Summary The RNA helicase eIF4A plays a key role in unwinding of mRNA and scanning during translation initiation. Free eIF4A is a poor helicase and requires the accessory proteins eIF4G and eIF4H. However, the structure of the helicase complex and the mechanisms of stimulation of eIF4A activity have remained elusive. Here we report the topology of the eIF4A/4G/4H helicase complex, which is built from multiple experimentally observed domain-domain contacts. Remarkably, some of the interactions are continuously rearranged during the ATP binding/hydrolysis cycle of the helicase. We show that the accessory proteins modulate the affinity of eIF4A for ATP by interacting simultaneously with both helicase domains and promoting either the closed, ATP-bound conformation or the open, nucleotide-free conformation. The topology of the complex and the spatial arrangement of the RNA-binding surfaces offer insights into their roles in stimulation of helicase activity and the mechanisms of mRNA unwinding and scanning.

Allosteric communication between two ligand-binding sites in a protein is a central aspect of biological regulation that remains mechanistically unclear. Here we show that perturbations in equilibrium picosecond-nanosecond motions impact zinc (Zn)-induced allosteric inhibition of DNA binding by the Zn efflux repressor CzrA (chromosomal zinc-regulated repressor). DNA binding leads to an unanticipated increase in methyl side-chain flexibility and thus stabilizes the complex entropically; Zn binding redistributes these motions, inhibiting formation of the DNA complex by restricting coupled fast motions and concerted slower motions. Allosterically impaired CzrA mutants are characterized by distinct nonnative fast internal dynamics "fingerprints" upon Zn binding, and DNA binding is weakly regulated. We demonstrate the predictive power of the wild-type dynamics fingerprint to identify key residues in dynamics-driven allostery. We propose that driving forces arising from dynamics can be harnessed by nature to evolve new allosteric ligand specificities in a compact molecular scaffold. Technological advances in structural biology have permitted insights (3-5) into how changes in protein structure and flexibility contribute to allostery (6-9). Allostery likely employs a continuum of mechanisms, from domain or subunit rearrangements to predominantly side-chain and backbone dynamics (6-8, 10, 11), to affect biological regulation (1). Although these motions clearly impact site-site communication via defined molecular pathways (9) or energy level perturbations at distant sites (12), an allosteric effect without conformational change remains largely a theoretical postulate (10,13,14). In this context, changes in dynamics upon ligand binding (8,(15)(16)(17)(18)(19)(20) have long been predicted to impact allostery (5, 14, 17, 21); however, obtaining a quantitative experimental demonstration of the role of conformational entropy in allosteric systems remains challenging. Here we test these ideas in the context of heterotropic linkage and pinpoint fast internal dynamics as a primary contributor to functional, structure-encoded dynamics. We report an example of allostery where side-chain rotamer degeneracy is largely responsible for coupling two ligand-binding events through perturbations in a dynamic network that is required for both entropic and enthalpic driving forces.Our model system for studying heterotropic allostery is the transcriptional regulator CzrA (chromosomal zinc-regulated repressor) from the bacterial pathogen Staphylococcus aureus (22-25) ( Fig. 1 and Fig. S1A). Zinc homeostasis is critical to the virulence of S. aureus (26) and of many other microbial pathogens, and allows the organism to adapt to host-imposed zinc toxicity or limitation (27, 28). CzrA is a member of the ubiquitous arsenic repressor (ArsR) family of metalloregulatory proteins (25, 29), individual members of which are capable of sensing a wide array of metal, metalloid, and nonmetal inducers on distinct sites on a relatively simple, homodimeric wi...

To obtain a comprehensive assessment of metabolite levels from extracts of leukocytes, we have recorded ultra-high-resolution 1 H-13 C HSQC NMR spectra of cell extracts, which exhibit spectral signatures of numerous small molecules. However, conventional acquisition of such spectra is time consuming and hampers measurements on multiple samples, which would be needed for statistical analysis of metabolite concentrations. Here we show that the measurement time can be dramatically reduced without loss of spectral quality when using non-linear sampling (NLS) and a new highfidelity Forward Maximum-entropy (FM) reconstruction algorithm. This FM reconstruction conserves all measured time domain data points and guesses the missing data points by an iterative process. This consists of discrete Fourier transformation of the sparse time-domain data set, computation of the spectral entropy, determination of a multidimensional entropy gradient, and calculation of new values for the missing time domain data points with a conjugate gradient approach. Since this procedure does not alter measured data points it reproduces signal intensities with high fidelity and does not suffer from a dynamic-range problem. As an example we measured a natural abundance 1 H-13 C HSQC spectrum of metabolites from granulocyte cell extracts. We show that a high-resolution 1 H-13 C HSQC spectrum with 4k complex increments recorded linearly within 3.7 days can be reconstructed from 1/7 th of the increments with nearly identical spectral appearance, indistinguishable signal intensities and comparable or even lower root mean square (rms) and peak noise patterns measured in signal-free areas. Thus, this approach allows recording of ultra-high resolution 1 H-13 C HSQC spectra in a fraction of the time needed for recording linearly sampled spectra.