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.

Programmed Cell Death 4 (PDCD4) is a protein known to bind eukaryotic initiation factor 4A (eIF4A), inhibit translation initiation, and act as a tumor suppressor. PDCD4 contains two C-terminal MA3 domains, which are thought to be responsible for its inhibitory function. Here, we analyze the structures and inhibitory functions of these two PDCD4 MA3 domains by x-ray crystallography, NMR, and surface plasmon resonance. We show that both MA3 domains are structurally and functionally very similar and bind specifically to the eIF4A N-terminal domain (eIF4A-NTD) using similar binding interfaces. We found that the PDCD4 MA3 domains compete with the eIF4G MA3 domain and RNA for eIF4A binding. Our data provide evidence that PDCD4 inhibits translation initiation by displacing eIF4G and RNA from eIF4A. The PDCD4 MA3 domains act synergistically to form a tighter and more stable complex with eIF4A, which explains the need for two tandem MA3 domains.apoptosis ͉ eIF4G ͉ protein ͉ NMR ͉ x-ray crystallography P DCD4 is a tumor-suppressor protein that is up-regulated on induction of apoptosis (1) and down-regulated in certain aggressive tumors (2). PDCD4 is controlled by protein kinase S6K1 and the ubiquitin ligase SCF ␤TRCP , and its degradation is necessary for efficient protein translation in vivo, which is a prerequisite for cell growth and, consequently, for cell proliferation (3).PDCD4 is known to bind two eukaryotic translation initiation factors eIF4A and eIF4G (4-6). eIF4A is an RNA helicase that works as a subunit of eIF4F, a complex composed of eIF4G and eIF4E. The helicase activity of eIF4A itself is weak but is enhanced upon binding to eIF4G (7,8). eIF4G has two independent binding sites for eIF4A (9), one in the conserved middle domain (eIF4G-m, HEAT1/MIF4G) ( Fig. 1), and the other in the adjacent second HEAT domain (eIF4G-MA3, HEAT2/MA3) (reviewed in ref. 10). NMR binding studies have shown that eIF4G-m interacts mainly with the C-terminal domain of eIF4A (eIF4A-CTD) (11), whereas eIF4G-MA3 binds to the N-terminal domain of eIF4A (eIF4A-NTD) and only weakly to eIF4A-CTD (A.M., C.S., K.A.E., and G.W., unpublished work). Mutation and deletion analysis indicates that the interaction of eIF4A with eIF4G-m is necessary for translation, whereas the interaction of eIF4A with eIF4G-c (eIF4G-MA3ϩHEAT3 domain) plays a modulatory role (12).PDCD4 contains two MA3 domains after an N-terminal segment of little known function (1). MA3 is a well conserved ␣-helical motif with typically 3-5 helical hairpins and is a subtype of HEAT domains. A single MA3 domain is found in eIF4G-c (eIF4G-MA3, Fig. 1), which has been reported to also bind eIF4A (10, 13). Recently, crystal and solution structures of mouse PDCD4's Cterminal MA3 domain (MA3-c) were reported (14, 15). MA3-c was shown to bind eIF4A, compete with eIF4G-c, and was sufficient to inhibit translation initiation. However, some questions remained. First, the function of the N-terminal MA3 domain (MA3-m) was unclear. Mutations of conserved amino acid residues in either MA3-c or ...

Bacterial transition metal homoeostasis or simply ‘metallostasis’ describes the process by which cells control the intracellular availability of functionally required metal cofactors, from manganese (Mn) to zinc (Zn), avoiding bothmetal deprivation and toxicity. Metallostasis is an emerging aspect of the vertebrate host–pathogen interface that is defined by a ‘tug-of-war’ for biologically essential metals and provides the motivation for much recent work in this area. The host employs a number of strategies to starve the microbial pathogen of essential metals, while for others attempts to limit bacterial infections by leveraging highly competitive metals. Bacteria must be capable of adapting to these efforts to remodel the transition metal landscape and employ highly specialized metal sensing transcriptional regulators, termed metalloregulatory proteins, and metallochaperones, that allocate metals to specific destinations, to mediate this adaptive response. In this essay, we discuss recent progress in our understanding of the structural mechanisms and metal specificity of this adaptive response, focusing on energy-requiring metallochaperones that play roles in the metallocofactor active site assembly in metalloenzymes and metallosensors, which govern the systems-level response to metal limitation and intoxication.

Hydrogen sulfide (H2S) is both a lethal gas and an emerging gasotransmitter in humans, suggesting that cellular H2S level must be tightly regulated. CstB is encoded by the cst operon of the major human pathogen Staphylococcus aureus (S. aureus) and is under the transcriptional control of the persulfide sensor CstR and H2S. Here we show that CstB is a multifunctional Fe(II)-containing persulfide dioxygenase (PDO), analogous to the vertebrate protein ETHE1 (Ethylmalonic Encephalopathy Protein 1). Chromosomal deletion of ethe1 is fatal in vertebrates. In the presence of molecular oxygen (O2), hETHE1 oxidizes glutathione persulfide (GSSH) to generate sulfite and reduced glutathione. In contrast, CstB oxidizes major cellular low molecular weight (LMW) persulfide substrates from S. aureus, coenzyme A persulfide (CoASSH) and bacillithiol persulfide (BSSH), directly to generate thiosulfate (TS) and reduced thiols, thereby avoiding the cellular toxicity of sulfite. Both Cys201 in the N-terminal PDO domain (CstBPDO) and Cys408 in the C-terminal rhodanese domain (CstBRhod) strongly enhance the TS generating activity of CstB. CstB also possesses persulfide transferase (PT; reverse rhodanese) activity which generates TS when provided with LMW persulfides and sulfite, as well as conventional thiosulfate transferase (TST; rhodanese) activity; both activities require Cys408. CstB protects S. aureus against H2S toxicity with C201S and C408S cstB genes unable to rescue a NaHS-induced ΔcstB growth phenotype. Induction of the cst operon by NaHS reveals that functional CstB impacts the cellular TS concentrations. These data collectively suggest that CstB may have evolved to facilitate the clearance of LMW persulfides that occur upon the elevation of the level of cellular H2S and hence may have an impact on bacterial viability under H2S stress, in concert with the other enzymes encoded by the cst operon.

Hydrogen sulfide (H2S) is a toxic molecule and a recently described gasotransmitter in vertebrates whose function in bacteria is not well understood. In this work, we describe the transcriptomic response of the major human pathogen Staphylococcus aureus to quantified changes in levels of cellular organic reactive sulfur species, which are effector molecules involved in H2S signaling. We show that nitroxyl (HNO), a recently described signaling intermediate proposed to originate from the interplay of H2S and nitric oxide, also induces changes in cellular sulfur speciation and transition metal homeostasis, thus linking sulfide homeostasis to an adaptive response to antimicrobial reactive nitrogen species.

Acinetobacter baumannii is an opportunistic nosocomial pathogen that is the causative agent of several serious infections in humans, including pneumonia, sepsis, and wound and burn infections. A. baumannii is also capable of forming proteinaceous biofilms on both abiotic and epithelial cell surfaces. Here, we investigate the response of A. baumannii toward sodium sulfide (Na2S), known to be associated with some biofilms at oxic/anoxic interfaces. The addition of exogenous inorganic sulfide reveals that A. baumannii encodes two persulfide-sensing transcriptional regulators, a primary σ54-dependent transcriptional activator (FisR), and a secondary system controlled by the persulfide-sensing biofilm growth-associated repressor (BigR), which is only induced by sulfide in a fisR deletion strain. FisR activates an operon encoding a sulfide oxidation/detoxification system similar to that characterized previously in Staphylococcus aureus, while BigR regulates a secondary persulfide dioxygenase (PDO2) as part of yeeE-yedE-pdo2 sulfur detoxification operon, found previously in Serratia spp. Global S-sulfuration (persulfidation) mapping of the soluble proteome reveals 513 persulfidation targets well beyond FisR-regulated genes and includes five transcriptional regulators, most notably the master biofilm regulator BfmR and a poorly characterized catabolite regulatory protein (Crp). Both BfmR and Crp are well known to impact biofilm formation in A. baumannii and other organisms, respectively, suggesting that persulfidation of these regulators may control their activities. The implications of these findings on bacterial sulfide homeostasis, persulfide signaling, and biofilm formation are discussed. IMPORTANCE Although hydrogen sulfide (H2S) has long been known as a respiratory poison, recent reports in numerous bacterial pathogens reveal that H2S and more downstream oxidized forms of sulfur collectedly termed reactive sulfur species (RSS) function as antioxidants to combat host efforts to clear the infection. Here, we present a comprehensive analysis of the transcriptional and proteomic response of A. baumannii to exogenous sulfide as a model for how this important human pathogen manages sulfide/RSS homeostasis. We show that A. baumannii is unique in that it encodes two independent persulfide sensing and detoxification pathways that govern the speciation of bioactive sulfur in cells. The secondary persulfide sensor, BigR, impacts the expression of biofilm-associated genes; in addition, we identify two other transcriptional regulators known or projected to regulate biofilm formation, BfmR and Crp, as highly persulfidated in sulfide-exposed cells. These findings significantly strengthen the connection between sulfide homeostasis and biofilm formation in an important human pathogen.