Molecular dynamics of EF-G during translocation

A study published in Proteins has employed both traditional molecular dynamics simulations and MDFF to study the dynamics of elongation factor G (EF-G). The work is a collaboration between the Schulten (University of Illinois at Urbana-Champaign, USA) and the Frank (Columbia University, USA) groups.

Molecular dynamics of EF-G during translocation.
Wen Li, Leonardo G. Trabuco, Klaus Schulten, and Joachim Frank. Proteins, 79, 1478-1486, 2011.

Elongation factor G (EF-G) plays a crucial role in two stages of mRNA-(tRNA)2 translocation. First, EF-G•GTP enters the pretranslocational ribosome in its intersubunit- rotated state, with tRNAs in their hybrid (P/E, A/P) positions. Second, a conformational change in EF-G’s domain IV induced by GTP hydrolysis disengages the mRNA-anticodon stem-loops of the tRNAs from the decoding center to advance relative to the small subunit when the ribosome undergoes a backward inter-subunit rotation. These events take place as EF-G undergoes a series of large conformational changes as visualized by cryo-EM and X-ray studies. The number and variety of these structures leave open many questions on how these different configurations form during the dynamic translocation process. To understand the molecular mechanism of translocation, we examined the molecular motions of EF-G in solution by means of molecular dynamics simulations. Our results show: (1) rotations of the super-domain formed by domains III-V with respect to the super-domain formed by I-II, and rotations of domain IV with respect to domain III; (2) flexible conformations of both 503- and 575-loops; (3) large conformational variability in the bound form provided by the interaction between domain V and the GTPase-associated center; (4) after GTP hydrolysis, the switch I region seems to be instrumental for effecting the conformational change at the end of domain IV implicated in the disengagement of the codon-anticodon helix from the decoding center.

Applications of the MDFF method reviewed

A special edition of the Journal of Structural Biology has been published with the theme "Combining computational modeling with sparse and low-resolution data." The special edition includes an article reviewing the first applications of the MDFF method, providing also an assessment of the accuracy of MDFF models.

Applications of the molecular dynamics flexible fitting method.
Leonardo G. Trabuco, Eduard Schreiner, James Gumbart, Jen Hsin, Elizabeth Villa, and Klaus Schulten. J. Struct. Biol., 173, 420-427, 2011.

In recent years, cryo-electron microscopy (cryo-EM) has established itself as a key method in structural biology, permitting the structural characterization of large biomolecular complexes in various functional states. The data obtained through single-particle cryo-EM has recently seen a leap in resolution thanks to landmark advances in experimental and computational techniques, resulting in sub-nanometer resolution structures being obtained routinely. The remaining gap between these data and revealing the mechanisms of molecular function can be closed through hybrid modeling tools that incorporate known atomic structures into the cryo-EM data. One such tool, molecular dynamics flexible fitting (MDFF), uses molecular dynamics simulations to combine structures from X-ray crystallography with cryo-EM density maps to derive atomic models of large biomolecular complexes. The structures furnished by MDFF can be used subsequently in computational investigations aimed at revealing the dynamics of the complexes under study. In the present work, recent applications of MDFF are presented, including the interpretation of cryo-EM data of the ribosome at different stages of translation and the structure of a membrane-curvature-inducing photosynthetic complex.

Atomic models of an eukaryotic ribosome

Two new back-to-back articles in PNAS describe atomic models of eukaryotic ribosomes, based on a cryo-EM reconstruction of a translating plant (Triticum aestivum) 80S ribosome at 5.5-Å resolution, together with a 6.1-Å map of a translating Saccharomyces cerevisiae 80S ribosome. MDFF was employed to refine the atomic models, in particular an interactive version that leverages the IMD interface in VMD.

Cryo-EM structure and rRNA model of a translating eukaryotic 80S ribosome at 5.5-Å resolution.
Jean-Paul Armache, Alexander Jarasch, Andreas M. Anger, Elizabeth Villa, Thomas Becker, Shashi Bhushan, Fabrice Jossinet, Michael Habeck, Gülcin Dindar, Sibylle Franckenberg, Viter Marquez, Thorsten Mielke, Michael Thomm, Otto Berninghausen, Birgitta Beatrix, Johannes Söding, Eric Westhof, Daniel N. Wilson, and Roland Beckmann Proc. Natl. Acad. Sci. USA, 107, 19748-19753, 2011.

Protein biosynthesis, the translation of the genetic code into polypeptides, occurs on ribonucleoprotein particles called ribosomes. Although X-ray structures of bacterial ribosomes are available, high-resolution structures of eukaryotic 80S ribosomes are lacking. Using cryoelectron microscopy and single-particle reconstruction, we have determined the structure of a translating plant (Triticum aestivum) 80S ribosome at 5.5-Å resolution. This map, together with a 6.1-Å map of a Saccharomyces cerevisiae 80S ribosome, has enabled us to model ∼98% of the rRNA. Accurate assignment of the rRNA expansion segments (ES) and variable regions has revealed unique ES–ES and r-protein–ES interactions, providing insight into the structure and evolution of the eukaryotic ribosome.

Localization of eukaryote-specific ribosomal proteins in a 5.5Å cryo-EM map of the 80S eukaryotic ribosome.
Jean-Paul Armache, Alexander Jarasch, Andreas M. Anger, Elizabeth Villa, Thomas Becker, Shashi Bhushan, Fabrice Jossinet, Michael Habeck, Gülcin Dindar, Sibylle Franckenberg, Viter Marquez, Thorsten Mielke, Michael Thomm, Otto Berninghausen, Birgitta Beatrix, Johannes Södina, Eric Westhof, Daniel N. Wilson, and Roland Beckmann. Proc. Natl. Acad. Sci. USA, 107, 19754-19759, 2011.

Protein synthesis in all living organisms occurs on ribonucleoprotein particles, called ribosomes. Despite the universality of this process, eukaryotic ribosomes are significantly larger in size than their bacterial counterparts due in part to the presence of 80 r proteins rather than 54 in bacteria. Using cryoelectron microscopy reconstructions of a translating plant (Triticum aestivum) 80S ribosome at 5.5-Å resolution, together with a 6.1-Å map of a translating Saccharomyces cerevisiae 80S ribosome, we have localized and modeled 74/80 (92.5%) of the ribosomal proteins, encompassing 12 archaeal/eukaryote-specific small subunit proteins as well as the complete complement of the ribosomal proteins of the eukaryotic large subunit. Near-complete atomic models of the 80S ribosome provide insights into the structure, function, and evolution of the eukaryotic translational apparatus.

Models of ribosomes in complex with tmRNA obtained by MDFF

Two new back-to-back articles in the EMBO Journal describe atomic models of ribosomes in complex with tmRNA, a molecule that rescues the ribosomes stalled at truncated messages. MDFF was employed in both studies to aid the interpretation of the cryo-EM reconstructions.

tmRNA–SmpB: a journey to the centre of the bacterial ribosome.
Félix Weis, Patrick Bron, Emmanuel Giudice, Jean-Paul Rolland, Daniel Thomas, Brice Felden, and Reynald Gillet. EMBO J., 29, 3810-3818, 2010.

Ribosomes mediate protein synthesis by decoding the information carried by messenger RNAs (mRNAs) and catalysing peptide bond formation between amino acids. When bacterial ribosomes stall on incomplete messages, the trans-translation quality control mechanism is activated by the transfer-messenger RNA bound to small protein B (tmRNA–SmpB ribonucleoprotein complex). Trans-translation liberates the stalled ribosomes and triggers degradation of the incomplete proteins. Here, we present the cryo-electron microscopy structures of tmRNA–SmpB accommodated or translocated into stalled ribosomes. Two atomic models for each state are proposed. This study reveals how tmRNA–SmpB crosses the ribosome and how, as the problematic mRNA is ejected, the tmRNA resume codon is placed onto the ribosomal decoding site by new contacts between SmpB and the nucleotides upstream of the tag-encoding sequence. This provides a structural basis for the transit of the large tmRNA–SmpB complex through the ribosome and for the means by which the tmRNA internal frame is set for translation to resume.

Visualizing the transfer-messenger RNA as the ribosome resumes translation.
Jie Fu, Yaser Hashem, Iwona Wower, Jianlin Lei, Hstau Y Liao, Christian Zwieb, Jacek Wower, and Joachim Frank. EMBO J., 29, 3819-3825, 2010.

Bacterial ribosomes stalled by truncated mRNAs are rescued by transfer-messenger RNA (tmRNA), a dual-function molecule that contains a tRNA-like domain (TLD) and an internal open reading frame (ORF). Occupying the empty A site with its TLD, the tmRNA enters the ribosome with the help of elongation factor Tu and a protein factor called small protein B (SmpB), and switches the translation to its own ORF. In this study, using cryo-electron microscopy, we obtained the first structure of an in vivo-formed complex containing ribosome and the tmRNA at the point where the TLD is accommodated into the ribosomal P site. We show that tmRNA maintains a stable ‘arc and fork’ structure on the ribosome when its TLD moves to the ribosomal P site and translation resumes on its ORF. Based on the density map, we built an atomic model, which suggests that SmpB interacts with the five nucleotides immediately upstream of the resume codon, thereby determining the correct selection of the reading frame on the ORF of tmRNA.

Formation of salt bridges mediates internal dimerization of myosin VI medial tail domain

A new article by the Schulten group (University of Illinois at Urbana-Champaign, USA) employs MDFF in an novel way, namely to retrieve atomistic information from a coarse-grained model of myosin.

Formation of salt bridges mediates internal dimerization of myosin VI medial tail domain.
HyeongJun Kim, Jen Hsin, Yanxin Liu, Paul R. Selvin, and Klaus Schulten. Structure, 18, 1443-1449, 2010.

The unconventional motor protein, myosin VI, is known to dimerize upon cargo-binding to its C-terminal end. It has been shown that one of its tail domains, called the medial tail domain, is a dimerization region. The domain contains an unusual pattern of alternating charged residues and a few hydrophobic residues. To reveal the unknown dimerization mechanism of the medial tail domain, we employed molecular dynamics and single-molecule experimental techniques. Both techniques suggest that the formation of electrostatic-based inter-helical salt bridges between oppositely-charged residues is a key dimerization factor. For the dimerization to occur, the two identical helices within the dimer don't bind in a symmetric fashion, but rather with an off-set of about one helical repeat. Calculations of the dimer-dissociation energy find the contribution of hydrophobic residues to the dimerization process to be minor; they also find that the asymmetric homodimer state is energetically favorable over a state of separate helices.

Structural basis for translational stalling by human cytomegalovirus and fungal arginine attenuator peptide

Another MDFF application has been recently published by the Roland Beckmann group (University of Munich, Germany). Their publication describes two cryo-EM structures of eukaryotic ribosomes stalled by regulatory peptides.

Structural basis for translational stalling by human cytomegalovirus and fungal arginine attenuator peptide. Shashi Bhushan, Helge Meyer, Agata L. Starosta, Thomas Becker, Thorsten Mielke, Otto Berninghausen, Michael Sattler, Daniel N. Wilson, and Roland Beckmann. Mol. Cell, 40, 138-146, 2010.

Specific regulatory nascent chains establish direct interactions with the ribosomal tunnel, leading to translational stalling. Despite a wealth of biochemical data, structural insight into the mechanism of translational stalling in eukaryotes is still lacking. Here we use cryo-electron microscopy to visualize eukaryotic ribosomes stalled during the translation of two diverse regulatory peptides: the fungal arginine attenuator peptide (AAP) and the human cytomegalovirus (hCMV) gp48 upstream open reading frame 2 (uORF2). The C terminus of the AAP appears to be compacted adjacent to the peptidyl transferase center (PTC). Both nascent chains interact with ribosomal proteins L4 and L17 at tunnel constriction in a distinct fashion. Significant changes at the PTC were observed: the eukaryotic-specific loop of ribosomal protein L10e establishes direct contact with the CCA end of the peptidyl-tRNA (P-tRNA), which may be critical for silencing of the PTC during translational stalling. Our findings provide direct structural insight into two distinct eukaryotic stalling processes.

The actin-myosin interface

MDFF has recently been applied by Michael Lorenz and Kenneth C. Holmes (Max Planck Institute for Medical Research, Heidelberg, Germany) to obtain an atomic model of an actin-myosin complex. Here's the abstract of their publication:

The actin-myosin interface. Michael Lorenz and Kenneth C. Holmes. Proc. Natl. Acad. Sci. USA, 107, 12529-12534, 2010.

In order to understand the mechanism of muscle contraction at the atomic level, it is necessary to understand how myosin binds to actin in a reversible way. We have used a novel molecular dynamics technique constrained by an EM map of the actin-myosin complex at 13-A resolution to obtain an atomic model of the strong-binding (rigor) actin-myosin interface. The constraining force resulting from the EM map during the molecular dynamics simulation was sufficient to convert the myosin head from the initial weak-binding state to the strong-binding (rigor) state. Our actin-myosin model suggests extensive contacts between actin and the myosin head (S1). S1 binds to two actin monomers. The contact surface between actin and S1 has increased dramatically compared with previous models. A number of loops in S1 and actin are involved in establishing the interface. Our model also suggests how the loop carrying the critical Arg 405 Glu mutation in S1 found in a familial cardiomyopathy might be functionally involved.