The Lsm Proteins: Ring Architectures for RNA Capture

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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.18 page(s

snRNP assemblies: defining subunit interactions of Lsm heptamer rings

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Assembling the core LSM protein complex for RNA processing

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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 Å wide, 20 Å pores) in discrete tetrameric and octameric forms. Following immobilization, the polyproteins successfully act as affinity pulldown ligands for proteins within yeast lysáte, 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 U10. 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

The series of conformational states adopted by rotorless F1-ATPase during its hydrolysis cycle

F1Fo ATP synthase interchanges phosphate transfer energy and proton motive force via a rotary catalytic mechanism and isolated F1-ATPase subcomplexes can also hydrolyze ATP to generate rotation of their central γ rotor subunit. As ATP is hydrolyzed, the F1-ATPase cycles through a series of conformational states that mediates unidirectional rotation of the rotor. However, even in the absence of a rotor, the α and β subunits are still able to pass through a series of conformations, akin to those that generate rotation. Here, we use cryoelectron microscopy to establish the structures of these rotorless states. These structures indicate that cooperativity in this system is likely mediated by contacts between the β subunit lever domains, irrespective of the presence of the γ rotor subunit. These findings provide insight into how long-range information may be transferred in large biological systems

Molecular organisation by small angle scattering of stacked LSM particles

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Cryo-EM reveals distinct conformations of E. coli ATP synthase on exposure to ATP

ATP synthase produces the majority of cellular energy in most cells. We have previously reported cryo-EM maps of autoinhibited E. coli ATP synthase imaged without addition of nucleotide (Sobti et al. 2016), indicating that the subunit ε engages the α, β and γ subunits to lock the enzyme and prevent functional rotation. Here we present multiple cryo-EM reconstructions of the enzyme frozen after the addition of MgATP to identify the changes that occur when this ε inhibition is removed. The maps generated show that, after exposure to MgATP, E. coli ATP synthase adopts a different conformation with a catalytic subunit changing conformation substantially and the ε C-terminal domain transitioning via an intermediate \u27half-up\u27 state to a condensed \u27down\u27 state. This work provides direct evidence for unique conformational states that occur in E. coli ATP synthase when ATP binding prevents the ε C-terminal domain from entering the inhibitory \u27up\u27 state

Changes within the central stalk of E. coli F1Fo ATP synthase observed after addition of ATP

F1Fo ATP synthase functions as a biological generator and makes a major contribution to cellular energy production. Proton flow generates rotation in the Fo motor that is transferred to the F1 motor to catalyze ATP production, with flexible F1/Fo coupling required for efficient catalysis. F1Fo ATP synthase can also operate in reverse, hydrolyzing ATP and pumping protons, and in bacteria this function can be regulated by an inhibitory ε subunit. Here we present cryo-EM data showing E. coli F1Fo ATP synthase in different rotational and inhibited sub-states, observed following incubation with 10 mM MgATP. Our structures demonstrate how structural transitions within the inhibitory ε subunit induce torsional movement in the central stalk, thereby enabling its rotation within the Fο motor. This highlights the importance of the central rotor for flexible coupling of the F1 and Fo motors and provides further insight into the regulatory mechanism mediated by subunit ε

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.MOE (Min. of Education, S’pore)Published versio