MEMO1 binds iron and modulates iron homeostasis in cancer cells.

Mediator of ERBB2-driven cell motility 1 (MEMO1) is an evolutionary conserved protein implicated in many biological processes; however, its primary molecular function remains unknown. Importantly, MEMO1 is overexpressed in many types of cancer and was shown to modulate breast cancer metastasis through altered cell motility. To better understand the function of MEMO1 in cancer cells, we analyzed genetic interactions of MEMO1 using gene essentiality data from 1028 cancer cell lines and found multiple iron-related genes exhibiting genetic relationships with MEMO1. We experimentally confirmed several interactions between MEMO1 and iron-related proteins in living cells, most notably, transferrin receptor 2 (TFR2), mitoferrin-2 (SLC25A28), and the global iron response regulator IRP1 (ACO1). These interactions indicate that cells with high-MEMO1 expression levels are hypersensitive to the disruptions in iron distribution. Our data also indicate that MEMO1 is involved in ferroptosis and is linked to iron supply to mitochondria. We have found that purified MEMO1 binds iron with high affinity under redox conditions mimicking intracellular environment and solved MEMO1 structures in complex with iron and copper. Our work reveals that the iron coordination mode in MEMO1 is very similar to that of iron-containing extradiol dioxygenases, which also display a similar structural fold. We conclude that MEMO1 is an iron-binding protein that modulates iron homeostasis in cancer cells.

Helicobacter pylori FlgN binds its substrate FlgK and the flagellum ATPase FliI in a similar manner observed for the FliT chaperone.

In bacterial flagellum biogenesis, secretion of the hook-filament junction proteins FlgK and FlgL and completion of the flagellum requires the FlgN chaperone. Similarly, the related FliT chaperone is necessary for the secretion of the filament cap protein FliD and binds the flagellar export gate protein FlhA and the flagellum ATPase FliI. FlgN and FliT require FliJ for effective substrate secretion. In Helicobacter pylori, neither FlgN, FliT, nor FliJ have been annotated. We demonstrate that the genome location of HP1120 is identical to that of flgN in other flagellated bacteria and that HP1120 is the homolog of Campylobacter jejuni FlgN. A modeled HP1120 structure contains three α-helices and resembles the FliT chaperone, sharing a similar substrate-binding pocket. Using pulldowns and thermophoresis, we show that both HP1120 and a HP1120Δ126-144 deletion mutant bind to FlgK with nanomolar affinity, but not to the filament cap protein FliD, confirming that HP1120 is FlgN. Based on size-exclusion chromatography and multi-angle light scattering, H. pylori FlgN binds to FlgK with 1:1 stoichiometry. Overall structural similarities between FlgN and FliT suggest that substrate recognition on FlgN primarily involves an antiparallel coiled-coil interface between the third helix of FlgN and the C-terminal helix of the substrate. A FlgNΔ126-144 N100A, Y103A, S111I triple mutant targeting this interface significantly impairs the binding of FlgK. Finally, we demonstrate that FlgNΔ126-144 , like FliT, binds with sub-micromolar affinity to the flagellum ATPase FliI or its N-terminal domain. Hence FlgN and FliT likely couple delivery of low-abundance export substrates to the flagellum ATPase FliI.

N-terminal tyrosine of ISCU2 triggers [2Fe-2S] cluster synthesis by ISCU2 dimerization.

Synthesis of iron-sulfur (Fe/S) clusters in living cells requires scaffold proteins for both facile synthesis and subsequent transfer of clusters to target apoproteins. The human mitochondrial ISCU2 scaffold protein is part of the core ISC (iron-sulfur cluster assembly) complex that synthesizes a bridging [2Fe-2S] cluster on dimeric ISCU2. Initial iron and sulfur loading onto monomeric ISCU2 have been elucidated biochemically, yet subsequent [2Fe-2S] cluster formation and dimerization of ISCU2 is mechanistically ill-defined. Our structural, biochemical and cell biological experiments now identify a crucial function of the universally conserved N-terminal Tyr35 of ISCU2 for these late reactions. Mixing two, per se non-functional ISCU2 mutant proteins with oppositely charged Asp35 and Lys35 residues, both bound to different cysteine desulfurase complexes NFS1-ISD11-ACP, restores wild-type ISCU2 maturation demonstrating that ionic forces can replace native Tyr-Tyr interactions during dimerization-induced [2Fe-2S] cluster formation. Our studies define the essential mechanistic role of Tyr35 in the reaction cycle of de novo mitochondrial [2Fe-2S] cluster synthesis.

Structure and functional dynamics of the mitochondrial Fe/S cluster synthesis complex.

Iron-sulfur (Fe/S) clusters are essential protein cofactors crucial for many cellular functions including DNA maintenance, protein translation, and energy conversion. De novo Fe/S cluster synthesis occurs on the mitochondrial scaffold protein ISCU and requires cysteine desulfurase NFS1, ferredoxin, frataxin, and the small factors ISD11 and ACP (acyl carrier protein). Both the mechanism of Fe/S cluster synthesis and function of ISD11-ACP are poorly understood. Here, we present crystal structures of three different NFS1-ISD11-ACP complexes with and without ISCU, and we use SAXS analyses to define the 3D architecture of the complete mitochondrial Fe/S cluster biosynthetic complex. Our structural and biochemical studies provide mechanistic insights into Fe/S cluster synthesis at the catalytic center defined by the active-site Cys of NFS1 and conserved Cys, Asp, and His residues of ISCU. We assign specific regulatory rather than catalytic roles to ISD11-ACP that link Fe/S cluster synthesis with mitochondrial lipid synthesis and cellular energy status.

Molecular basis for the binding and modulation of V-ATPase by a bacterial effector protein.

Intracellular pathogenic bacteria evade the immune response by replicating within host cells. Legionella pneumophila, the causative agent of Legionnaires' Disease, makes use of numerous effector proteins to construct a niche supportive of its replication within phagocytic cells. The L. pneumophila effector SidK was identified in a screen for proteins that reduce the activity of the proton pumping vacuolar-type ATPases (V-ATPases) when expressed in the yeast Saccharomyces cerevisae. SidK is secreted by L. pneumophila in the early stages of infection and by binding to and inhibiting the V-ATPase, SidK reduces phagosomal acidification and promotes survival of the bacterium inside macrophages. We determined crystal structures of the N-terminal region of SidK at 2.3 Å resolution and used single particle electron cryomicroscopy (cryo-EM) to determine structures of V-ATPase:SidK complexes at ~6.8 Å resolution. SidK is a flexible and elongated protein composed of an α-helical region that interacts with subunit A of the V-ATPase and a second region of unknown function that is flexibly-tethered to the first. SidK binds V-ATPase strongly by interacting via two α-helical bundles at its N terminus with subunit A. In vitro activity assays show that SidK does not inhibit the V-ATPase completely, but reduces its activity by ~40%, consistent with the partial V-ATPase deficiency phenotype its expression causes in yeast. The cryo-EM analysis shows that SidK reduces the flexibility of the A-subunit that is in the 'open' conformation. Fluorescence experiments indicate that SidK binding decreases the affinity of V-ATPase for a fluorescent analogue of ATP. Together, these results reveal the structural basis for the fine-tuning of V-ATPase activity by SidK.

Structural and Functional Investigations of the Effector Protein LpiR1 from Legionella pneumophila.

Legionella pneumophila is a causative agent of a severe pneumonia, known as Legionnaires' disease. Legionella pathogenicity is mediated by specific virulence factors, called bacterial effectors, which are injected into the invaded host cell by the bacterial type IV secretion system. Bacterial effectors are involved in complex interactions with the components of the host cell immune and signaling pathways, which eventually lead to bacterial survival and replication inside the mammalian cell. Structural and functional studies of bacterial effectors are, therefore, crucial for elucidating the mechanisms of Legionella virulence. Here we describe the crystal structure of the LpiR1 (Lpg0634) effector protein and investigate the effects of its overexpression in mammalian cells. LpiR1 is an α-helical protein that consists of two similar domains aligned in an antiparallel fashion. The hydrophilic cleft between the domains might serve as a binding site for a potential host cell interaction partner. LpiR1 binds the phosphate group at a conserved site and is stabilized by Mn(2+), Ca(2+), or Mg(2+) ions. When overexpressed in mammalian cells, a GFP-LpiR1 fusion protein is localized in the cytoplasm. Intracellular signaling antibody array analysis revealed small changes in the phosphorylation state of several components of the Akt signaling pathway in HEK293T cells overexpressing LpiR1.

Leucyl-tRNA synthetase editing domain functions as a molecular rheostat to control codon ambiguity in Mycoplasma pathogens.

Mycoplasma leucyl-tRNA synthetases (LeuRSs) have been identified in which the connective polypeptide 1 (CP1) amino acid editing domain that clears mischarged tRNAs are missing (Mycoplasma mobile) or highly degenerate (Mycoplasma synoviae). Thus, these enzymes rely on a clearance pathway called pretransfer editing, which hydrolyzes misactivated aminoacyl-adenylate intermediate via a nebulous mechanism that has been controversial for decades. Even as the sole fidelity pathway for clearing amino acid selection errors in the pathogenic M. mobile, pretransfer editing is not robust enough to completely block mischarging of tRNA(Leu), resulting in codon ambiguity and statistical proteins. A high-resolution X-ray crystal structure shows that M. mobile LeuRS structurally overlaps with other LeuRS cores. However, when CP1 domains from different aminoacyl-tRNA synthetases and origins were fused to this common LeuRS core, surprisingly, pretransfer editing was enhanced. It is hypothesized that the CP1 domain evolved as a molecular rheostat to balance multiple functions. These include distal control of specificity and enzyme activity in the ancient canonical core, as well as providing a separate hydrolytic active site for clearing mischarged tRNA.

In vitro assays for the determination of aminoacyl-tRNA synthetase editing activity.

Aminoacyl-tRNA synthetases are essential enzymes that help to ensure the fidelity of protein translation by accurately aminoacylating (or "charging") specific tRNA substrates with cognate amino acids. Many synthetases have an additional catalytic activity to confer amino acid editing or proofreading. This activity relieves ambiguities during translation of the genetic code that result from one synthetase activating multiple amino acid substrates. In this review, we describe methods that have been developed for assaying both pre- and post-transfer editing activities. Pre-transfer editing is defined as hydrolysis of a misactivated aminoacyl-adenylate prior to transfer to the tRNA. This reaction has been reported to occur either in the aminoacylation active site or in a separate editing domain. Post-transfer editing refers to the hydrolysis reaction that cleaves the aminoacyl-ester linkage formed between the carbonyl carbon of the amino acid and the 2' or 3' hydroxyl group of the ribose on the terminal adenosine. Post-transfer editing takes place in a hydrolytic active site that is distinct from the site of amino acid activation. Here, we focus on methods for determination of steady-state reaction rates using editing assays developed for both classes of synthetases.