Meghna Sobti

Cryo-EM structures of the autoinhibited E. coli ATP synthase in three rotational states.

A molecular model that provides a framework for interpreting the wealth of functional information obtained on the E. coli F-ATP synthase has been generated using cryo-electron microscopy. Three different states that relate to rotation of the enzyme were observed, with the central stalk’s ε subunit in an extended autoinhibitory conformation in all three states. The Fo motor comprises of seven transmembrane helices and a decameric c-ring and invaginations on either side of the membrane indicate the entry and exit channels for protons. The proton translocating subunit contains near parallel helices inclined by ~30° to the membrane, a feature now synonymous with rotary ATPases. For the first time in this rotary ATPase subtype, the peripheral stalk is resolved over its entire length of the complex, revealing the F1 attachment points and a coiled-coil that bifurcates toward the membrane with its helices separating to embrace subunit a from two sides. DOI: http://dx.doi.org/10.7554/eLife.21598.001

Rotary ATPases--dynamic molecular machines.

Recent work has provided the detailed overall architecture and subunit composition of three subtypes of rotary ATPases. Composite models of F-type, V-type and A-type ATPases have been constructed by fitting high-resolution X-ray structures of individual components into electron microscopy derived envelopes of the intact enzymes. Electron cryo-tomography has provided new insights into the supra-molecular arrangement of eukaryotic ATP synthases within mitochondria. An inherent flexibility in rotary ATPases observed by different techniques suggests greater dynamics during operation than previously envisioned. The concerted movement of subunits within the complex might provide means of regulation and information transfer between distant parts of rotary ATPases thereby fine tuning these molecular machines to their cellular environment, while optimizing their efficiency.

Rotary ATPases: models, machine elements and technical specifications.

Rotary ATPases are molecular rotary motors involved in biological energy conversion. They either synthesize or hydrolyze the universal biological energy carrier adenosine triphosphate. Recent work has elucidated the general architecture and subunit compositions of all three sub-types of rotary ATPases. Composite models of the intact F-, V- and A-type ATPases have been constructed by fitting high-resolution X-ray structures of individual subunits or sub-complexes into low-resolution electron densities of the intact enzymes derived from electron cryo-microscopy. Electron cryo-tomography has provided new insights into the supra-molecular arrangement of eukaryotic ATP synthases within mitochondria and mass-spectrometry has started to identify specifically bound lipids presumed to be essential for function. Taken together these molecular snapshots show that nano-scale rotary engines have much in common with basic design principles of man made machines from the function of individual “machine elements” to the requirement of the right “fuel” and “oil” for different types of motors.

Crystal Structure of Lsm3 Octamer from Saccharomyces cerevisiae: Implications for Lsm Ring Organisation and Recruitment

Sm and Sm-like (Lsm) proteins are core components of the ribonucleoprotein complexes essential to key nucleic acid processing events within the eukaryotic cell. They assemble as polyprotein ring scaffolds that have the capacity to bind RNA substrates and other necessary protein factors. The crystal structure of yeast Lsm3 reveals a new organisation of the L/Sm beta-propeller ring, containing eight protein subunits. Little distortion of the characteristic L/Sm fold is required to form the octamer, indicating that the eukaryotic Lsm ring may be more pliable than previously thought. The homomeric Lsm3 octamer is found to successfully recruit Lsm6, Lsm2 and Lsm5 directly from yeast lysate. Our crystal structure shows the C-terminal tail of each Lsm3 subunit to be engaged in connections across rings through specific beta-sheet interactions with elongated loops protruding from neighbouring octamers. While these loops are of distinct length for each Lsm protein and generally comprise low-complexity polar sequences, several Lsm C-termini comprise hydrophobic sequences suitable for beta-sheet interactions. The Lsm3 structure thus provides evidence for protein-protein interactions likely utilised by the highly variable Lsm loops and termini in the recruitment of RNA processing factors to mixed Lsm ring scaffolds. Our coordinates also provide updated homology models for the active Lsm[1-7] and Lsm[2-8] heptameric rings.

Engineered rings of mixed yeast Lsm proteins show differential interactions with translation factors and U-rich RNA.

The Lsm proteins organize as heteroheptameric ring assemblies capable of binding RNA substrates and ancillary protein factors. We have constructed simplified Lsm polyproteins that organize as multimeric ring structures as analogues of the functional Lsm complexes. Polyproteins Lsm[2+3], Lsm[4+1], and Lsm[5+6] incorporate natural sequence extensions as linker peptides between the core Lsm domains. In solution, the recombinant products organize as stable ring oligomers (75 A wide, 20 A pores) in discrete tetrameric and octameric forms. Following immobilization, the polyproteins successfully act as affinity pull-down ligands for proteins within yeast lysate, including native Lsm proteins. Interaction partners were consistent with current models of the mixed Lsm ring assembly in vivo but also suggest that dynamic rearrangements of Lsm protein complexes can occur. The Lsm polyprotein ring complexes were seen in gel shift assays to have a preference for U-rich RNA sequences, with tightest binding measured for Lsm[2+3] with U(10). Polyprotein rings containing truncated forms of Lsm1 and Lsm4 were found to associate with translation, initiation, and elongation protein factors in an RNA-dependent manner. Our findings suggest Lsm1 and/or Lsm4 can interact with translationally active mRNA.

Rational-based protein engineering: tips and tools.

The rational engineering of proteins is driven by contemporary needs for new and altered biomolecular forms. Utilizing manipulative procedures of molecular biology, it is relatively straightforward to alter protein structure and function to create mutated or fused sequences. We here give an overview of procedures and strategies for site-directed mutagenesis, construction of fusion proteins, and insertion of tags. The design of new protein constructs as well as their over-expression as recombinant products is considered. We also summarize approaches for the engineering of protein complexes by co-expression, a valuable route to generate bioactive multicomponent systems.