Structural studies of the eIF4E-VPg complex reveal a direct competition for capped RNA: Implications for translation.

Viruses have transformed our understanding of mammalian RNA processing, including facilitating the discovery of the methyl-7-guanosine (m7G) cap on the 5' end of RNAs. The m7G cap is required for RNAs to bind the eukaryotic translation initiation factor eIF4E and associate with the translation machinery across plant and animal kingdoms. The potyvirus-derived viral genome-linked protein (VPg) is covalently bound to the 5' end of viral genomic RNA (gRNA) and associates with host eIF4E for successful infection. Divergent models to explain these observations proposed either an unknown mode of eIF4E engagement or a competition of VPg for the m7G cap-binding site. To dissect these possibilities, we resolved the structure of VPg, revealing a previously unknown 3-dimensional (3D) fold, and characterized the VPg-eIF4E complex using NMR and biophysical techniques. VPg directly bound the cap-binding site of eIF4E and competed for m7G cap analog binding. In human cells, VPg inhibited eIF4E-dependent RNA export, translation, and oncogenic transformation. Moreover, VPg formed trimeric complexes with eIF4E-eIF4G, eIF4E bound VPg-luciferase RNA conjugates, and these VPg-RNA conjugates were templates for translation. Informatic analyses revealed structural similarities between VPg and the human kinesin EG5. Consistently, EG5 directly bound eIF4E in a similar manner to VPg, demonstrating that this form of engagement is relevant beyond potyviruses. In all, we revealed an unprecedented modality for control and engagement of eIF4E and show that VPg-RNA conjugates functionally engage eIF4E. As such, potyvirus VPg provides a unique model system to interrogate eIF4E.

Unorthodox PCNA Binding by Chromatin Assembly Factor 1.

The eukaryotic DNA replication fork is a hub of enzymes that continuously act to synthesize DNA, propagate DNA methylation and other epigenetic marks, perform quality control, repair nascent DNA, and package this DNA into chromatin. Many of the enzymes involved in these spatiotemporally correlated processes perform their functions by binding to proliferating cell nuclear antigen (PCNA). A long-standing question has been how the plethora of PCNA-binding enzymes exert their activities without interfering with each other. As a first step towards deciphering this complex regulation, we studied how Chromatin Assembly Factor 1 (CAF-1) binds to PCNA. We demonstrate that CAF-1 binds to PCNA in a heretofore uncharacterized manner that depends upon a cation-pi (π) interaction. An arginine residue, conserved among CAF-1 homologs but absent from other PCNA-binding proteins, inserts into the hydrophobic pocket normally occupied by proteins that contain canonical PCNA interaction peptides (PIPs). Mutation of this arginine disrupts the ability of CAF-1 to bind PCNA and to assemble chromatin. The PIP of the CAF-1 p150 subunit resides at the extreme C-terminus of an apparent long α-helix (119 amino acids) that has been reported to bind DNA. The length of that helix and the presence of a PIP at the C-terminus are evolutionarily conserved among numerous species, ranging from yeast to humans. This arrangement of a very long DNA-binding coiled-coil that terminates in PIPs may serve to coordinate DNA and PCNA binding by CAF-1.

A biochemical framework for eIF4E-dependent mRNA export and nuclear recycling of the export machinery.

The eukaryotic translation initiation factor eIF4E acts in the nuclear export and translation of a subset of mRNAs. Both of these functions contribute to its oncogenic potential. While the biochemical mechanisms that underlie translation are relatively well understood, the molecular basis for eIF4E's role in mRNA export remains largely unexplored. To date, over 3000 transcripts, many encoding oncoproteins, were identified as potential nuclear eIF4E export targets. These target RNAs typically contain a ∼50-nucleotide eIF4E sensitivity element (4ESE) in the 3' UTR and a 7-methylguanosine cap on the 5' end. While eIF4E associates with the cap, an unknown factor recognizes the 4ESE element. We previously identified cofactors that functionally interacted with eIF4E in mammalian cell nuclei including the leucine-rich pentatricopeptide repeat protein LRPPRC and the export receptor CRM1/XPO1. LRPPRC simultaneously interacts with both eIF4E bound to the 5' mRNA cap and the 4ESE element in the 3' UTR. In this way, LRPPRC serves as a specificity factor to recruit 4ESE-containing RNAs within the nucleus. Further, we show that CRM1 directly binds LRPPRC likely acting as the export receptor for the LRPPRC-eIF4E-4ESE RNA complex. We also found that Importin 8, the nuclear importer for cap-free eIF4E, imports RNA-free LRPPRC, potentially providing both coordinated nuclear recycling of the export machinery and an important surveillance mechanism to prevent futile export cycles. Our studies provide the first biochemical framework for the eIF4E-dependent mRNA export pathway.

Importin 8 mediates m7G cap-sensitive nuclear import of the eukaryotic translation initiation factor eIF4E.

Regulation of nuclear-cytoplasmic trafficking of oncoproteins is critical for growth homeostasis. Dysregulated trafficking contributes to malignancy, whereas understanding the process can reveal unique therapeutic opportunities. Here, we focus on eukaryotic translation initiation factor 4E (eIF4E), a prooncogenic protein highly elevated in many cancers, including acute myeloid leukemia (AML). Typically, eIF4E is localized to both the nucleus and cytoplasm, where it acts in export and translation of specific methyl 7-guanosine (m(7)G)-capped mRNAs, respectively. Nuclear accumulation of eIF4E in patients who have AML is correlated with increased eIF4E-dependent export of transcripts encoding oncoproteins. The subcellular localization of eIF4E closely correlates with patients' responses. During clinical responses to the m(7)G-cap competitor ribavirin, eIF4E is mainly cytoplasmic. At relapse, eIF4E reaccumulates in the nucleus, leading to elevated eIF4E-dependent mRNA export. We have identified importin 8 as a factor that directly imports eIF4E into the nucleus. We found that importin 8 is highly elevated in untreated patients with AML, leading to eIF4E nuclear accumulation. Importin 8 only imports cap-free eIF4E. Cap-dependent changes to the structure of eIF4E underpin this selectivity. Indeed, m(7)G cap analogs or ribavirin prevents nuclear entry of eIF4E, which mirrors the trafficking phenotypes observed in patients with AML. Our studies also suggest that nuclear entry is important for the prooncogenic activity of eIF4E, at least in this context. These findings position nuclear trafficking of eIF4E as a critical step in its regulation and position the importin 8-eIF4E complex as a novel therapeutic target.

The eukaryotic translation initiation factor eIF4E in the nucleus: taking the road less traveled.

The eukaryotic translation initiation factor eIF4E is a potent oncogene. Although eIF4E has traditional roles in translation initiation in the cytoplasm, it is also found in the nucleus, suggesting that it has activities beyond its role in protein synthesis. The road less traveled has been taken to study these nuclear activities and to understand their contribution to the oncogenic potential of eIF4E. The molecular features and biological pathways underpinning eIF4E's nuclear mRNA export are described. New classes of eIF4E regulators have been identified and their relevance to cancer shown. The studies presented here reveal the molecular, biophysical, and structural bases for eIF4E regulation. Finally, recent clinical work targeting eIF4E in acute myeloid leukemia patients with ribavirin is discussed. In summary, these findings provide a novel paradigm for eIF4E function and the molecular basis for targeting it in leukemia patients.

eIF4E3 acts as a tumor suppressor by utilizing an atypical mode of methyl-7-guanosine cap recognition.

Recognition of the methyl-7-guanosine (m(7)G) cap structure on mRNA is an essential feature of mRNA metabolism and thus gene expression. Eukaryotic translation initiation factor 4E (eIF4E) promotes translation, mRNA export, proliferation, and oncogenic transformation dependent on this cap-binding activity. eIF4E-cap recognition is mediated via complementary charge interactions of the positively charged m(7)G cap between the negative π-electron clouds from two aromatic residues. Here, we demonstrate that a variant subfamily, eIF4E3, specifically binds the m(7)G cap in the absence of an aromatic sandwich, using instead a different spatial arrangement of residues to provide the necessary electrostatic and van der Waals contacts. Contacts are much more extensive between eIF4E3-cap than other family members. Structural analyses of other cap-binding proteins indicate this recognition mode is atypical. We demonstrate that eIF4E3 relies on this cap-binding activity to act as a tumor suppressor, competing with the growth-promoting functions of eIF4E. In fact, reduced eIF4E3 in high eIF4E cancers suggests that eIF4E3 underlies a clinically relevant inhibitory mechanism that is lost in some malignancies. Taken together, there is more structural plasticity in cap recognition than previously thought, and this is physiologically relevant.

Medicinal Chemistry and NMR Driven Discovery of Novel UDP-glucuronosyltransferase 1A Inhibitors That Overcome Therapeutic Resistance in Cells.

The UDP glucuronosyltransferases (UGT) deactivate many therapeutics via glucuronidation while being required for clearance of normal metabolites and xenobiotics. There are 19 UGT enzymes categorized into UGT1A and UGT2B families based on sequence conservation. This presents a challenge in terms of targeting specific UGTs to overcome drug resistance without eliciting overt toxicity. Here, we identified for the first time that UGT1A4 is highly elevated in acute myeloid leukemia (AML) patients and its reduction corresponded to objective clinical responses. To develop inhibitors to UGT1A4, we leveraged previous NMR-based fragment screening data against the C-terminal domain of UGT1A (UGT1A-C). NMR and medicinal chemistry strategies identified novel chemical matter based on fragment compounds with the capacity to bind ∼20 fold more tightly to UGT1A-C (Kd âˆ¼ 600 µM vs ∼30 µM). Some compounds differentially inhibited UGT1A4 versus UGT1A1 enzyme activity and restored drug sensitivity in resistant human cancer cells. NMR-based NOE experiments revealed these novel compounds recognised a region distal to the catalytic site suggestive of allosteric regulation. This binding region is poorly conserved between UGT1A and UGT2B C-terminal sequences, which otherwise exhibit high similarity. Consistently, these compounds did not bind to the C-terminal domain of UGT2B7 nor a triple mutant of UGT1A-C replaced with UGT2B7 residues in this region. Overall, we discovered a site on UGTs that can be leveraged to differentially target UGT1As and UGT2Bs, identified UGT1A4 as a therapeutic target, and found new chemical matter that binds the UGT1A C-terminus, inhibits glucuronidation and restores drug sensitivity.

Conformational changes induced in the eukaryotic translation initiation factor eIF4E by a clinically relevant inhibitor, ribavirin triphosphate.

The eukaryotic translation initiation factor eIF4E is highly elevated in human cancers including acute myeloid leukemia (AML). A potential anticancer agent, ribavirin, targets eIF4E activity in AML patients corresponding to clinical responses. To date, ribavirin is the only direct inhibitor of eIF4E to reach clinical trials. We showed that ribavirin acts as a competitive inhibitor of the methyl 7-guanosine (m(7)G) cap, the natural ligand of eIF4E. Here we examine the conformational changes occurring in human eIF4E upon binding the active metabolite of ribavirin, ribavirin triphosphate (RTP). Our NMR data revealed an unexpected concentration dependence on RTP affinity for eIF4E. We observed NMR spectra characteristic of tight binding at low micromolar concentrations (2-5 µM eIF4E) but much weaker affinity at more typical NMR concentrations (50- ). Comparison of chemical shift perturbation and line broadening suggest that the two eIF4E-RTP complexes differ in the precise positioning of RTP within the cap binding pocket, with the high affinity complex showing more extensive changes to the central ß-sheet and dorsal surface of eIF4E, similar to m(7)G cap. The differences between high and low affinity complexes arise due to concentration dependent aggregation of eIF4E and RTP. Given the intracellular concentrations of eIF4E and RTP and the differential binding toward the W56A eIF4E mutant the high affinity complex is the most physiologically relevant. In summary, these findings demonstrate that RTP binds in the cap-binding site but also suggests new features of this pocket that should be considered in drug design efforts and reveal new insights into ligand eIF4E recognition.

Structural characterization of the Z RING-eIF4E complex reveals a distinct mode of control for eIF4E.

The eukaryotic translation initiation factor eIF4E, a potent oncogene, is highly regulated. One class of eIF4E regulators, including eIF4G and the 4E-binding proteins (4E-BPs), interact with eIF4E using a conserved YXXXXLPhi-binding site. The structural basis of this interaction and its regulation are well established. Really Interesting New Gene (RING) domain containing proteins, such as the promyelocytic leukemia protein PML and the arenaviral protein Z, represent a second class of eIF4E regulators that inhibit eIF4E function by decreasing eIF4E's affinity for its m(7)G cap ligand. To elucidate the structural basis of this inhibition, we determined the structure of Z and studied the Z-eIF4E complex using NMR methods. We show that Z interacts with eIF4E via a novel binding site, which has no homology with that of eIF4G or the 4E-BPs, and is different from the RING recognition site used in the ubiquitin system. Z and eIF4G interact with distinct parts of eIF4E and differentially alter the conformation of the m(7)G cap-binding site. Our results provide a molecular basis for how PML and Z RINGs reduce the affinity of eIF4E for the m(7)G cap and thereby act as key inhibitors of eIF4E function. Furthermore, our findings provide unique insights into RING protein interactions.

1H, 13C, 15N Backbone and sidechain chemical shift assignments of the C-terminal domain of human UDP-glucuronosyltransferase 2B17 (UGT2B17-C).

UDP-glucuronosyltransferases are the principal enzymes involved in the glucuronidation of metabolites and xenobiotics for physiological clearance in humans. Though glucuronidation is an indispensable process in the phase II metabolic pathway, UGT-mediated glucuronidation of most prescribed drugs (> 55%) and clinical evidence of UGT-associated drug resistance are major concerns for therapeutic development. While UGTs are highly conserved enzymes, they manifest unique substrate and inhibitor specificity which is poorly understood given the dearth of experimentally determined full-length structures. Such information is important not only to conceptualize their specificity but is central to the design of inhibitors specific to a given UGT in order to avoid toxicity associated with pan-UGT inhibitors. Here, we provide the 1H, 13C and 15N backbone (~ 90%) and sidechain (~ 62%) assignments for the C-terminal domain of UGT2B17, which can be used to determine the molecular binding sites of inhibitor and substrate, and to understand the atomic basis for inhibitor selectivity between UGT2B17 and other members of the UGT2B subfamily. Given the physiological relevance of UGT2B17 in the elimination of hormone-based cancer drugs, these assignments will contribute towards dissecting the structural basis for substrate specificity, selective inhibitor recognition and other aspects of enzyme activity with the goal of selectively overcoming glucuronidation-based drug resistance.

Identification and Characterization of the Interaction Between the Methyl-7-Guanosine Cap Maturation Enzyme RNMT and the Cap-Binding Protein eIF4E.

The control of RNA metabolism is an important aspect of molecular biology with wide-ranging impacts on cells. Central to processing of coding RNAs is the addition of the methyl-7 guanosine (m7G) "cap" on their 5' end. The eukaryotic translation initiation factor eIF4E directly binds the m7G cap and through this interaction plays key roles in many steps of RNA metabolism including nuclear RNA export and translation. eIF4E also stimulates capping of many transcripts through its ability to drive the production of the enzyme RNMT which methylates the G-cap to form the mature m7G cap. Here, we found that eIF4E also physically associated with RNMT in human cells. Moreover, eIF4E directly interacted with RNMT in vitro. eIF4E is only the second protein reported to directly bind the methyltransferase domain of RNMT, the first being its co-factor RAM. We combined high-resolution NMR methods with biochemical studies to define the binding interfaces for the RNMT-eIF4E complex. Further, we found that eIF4E competes for RAM binding to RNMT and conversely, RNMT competes for binding of well-established eIF4E-binding partners such as the 4E-BPs. RNMT uses novel structural means to engage eIF4E. Finally, we observed that m7G cap-eIF4E-RNMT trimeric complexes form, and thus RNMT-eIF4E complexes may be employed so that eIF4E captures newly capped RNA. In all, we show for the first time that the cap-binding protein eIF4E directly binds to the cap-maturation enzyme RNMT.

1H, 13C and 15N chemical shift assignments of the C-terminal domain of human UDP-Glucuronosyltransferase 2B7 (UGT2B7-C).

The human UDP-glucuronosyltransferase (UGT) family of enzymes catalyze the covalent addition of glucuronic acid to a wide range of compounds, generally rendering them inactive. Although important for clearance of environmental toxins and metabolites, UGT activation can lead to inappropriate glucuronidation of therapeutics underlying drug resistance. Indeed, 50% of medications are glucuronidated. To better understand this mode of resistance, we studied the UGT2B7 enzyme associated with glucuronidation of cancer drugs such as Tamoxifen and Sorafenib. We report 1H, 13C and 15N backbone (> 90%) and side-chain assignments (~ 78% completeness according to CYANA) for the C-terminal domain of UGT2B7 (UGT2B7-C). Given the biomedical importance of this family of enzymes, our assignments will provide a key tool for improving understanding of the biochemical basis for substrate selectivity and other aspects of enzyme activity. This in turn will inform on drug design to overcome UGT-related drug resistance.

Production of functionalized polyhydroxyalkanoates by genetically modified Methylobacterium extorquens strains.

BACKGROUND: Methylotrophic (methanol-utilizing) bacteria offer great potential as cell factories in the production of numerous products from biomass-derived methanol. Bio-methanol is essentially a non-food substrate, an advantage over sugar-utilizing cell factories. Low-value products as well as fine chemicals and advanced materials are envisageable from methanol. For example, several methylotrophic bacteria, including Methylobacterium extorquens, can produce large quantities of the biodegradable polyester polyhydroxybutyric acid (PHB), the best known polyhydroxyalkanoate (PHA). With the purpose of producing second-generation PHAs with increased value, we have explored the feasibility of using M. extorquens for producing functionalized PHAs containing C-C double bonds, thus, making them amenable to future chemical/biochemical modifications for high value applications. RESULTS: Our proprietary M. extorquens ATCC 55366 was found unable to yield functionalized PHAs when fed methanol and selected unsaturated carboxylic acids as secondary substrates. However, cloning of either the phaC1 or the phaC2 gene from P. fluorescens GK13, using an inducible and regulated expression system based on cumate as inducer (the cumate switch), yielded recombinant M. extorquens strains capable of incorporating modest quantities of C-C double bonds into PHA, starting from either C6= and/or C8=. The two recombinant strains gave poor results with C11=. The strain containing the phaC2 gene was better at using C8= and at incorporating C-C double bonds into PHA. Solvent fractioning indicated that the produced polymers were PHA blends that consequently originated from independent actions of the native and the recombinant PHA synthases. CONCLUSIONS: This work constitutes an example of metabolic engineering applied to the construction of a methanol-utilizing bacterium capable of producing functionalized PHAs containing C-C double bonds. In this regard, the PhaC2 synthase appeared superior to the PhaC1 synthase at utilizing C8= as source of C-C double bonds and at incorporating C-C double bonds into PHA from either C6= or C8=. The M. ex-phaC2 strain is, therefore, a promising biocatalyst for generating advanced (functionalized) PHAs for future high value applications in various fields.

Biochemical and Structural Insights into the Eukaryotic Translation Initiation Factor eIF4E.

A major question in cell and cancer biology is concerned with understanding the flow of information from gene to protein. Indeed, many studies indicate that the proteome can be decoupled from the transcriptome. A major source of this decoupling is post-transcriptional regulation. The eukaryotic translation initiation factor eIF4E serves as an excellent example of a protein that can modulate the proteome at the post-transcriptional level. eIF4E is elevated in many cancers thus highlighting the relevance of this mode of control to biology. In this review, we provide a brief overview of various functions of eIF4E in RNA metabolism e.g. in nuclear-cytoplasmic RNA export, translation, RNA stability and/or sequestration. We focus on the modalities of eIF4E regulation at the biochemical and particularly structural level. In this instance, we describe not only the importance for the m7Gcap eIF4E interaction but also of recently discovered non-traditional RNA-eIF4E interactions as well as cap-independent activities of eIF4E. Further, we describe several distinct structural modalities used by the cell and some viruses to regulate or co-opt eIF4E, substantially extending the types of proteins that can regulate eIF4E from the traditional eIF4E-binding proteins (e.g. 4E-BP1 and eIF4G). Finally, we provide an overview of the results of targeting eIF4E activity in the clinic.

Chemical shift assignment of the viral protein genome-linked (VPg) from potato virus Y.

The dysregulation of translation contributes to many pathogenic conditions in humans. Discovering new translational mechanisms is important to understanding the diversity of this process and its potential mechanisms. Such mechanisms can be initially observed in viruses. With this in mind, we studied the viral protein genome-linked VPg factor from the largest genus of plant viruses. Studies in plants show that VPg binds to the eukaryotic translation initiation factor eIF4E for translation of viral RNAs. VPg contains no known eIF4E binding motifs and no sequence homology to any known proteins. Thus, as a first step in understanding the structural basis of this interaction, we carried out NMR assignments of the VPg from the potato virus Y potyvirus protein.

Overcoming Drug Resistance through the Development of Selective Inhibitors of UDP-Glucuronosyltransferase Enzymes.

Drug resistance is a major cause of cancer-related mortality. Glucuronidation of drugs via elevation of UDP-glucuronosyltransferases (UGT1As) correlates with clinical resistance. The nine UGT1A family members have broad substrate specificities attributed to their variable N-terminal domains and share a common C-terminal domain. Development of UGT1As as pharmacological targets has been hampered by toxicity of pan-UGT inhibitors and by difficulty in isolating pure N-terminal domains or full-length proteins. Here, we developed a strategy to target selected UGT1As which exploited the biochemical tractability of the C-domain and its ability to allosterically communicate with the catalytic site. By combining NMR fragment screening with in vitro glucuronidation assays, we identified inhibitors selective for UGT1A4. Significantly, these compounds selectively restored sensitivity in resistant cancer cells only for substrates of the targeted UGT1A. This strategy represents a crucial first step toward developing compounds to overcome unwanted glucuronidation thereby reversing resistance in patients.

The structurally disordered KRAB repression domain is incorporated into a protease resistant core upon binding to KAP-1-RBCC domain.

The KRAB domain is a 75 amino acid transcriptional repression module that is encoded by more than 400 zinc finger protein genes, making it the most abundant repression domain in the human proteome. KRAB-mediated gene silencing requires a direct high affinity interaction with the RBCC domain of KAP-1 co-repressor. The structures of the free KRAB domain or the KRAB-RBCC complex are unknown. To address this, we have performed a systematic biophysical analysis of all KRAB isoforms using purified recombinant proteins. All KRAB domains are predominantly monomeric either alone or in a complex with KAP-1-RBCC protein, while a KRAB-SCAN isoform exists as a stable dimer. The KRAB:KAP-1-RBCC interaction requires only the A box in the context of the KRAB(Ab) or KRAB(AC) but both A and B boxes in the context of KRAB(AB). All isoforms bind the KAP-1-RBCC in a stable, zinc dependent fashion with a stoichiometry of KRAB1:3 RBCC with a zinc content of four atoms per RBCC monomer. Limited proteolysis, mass spectrometry and N-terminal sequence analyses suggest that a core complex comprises the entire RBCC domain of KAP-1 and the AB box of the KRAB domain rendering it resistant to proteolysis. NMR spectroscopy showed that unbound KRAB domain does not exist as a well-folded globular protein in solution but may fold into an ordered structure upon binding to the KAP-1-RBCC protein. This is the first example of a structurally disordered repressor domain that is the most widely conserved silencing domain in tetrapods.

Backbone assignment of the apo-form of the human C-terminal domain of UDP-glucuronosyltransferase 1A (UGT1A).

A major component of phase II drug metabolism is the covalent addition of glucuronic acid to metabolites and xenobiotics. This activity is carried out by UDP-glucuronosyltransferases (UGT) which bind the UDP-glucuronic acid donor and catalyze the covalent addition of glucuronic acid sugar moieties onto a wide variety of substrates. UGTs play important roles in drug detoxification and were recently shown to act in an inducible form of multi-drug resistance in cancer patients. Despite their biological importance, structural understanding of these enzymes is limited. The C-terminal domain is identical for all UGT1A family members and required for binding to UDP-glucuronic acid as well as involved in contacts with substrates. Here, we report the backbone assignments for the C-terminal domain of UGT1A. These assignments are a critical tool for the development of a deeper biochemical understanding of substrate specificity and enzymatic activity.

The solution structure of the oxidative stress-related protein YggX from Escherichia coli.

YggX is a highly conserved protein found only in eubacteria and is proposed to be involved in the bacterial response to oxidative stress. Here we report the solution structure of YggX from Escherichia coli determined by nuclear magnetic resonance spectroscopy. The structure of YggX displays a fold consisting of two N-terminal antiparallel beta-sheets and three alpha-helices, which shares significant structural similarity to the crystal structure of a hypothetical protein PA5148 from Pseudomonas aeruginosa. Previous studies propose YggX as an iron binding protein that is involved in cellular iron trafficking. Our data indicate that the protein alone does not bind iron in vitro, suggesting other cofactors or different conditions may be necessary for metal binding.

Structural dynamics of the membrane translocation domain of colicin E9 and its interaction with TolB.

In order for the 61 kDa colicin E9 protein toxin to enter the cytoplasm of susceptible cells and kill them by hydrolysing their DNA, the colicin must interact with the outer membrane BtuB receptor and Tol translocation pathway of target cells. The translocation function is located in the N-terminal domain of the colicin molecule. (1)H, (1)H-(1)H-(15)N and (1)H-(13)C-(15)N NMR studies of intact colicin E9, its DNase domain, minimal receptor-binding domain and two N-terminal constructs containing the translocation domain showed that the region of the translocation domain that governs the interaction of colicin E9 with TolB is largely unstructured and highly flexible. Of the expected 80 backbone NH resonances of the first 83 residues of intact colicin E9, 61 were identified, with 43 of them being assigned specifically. The absence of secondary structure for these was shown through chemical shift analyses and the lack of long-range NOEs in (1)H-(1)H-(15)N NOESY spectra (tau(m)=200 ms). The enhanced flexibility of the region of the translocation domain containing the TolB box compared to the overall tumbling rate of the protein was identified from the relatively large values of backbone and tryptophan indole (15)N spin-spin relaxation times, and from the negative (1)H-(15)N NOEs of the backbone NH resonances. Variable flexibility of the N-terminal region was revealed by the (15)N T(1)/T(2) ratios, which showed that the C-terminal end of the TolB box and the region immediately following it was motionally constrained compared to other parts of the N terminus. This, together with the observation of inter-residue NOEs involving Ile54, indicated that there was some structural ordering, resulting most probably from the interactions of side-chains. Conformational heterogeneity of parts of the translocation domain was evident from a multiplicity of signals for some of the residues. Im9 binding to colicin E9 had no effect on the chemical shifts or other NMR characteristics of the region of colicin E9 containing the TolB recognition sequence, though the interaction of TolB with intact colicin E9 bound to Im9 did affect resonances from this region. The flexibility of the translocation domain of colicin E9 may be connected with its need to recognise protein partners that assist it in crossing the outer membrane and in the translocation event itself.