ABC-F Translation Factors: from Antibiotic Resistance to Immune Response Corentin Fostier, Laura Monlezun, Farès Ousalem, Shikha Singh, John Hunt, Grégory Boël

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ABC-F translation factors: from antibiotic resistance to immune response Corentin Fostier, Laura Monlezun, Farès Ousalem, Shikha Singh, John Hunt, Grégory Boël

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Corentin Fostier, Laura Monlezun, Farès Ousalem, Shikha Singh, John Hunt, et al.. ABC-F translation factors: from antibiotic resistance to immune response. FEBS Letters, Wiley, 2020, 10.1002/1873- 3468.13984. hal-02998850

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HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. ABC-F translation factors: from antibiotic resistance to immune response

Corentin R. Fostiera, Laura Monlezuna, Farès Ousalema, Shikha Singhb, John F. Huntb†,

Grégory Boëla†

a UMR 8261, CNRS, Université de Paris, Institut de Biologie Physico-Chimique, 75005 Paris, France. b Department of Biological Sciences, 702A Sherman Fairchild Center, Columbia University, New York, NY 10027, United States.

† To whom correspondence may be addressed: Grégory Boël, Institut de Biologie Physico-Chimique, 13 rue Pierre et Marie Curie, 75005 Paris, France, Tel.: +33-1-58415121; E-mail: [email protected]. John F. Hunt, Department of Biological Sciences, 702A Fairchild Center, MC2434, Columbia University, New York, NY 10027-6601, USA, Tel.: (212)-854-5443; Fax: (212)-865-8246; E- mail: [email protected].

Abstract Energy-dependent Translational Throttle A (EttA) from E. coli is a paradigmatic ABC-F protein that controls the first step in polypeptide elongation on the ribosome according to the cellular energy status. Biochemical and structural studies have established that ABC-F proteins generally function as translation factors that modulate the conformation of the peptidyl transferase center upon binding to the ribosomal tRNA exit site. These factors, present in both prokaryotes and eukaryotes but not in archaea, use related molecular mechanisms to modulate protein synthesis for heterogenous purposes, ranging from antibiotic resistance and rescue of stalled ribosomes to modulation of the mammalian immune response. Here we review the canonical studies characterizing the phylogeny, regulation, ribosome interactions, and mechanisms of action of the bacterial ABC-F proteins, and discuss the implications of these studies for the molecular function of eukaryotic ABC-F proteins, including the three human family members.

(142 words)

Keywords: ABC ATPase, ABC-F protein family, protein synthesis, mRNA translation, antibiotic resistance, infection, immune response.

Abbreviations: A/P/E sites: Ribosomal amino-acyl-tRNA (A), peptidyl-tRNA (P), exit (E) sites ARE: Antibiotic resistance Cryo-EM: Cryo-electron microscopy NPET: Nascent peptide exit tunnel PTC: Peptidyl transferase center

Introduction The ATP Binding Cassette (ABC) superfamily is one of the largest protein families. Most of its members are transmembrane transporters using adenosine triphosphate (ATP) hydrolysis to transport molecules across phospholipid membranes. There is nevertheless a large number of cytosolic proteins within this superfamily that use ATP binding and hydrolysis to drive processes unrelated to transmembrane transport. These cytosolic ABC proteins have diverse cellular functions ranging from DNA repair to regulation and modulation of messenger RNA (mRNA) translation. The bacterial enzyme UvrA is a component of a nucleotide excision DNA repair complex [1,2], while the eukaryotic enzyme Rad50 [3] is part of a complex involved in double-stranded DNA break repair. Both of these enzymes use ATP-driven mechanical force to rearrange DNA strands [4,5]. Yeast expresses several translation factors in the ABC superfamily, including two homologous enzymes called eukaryotic Elongation Factor 3 (eEF3) [6] and New1 [7] that form a unique branch of the ABC superfamily. These proteins promote efficient translation termination [7–9] and release of transfer RNA (tRNA) from the ribosomal exit (E) site. Similarly, a homologous group of ABC proteins present only in eukaryotes and archaea is involved in ribosome recycling and referred to as the ABC-E family [10–13]. A very large group of homologous proteins found in bacteria and eukaryotes forms a unique family within the ABC protein superfamily known as the ABC-F family. These proteins, which comprise the largest family of soluble proteins within the ABC superfamily, have pleotropic functions related to mRNA translation.

ABC-F proteins are absent from archaea but widespread among prokaryotes and eukaryotes, most of which encode multiple ABC-F family members. All members of this family that have been rigorously characterized to date are translation factors that bind to the tRNA E site on the ribosome and modulate the conformation of its peptidyl transferase center (PTC) [14–17]. These proteins regulate protein expression at different stages of the translation elongation cycle, generally in response to a wide variety of stresses. Noteworthy examples include yeast GCN20, which is involved in regulating translation initiation during amino acids starvation [18], yeast Arb1, an essential protein involved in multiple steps of ribosome biogenesis [19], human ABCF1 (ABC50), which interacts with eukaryotic initiation factor 2 (eIF2) in initiating ribosomes [20], and Escherichia coli Energy-dependent Translational Throttle A (EttA), an ABC-F protein that binds preferentially to the 70S ribosome initiation complex (IC) to control the first step in polypeptide elongation dependent on cellular ATP/ADP ratio [14,21].

Several subfamilies of bacterial ABC-F proteins (paralog groups) mediate antibiotic resistance (ARE) specifically to compounds that target the 50S subunit of the ribosome, including lincosamides, pleuromutilins, and macrolides [22,15]. ARE ABC-Fs are encoded on the chromosome or on mobile genetic elements in many clinical isolates of bacterial pathogens, including the ESKAPE species (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) [23–27]. We will review below the importance and possible mechanism of action of the ARE ABC-F proteins with a focus on two proteins for which ribosome-bound structures are available, MsrE from P. aeruginosa and VmlR from Bacillus subtilis.

Despite these distinct and broad functions completely unrelated to transmembrane transport, ABC-F proteins still share the canonical architecture and mechanical chemistry of the ABC superfamily, which is characterized by homo- or hetero-dimerization of two ABC ATP-binding domains (also called nucleotide binding domains, NBDs) upon ATP binding. The “ATP-sandwich” dimer of these domains encapsulates two bound Mg-ATP molecules between the Walker A/B motifs in one ABC domain and the “LSGGQ” Signature Sequence [28] in the dimer-related ABC domain [14–17]. The Walker A/B motifs are shared with a much larger group of ATPases including the F1 ATPase and the AAA+ ATPases, but the Signature Sequence is specific to ABC ATPases [29]. ABC-F proteins all contain a unique and characteristic PtIM (P-site tRNA Interaction Motif, PF12848) domain that links its tandem ABC domains [14]. This domain, which is designated PF12848 by the PFAM conserved domain database, is unique

3 to ABC-F proteins and absent from all other ABC superfamily proteins including the eEF3, New1, and ABC-E proteins that also interact with ribosomes but perform unrelated functions [7,8,13,30]. Other structural features found in many, but not all, ABC-F proteins include the arm motif in the a-helical subdomain in the first ABC domain [14,21], the toe motif at the equivalent position in the second ABC domain, and a C-terminal extension [31–33].

This review will describe the phylogeny and structures of ABC-F proteins and subsequently their roles in regulating mRNA translation regulation and mediating antibiotic resistance. We conclude by considering the possible roles of eukaryotic ABC-F proteins in quality control during protein synthesis and in immune regulation. In brief, key points about the ABC-F proteins are summarized in Text Box 1.

Structure and phylogeny of ABC-F translation factors

Distribution within the tree of life The ABC-F family comprises cytosolic proteins containing two tandem ATP-binding cassette (ABC) domains joined by a 60-to-100 residues linker [14,34]. The ABC domains belong to conserved domain group ABC_tran (PF00005), which is the second most common protein domain in nature according to the Pfam database [35] and the defining feature of ABC superfamily proteins [28,36,37]. The linker in ABC-F proteins is also identified by the Pfam database as a conserved structural domain called ABC_tran_Xtn (PF12848). To date, all the ABC-F proteins that have had their biochemical activities characterized play a role in protein synthesis and directly interact with ribosomes. For some of these, the structure of the ABC-F protein in complex with the ribosome has been resolved by cryo-electron microscopy (cryo-EM) [14,16,17] (Figure 1). These structures all show the ABC-F protein binding in a similar geometry in the ribosomal E site.

ABC-F family proteins can be identified based on the presence of three distinct sequence features. As indicated above, the first is the presence of two tandem ABC domain sequences (called ABC1 and ABC2) connected by a linker. The second is the presence in both of the ABC domains of the ABC “Signature Sequence” (LSGGQ/E) [28], which is a hallmark of domains in the ABC superfamily. Finally, the linker between the tandem ABC domains must have a sequence profile that matches the ABC_tran_Xtn (PF12848) domain. Notably, this Pfam [35] domain was identified based on systematic sequence analyses that were conducted before any functional or structural information was available about ABC-F proteins or their interaction with ribosomes. The name ABC_tran_Xtn, which stands for ABC transporter extension, was therefore based only on the knowledge available at that time about other proteins in the ABC transporter superfamily, and no ABC protein containing the ABC_tran_Xtn linker domain has ever been shown to have any involvement in transmembrane transport. Later, when the first structure of an ABC-F protein in complex with the ribosome was reported, the ABC_tran_Xtn domain was observed to form an a-helical hairpin that interacts directly with the tRNA in the peptidyl- tRNA binding P site in the ribosome, so it was named the P-site tRNA interaction motif (PtIM) based on this structural observation [14,21]. The structure and interactions of the PtIM are discussed in the next section.

Some cytosolic ABC proteins like the E. coli protein Ribosomal-Bound ATPase (RbbA) [38,39], the eukaryotic ABC-E protein family [10–13,40], the yeast specific eEF3 protein [8] and its homologue New1 [7] have a similar architecture with two ABC domains in tandem, and these proteins also interact with the mRNA translation machinery (Figure 1). However, importantly, none of them have the PtIM motif, and they all have specific domains that are not present in the ABC-F family, like an iron-sulfur cluster in the ABC-E family [10] and a chromo domain in the eEF3 like family [8]. Moreover, these proteins all interact with different sites on the ribosome than ABC-F family proteins (right in Figure 1).

ABC-F proteins are found in all eukaryotes and the vast majority of eubacteria, and these organisms generally have multiple representatives. However, they are absent from archaea (Figure 1). In contrast, the ABC-E family, which functions in ribosome-recycling in archaea and eukaryotes, has only one representative per organism (aside from the plants) and is present only in those two kingdoms [11–13,41]. In eubacteria, the biochemical function of the ABC-E family is carried out by an unrelated protein family, called the Ribosome-Recycling Factor protein family [42,43], which functions in conjunction with Elongation Factor G (EF-G), to dissociate ribosomal subunits, as the ABC-E proteins do in the other kingdoms. Based on this example, it is possible that the biochemical functions performed by the ABC-F proteins in eubacteria and eukaryotes are fulfilled by a different protein family in archaea that is unrelated in sequence but performs similar mechanical work to the ABC-F family.

With the exception of a set of obligate intracellular pathogens, ABC-F proteins are encoded in the genomes of all other eubacteria. The model eubacterium E. coli K12 expresses four representatives of the ABC-F family (EttA, Uup, YbiT, YheS), while the yeast Saccharomyces cerevisiae expresses two (Arb1 and GCN20), the plant Arabidopsis thaliana expresses five (ABC-F1 to 5), and Homo sapiens express three (ABCF1, ABCF2 and ABCF3) (Figure 1). The large ABC-F family has more than 25 different paralog groups [34], all of which have two tandem ABC domains separated by the PtIM (PF12848 domain), but there is only 25%-35% sequence identity between the proteins in the different paralog groups.

As indicted in the Introduction, one of the paralog groups named EttA controls the first step in polypeptide elongation on the ribosome dependent on cellular ATP/ADP ratio [14,21], while several others called ARE ABC-Fs mediate resistance to some ribosomally-directed antibiotics. EttA and two of the AREs, MsrE and VmlR, have had their 70S-ribosome-bound structures determined by cryo-EM [15,16] (Figure 1). These structures and the mechanism-of-action of the ARE proteins are discussed below. The Saccharomyces protein Arb1 has also had its structure bound to the 60S large ribosomal subunit determined by cryo-EM [17]. This last protein presumably contributes to recycling stalled ribosomes although its exact mechanism-of-action has yet to be defined. The available cryo-EM structures all show the ABC-F proteins bound in an equivalent location in the E site of the ribosome. Furthermore, as discussed further below, the PtIMs make equivalent interactions with the ribosomal RNA (rRNA) from the large ribosomal subunit that positions their Tips near or directly in the PTC of the ribosome. While the biochemical function is not yet known for the other ABC-F paralog groups, they probably all interact with ribosomes in this conserved manner.

Structural conservation of the family Both of the tandem ABC domains in ABC-F proteins contain three subdomains that are present in all ABC superfamily ATPases (Figure 2A). The F1-like-core a/� subdomain forms the central part of the domain, and its topology is conserved in many other ATPases, including F1 ATPase and the AAA+ ATPase superfamily. The N-terminal antiparallel �-sheet (ABC�) subdomain combined with the F1- like-core subdomain forms a rigid ATP-binding structure shared by all ABC superfamily members. Finally, the a-helical (ABCa) subdomain is inserted between two b-strands in the F1-like-core subdomain on its opposite side from the location of the ABC� subdomain (Figure 2A). The ABCa subdomain contains the ABC superfamily Signature Sequence, which is a five amino acid motif with consensus LSGGQ/E [28]. This sequence, which is considered as the hallmark of ABC superfamily proteins, is an ATP-binding motif, as explained below. In contrast to the rigid interaction of the ABC� subdomain with the F1-like-core subdomain, the ABCa subdomain rotates significantly relative to the F1-like-core in different nucleotide-bound conformational states of ABC superfamily domains [44].

The F1-like-core subdomain contains the Walker A and B ATP-binding motifs first identified in F1 ATPase [45]. The Walker A motif is a phosphate-binding loop or “P-loop” that ligates the a- and b-

5 phosphates of ATP. The Walker B motif is a hydrophobic �-strand that terminates with an aspartic acid residue that participates in coordinating the Mg2+ cofactor of ATP [14,16,29]. The g-phosphate switch or Q-loop motif is a short linker between the F1-like-core subdomain and the N-terminus of the ABCa subdomain. The first residue in this motif, which is at the end of the final b-strand in the topology of the F1-like-core subdomain, is a conserved glutamine that interacts with the γ-phosphate group of ATP [28,29].

The fundamental mechanistic characteristic of ABC superfamily proteins is that pairs of ABC domains function together as an ATP-driven mechanical clamp. Concomitant with binding of two ATP molecules, two ABC domains are drawn together to form a geometrically conserved complex in which the LSGGQ/E Signature Sequence in the ABCa subdomain in one of the ABC domains encapsulates the ATP molecule bound to the Walker A/B and Q-loop motifs in the other ABC domain [28] (Figure 2A and B, EttA, VmlR, MsrE). The resulting structure is called an “ATP-sandwich” dimer because the same interactions are formed reciprocally to the ATP molecules bound to each of the two interacting ABC domains. In some ABC superfamily members, two identical ABC domains interact in this conserved geometry (ATP-sandwich homodimers), while in others, the interaction involves two homologous ABC domains with different sequences (ATP-sandwich heterodimers). In ABC-F proteins, the two tandem ABC domains in a single polypeptide chain form an ATP-sandwich heterodimer in the ATP-bound state [14].

The ATP-sandwich dimer conformation of ABC superfamily proteins can be trapped by inhibiting ATP hydrolysis in a manner that preserves ATP-binding affinity [14,16,21]. A generally effective way to achieve this goal is to introduce dual glutamate-to-glutamine (EQ2) mutations in both interacting ABC domains in the catalytic base residue that is located at the C-terminus of the Walker B motif [29]. The native glutamate residue at this position abstracts a hydrogen atom from a water molecule to initiate the hydrolytic attack on the g-phosphate group of ATP, and mutation of this residue to a glutamine preserves the local stereochemistry while preventing activation of the hydrolytic water molecule. Such EQ2 mutations have been extensively used to obtain structures of closed ATP-sandwich conformations of ABC dimers [14,16,21]. In case of the ABC-F proteins, these mutations were first used to study the function of EttA, which showed that inhibition of the ATPase activity inhibits the release of the protein from the 70S ribosome [14]. Consequently, a stable ribosomal complex is formed, which was used to determine the structure of EttA bound to a 70S IC using cryo-EM image reconstruction (Figure 1 and 2A,). This same approach was subsequently used to determine cryo-EM structures of EQ2 mutants of VmlR in complex with 70S ribosome, the structure of MsrE complex was obtained with a wild-type MsrE trapped by a non-hydrolysable ATP homolog (AMPNP) on the Thermus thermophilus 70S ribosome (Figure 1, MsrE, VmlR).

The cryo-EM structure of the EttA in complex, together with biochemical experiments demonstrated that EttA interacts with the E site of ribosomes bearing the initiator tRNA in the P site [21] (Figure 1, EttA). The PtIM of EttA forms an α-helical hairpin that covalently links the tandem ABC domains (Figure 2A), and the Tip of the PtIM, which links the two a-helices forming the hairpin, makes contacts near the PTC in the ribosome, as discussed below.

Figure 2B shows all structures determined to date for ABC-F proteins. The first to be elucidated was EttA, for which a crystal structure was determined that showed a dimer of the protein with its two protomers forming two full ABC domain heterodimer complexes [14]. Each such complex contains the N-terminal ABC domain from one protomer and the C-terminal ABC domain from the other protomer in the dimer. In contrast, the cryo-EM structure of the EQ2 variant of EttA in complex with the 70S IC shows only a monomer of EttA with its two tandem ABC domains interacting with each other in the classic ATP-sandwich conformation (Figure 2A). Biochemical experiments revealed that EttA undergoes a slow but well-behaved reversible dimerization reaction in solution. The EttA dimer forms

6 preferentially at high protein concentration and may be an inactive storage form of the protein, but the activity of the protein on the ribosome is clearly carried out by the monomer [14].

Higher resolution cryo-EM structures were obtained later for the ARE ABC-F proteins MsrE and VmlR [15,16] at 3.5 and 3.6 Å respectively. These proteins bind in a very similar geometry to EttA within the ribosomal E site, where they make equivalent interactions with the P tRNA, and their PtIMs, which are longer than that in EttA, extend directly into the PTC (Figure 1 and 2, VmlR, MsrE). The PtIM in VmlR was called the antibiotic resistance domain (ARD) by the authors of that paper. Furthermore, as noted above, all of these domains belong to PF12848, indicating they have a related sequence profile and likely share a common evolutionary origin, which is supported by their conserved structural interactions with the large ribosomal subunit and the P tRNA. Therefore, the ARD nomenclature is inconsistent with the biochemical activities of evolutionarily related domains, while the PtIM nomenclature accurately captures their conserved structural interactions on the ribosome.

Structures have also been determined for two eukaryotic ABC-F proteins. The first is a crystal structure of human ABCF1 [46]. While its tandem ABC domains have essentially canonical structures, they are bound to ATP in the crystal structure, but they do not adopt the ATP-sandwich heterodimer conformation observed in all of the ribosome-bound ABC-F structures published to date, which is equivalent to the active ATP-bound conformations observed for a wide variety of ABC transporter transmembrane proteins [47–49]. The ABCF1 crystal structure therefore suggests that ABC-F proteins have to be allosterically activated by ribosome binding to form the ATP-sandwich complex and hydrolyze ATP, as discussed further below.

The second structure available for a eukaryotic ABC-F protein is a cryo-EM structure of a yeast ribosome-associated quality control (RQC) complex [50–52] with the large 60S subunit in a pre- peptidyl-tRNA cleavage state (Figure 1 and 2, Arb1) [17]. This complex unexpectedly showed the ABC- F protein Arb1 bound in the E site, where its PtIM contacts the peptidyl-tRNA in the P site and extends toward the PTC [17] in a geometry equivalent to that first observed in the cryo-EM structure of ribosome-bound EttA. The Arb1 complex structure represents an intermediate in the ribosome-rescue pathway that processes faulty nascent proteins after translational stalling. The small subunit of the stalled ribosome is first dissociated via an uncharacterized mechanism that could potentially involve the ATPase activity of Arb1. The bound peptidyl-tRNA remains associated with the 60S subunit, and ribosome associated quality control complex subunit 2 (Rqc2) then catalyzes the addition of C- terminal alanyl and threonyl residues (CAT tail) to the tRNA-bound peptide [50–52]. Finally, the Vms1 protein catalyzes the cleavage of the peptidyl-tRNA to complete the rescue/recycling process.

Least-squares alignment of the N-terminal ABC domain (ABC1) in the five published ABC-F protein structures (Figure 2C) shows that the conformation of this domain is tightly conserved. Furthermore, the structures of EttA, VmlR, and MsrE all show their tandem ABC domains in a similar closed ATP- sandwich heterodimer conformation with two ATP molecules bound [15,16]. This result is consistent with the fact that all of these structures were produced using EQ2 mutant proteins (EttA, VmlR) or a non-hydrolysable ATP analog (AMPPNP-bound MsrE), constituting conditions where ATP hydrolysis by ABC proteins is severely inhibited.

The cryo-EM structure containing Arb1 was determined from an affinity-purified complex formed in vivo in a strain bearing the wild-type Arb1 gene [17]. The resolution of the density for the ABC domains in this structure is lower than the overall resolution of the map, suggesting that the ABC domains are more dynamic than in the cryo-EM structures containing EQ2 mutants of ABC-F proteins. According to the authors of the paper describing the Arb1 complex structure, its ABC domains adopt a flexible but clearly open-state conformation. The ABC1 domain alignment of the available ABC-F structures (Figure 2C and table) shows that the ABC domains in Arb1 are in an open conformation compared to the

7 classical catalytic ATP-sandwich dimer conformation observed for EttA and VmlR. Both ATP-binding sites are also open in the ABCF1 structure. The ABC domains in this structure are rotated away from one another in the same direction observed in the apo structure of EttA [14] and Arb1 (Figure 2C and table), although not to the same extent. This structure may represent a “pre-activated” conformation that cannot close fully to form the composite ATP-binding sites at the ABC1-ABC2 interface until bound to the ribosome, suggesting that the ATPase activity of ABC-F is allosterically activated by interaction with the ribosome.

The ABC1 domain alignment of the available ABC-F structures shows their PtIMs in somewhat different locations and orientations projecting laterally outward from the ABC1-ABC2 interface (Figure 2C). However, when the structures of the corresponding ABC-F ribosome complexes are aligned based on least-squares superposition of the 23S/25S rRNA in the large subunit of the ribosome, all the PtIMs occupy a similar binding site on the ribosome and project in the same direction directly towards the PTC (Figure 2D). This observation implies that the ABC domains in each ABC-F protein have a slightly different orientation in the ribosomal E site but that the geometry of the PtIM in each protein is optimized to maintain its location and orientation relative to the PTC. This evolutionary conservation in interaction geometry demonstrates functional selection in the ABC-F protein family for PtIM interaction with the PTC. Moreover, it implies the PtIM is the functional fulcrum for ABC-F protein interaction with the ribosome.

Variable domains of the family The previous section emphasized the strong homology in the ABC domains and PtIMs of ABC-F proteins. However, sequence analyses reveal some features that are conserved in specific paralog groups (functional subfamilies) but not shared by all ABC-F family members.

Some ABC-F paralog groups have variant sequence patterns in the Signature Sequence motif in ABC1. In E. coli YbiT, it has the sequence AVPGW, far from the classical LSGGQ/E sequence. The E. coli proteins YheS and Uup have a tryptophan residue instead of an E/Q residue at the end of otherwise canonical Signature Sequence motifs in their ABC1 domains, and a tryptophan also occurs at this position in most of the eukaryotic ABC-F proteins. These proteins may adopt variant and less symmetrical ATP-sandwich heterodimer conformations on the ribosome. Future structural and biochemical work should address the functional implications of the observed sequence degeneracy in the ABC1 domains of some ABC-F paralog groups.

EttA contains an a-helical hairpin called the Arm motif inserted in the ABCa subdomain in ABC1 at its contact site with the transmembrane domains in ABC transporter proteins. The Arm motif contacts the uL1 stalk of the 50S ribosomal subunit in the cryo-EM structure of ribosome-bound EttA [21]. It is found in some other ABC-F paralog groups but not all of them, and it is not found in other ABC superfamily proteins [30,34] (Table 1). The X-ray crystal structure of human ABCF1, which contains the Arm motif in ABC1, shows two protein conformations in the asymmetric unit. One of these has well-resolved electron density for the PtIM, while the Arm motif is disordered (Figure 2B, ABCF1), and the other has weak electron density for the PtIM, while the Arm motif has well-resolved electron density [46]. These observations suggest that there is some degree of flexibility in the attachment of these structures to the ABC domains and that crystal-packing interactions possibly mimicking interactions with the ribosome control their exact orientation in the crystal lattice. Yeast Arb1 also contains the Arm motif, but it is not observed in the cryo-EM structure of Arb1 in complex with the 60S ribosomal subunit. The density for the ABC domains is generally weak in these structures, possibly due to the presence of wild-type ATPase-active Arb1. The lack of density for the Arm motif suggests that ATPase activity may increase its dynamics.

Some ABC-F proteins have a C-terminal domain (CTD) that is not present in all paralog groups (Table

1). This domain was first characterized in the protein Uup as a two-stranded a-helical coiled-coil that can bind DNA [31,32]. It is identified by Pfam as a conserved structural domain called ABC_tran_CTD (PF16326). The CTD is required for proper function of ARE ABC-Fs based on studies of VmlR and Vga(A) [16,53].

The Tip of the PtIM, which is the loop between the two α-helices in the PtIM, varies in length and sequence profile between different ABC-F paralog groups [30]. The Tip length is significantly longer in the two ARE ABC-F proteins VmlR and MsrE compared to the other proteins of known structure (Figure 2B). This extension allows the Tip to penetrate directly into the PTC, which displaces the P site tRNA and is likely to play a role in excluding or ejecting antibiotics that bind to this region of the ribosome [15,16,22,30], as discussed further below. The size of the PtIM varies among the different ARE ABC-F subfamilies (Table 1), but there is no clear correlation between its size and the spectrum of antibiotic resistance mediated by the subfamilies [30]. Some individual residues in the Tip have been identified as critical for antibiotic specificity [16,54], which likely contributes to the sequence variability in this region.

In the cryo-EM reconstruction of the yeast ribosome rescue complex, density for Arb1 was only found in 60S ribosomal subunits bearing a peptidyl-tRNA in the pre-cleavage state. Tyrosine-346 in the Tip of the PtIM in Arb1 participates in forming a triple base-stacking interaction with nucleotide A73 of the peptidyl-tRNA and nucleotide A2971 in the 25S rRNA (equivalent to A2602 in E. coli 23S rRNA). Tryptophan-223 in VmlR, which is in an equivalent location in the Tip of the PtIM to tyrosine 346 in Arb1, forms a base-stacking with nucleotide A2602 in the 23S rRNA. In both cases, the interaction with an aromatic residue in the PtIM produces a 180° flip in the orientation of the adenine base of nucleotide A2602 that moves it in the direction of the E site [16,17]. The ABC domains in Arb1 are in the open conformation, while those in VmlR are in the closed ATP-sandwich heterodimer conformation [16,17], which implies that such a base stacking interaction can be maintained in both conformational states of the ABC domains. Notably, these observations suggest that, despite significant sequence variations in this region, the Tip of the PtIM can maintain similar functional interactions with the ribosomal PTC.

Post-translational modifications A global survey of the phosphoproteomes in two E. coli strains, the commonly used laboratory strain K12 and the pathogenic human enterohemorrhagic strain O157:H7, both identified tyrosine-108 in the Arm motif of EttA as a phosphorylated residue [55]. In the ribosome-bound state, the Arm of EttA contacts the L1 stalk of the ribosome which is formed by ribosomal RNA and the uL1 protein. Phosphorylation of tyrosine-108 could alter this interaction and thereby modulate the activity of EttA on the ribosome. In eukaryotes, the ABCF1 protein is phosphorylated on serine-109 and serine-140 by the CK2 kinase, both of which are located in an N-terminal domain upstream of its first ABC domain. These phosphorylation sites are not likely to change the reported interaction of ABCF1 with eIF2, which involves primarily the first 42 amino-acids in ABCF1, but they may modulate the association of ABCF1-eIF2 complex with ribosomes [56].

ABC-F translation factors as regulator of protein expression

ABC-F protein involvement in stress responses in bacteria Starvation is a common environmental stress encountered by bacteria that has shaped their evolution. Efficient regulatory mechanisms govern the adaptation of bacteria to the low nutriment condition by reducing their growth and energy consumption [57]. They adapt using different mechanisms, but reducing and reorienting protein synthesis is particularly important for survival and evolutionary competitiveness because protein synthesis accounts for more than a third of the cellular energy consumption. One important mechanism is the stringent response that involves generating the

9 alarmone guanosine pentaphosphate or tetraphosphate (pppGpp or ppGpp) that modulates both transcription and translation [58]. Other mechanisms involve direct arrest of translation by toxin- antitoxin systems [59].

The ABC-F protein EttA is a translation factor that controls synthesis of the first peptide bond in a nascent protein dependent on cellular ADP:ATP ratio [14]. Using an in vitro translation system, it was shown that the EttA protein has two actions. In the presence of ATP, EttA stabilizes a productive conformation of the CCA acceptor stem of the initiator P site fMet-tRNAfMet in the PTC of the ribosome, and it stimulates slightly the formation of the first peptide bond in a reporter peptide. In contrast, in the presence of a high concentration of ADP compared to ATP, EttA inhibits the formation of the first peptide bond [14] presumably by stabilizing a different conformation of the CCA acceptor stem of the initiator P site fMet-tRNAfMet, although this structural hypothesis has not yet been proven. In accordance with a model where EttA play the role of a thermostat sensing ATP/ADP ratio to regulate protein synthesis, a strain deleted of the ettA gene lose its viability in a co-culture with the wild strain of E. coli during prolonged stationary phase [14]. This suggests that the ettA gene is important for growth in prolonged stationary phase when the cells are starved.

As described in the first section, E. coli K12 contains three EttA paralogs, YheS, YbiT and Uup, and these proteins also interact with the ribosome and modulate protein translation [14,34]. The uup gene is also important for competitive fitness. During co-culture of wild-type and uup deletion strains, the latter is depleted in stationary phase [60]. The other two E. coli paralogs do not show the same phenotype [60], suggesting that there is some specificity in the role of each paralog. Overexpression of the yheS gene in vivo attenuates the cold-sensitive phenotype of an E. coli strain harboring a deletion of the bipA gene, which is believed to be involved in assembling the 50S ribosomal subunit at low temperatures [34]. Earlier work on uup established a different phenotype in vivo, which was the first demonstration of any physiological activity for any ABC-F protein. Deletion of the uup gene in E. coli increases the frequency of precise excision of the Tn5 and Tn10 transposons, indicating that the Uup protein plays a role either directly or indirectly in the excision of transposons. The introduction of mutations in the catalytic site for ATP hydrolysis produces the same phenotype in vivo. The C- terminal domain of Uup following its second ABC domain is necessary for a direct interaction with DNA that has been characterized in vitro, but to date, no activity related to transposition has been demonstrated for the protein in vitro. [33,61]. Uup is also implicated in DNA repair, function that also request DNA recombination like transposon excision [62]. Uup binds to branched DNAs in vitro and can prevents nucleoid mis-segregation during DNA repair [62]. A recent publication [63] proposes based on genetic results that uup is implicated in the stabilization of possible toxic branched DNA intermediates produces in a strain deleted of two gene DNA repair genes radD and recG. Deletion of uup restores the defect of growth of the strain deleted of radD and recG. The connection between the transposon excision phenotype caused by uup deletion, its involvement in DNA repair and its implication in protein synthesis has yet to be elucidated, so it is not known whether it derives from modulation of the translation of enzymes involved in DNA recombination or from a direct binding interaction with such enzymes. In either case, control of genome plasticity in parallel with protein synthesis could potentially enhance bacterial survival under starvation conditions. It also possible that Uup connects DNA recombination (in DNA repair and transposon excision) with mRNA translation to coordinate these different events in the cell.

ABC-F proteins involved in cell development in eukaryotes Some genes encoding proteins belonging to the ABC-F protein family have been demonstrated to be essential for eukaryotic development, which presumably involves modulation of mRNA translation. Lethality is observed in mice in which the ABCF1 gene is inactivated. This gene is expressed in all organs and at higher levels in blastocysts and embryos [64]. The activity of ABCF1 has been linked to E2 ubiquitin-conjugating enzyme activity involved in the macrophage development, which will be

10 discussed below (see section ‘Eukaryotic ABC-F translation factors – protein quality control and modulation of immune response’). In insects, repression of the expression of one of the two genes TcABCF-2A or TcABCF-3A stops development of the beetle Tribolium castaneum, resulting in mortality [65]. ABCF2, the human paralog of yeast Arb1, is engaged in protein aggregate processing during the earliest stages of animal development. It is therefore possible that the activity of ABCF2 in development involves a similar function in translational quality control to that described for Arb1. The human ABCF2 protein has been shown to be a component of an interaction pathway that controls the regulation of cell volume decrease during hypotonic stress in epithelial cells [66]

ABC-F genes are also important for plant development. The AtABCF3 gene in Arabidopsis thaliana is involved in the growth and development of roots. Deletion of AtABCF3 leads both to formation of short roots [67] and reduction in meristem size [68]. In addition, the AtABCF3 gene is important in repairing damaged DNA because its deletion shows a high sensitivity to treatment with methyl methanesulfonate, mitomycin C, or UV-C light [68].

Bacterial ABC-F translation factors and antibiotic resistance

The ribosome as a major target for antibiotics Antibiotics are chemically diverse molecules of natural or synthetic origin that act at low concentration to kill bacteria or inhibit their growth. Antibiotics interfere with essential cellular processes like cell wall synthesis and integrity, nucleic acids synthesis, and protein synthesis [69,70]. Many antibiotics act on the ribosome, hampering polypeptide synthesis or increasing the error rate in decoding mRNA [71]. With a few exceptions, ribosomally-directed antibiotics bind to well-characterized functional sites namely the decoding center (DC), the mRNA channel in the 30S subunit, and, most frequently, the PTC and the nascent polypeptide exit tunnel (NPET) in the 50S subunit [72]. Bacteria have developed antibiotic resistance to ensure population persistence, with resistance being defined as the capacity of a bacterium to tolerate and resist the growth-inhibition or lethal effects of a given antimicrobial compound. The most common mechanisms mediating antibiotic resistance include: i) reducing the permeability of the bacterial cell wall to prevent antibiotic entry; ii) antibiotic ejection from the cell through drug efflux pumps; iii) enzymatic modification of the antibiotic; iv) degradation of the antibiotic; v) modification of the antibiotic target to prevent binding; vi) antibiotic sequestration in an inactive complex; and vii) protection of the antibiotic target (factor-associated protection) [73,74]. The bacterial ARE ABC-F proteins provide ribosome protection to most of the antibiotics targeting the PTC/NPET. The different antibiotic families to which ARE ABC-Fs mediate resistance as well as their binding sites and mode of action are described below and in Figure 3. Unless otherwise indicated, the numbering of ribosomal interaction sites refers to the E. coli ribosome.

Phenicols, sometimes referred to amphenicols, are chlorinated broad-spectrum compounds comprised of chloramphenicol, florfenicol and thiamphenicol. Phenicols bind to the A site crevice which is a wedge-shaped pocket formed by 23S rRNA nucleobases A2451, C2452 and U2504, through a π-stacking interaction of their nitrobenzyl ring with nucleobase C2452 [75,76] (Figure 3A). Although it was believed that phenicols are universal transpeptidation reaction inhibitors that compete with the amino acid side chains of incoming aminoacyl-tRNAs, recent studies indicate that inhibition is context- dependent [77].

Oxazolidinones, sometimes referred to “last-resort drugs”, are synthetic antibiotics comprised of compounds such as linezolid and tedizolid characterized by a 2-oxazolidone ring substituted at its 3- position by a 3-fluorophenyl moiety [78]. Similar to chloramphenicol, linezolid binds to the A site crevice through a π-stacking interaction of its 2-oxazolidone ring with nucleobase U2504 and a π- stacking of the 3-fluorophenyl moiety with nucleobase C2452 (Figure 3A). In this conformation, the

11 remaining morpholino moiety interferes with positioning of the CCA acceptor stem on the incoming aminoacyl-tRNA and/or the sidechain of its bound amino acid [79–81]. Recent works demonstrated that oxazolidinones are not universal inhibitors of transpeptidation reaction but instead inhibit peptide bond formation in a context-dependent manner, leading to unproductive binding-dissociation cycles of incoming aminoacyl-tRNAs [77,82,83].

Macrolides constitute a heterogeneous and broad spectrum-group of naturally occurring antibiotics containing a 12-, 14-, 15- or 16-membered lactone ring substituted by hydroxyl and alkyl groups, with the ring linked to one or more sugar moieties [84–86]. Macrolides bind at the entrance of the NPET with the lactone ring interacting with the wall of the exit tunnel through hydrophobic interactions, while the hydrophilic face of the ring is exposed to the solvent. All macrolide antibiotics possess a sugar moiety essential for drug binding attached to C5 position of the ring; this sugar moiety forms hydrogen bonds with 23S rRNA nucleobases A2058 and A2059 (Figure 3A) [76,87,88]. The C5 mycaminose-mycarose moiety at the C5 position of 16-membered macrolides interferes with proper accommodation of tRNAs or the transpeptidation reaction in the PTC [87]. This mechanism is consistent with the observation that carbomycin A inhibits almost one hundred percent of peptide bond formation likely due to its isobutyrate extension on the C5-disaccharide [89]. The mechanism is more complex for 14- and 15-membered macrolides, as first structural studies hypothesized that they would block the egress of elongating polypeptides by clogging the NPET [87,90]. However, in E. coli cells, around 5% of proteins are still synthesized when treated with erythromycin, 25% with ketolide telithromycin and 40% with pikromycin [91,92]. Biochemical and structural experiments demonstrated that ribosome stalling occurs when specific amino acid motifs are encountered at the PTC in macrolide-bound ribosomes because it is unable to polymerize incoming amino acid. Thus, macrolides and ketolides should be considered as context-specific modulators of peptide bond formation [83,93–97].

Streptogramins are a class of antibiotics containing two chemically distinct divisions called Group A (SA) and Group B (SB) streptogramins (Figure 3A). These antibiotics are produced naturally by several Streptomyces strains [98]. The group A streptogramins (SA), containing drugs such as virginiamycin M (also known as pristinamycin IIA) and dalfopristin, are nonribosomal peptides, specifically 23- membered macrocyclic polyketide hybrids. The group B streptogramins (SB), containing drugs such as pristinamycin IA, virginiamycin S1, and quinupristin, have a different chemical structure, with their backbone being a 19-membered macrocyclic depsipeptide [98,99]. Group A streptogramins bind in a tight pocket within the PTC overlapping the binding site of chloramphenicol and oxazolidinone. Their macrocyclic ring forms hydrogen bonds with nucleotides G2505 and G2061. Binding at this site interferes with correct positioning of the CCA acceptor stems of the A and P tRNAs, making peptide bond formation impossible [100]. Group B streptogramins bind to the NPET at an adjacent but not overlapping site, where they form extensive hydrophobic interactions with the walls of the tunnel and hydrogen bonds with nucleotides A2062 and C2586 (Figure 3A). Although their mechanism of action is not known with confidence, it is assumed that they hamper egress of nascent peptides [71,100]. Lincosamides, comprising clindamycin and lincomycin, are mostly indicated for the treatment of Gram- positive pathogens, but are also effective against some Gram-negative bacteria [101]. Lincosamides bind near the PTC with the propyl group of pyrrolidinyl propyl moiety forming van der Waals contacts with nucleobases C2452 and U2506 (Figure 3A) [76,90]. In this position, the pyrrolidinyl propyl moiety clashes with the sidechain of the aminoacyl group on the tRNA entering the aminoacyl-tRNA binding (A) site on the ribosome, leading to inhibition of protein synthesis [102].

Pleuromutilins, comprising tiamulin, retapamulin, and lefamulin, are highly potent drugs against multidrug resistant Gram-positive bacteria and some pathogenic Gram-negative bacteria [103,104]. Pleuromutilins consist of a tricyclic terpene core that forms hydrophobic interactions with nucleotides A2451, C2452, A2503, U2504 and G2505 in the PTC, and a glycolic ester moiety at the

C14-position prevents proper placement of both A and P tRNAs leading to inhibition of protein synthesis (Figure 3) [105–107]. Moreover, pleuromutilins seem to preferentially inhibit protein synthesis at initiation codons, so they have been used to evaluate the locations of start codons and alternative start sites [108,109].

Spectrum of antibiotics to which ABC-Fs confer resistance As indicated above, the PTC and NPET in the 50S ribosomal subunit both provide binding sites for chemically distinct families of antibiotic, some being critical for human health. Moreover, several of them (i.e. macrolides, phenicols, oxazolidinones) cannot be considered strict inhibitors of protein synthesis, but rather modulators because their action is context-specific, depending on the nature of amino acids in the elongating polypeptide. Different bacterial ARE ABC-F proteins provide factor- associated antibiotic protection to virtually all antimicrobial drugs targeting the PTC/NPET. At least three different resistance phenotypes can be defined based on the locations of the overlapping antibiotic binding sites [30,110], as shown on figure 3B and C: i) the MKSB phenotype (Macrolides, Ketolides, group B Streptogramins) exemplified by the msr family; ii) the PhO phenotype (Phenicols and Oxazolidinones) exemplified by optrA and poxtA; and iii) the PLSA phenotype (Pleuromutilins, Lincosamides, group A Streptogramins) exemplified by the lsA family.

Observation of the spectrum of antibiotic-resistance provided by ARE ABC-F proteins leads to the conclusion that the phenotypes produced by different groups are not mutually exclusive, and some inconsistencies can be found, as outlined in table 1. First, as described by Reynolds and collaborators, msr homologs, which produce the MKSB phenotype, provide resistance to the 14-membered macrolide erythromycin and the 14-membered ketolide telithromycin but not to the 16-membered macrolide tylosin [111]. Tylosin possesses a mycaminose-mycarose moiety that is absent in erythromycin and telithromycin and that protrudes further forward into the PTC, where it overlaps the binding site of the lincosamide clindamycin. Notably, msr homologs also do not provide resistance to this latter antibiotic, suggesting that they cannot offset binding interactions in this region in the PTC. An inconsistency in activity is similarly observed with vgaA, which exhibits the PLSA phenotype. Extensive screening of resistance profile shows that the factor provides resistance to the pleuromutilin drug retapamulin, but not to pleuromutilin drug tiamulin, even though they share the same binding site on the ribosome [22]. Even more striking, vgaA provides resistance to some non-related PLSA antibiotics such as the 16-membered macrolides carbomycin A and leucomycin, although not to the 16- membered macrolide tylosin and spiramycin.

Antibiotic resistance activity could be explained by a molecular mechanism involving direct displacement of bound drug upon ARE ABC-F protein binding to the ribosome. This mechanism-of- action has previously established for the unrelated TetM protein [112]. However, this direct- displacement hypothesis is naïvely inconsistent with the description above of the overlapping and inconsistent specificity profiles of different ARE ABC-F proteins.

A good example of the serious challenges facing the direct-displacement hypothesis is provided by studies of the well-characterized B. subtilis protein VmlR, which produces the PLSA resistance phenotype [16]. As illustrated in Figure 4A and B, the cryo-EM structure of VmlR shows that it binds in the ribosomal E site where its PtIM points towards the PTC, as observed for all ABC-F proteins. The Tip of its PtIM protrudes directly inside the PTC, and this interaction along with direct binding interactions with VmlR displace the P tRNA from its canonical binding location. These interactions move segments of this tRNA towards the A site by distances up to 37 Å, and this non-canonical ribosome conformation has been called the P/V-tRNA conformation (with the V in this name derived from the first letter in VmlR). Structural and biochemical analyses [16] lead to the conclusion that a critical residue for the antibiotic-resistance activity of VmlR is phenylalanine-237 (F237), which is located at the end of the Tip of the PtIM. The observed location of this sidechain in the cryo-EM structure of ribosome-bound

VmlR directly overlaps the known binding sites for virginiamycin M, tiamulin, and lincomycin, three antibiotics to which VmlR provides resistance. Therefore, steric overlap with these antibiotics upon VmlR binding to the ribosome could directly displace them from their binding sites.

However, the Tip of the PtIM in VmlR also directly overlaps the known ribosomal binding sites of chloramphenicol, and linezolid, but VmlR does not mediate resistance to any of these antibiotics. Steric overlap would also be expected to directly displace these antibiotics upon VmlR binding to ribosomes, but the widely-validated observations showed that it does not represents a significant problem for the direct displacement hypothesis. It has been suggested that the PtIM could indirectly displace antibiotics bound in the PTC and NPET by allosterically modulating their conformation upon ribosome binding [16], but it is not clear how an allosteric mechanism can explain the overlapping and inconsistent specificity profiles of different ARE ABC-F proteins, which remains the fundamental challenge for the direct-displacement hypothesis (Figure 4C). Moreover, a displacement mechanism would also imply that the displaced antibiotic will not rebind to the ribosome, this point will be discussed below.

Possible mechanisms-of-action of ABC-F antibiotic-resistance proteins As illustrated by numerous articles in this special issue of FEBS Letters, most ABC superfamily proteins are transmembrane transporters that mediate ATP-powered import and export of a wide variety of drugs, nutrients, and even polymers. The most prominent exceptions are the ABC-E and ABC-F families that lack transmembrane domains and instead modulate mRNA translation by ribosomes in diverse ways. The prevalence of bacterial ABC drug transport systems such as the tripartite antibiotic exporters EmrAB-TolC or MacAB-TolC led to numerous erroneous inferences that ARE ABC-Fs proteins function as part of transmembrane transporter systems, including some recent assumptions that they function as drug efflux pumps [113,114]. However, extensive research has demonstrated that ARE ABC-Fs are exclusively cytosolic proteins that act directly on the ribosome by binding to its E site [14,21]. The first concrete evidence that ARE ABC-F proteins directly mediate antibiotic resistance came from in vitro experiments in which purified VgaA and LsaA were able to rescue translation and displace bound antibiotics from the ribosome [22].

As explained in detail above, all ABC-F factors bind to the ribosomal E site in a similar geometry with their PtIMs extending towards the PTC in the 50S subunit. For binding to occur, ABC-F factors require an empty E site. When polypeptide elongation is proceeding normally on the ribosome, the deacylated tRNA is ejected from the E site simultaneously with the movement of the next deacylated tRNA from the P site to the E site. Therefore, the E site of a translating ribosome is rarely empty and available for ABC-F protein binding. It is only systematically empty in the 70S IC before the first step in polypeptide elongation and in ribosomes that are stalled due to antibiotic binding or some other problem impeding polypeptide elongation. Therefore, these species should be the primary targets for all ABC-F proteins. EttA interacts preferentially with the initiating ribosome [14], while the ARE ABC-Fs function on antibiotic-stalled ribosomes [15,16].

Notably the cryo-EM structures of ribosome-bound VmlR and MsrE both show alternative conformations of the P tRNA with its CCA acceptor stem substantially shifted out of the PTC [15,16] (Figure 4), as noted above for VmlR. In contrast, EttA, which interacts preferentially with the 70S IC and does not mediate antibiotic resistance, stabilizes the CCA acceptor stem of the P tRNA in its canonical location in the PTC in the proper geometry for peptide bond synthesis. The VmlR and MsrE structures both also show direct penetration of the PtIM into the PTC, resulting in rearrangement of several nucleotides in 23S rRNA (U2585, U2506, and A2062) proximal to the binding sites for the antibiotics to which they confer resistance (Figure 4C). Therefore, the cryo-EM data highlight two related but distinct structural effects specific to the interaction of ARE ABC-Fs with ribosomes: steric interactions changing the conformation of the PTC and spatial displacement of the acceptor stem of

14 the P tRNA from its canonical binding site in the PTC. These effects provide critical guidance for the development and testing of models for the mechanism by which ABC-F proteins mediate antibiotic resistance. However, even with structural data available at this level of detail, the mechanism by which the ARE ABC-F proteins mediate antibiotic resistance is unclear, and several important issues remain unresolved.

As discussed in the last section, the overlapping binding sites of the antibiotics targeting the PTC/NPET and the inconsistent resistance profiles of the ARE ABC-Fs are incompatible with a simple mechanism- of-action based on direct steric displacement of the antibiotic (Figure 3 and 4). It remains possible that ARE ABC-Fs allosterically displace antibiotics via induction of conformational changes in the PTC/NPET that reduce their binding energy and thereby promote their dissociation from the ribosome. This mechanism-of-action is consistent with the observation that ABC-F proteins are able to reset the conformation of the PTC in antibiotic-stalled ribosomes [115]. However, it does not explain in a straightforward manner the complex specificity profile of the ARE ABC-F proteins or their ability to discriminate between antibiotics with overlapping binding sites in the PTC/NPET of the ribosome. It is also unclear why this mechanism would require the very substantial shift of the CCA acceptor stem of the P tRNA away from the PTC as observed in both the VmlR and MsrE structures.

Another open question concerns the function of the CTD extensions present in some ARE ABC-Fs including VmlR and VgaA (Figure 2B). This domain binds to the ribosome near the mRNA exit channel. Its presence is required to mediate antibiotic resistance in vivo, but its role remains unexplained [16,53].

One critical point that remains unresolved concerns the role of the ATPase activity of the ABC domains in ARE ABC-F proteins in mediating antibiotic resistance. As described above, ATP-hydrolysis deficient EQ2 mutations generally trap ABC-F proteins in their ATP-bound conformations and block their release from the ribosome [14,16,21,34,115]. It is therefore unsurprising that EQ2 mutants of ARE ABC-F factors are unable to reset the PTC of an antibiotic-bound ribosome to its active state [115]. However, the EQ2 mutations symmetrically lock both of the tandem ATPase active sites in the ATP-bound conformation, which could potentially obscure differential effects of ATP binding and hydrolysis at the two different active sites. An asymmetric role for each ATPase active site therefore remains possible. For instance, ATP hydrolysis at one site could mediate some kind of “power stroke” that moves the CCA acceptor stem of the P tRNA or even ejects the antibiotic from the PTC, while ATP hydrolysis at the other site could induce release of the ARE ABC-F protein from the ribosome.

Recent work on ABCE1 supports an asymmetrical mechanism of this kind in which its two ATPase active sites play different roles during ribosome recycling [116,117]. The current model for the mechanism of this protein posits that a low-turnover site (site II, the ATP binding site that contains the walker B motif of the ABC2 domain) acts as a checkpoint in the overall conformational reaction cycle. ATP binding to this site upon ribosome recognition induces formation of a semi-closed conformation of the tandem ABC domains that does not lead to ATP hydrolysis in this active site but instead allosterically activates ATP binding to the other active site (site I, the ATP binding site that contains the walker B motif of the ABC1 domain). The second site then undergoes multiple cycles of uncoupled ATP hydrolysis until a conformational change leads to formation of the fully closed ATP-sandwich heterodimer conformation of the tandem ABC domains, and this second conformational change is believed to push an N-terminal FeS cluster domain in ABCE1 along a trajectory that forces the ribosomal subunits to dissociate. ABCE1 may remain in its symmetrically occluded ATP-sandwich heterodimer conformation until translation initiation factor binding to the dissociated ribosomal subunits triggers ATP hydrolysis and thereby dissociation of ABCE1 [118]. The similar architecture of the tandem ABC domains in ABCE1 and the ABC-F proteins suggests the latter could potentially employ a similarly Byzantine multistep molecular mechanism.

One potential model along these lines involves binding of the ARE ABC-F in ATP-bound occluded state to the empty E site in an antibiotic-stalled ribosome triggering ATP hydrolysis in one of the two ATPase active sites in an ARE ABC-F protein. This initial hydrolysis event could transmit a chemomechanical power stroke to its PtIM that results in displacement of the CCA acceptor stem of the P site tRNA from the PTC on the ribosome, a motion that will pull the nascent polypeptide bound to that tRNA out of the PTC/NPET [30]. Such a motion is consistent with the substantially displaced location of the CCA acceptor stem of this tRNA observed in the cryo-EM structures of ribosomes bound to both VmlR and MsrE. Because the CCA acceptor stem of the P site tRNA is covalently bound to the nascent polypeptide in actively translating ribosomes, this motion would pull the nascent polypeptide out of the NPET. Furthermore, given the tight steric interactions in the PTC/NPET in actively translating ribosomes, such a motion of the nascent polypeptide could dynamically displace or “unplug” an antibiotic bound in this region of the ribosome. The reaction cycle could be completed by ATP hydrolysis by the second ATPase active site inducing release of the ARE ABC-F protein from the ribosome and retraction of the CCA acceptor stem of the P site tRNA stem and its covalently bound peptidyl-tRNA back into the PTC/NPET [30].

Cycles of ATP hydrolysis by one or both ATPase active sites could thus exert reciprocating mechanical forces on the peptidyl-tRNA and the nascent peptide in the P site of the ribosome, repetitively pulling them out of and letting them retract back into the PTC/NPET [30]. This kind of reciprocating motion is mechanically analogous to that of a “drain snake”, a device widely used by plumbers to unclog drains. It seems likely to drive displacement of antibiotics bound in this region, although its effectiveness in producing antibiotic resistance would depend on complex structural and kinetic factors including steric interactions between the PtIM and the antibiotic/PTC/NPET, the distance of displacement of the nascent polypeptide from the PTC/NPET, and the rate/mechanism of antibiotic rebinding in the PTC/NPET. These complexities could explain the overlapping and inconsistent specificity profiles of different ARE ABC-F proteins. However, while this drain-snake model has interesting features, it has yet to be tested experimentally.

Another unresolved but important issue concerning ARE ABC-F protein function concerns the kinetics of drug rebinding to the ribosome. Some antibiotic-resistance systems have enzymes that chemically modify antibiotics to block their growth-inhibiting binding interactions. However, the ARE ABC-Fs only displace drugs from the ribosome without inactivating them. It is unclear whether their drug- displacement activity must be coupled to functional partners that degrade the drugs or pump them out of the cell or whether, alternatively, continual dynamic displacement of antibiotics driven by the ATPase activity of the ARE ABC-Fs is sufficient to enable translation to proceed at a sufficient rate to support cell growth.

Genetic regulation of ABC-F antibiotic-resistance proteins Constitutive expression of antibiotic resistance genes in the absence of antibiotic imposes a burden on cellular fitness, and this burden is enhanced in the case of the ARE ABC-F proteins given the relatively high concentration of these proteins needed to mediate effective resistance to antibiotics acting on the ribosome. This factor has led to the evolution of regulatory systems that induce the expression of some antibiotic resistance genes only when a target drug is present [119]. Bacteria have developed several cis-acting post-transcriptional regulatory mechanisms that control mRNA translation in response to small molecules [120]. These mechanisms include metabolite/antibiotic-sensing riboswitches and antibiotic-mediated ribosomal stalling at specific nascent polypeptide sequences [121,122], both of which can be coupled to transcriptional attenuation via Rho-independent or Rho- dependent pathways (Figure 5).

Sophisticated biochemical and structural investigations have been used to elucidate how specific nascent polypeptides are able to sense ribosome-bound drugs (antibiotics and metabolites), leading the translational machinery to stop [93,94,123,124]. This phenomenon known as programmed drug- dependent ribosomal stalling is widely used by antibiotic-resistant bacteria to induce expression of resistance genes in the presence of a particular antibiotic. One well-characterized system is found in macrolide-resistant Gram-positive bacteria that carry the ermC gene, which encodes a methyltransferase enzyme that dimethylates the N6-position of nucleotide A2058 in 23S rRNA to block macrolide binding to the ribosome [125,126]. Induction of ermC is controlled by translational attenuation dependent on macrolide-dependent translational stalling in a short upstream regulatory ORF called ermCL (ermC leader) that encodes a 19-residue “leader peptide” with sequence MGIFSIFVISTVHYQPNKK [127,128]. The most stable conformation of the bicistronic ermCL-ermC mRNA has two stem-loops, one overlapping the ermCL upstream ORF and one overlapping the ribosome- binding site and first two codons in the ermC gene encoding the methyltransferase enzyme, and this second stem-loop prevents initiation of translation of this antibiotic resistance enzyme (Figure 5A). Translation of the ermCL upstream ORF in presence of 14- or 15-membered macrolides bound to the NPET leads to translational stalling specifically at residues 6 to 9, which have sequence IFVI, due to induction of a non-productive conformation of the PTC that prevents catalysis of peptide-bond formation [93].⁠ Ribosome stalling at this site disrupts the stem-loop that overlaps the ermCL upstream ORF and enables its 5’ segment to form an alternative stem-loop with the 3’ segment of the stem-loop sequestering the translational start site of the downstream ermC gene. Formation of this alternative mRNA structure frees the ribosome binding site of the ermC gene, allowing efficient translation of the encoded antibiotic-resistance enzyme.

As summarized in table 2, putative upstream regulatory ORFs encoding leader peptides have been identified in several ARE ABC-F genes including msr homologs, which produce the MKSB resistance phenotype, and lsa, which produces the PLSA resistance phenotype. However, to date, msrA and vmlR are the only ARE ABC-F genes that have been found to be inducible by antibiotics to which they mediate resistance [129,130].

The msr homologs msrA, msrC, and msrSA are expressed from transcripts containing upstream ORFs (msrAL, msrCL and msrSAL) that encode leader peptides sharing strong sequence similarity with ermDL (MTHSMRLRFPTLNQ) [96], an upstream ORF encoding a leader peptide that controls translation of another ribosomal methyltransferase mediating macrolide resistance. All of these leader peptides share an Arg/Lys-X-Arg/Lys sequence motif shown by Sothiselvam et al. to be responsible for macrolide-dependent ribosomal stalling [97]. Given that the msrA, msrC, and msrSA gene products mediate resistance to related antibiotics, the presence of this sequence motif in the leader peptides encoded in their upstream ORFs suggest that their translation is likely to be induced by macrolide- induced ribosome stalling in these upstream ORFs.

The msrDL leader peptide encoded upstream of the msrD ARE ABC-F gene does not contain any known macrolide-dependent stalling motifs. However, these genes are transcribed as part of a larger mefAL- mefA-msrDL-msrD operon that is known to be induced by macrolides due to suppression of Rho- independent transcription termination (Figure 5B). The mefA gene encodes a transmembrane transporter in the major facilitator family, while the mefAL upstream ORF encodes a leader peptide with sequence MTASMRLR that includes the Arg/Lys-X-Arg/Lys macrolide-dependent ribosome stalling motif [131]. Stalling at this motif mediates the macrolide-dependent transcriptional anti-termination effect that induces expression of the entire operon, while stalling in the internal msrDL leader peptide is hypothesized to tune translation of the downstream msrD gene. While the substrate specificity of the MefA protein is not yet known, based on the specificity of the leader peptide in its upstream ORF, it seems likely to transport macrolides, which would enable it to act synergistically with the ARE ABC- F protein MsrD to dislodge macrolides from the ribosome and then pump them out of the cell [30].

Virginiamycin M or lincomycin induce expression of vmlR due to suppression of Rho-independent transcriptional attenuation. However, no upstream regulatory ORF has been identified in the vmlR transcript, and this regulatory effect has been hypothesized to be mediated either by antibiotic- dependent RNA polymerase read-through or by a trans-acting factor, although experimental data is lacking on the molecular mechanism. Interestingly, a putative upstream regulatory ORF has been identified in the transcript for lsaB, which shares the same resistance phenotype as vmlR, but its expression has not yet been shown to be induced by an antibiotic to which it mediates resistance [132].

The PhO resistance phenotype produced by ABC-F genes such as optrA or poxtA has raised concerns about their potential spread in hospitals and in livestock [133,134]. Although some chloramphenicol resistance genes are induced upon exposure to that antibiotic (e.g., catA86 regulated by the catA86L upstream ORF), no PhO ARE ABC-F protein has yet been found to be inducible by antibiotic exposure [135].

Eukaryotic ABC-F translation factors – protein quality control and modulation of immune response

ABC-F proteins and translational quality control The numerous eukaryotic ABC-F proteins are all believed to be involved in the protein synthesis process, although a limited amount of detailed biochemical data is available.

The yeast Saccharomyces cerevisiae expresses two ABC-F paralogs: GCN20 and Arb1. GCN20 has been demonstrated to interact with another translation factor called GCN1 to downregulate translation initiation during amino acid starvation [18]. This regulation relies on the binding of the GCN20-GCN1 complex to the N-terminus of the GCN2 protein kinase [136], which stimulates the phosphorylation of eukaryotic initiation factor 2a (eIF2a) on serine-51 after activation by uncharged tRNA. This modification stimulates the translation of the transcriptional activator GCN4 by inhibiting initiation of translation of a small upstream ORF encoded in the same mRNA that causes ribosome release from the mRNA without translation of GCN4. When this upstream ORF is not translated, ribosomes scan through to the start codon of the GCN4 ORF and efficiently initiate translation there. The resulting induction of GCN4 translation leads to strongly increased transcription of more than 30 genes encoding amino acid biosynthetic enzymes [137]. It has been shown that the GCN20-GCN1 complex interacts with 80S elongating ribosomes and that ATP hydrolysis stimulates this binding. However, genetic studies demonstrate that the ABC domains of GCN20 are dispensable for its well-characterized regulatory function activating the GCN2 kinase in amino-acid-starved cells [137].

Arb1 is an essential protein in yeast. Its depletion leads to a reduction in the steady-state level of mature 18S rRNA and 40S ribosomal subunits, which was originally interpreted to reflect a function in ribosome biogenesis [19]. However, the complex regulation of the expression of the translational apparatus can cause defects in the dynamics of mRNA translation by the ribosome to indirectly feedback to deficits in ribosome biogenesis. As discussed above, Arb1 functions in translational quality control during recycling of stalled ribosomes by RQC [50–52]. It inhibits the Rqc2 protein from elongating so-called “CAT-tails” [50–52], non-mRNA-encoded alanine/threonine extensions, on peptidyl-tRNAs bound to dissociated 60S ribosomal subunits, and it simultaneously stimulates the peptidyl-tRNA-hydrolase activity of Vms1 in such complexes. Both of these activities promote efficient recycling of stalled ribosomes, and inhibition of Rqc2 also reduces toxic aggregation of CAT-tailed proteins, which causes mitochondrial stress [20]. Arb1 may also function in initial recognition of stalled ribosomes by detecting an empty E site, which is a sign of reduced elongation rate in assembled 80S ribosome complexes, as also discussed above. It could furthermore function in dissociating the P site tRNA from dissociated 60S subunits. However, these last two activities have yet to be demonstrated

18 experimentally. It is unclear whether the previously observed ribosome biogenesis defect observed upon Arb1 depletion in yeast reflects a direct involvement in ribosome biogenesis or an indirect effect caused by defective recycling of translationally stalled ribosomes.

Humans express three ABC-F paralogs: ABCF1 (ABC50), ABCF2 and ABCF3 (Figure 1 and 6). However, only ABCF1 has been extensively studied, and this protein has also been demonstrated to play a role in translational quality control. ABCF1 is a close relative of yeast GCN20 and similarly interacts with eIF2, but it has distinct functions, presumably due to marked differences in their N-terminal domains. ABCF1 stimulates the formation of the ternary complex between eIF2, GTP, and the initiator tRNAMet. Sucrose gradients experiments have shown, like GCN20, ABCF1 associates mainly with the 40 and 60S subunits rather than polysomes. Using bicistronic reporters, it has been shown that knockdown of ABCF1 impaired translation of reporter genes/proteins irrespective of the dependency of their translation on the 5’ mRNA cap. As eIF2 is required for both cap-dependent and cap-independent translation initiation, this finding strengthens the case for the physiological significance of the interaction between ABCF1 and eIF2. Moreover, the integrity of the ABC domains of ABCF1 are essential for its function in stimulating translation initiation because mutations in the Walker ATP binding/hydrolysis motifs described above reduce polyribosomes levels [138]. Recently, the role of ABCF1 in the translation initiation has been dissected more precisely by Stewart et al. who have shown that ABCF1 ensures accurate recognition of the start codon. They demonstrated that mutations impairing ATP binding or hydrolysis increase use of non-AUG start codons in addition to decreasing overall initiation [139]. A recent study proposed an involvement of ABCF1 in m6A-mediated translation initiation. This cap-independent and IRES-independent translation initiation mechanism involves the methyltransferase METTL3, and it is believed to mediate the translation of “privileged” mRNAs to produce proteins important for cell maintenance and survival [140].

An indirect connection between the established properties of yeast Arb1 and human ABCF1 in protein quality control points to potentially unifying principles and pathways related to eukaryotic ABC-F protein function. As discussed above, Arb1 has been found to be part of a complex involving several members of the ribosome-associated quality control (RQC) system that rescue stalled-ribosomes and then processes faulty nascent proteins. In the RQC, Rqc2 polymerizes C-terminal alanyl and threonyl residues (CAT-tail) on the translationally stalled peptidyl-tRNA, and the E3 ligase Ltn1 ubiquitylates that nascent polypeptide. It is extracted from the 60S subunit by the AAA-ATPase Cdc48 and sent to the proteasome for degradation. However, CAT-tailed polypeptides may aggregate and lead to mitochondrial toxicity. Rqc2 activity is antagonized by Vms1, which cleaves the peptidyl-tRNA to release the nascent polypeptide from the CCA acceptor stem of that tRNA [17]. By promoting Vms1 activity, Arb1 plays an important role in preventing CAT-tail-driven aggregation and toxicity. Recent observations suggested a larger regulatory role for human ABCF1. This protein has an N-terminal E2 ubiquitin-conjugating-enzyme-like domain that regulates the inflammatory response in macrophages [141]. ABCF1 has been shown to play a role in protein disaggregation in vivo [142], which could be mediated by activity of its E2-like domain in promoting ubiquitin-dependent degradation of aberrant proteins. If this activity includes prematurely terminated nascent polypeptides, its function would parallel at least to an extent the role of Arb1 in rescuing and recycling stalled ribosomes and degrading their prematurely terminated nascent polypeptides.

ABC-F modulation of immunity and infection Numerous studies have pointed to a linkage between ABCF1 and human autoimmune diseases including rheumatoid arthritis, autoimmune pancreatitis, gout, and Crohn’s disease [143–146], but the mechanism(s) underlying such effects remained obscure until recently. Three papers have presented results that have begun to elucidate molecular mechanisms by which ABCF1 can regulate the mammalian immune response. The first showed that ABCF1 interacts with proteins in the SET complex and notably HMGB2, which senses cytoplasmic DNA of viral origin or, in the case of

19 autoimmune diseases such as Lupus, self-DNA [147]. More recently, using a new technique derived from phage display coupled with next-generation sequencing, Guo et al. identified ABCF1 as a new extracellular phagocytotic ligand [148]. They showed that whereas cytoplasmic ABCF1 is absent in the culture medium of healthy cells, it is released from and binds to photoreceptor outer segments (POSs) that are shed in an autocrine manner. This process facilitates their clearance by retinal pigment epithelial cells (RPE), a type of specialized phagocytes that maintain retinal homeostasis. It seems that this pathway also occurs in apoptotic microglial neurons [148]. Finally, last year, ABCF1 was identified as a class IV E2 ubiquitin-conjugating enzyme based on the catalytic activity of cysteine-647, which is conserved in ABCF1 paralogs. ABCF1 is also involved in TLR4 signaling in the innate immunity system. It participates in a molecular switch that mediates the transition from MyD88-dependent to TRIF- dependent signaling in macrophages, which facilitates production of IFN-b, a protective cytokine involved in LPS-induced sepsis [141]. If ABCF1 indeed regulates innate immune responses in many autoimmune disorders, it would be an interesting target for pharmacological drug development. A recent study identified it as the target of dicarboximides compounds tested in the treatment of leukemia cells [149], supporting investigation of ABCF1 as a potential human drug target.

ABCF2 has been identified as an anti-apoptotic factor and associated with several human cancers. High expression has been detected in various types of ovarian cancer and particularly in clear-cells adenocarcinomas associated with a poor prognosis [150,151]. ABCF2 could therefore be a good biomarker to detect this type of cancer at early stages. Hepatocellular carcinoma (HCC) is another kind of cancer with a poor prognosis associated with ABCF2 expression. Increased ABCF2 expression is associated with progression of HCC and seems to be linked to high levels of the circular RNA circ- TCF4.85. ABCF2 is normally inhibited by the miRNA miR-486-5p, which promotes apoptosis, but overexpression of cric-TCF4.85 reverses this inhibition, thereby promoting cell proliferation, migration, and invasion [152]. Moreover, the anti-apoptotic function ABCF2 has also been demonstrated in the context of infection by enteropathogenic E. coli (EPEC). Nougayrede et al. provided evidence that the type III secretion system effector EspF from EPEC binds to ABCF2 and decreases its level in host cells concomitantly with an increase in caspase 9 and caspase 3 cleavage, a characteristic of the mitochondrial death pathway [153]. Thus, EspF seems to target ABCF2 to promote or facilitate apoptosis, but it remains unknown if the binding to EspF induces the degradation of ABCF2 through ubiquitination or via another pathway [153].

A yeast two-hybrid screen of a mouse brain library has identified ABCF3 as an interacting partner of Oas1b (Inactive 2'-5'-oligoadenylate synthase 1B), a protein which is involved in a specific resistance mechanism against flaviviruses. Both Oas1b and ABCF3 localize to the endoplasmic reticulum (ER) membrane, and this localization is required for the resistance phenotype [154]. A recent study has shown that ABCF3 is an active ATPase whose activity can be modulated by several lipids, and its activity is notably increased by sphingosine and sphingomyelin. Point mutations in the ABC1 and ABC2 domains of ABCF3 support asymmetric function of those domains. A mechanistic model was proposed in which pocket 1 (Walker A/B motifs from ABC1 together with the Signature Sequence of ABC2) is the site of basal catalysis, while pocket 2 is engaged in Oas1b-stimulated ATP hydrolysis. Interaction between ABCF3 and Oas1b was also confirmed in this study using a heterologous expression system in E. coli. Expression of Oas1b alone is toxic in bacterial cells, but this toxicity is alleviated by co- expression with ABCF3. The mechanism by which Oas1b confers resistance to flaviviruses is still unknown, but a genetic screen using a toxicity-rescue approach could help to decipher the role of ABCF3 [155]. The human version of ABCF3 (hABCF3) is highly expressed in liver, heart, and pancreas, and it is associated with cell proliferation. An interaction with the protein TPD52L2 (tumor protein D52-like 2) involving the first 200 residues in ABCF3 is required for its observed activity in cell proliferation [156].

Conclusions and Perspectives

ABC-F proteins have been shown to be E site ribosome translation factors that modulate the conformation of the PTC. They control the initiation of translation and the rescue of the ribosome during abortive translation, and they provide protection against a wide spectrum of ribosome- targeting antibiotics. ABC-F proteins can thus be considered analogous to a “Swiss Army knife” for the ribosomal E site based on their observed multiplicity of functions in a single location. They are conserved from eubacteria to humans but absent in archaea. This pattern of distribution among the kingdoms of life is consistent with the evolution of the eukaryotic translation apparatus, a hybrid between the archaebacterial and eubacterial translation apparatus. Interest in ABC-F proteins has been growing rapidly based on their key roles in the environmental spread of bacterial antibiotic resistance and in the modulation of the human immune response. They furthermore play roles in cellular development and regulation of gene expression in both eubacteria and eukaryotes.

The protein architecture of the ABC superfamily has evolved to protect bacteria from antibiotics using two completely different biochemical mechanisms – direct ribosome protection by ARE ABC-F proteins and extrusion of antibiotics across the cell membrane by ABC exporter proteins. These two different processes, protein synthesis and transmembrane transport, are also both critical for the function of antibacterial peptides. The possible role of ABC-F proteins immunity against antimicrobial peptides that target the ribosome remains an open question.

In conclusion, the role of the ABC-F proteins in mRNA translation has only been elucidated during the past decade [14,21]. Yet, these fundamental studies have critical implications for human health, given the ongoing spread of antibiotic resistance factors in this protein family among gram positive and negative bacteria [15,16,22,54,115,157] and the involvement of the human homologs in immune responses [64,143,147,149], and apoptosis in cancer cells [152,156].

Acknowledgements We thank Tina Wang for comments and edits on the manuscript.

Funding sources and disclosure of conflicts of interest C.R.F, F.O, L. M. and G.B. are supported by the LABEX program (DYNAMO ANR-11-LABX-0011) and the ANR grants EZOtrad (ANR-14-ACHN-0027) and ABC-F_AB (ANR-18-CE35-0010), while S.S. and J.F. are supported by grant 1R01GM120579 from the US National Institutes of Health.

The authors declare that there is no conflict of interest regarding the publication of this article.

Authors contributions C.R.F., L.M., F.O., S.S., J.F.H., and G.B. wrote the manuscript. C.R.F. generated the structure illustrations, while F.O. composed some of the other figures.

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Figures legends

Figure 1: Phylogeny and structure of ABC-F factors.

Cladogram labeled with Swiss-Prot (SP) or TrEMBL (TR) accession codes for representative ABC-F proteins as well as proteins from two non-ABC-F families containing ABC domains (the eEF3 and ABCE families), and their corresponding structures. Identifiers correspond to the following species: Escherichia coli (ECOLI), Bacillus subtilis (BACSU), Pseudomonas aeruginosa (PSEAI), Saccharomyces cerevisiae (YEAST), Arabidopsis thaliana (ARATH) and Homo sapiens (HUMAN). Structure of factors were aligned based on least-squares superposition of ABC1, and missing residues were added as dotted lines. The corresponding cryo-EM structures were aligned based on least-squares superposition the 23S (prokaryotes) 25S/28S (eukaryotes) rRNA in the large ribosomal subunit. Orientation and major landmarks are indicated on the schematic ribosome on the top of the figure (A, A tRNA; P, P tRNA; E, E tRNA; CP, central protuberance). Cryo-EM density segments are colored as follows: ribosomal small subunit in sand, ribosomal large subunit in transparent grey, and P tRNA in yellow. Note that the ribosomal small subunit is absent in the Arb1 structure, and no cryo-EM structure has been solved yet for ABCF1. ABC-F factors exclusively bind to the ribosomal E site, while ABCE factors bind near the A site, and EF3-related factors bind near the central protuberance at the junction of small and large ribosomal subunits. PDB accession codes are as follows: EttA (3J5S - Cryo- EM, resolution: 7.5 Å ), MsrE (5ZLU - Cryo-EM, resolution: 3.6 Å), VmlR (6HA8 - Cryo-EM, resolution: 3.5 Å), Arb1 (6R84 - Cryo-EM, resolution: 3.6 Å), ABCF1 (5ZXD - X-ray diffraction, resolution: 2.29 Å), ABCE1 (5LZV - Cryo-EM, resolution: 3.35 Å), NEW1 (6S47 - Cryo-EM, resolution: 3.28 Å) [7,15– 17,21,46,158].

Figure 2: General structure of ABC-F factors and their binding site on the ribosome. (A) Architecture of ribosome-bound EttA, showing its domains and subdomains. Structural features are colored as follows: ABC1 in dark red shade, PtIM in bright red shade and ABC2 in light red shade. Table with the distances in Å between serine Ca of the signature motif of the ABC1 domain and the Lysine Ca of the walker A of the ABC2 domain (ATP binding site I) and the serine Ca of the signature motif of the ABC2 domain and the Lysine Ca of the walker A of the ABC1 domain (ATP binding site II). (B, C) Structural alignment of several ABC-F factors. Factors were aligned based on least-squares superposition of ABC1, and their domains are shown in different shades of the same color: ABC1 in a dark shade, the PtIM in a bright shade, and ABC2 in a light shade. Missing residues are represented as dotted lines, and additional sequence motifs and domains are labeled (e.g., the Arm in EttA and the C-terminal domain in VmlR. Structural superposition shows strong structural conservation of ABC1 and ABC2 varying alignment of the PtIM. (D) Structural alignment of ribosome-bound ABC-Fs factors. Cryo-EM structures were aligned on the large subunit 23S rRNA (or the 25S rRNA for Arb1), and the corresponding cryo-EM densities were extracted and colored as in Figure 1. Alignment of ribosome- bound ABC-Fs proteins based on the rRNA in the large subunit shows that PtIMs superimpose well and point in the same direction, while the ABC cassettes are less closely aligned and tending to oscillate in the ribosomal E site as indicated by the arrows. PDB accession codes are the same as in Figure 1.

Figure 3: ABC-Fs factors and resistance to ribosomally-directed antibiotics. (A) Binding site of some major representatives of ribosomally-directed antibiotic families to which ARE ABC-Fs provide resistance. Antibiotic families are grouped according to ARE ABC-Fs resistance phenotypes: MKSB antibiotics (Macrolides, Ketolides, group B Streprogramins in purple), PLSA antibiotics (Pleuromutilins, Lincosamides, group A Streptogramins in yellow) and PhO antibiotics (Phenicols and Oxazolidinones in green). (B, C) Resistance phenotypes are correlated but not strictly specified by the location of the antibiotic binding sites in the PTC/NPET. A transverse section of the PTC/NPET indicates the relative position of some antibiotics shown on Figure 3A, namely

33 chloramphenicol, lincomycin and erythromycin as well as A tRNA in red and P tRNA in yellow. Antibiotics belonging to MKSB, PLSA and PhO resistance phenotypes are also depicted as surface to show their relative occupancy and binding promiscuity. Note that PhO antibiotics would sterically clash A tRNA while PLSA antibiotics would clash both A and P tRNAs. Structures were aligned on the 23S rRNA domain V of a high-resolution structure of E. coli ribosome (PDB 4YBB) [159]. PDB accession codes are as follows: Azithromycin (4V7Y) [88], Carbomycin A (1K8A) [87], Chloramphenicol (6ND5) [160], Clindamycin (4V7V) [76], Dalfopristrin (4U24) [161], Erythromycin (4V7U) [76], Lincomycin (5HKV), [162], Linezolid (3CPW) [79], Quinupristrin (4U1U) [161], Retapamulin (2OGO) [106], Spiramycin (1KD1) [87], Telithromycin (4V7S) [76], Tiamulin (3G4S) [163], Tylosin (1K9M) [87], Virginiamycin M (4U25) [161], Pre-catalysis A and P tRNAs (1VY4), [164].

Figure 4: VmlR structure and possible allosteric mechanism. (A) Cryo-EM density of ribosome-bound VmlR (PDB 6HA8) [16]. Density segments were extracted and colored as follows: ribosomal small subunit in sand, ribosomal large subunit in transparent grey, P site tRNA in yellow, VmlR in dark cyan. The labels indicate functional sites on the ribosome (A, A site; P, P site; E, E site; PTC, peptidyl transferase center; NPET, nascent polypeptide exit tunnel). VmlR binds in the ribosomal E site and displaces the CCA acceptor stem of the P site tRNA CCA by 37 Å in the direction of the A site. The Tip of its PtIM penetrates directly into the PTC. Equivalent structural effects are observed in the cryo-EM structure of ribosome-bound MsrE from Pseudomonas aeruginosa MsrE (PDB 5ZLU) [15]. (B) Direct steric drug displacement by the PtIM in ARE ABC-Fs cannot fully explain the resistance mechanism. Cryo-EM structures of VmlR-bound and antibiotic-bound ribosomes were aligned based on least-squares superposition of the 23S rRNA in the large subunit, demonstrating that the residue F237 in the Tip of the PtIM in VmlR (dark cyan) can interact with several antibiotics to which it provides resistance, namely virginiamycin M (VgM), tiamulin (Tia) and lincomycin (Lnc). However, VmlR does not provide resistance to chloramphenicol (Cam) nor linezolid (Lnz) even though F237 would also sterically clash those two antibiotics. VmlR furthermore does not provide resistance to erythromycin (Ery). (C) 23S rRNA nucleotides rearrangement at the PTC upon VmlR binding would suggest an allosteric mechanism. Nucleotides with lincomycin-, virginiamycin M- and tiamulin-bound in absence of VmlR are shown in grey while they are shown in cyan in presence of VmlR. Relative rearrangements are indicated by red arrows. PDB accession codes are the same as in Figure 3.

Figure 5: Ligand-induced translational stalling regulates gene expression. Top, translational attenuators sequester the ribosome binding site (RBS) of a regulated ORF in a structured stem-loop that blocks ribosome binding and initiation of translation. Drug-induced ribosomal stalling on an upstream ORF encoding a short “leader peptide” induces formation of an alternative stem-loop that opens RBS to promote efficient translation initiation and thereby higher protein expression. This mechanism is exemplified by the erythromycin-sensing ErmBL and ErmCL leader peptides [93,94]. Middle, rho-independent transcriptional attenuators fold into their transcription-attenuating conformation in absence of an inducer compound, leading RNA polymerase terminate transcription prematurely and produce a truncated mRNA. Drug-induced ribosomal stalling on the leader peptide encoded in an upstream ORF stabilizes an alternative “anti-attenuator” conformation that prevents RNA polymerase drop-off and allows transcription of a full-length mRNA. This mechanism is exemplified by the erythromycin-sensing MefAL leader peptide [131]. Bottom, in Rho-dependent transcriptional attenuators, the Rho helicase enzyme is able to terminate transcription by binding to a Rho utilization site (rut), leading RNA polymerase to drop off and produce a truncated mRNA. Drug-induced ribosomal stalling on an ORF encoding a leader peptide could directly mask the rut site or stabilize an alternative mRNA conformation that prevents Rho binding and transcription termination. This mechanism is exemplified by the L-tryptophan-sensing TnaC and the L-ornithine- sensing SpeFL leader peptides [123,124]. Note that, to date, no mechanism involving antibiotic- dependent regulation of a Rho-dependent transcriptional attenuator has been described, making the mechanism illustrated in the bottom panel hypothetical.

Figure 6: Known functions of eukaryotic ABC-F factors. The different roles of ABC-F factors in yeast, human, and plants are presented and distinguished by different colors: protein synthesis and quality control in yellow, physiological stress response in grey, bacterial or viral infection response in light blue, cellular development in green, and diseases processes in pink.

Text Boxes

Text Box 1: Key points about the ABC-F proteins

• Protein family that belong to the ABC superfamily • The ABC-F protein family is defined by the present of 2 ABC domains in tandem separated by a linker region that contain the P-site tRNA interaction motif (PtIM, Pfam domain PF12848) • ABC-F proteins are translation factors • ARE ABC-F proteins protect ribosomes against antibiotics that bind to the peptidyl- transferase center (PTC) or nascent protein exit tunnel (NPET) • The human ABC-Fs modulated the immune response and influence apoptosis in cancer cells

Figure 1

50/60S L1 L7/L12 CP E P A

30/40S

Figure 2 A EttA

F1-like Arm Core1 Distance between domains ABC1 and ABC2 (values in Å) ABC1 ABC-F protein ATP Site I ATP Site II ABC 1 domain α EttA 8,5 9,6 EttA Apo (4FIN) 13,3 17,5 ABCβ1 VmlR 9,4 9 ATP MsrE 10,8 11,5 ABC 2 β Arb1 14,9 17,8 ABCα2 ABCF1 12 16,4 ABC2 PtIM: domain Toe P-site tRNA Interaction Motif F1-like Core2 B N-terminal domain N-terminal domain Arm domain (84 amino acids) (299 amino acids) ABC1 ABC1 ABC1 ABC1 ABC1

PtIM PtIM PtIM PtIM PtIM

ABC2 ABC2 ABC2 ABC2 ABC2 C-terminal EttA domain VmlR MsrE Arb1 ABCF1 C D

Figure 3

A MKSB antibiotics PhO antibiotics PLSA antibiotics

Macrolides Phenicols Oxazolidinones Lincosamides

C2452 U2504 U2504 A2059 U2504 A2058 C2452 C2452

U2506

A2451 A2451 C2611 G2505

Erythromycin Chloramphenicol Linezolid Lincomycin

Streptogramins Pleuromutilins Group B Group A U2504 G2505 A2062 C2452

U2506 U2585 G2061 A2503 U2585 A2451 G2505 U2586 U2609 A2062

Quinupristrin Virginiamycin M Retapamulin B C Pleuromutilins Lincosamides Streprogramins (Group A) Lincomycin Erythromycin

P-tRNA

CLASH

Chloramphenicol Macrolides Ketolides A-tRNA Streprogramins (Group B) Phenicols CLASH Oxazolidinones

•

Figure 4

A 50S

NPET PTC 37 Å PTC

P-tRNA VmlR E P VmlR A-site A

P-tRNA 30S B Resistance No resistance

F237 F237 VgM Ery Tia Cam Lnc Lnz

C Lincomycin Virginiamycin M Tiamulin

A2062 A2062 U2585 U2585

U2506 F237 F237 F237 G2505 U2506

Figure 5

Non-inducing conditions Inducing conditions A Translational attenuation

Ribosome Binding Site

RBS

leader ORF ORF leader ORF ORF B Rho-independent transcriptional attenuation Anti-attenuator conformation Attenuator conformation

UUUU leader ORF leader ORF ORF

Rho factor RNA polymerase C Rho-dependent transcriptional attenuation

Rho factor

leader ORF rho RNA polymerase leader ORF rut ORF utilization site

Figure 6

Tables

Table 1: List of some ARE ABC-F genes and their resistance spectrum.

ARE ABC-F genes, their host organism, as well as their extensive antibiotic resistance spectrum and some structural features are shown. Structural features have been adapted from [30]. (* For LsA(E) and msr homologs, antibiotic susceptibility has been tested in Staphylococcus aureus and not in the endogenous organism).

Gene Species Resistance Provides Doesn’t provide ABC1 “arm” PtIM tip CTD Reference phenotype resistance to resistance to lsA E. faecium PLSA Clindamycin Erythromycin Yes (shorter) Medium No [165] Dalfopristin Quinupristin lsA(E) S. aureus* PLSA Clindamycin Erythromycin Yes (shorter) Medium No [166,167] Dalfopristin Lincomycin Pirlimycin Quinupristin Tiamulin Virginamycin M1 msr homologs S. aureus* MKSB Erythromycin Clindamycin No Longest No [111] Telithromycin Tylosin Streptogramins B Streptogramins A

msrE staphylococci, MKSB Azithromycin N.D. No Longest No [15] streptococci, enterococci, Pseudomonas aeruginosa msrD S. pneumoniae MKSB Azithromycin Clindamycin No Longest No [168–170] S. pyogenes Clarithromycin Streptogramins A Erythromycin Streptogramins B optrA E. faecalis PhO Chloramphenicol Erythromycin Yes Shortest Yes [171] Florphenicol Dalfopristin (longest) Linezolid Quinupristin vgaA E. faecalis PLSA Carbomycin Blasticidin S No Long Yes [22,172] Clindamycin Erythromycin Leucomycin Florfenicol Lincomycin Linezolid Retapamulin Puromycin Virginiamycin M1 Sparsomycin Spiramycin Tiamulin Tylosin vmlR B. subtilis PLSA Lincomycin Chloramphenicol No Medium Yes [16] Virginiamycin M1 Linezolid Tiamulin Erythromycin

Table 2: List of known and probable antibiotic-inducible ARE ABC-F genes. For each ARE ABC-F gene, the resistance phenotype, the inducibility, the regulatory feature, the presence of a transcriptional attenuator (i.e. Rho-independent or Rho-dependent transcriptional attenuation), the putative ORF, its sequence and the Genbank accession number have been summarized. This table was compiled as previously described [129,130,132,173].

ABC-F Resistance Inducibility Regulatory Transcriptional Putative Putative regulatory Accession No. gene phenotype feature attenuation regulatory ORF ORF sequence lsaB PLSA Not Programmed Unknown lsaBL MVLELDVTHELLRISR AJ579365 demonstrated yet drug-dependent YNLSNIYGVI ribosomal stalling msrA MKSB Yes Programmed Unknown – Probably no msrAL MTASMRLK AB013298 drug-dependent transcription attenuation ribosomal stalling msrC MKSB Not Programmed Unknown msrCL MTASMKLRFELLNN AY004350 demonstrated yet drug-dependent N ribosomal stalling msrD MKSB Not Programmed Unknown msrDL MYLIFM AF274302 demonstrated yet drug-dependent ribosomal stalling msrSA MKSB Not Programmed Unknown msrSAL MTASMRLK AB016613 demonstrated yet drug-dependent ribosomal stalling vmlR PLSA Yes Unknown Rho-independent Unknown Unknown CP053102.1 transcription attenuation