytically important residues. Collectively, our biochemical and structural findings yielded detailed insights into the catalytic core of the EctA enzyme that laid the foundation for unraveling its reaction mechanism.

Effect of Nascent Peptide Steric Bulk on Elongation Kinetics in the Ribosome Exit Tunnel