NMR Chemical Shift Mapping Research Articles

Fragment-based drug discovery of small molecule ligands for the human chemokine CCL28.

The mucosal chemokine CCL28 is a promising target for immunotherapy drug development due to its elevated expression level in epithelial cells and critical role in creating and maintaining an immunosuppressive tumor microenvironment. Using sulfotyrosine as a probe, NMR chemical shift mapping identified a potential receptor-binding hotspot on the human CCL28 surface. CCL28 was screened against 2,678 commercially available chemical fragments by 2D NMR, yielding thirteen verified hits. Computational docking predicted that two fragments could occupy adjoining subsites within the sulfotyrosine recognition cleft. Dual NMR titrations confirmed their ability to bind CCL28 simultaneously, thereby validating an initial fragment pair for linking and merging strategies to design high-potency CCL28 inhibitors.

Cyp33 binds AU-rich RNA motifs via an extended interface that competitively disrupts the gene repressive Cyp33-MLL1 interaction in vitro.

Cyp33 is an essential human cyclophilin prolyl isomerase that plays myriad roles in splicing and chromatin remodeling. In addition to a canonical cyclophilin (Cyp) domain, Cyp33 contains an RNA-recognition motif (RRM) domain, and RNA-binding triggers proline isomerase activity. One prominent role for Cyp33 is through a direct interaction with the mixed lineage leukemia protein 1 (MLL1, also known as KMT2A) complex, which is a histone methyltransferase that serves as a global regulator of human transcription. MLL activity is regulated by Cyp33, which isomerizes a key proline in the linker between the PHD3 and Bromo domains of MLL1, acting as a switch between gene activation and repression. The direct interaction between MLL1 and Cyp33 is critical, as deletion of the MLL1-PHD3 domain responsible for this interaction results in oncogenesis. The Cyp33 RRM is central to these activities, as it binds both the PHD3 domain and RNA. To better understand how RNA binding drives the action of Cyp33, we performed RNA-SELEX against full-length Cyp33 accompanied by deep sequencing. We have identified an enriched Cyp33 binding motif (AAUAAUAA) broadly represented in the cellular RNA pool as well as tightly binding RNA aptamers with affinities comparable and competitive with the Cyp33 MLL1-PHD3 interaction. RNA binding extends beyond the canonical RRM domain, but not to the Cyp domain, suggesting an indirect mechanism of interaction. NMR chemical shift mapping confirms an overlapping, but not identical, interface on Cyp33 for RNA and PHD3 binding. This finding suggests RNA can disrupt the gene repressive Cyp33-MLL1 complex providing another layer of regulation for chromatin remodeling by MLL1.

Demonstration that Small Molecules can Bind and Stabilize Low-abundance Short-lived RNA Excited Conformational States

Many promising RNA drug targets have functions that require the formation of RNA-protein complexes, but inhibiting RNA-protein interactions can prove difficult using small molecules. Regulatory RNAs have been shown to transiently form excited conformational states (ESs) that remodel local aspects of secondary structure. In some cases, the ES conformation has been shown to be inactive and to be poorly recognized by protein binding partners. In these cases, specifically targeting and stabilizing the RNA ES using a small molecule provides a rational structure-based strategy for inhibiting RNA activity. However, this requires that a small molecule discriminates between two conformations of the same RNA to preferentially bind and stabilize the short-lived low-abundance ES relative to the long-lived more abundant ground state (GS). Here, we tested the feasibility of this approach by designing a mutant that inverts the conformational equilibrium of the HIV-1 transactivation response element (TAR) RNA, such that the native GS conformation becomes a low-abundance ES. Using this mutant and NMR chemical shift mapping experiments, we show that argininamide, a ligand mimic of TAR's cognate protein binding partner Tat, is able to restore a native-like conformation by preferentially binding and stabilizing the transient and low-populated ES. A synthetic small molecule optimized to bind the TAR GS also partially stabilized the ES, whereas an aminoglycoside molecule that binds RNAs nonspecifically did not preferentially stabilize the ES to a similar extent. These results support the feasibility of inhibiting RNA activity using small molecules that preferentially bind and stabilize the ES.

Structural and functional characterization of the PDZ domain of the human phosphatase PTPN3 and its interaction with the human papillomavirus E6 oncoprotein

The human protein tyrosine phosphatase non-receptor type 3 (PTPN3) is a PDZ (PSD-95/Dlg/ZO-1) domain-containing phosphatase with a tumor-suppressive or a tumor-promoting role in many cancers. Interestingly, the high-risk genital human papillomavirus (HPV) types 16 and 18 target the PDZ domain of PTPN3. The presence of a PDZ binding motif (PBM) on E6 confers interaction with a number of different cellular PDZ domain-containing proteins and is a marker of high oncogenic potential. Here, we report the molecular basis of interaction between the PDZ domain of PTPN3 and the PBM of the HPV E6 protein. We combined biophysical, NMR and X-ray experiments to investigate the structural and functional properties of the PDZ domain of PTPN3. We showed that the C-terminal sequences from viral proteins encompassing a PBM interact with PTPN3-PDZ with similar affinities to the endogenous PTPN3 ligand MAP kinase p38γ. PBM binding stabilizes the PDZ domain of PTPN3. We solved the X-ray structure of the PDZ domain of PTPN3 in complex with the PBM of the HPV E6 protein. The crystal structure and the NMR chemical shift mapping of the PTPN3-PDZ/peptide complex allowed us to pinpoint the main structural determinants of recognition of the C-terminal sequence of the E6 protein and the long-range perturbations induced upon PBM binding.

Structural characterization of B. subtilis m1A22 tRNA methyltransferase TrmK: insights into tRNA recognition.

1-Methyladenosine (m1A) is a modified nucleoside found at positions 9, 14, 22 and 58 of tRNAs, which arises from the transfer of a methyl group onto the N1-atom of adenosine. The yqfN gene of Bacillus subtilis encodes the methyltransferase TrmK (BsTrmK) responsible for the formation of m1A22 in tRNA. Here, we show that BsTrmK displays a broad substrate specificity, and methylates seven out of eight tRNA isoacceptor families of B. subtilis bearing an A22. In addition to a non-Watson–Crick base-pair between the target A22 and a purine at position 13, the formation of m1A22 by BsTrmK requires a full-length tRNA with intact tRNA elbow and anticodon stem. We solved the crystal structure of BsTrmK showing an N-terminal catalytic domain harbouring the typical Rossmann-like fold of Class-I methyltransferases and a C-terminal coiled-coil domain. We used NMR chemical shift mapping to drive the docking of BstRNASer to BsTrmK in complex with its methyl-donor cofactor S-adenosyl-L-methionine (SAM). In this model, validated by methyltransferase activity assays on BsTrmK mutants, both domains of BsTrmK participate in tRNA binding. BsTrmK recognises tRNA with very few structural changes in both partner, the non-Watson–Crick R13–A22 base-pair positioning the A22 N1-atom close to the SAM methyl group.

Increasing the length of poly-pyrimidine bulges broadens RNA conformational ensembles with minimal impact on stacking energetics.

Helical elements separated by bulges frequently undergo transitions between unstacked and coaxially stacked conformations during the folding and function of noncoding RNAs. Here, we examine the dynamic properties of poly-pyrimidine bulges of varying length (n = 1–4, 7) across a range of Mg2+ concentrations using HIV-1 TAR RNA as a model system and solution NMR spectroscopy. In the absence of Mg2+, helices linked by bulges with n ≥ 3 residues adopt predominantly unstacked conformations (stacked population <15%), whereas one-bulge and two-bulge motifs adopt predominantly stacked conformations (stacked population >74%). In the presence of 3 mM Mg2+, the helices predominantly coaxially stack (stacked population >84%), regardless of bulge length, and the midpoint for the Mg2+-dependent stacking transition is within threefold regardless of bulge length. In the absence of Mg2+, the difference between free energy of interhelical coaxial stacking across the bulge variants is estimated to be ∼2.9 kcal/mol, based on an NMR chemical shift mapping with stacking being more energetically disfavored for the longer bulges. This difference decreases to ∼0.4 kcal/mol in the presence of Mg2+. NMR RDCs and resonance intensity data show increased dynamics in the stacked state with increasing bulge length in the presence of Mg2+. We propose that Mg2+ helps to neutralize the growing electrostatic repulsion in the stacked state with increasing bulge length thereby increasing the number of coaxial conformations that are sampled. Energetically compensated interhelical stacking dynamics may help to maximize the conformational adaptability of RNA and allow a wide range of conformations to be optimally stabilized by proteins and ligands.

Identification of a new small ubiquitin-like modifier (SUMO)-interacting motif in the E3 ligase PIASy

Small ubiquitin-like modifier (SUMO) conjugation is a reversible post-translational modification process implicated in the regulation of gene transcription, DNA repair, and cell cycle. SUMOylation depends on the sequential activities of E1 activating, E2 conjugating, and E3 ligating enzymes. SUMO E3 ligases enhance transfer of SUMO from the charged E2 enzyme to the substrate. We have previously identified PIASy, a member of the Siz/protein inhibitor of activated STAT (PIAS) RING family of SUMO E3 ligases, as essential for mitotic chromosomal SUMOylation in frog egg extracts and demonstrated that it can mediate effective SUMOylation. To address how PIASy catalyzes SUMOylation, we examined various truncations of PIASy for their ability to mediate SUMOylation. Using NMR chemical shift mapping and mutagenesis, we identified a new SUMO-interacting motif (SIM) in PIASy. The new SIM and the currently known SIM are both located at the C terminus of PIASy, and both are required for the full ligase activity of PIASy. Our results provide novel insights into the mechanism of PIASy-mediated SUMOylation. PIASy adds to the growing list of SUMO E3 ligases containing multiple SIMs that play important roles in the E3 ligase activity.

Toxin‐antitoxin systems: novel mechanisms of toxin activity and antitoxin inhibition

Toxin‐antitoxin (TA) modules function as regulatory switches that allow bacteria to adapt to rapidly changing environments. Under conditions of unrestrained growth, these systems are repressed. However, under conditions of stress, these systems are activated, leading to growth arrest and dormancy. Their central role controlling bacterial growth has led to renewed efforts to understand the molecular basis of TA function in order to develop strategies to exploit these systems for societal benefit. The E. coli mqsR gene was originally discovered for its role in promoting biofilm formation and dormancy. During the past five years, we have used biochemistry and structural biology to show that mqsRA is a bona fide toxin‐antitoxin system, in which MqsR is ribonuclease (RNase) toxin, and MqsA is its cognate antitoxin. Unpublished results from our group now demonstrate that MqsR defines a new hybrid family of toxin RNases and that its MqsA antitoxin inhibits MqsR via a novel mechanism. Specifically, we are using NMR chemical shift mapping coupled with mutagenesis and biochemistry to identify the residues that mediate mRNA binding and cleavage and show that MqsR exhibits sequence specificity outside the primary GCU cleavage sequence. Furthermore, our work is revealing that MqsA inhibits MqsR activity via a completely novel mechanism for antitoxins; i.e. it potentially inhibits substrate binding by engaging loops outside the MqsR active site that are critical for the interaction with mRNA. Finally, we extending these studies to evolutionarily related families to elucidate the molecular basis of conservation and divergence of enzyme function in this family. Collectively, these studies are not only revealing that MqsRA defines a novel subfamily of TA systems but, more critically, also leading to the identification of the key molecular details that will allow these systems to be directed for societal benefit.Support or Funding InformationNSF‐CAREER award (MCB0952550) to RP.

Rapid Exchange Between Free and Bound States in RNA-Dendrimer Polyplexes: Implications on the Mechanism of Delivery and Release.

A combination of solution NMR, dynamic light scattering (DLS), and fluorescence quenching assays were employed to obtain insights into the dynamics and structural features of a polyplex system consisting of HIV-1 transactivation response element (TAR) and PEGylated generation 5 poly(amidoamine) dendrimer (G5-PEG). NMR chemical shift mapping and (13)C spin relaxation based dynamics measurements depict the polyplex system as a highly dynamic assembly where the RNA, with its local structure and dynamics preserved, rapidly exchanges (<ms) between free and polyplex-bound forms, at least for the probed N/P ratios. Recovery of quenched fluorescence shows that RNA in the polyplex can be competitively exchanged over a wide range of amine to phosphate (N/P) charge ratios. The rapid exchange between free and bound forms may contribute to the mechanism by which RNA is released into the cell upon delivery while being protected by the polyplex environment from immediate degradation by nucleases.

Regulation of Translation Initiation by a Network of Overlapping Intra‐ and Intermolecular Interactions Mediated by Natively Unfolded Protein Segments

Translation initiation requires a number of eukaryotic translation initiation factors (eIFs) and consists of several steps: initiation complex assembly; binding to mRNA; scanning; start codon recognition; and ribosomal subunit joining. The G-protein eIF2 and its GTPase-activating protein (GAP) eIF5 play central roles in start codon recognition. A second G-protein, eIF5B promotes ribosomal subunit joining. eIF1A, is involved in virtually all steps of translation initiation. We used a combination of NMR chemical shift mapping, fluorescence spectroscopy, site-directed mutagenesis, and deletion analysis to elucidate the interplay among intra- and intermolecular interactions of human translation initiation factors eIF5B, eIF1A and eIF5. We discovered multiple transient interactions, too weak to form in isolation, but sufficient to modulate the affinity of other interactions in the context of large complexes. These interactions are often mediated by natively unfolded protein segments. A common theme emerges, where natively unfolded protein segments are able to form weak transient interactions while remaining unfolded and flexible, thus apparently limiting the entropy cost of the interaction, whereas stronger interactions require at least partial folding and immobilization of the peptide. Based on the observed overlaps in interaction surfaces and the relative binding affinities, we propose a mechanism for formation and remodeling of ribosomal complexes through coordinated rearrangement of multiple relatively weak interactions.

Structure-function analysis reveals that the Pseudomonas aeruginosa Tps4 two-partner secretion system is involved in CupB5 translocation.

Pseudomonas aeruginosa is a Gram-negative opportunistic bacterium, synonymous with cystic fibrosis patients, which can cause chronic infection of the lungs. This pathogen is a model organism to study biofilms: a bacterial population embedded in an extracellular matrix that provide protection from environmental pressures and lead to persistence. A number of Chaperone-Usher Pathways, namely CupA-CupE, play key roles in these processes by assembling adhesive pili on the bacterial surface. One of these, encoded by the cupB operon, is unique as it contains a nonchaperone-usher gene product, CupB5. Two-partner secretion (TPS) systems are comprised of a C-terminal integral membrane β-barrel pore with tandem N-terminal POTRA (POlypeptide TRansport Associated) domains located in the periplasm (TpsB) and a secreted substrate (TpsA). Using NMR we show that TpsB4 (LepB) interacts with CupB5 and its predicted cognate partner TpsA4 (LepA), an extracellular protease. Moreover, using cellular studies we confirm that TpsB4 can translocate CupB5 across the P. aeruginosa outer membrane, which contrasts a previous observation that suggested the CupB3 P-usher secretes CupB5. In support of our findings we also demonstrate that tps4/cupB operons are coregulated by the RocS1 sensor suggesting P. aeruginosa has developed synergy between these systems. Furthermore, we have determined the solution-structure of the TpsB4-POTRA1 domain and together with restraints from NMR chemical shift mapping and in vivo mutational analysis we have calculated models for the entire TpsB4 periplasmic region in complex with both TpsA4 and CupB5 secretion motifs. The data highlight specific residues for TpsA4/CupB5 recognition by TpsB4 in the periplasm and suggest distinct roles for each POTRA domain.

Abstract 1641: Structure-based design and development of pyrazolopyridine-based inhibitors of Mcl-1

Abstract The anti-apoptotic myeloid cell leukemia protein Mcl-1, a member of the Bcl-2 family proteins, has emerged as a promising therapeutic target. Mcl-1 is overexpressed in many human cancers which has been associated with inferior survival, poor prognosis and resistance to chemotherapies. Disruption of Mcl-1 interaction with its pro-apoptotic partners through small molecules is a viable strategy to overcome Mcl-1 mediated resistance to apoptosis. Targeting Mcl-1 protein represents a promising strategy either alone or in combination with other therapies. Applying an integrated screening approach through combining high throughput and virtual screenings, several novel chemical classes were identified as Mcl-1 inhibitors. Compound 38 with a pyrazolopyridine scaffold was selected as a promising high throughput lead for medicinal chemistry efforts. Using reported chemistry a focused library of analogs of 38 was generated. Through HSQC protein-observed NMR studies and chemical shift mapping, the binding of the lead compound 38 was characterized and confirmed to be the BH3 binding pocket of Mcl-1. Computational modeling guided by NMR studies was applied in lead optimization and rational design of more potent analogs. Structure-activity relationship was established utilizing two different competitive platforms of fluorescent polarization and surface plasmon resonance, and confirmed by HSQC NMR spectroscopy. The binding affinity of this class of compounds was improved more than twenty fold in comparison with the lead compound 38. In vitro binding, functional and cell-based assays were performed in order to determine selectivity profile against five members of Bcl-2 family, mechanism of action and cellular activity of analogs with improved potency. The obtained results show promise for further chemical modification and development of pyrazolopyridine-based inhibitors of Mcl-1. Citation Format: Fardokht A. Abulwerdi, Ahmed S.A. Mady, Andrej Perdih, Jeanne A. Stuckey, Hollis D. Showalter, Zaneta Nikolovska-Coleska. Structure-based design and development of pyrazolopyridine-based inhibitors of Mcl-1. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1641. doi:10.1158/1538-7445.AM2014-1641

Characterization of RNA binding and chaperoning activities of HIV-1 Vif protein

The viral infectivity factor (Vif) is essential for the productive infection and dissemination of HIV-1 in non-permissive cells, containing the cellular anti-HIV defense cytosine deaminases APOBEC3 (A3G and A3F). Vif neutralizes the antiviral activities of the APOBEC3G/F by diverse mechanisms including their degradation through the ubiquitin/proteasome pathway and their translational inhibition. In addition, Vif appears to be an active partner of the late steps of viral replication by interacting with Pr55Gag, reverse transcriptase and genomic RNA. Here, we expressed and purified full-length and truncated Vif proteins, and analyzed their RNA binding and chaperone properties. First, we showed by CD and NMR spectroscopies that the N-terminal domain of Vif is highly structured in solution, whereas the C-terminal domain remains mainly unfolded. Both domains exhibited substantial RNA binding capacities with dissociation constants in the nanomolar range, whereas the basic unfolded C-terminal domain of Vif was responsible in part for its RNA chaperone activity. Second, we showed by NMR chemical shift mapping that Vif and NCp7 share the same binding sites on tRNALys3, the primer of HIV-1 reverse transcriptase. Finally, our results indicate that Vif has potent RNA chaperone activity and provide direct evidence for an important role of the unstructured C-terminal domain of Vif in this capacity.

Substrate-modulated Cytochrome P450 17A1 and Cytochrome b5 Interactions Revealed by NMR

The membrane heme protein cytochrome b5 (b5) can enhance, inhibit, or have no effect on cytochrome P450 (P450) catalysis, depending on the specific P450, substrate, and reaction conditions, but the structural basis remains unclear. Here the interactions between the soluble domain of microsomal b5 and the catalytic domain of the bifunctional steroidogenic cytochrome P450 17A1 (CYP17A1) were investigated. CYP17A1 performs both steroid hydroxylation, which is unaffected by b5, and an androgen-forming lyase reaction that is facilitated 10-fold by b5. NMR chemical shift mapping of b5 titrations with CYP17A1 indicates that the interaction occurs in an intermediate exchange regime and identifies charged surface residues involved in the protein/protein interface. The role of these residues is confirmed by disruption of the complex upon mutagenesis of either the anionic b5 residues (Glu-48 or Glu-49) or the corresponding cationic CYP17A1 residues (Arg-347, Arg-358, or Arg-449). Cytochrome b5 binding to CYP17A1 is also mutually exclusive with binding of NADPH-cytochrome P450 reductase. To probe the differential effects of b5 on the two CYP17A1-mediated reactions and, thus, communication between the superficial b5 binding site and the buried CYP17A1 active site, CYP17A1/b5 complex formation was characterized with either hydroxylase or lyase substrates bound to CYP17A1. Significantly, the CYP17A1/b5 interaction is stronger when the hydroxylase substrate pregnenolone is present in the CYP17A1 active site than when the lyase substrate 17α-hydroxypregnenolone is in the active site. These findings form the basis for a clearer understanding of this important interaction by directly measuring the reversible binding of the two proteins, providing evidence of communication between the CYP17A1 active site and the superficial proximal b5 binding site.

Bacillus subtilis RNA deprotection enzyme RppH recognizes guanosine in the second position of its substrates

The initiation of mRNA degradation often requires deprotection of its 5' end. In eukaryotes, the 5'-methylguanosine (cap) structure is principally removed by the Nudix family decapping enzyme Dcp2, yielding a 5'-monophosphorylated RNA that is a substrate for 5' exoribonucleases. In bacteria, the 5'-triphosphate group of primary transcripts is also converted to a 5' monophosphate by a Nudix protein called RNA pyrophosphohydrolase (RppH), allowing access to both endo- and 5' exoribonucleases. Here we present the crystal structures of Bacillus subtilis RppH (BsRppH) bound to GTP and to a triphosphorylated dinucleotide RNA. In contrast to Bdellovibrio bacteriovorus RppH, which recognizes the first nucleotide of its RNA targets, the B. subtilis enzyme has a binding pocket that prefers guanosine residues in the second position of its substrates. The identification of sequence specificity for RppH in an internal position was a highly unexpected result. NMR chemical shift mapping in solution shows that at least three nucleotides are required for unambiguous binding of RNA. Biochemical assays of BsRppH on RNA substrates with single-base-mutation changes in the first four nucleotides confirm the importance of guanosine in position two for optimal enzyme activity. Our experiments highlight important structural and functional differences between BsRppH and the RNA deprotection enzymes of distantly related bacteria.

Tyrosine Sulfation of Chemokine Receptor CCR2 Enhances Interactions with Both Monomeric and Dimeric Forms of the Chemokine Monocyte Chemoattractant Protein-1 (MCP-1)

Chemokine receptors are commonly post-translationally sulfated on tyrosine residues in their N-terminal regions, the initial site of binding to chemokine ligands. We have investigated the effect of tyrosine sulfation of the chemokine receptor CCR2 on its interactions with the chemokine monocyte chemoattractant protein-1 (MCP-1/CCL2). Inhibition of CCR2 sulfation, by growth of expressing cells in the presence of sodium chlorate, significantly reduced the potency for MCP-1 activation of CCR2. MCP-1 exists in equilibrium between monomeric and dimeric forms. The obligate monomeric mutant MCP-1(P8A) was similar to wild type MCP-1 in its ability to induce leukocyte recruitment in vivo, whereas the obligate dimeric mutant MCP-1(T10C) was less effective at inducing leukocyte recruitment in vivo. In two-dimensional NMR experiments, sulfated peptides derived from the N-terminal region of CCR2 bound to both the monomeric and dimeric forms of wild type MCP-1 and shifted the equilibrium to favor the monomeric form. Similarly, MCP-1(P8A) bound more tightly than MCP-1(T10C) to the CCR2-derived sulfopeptides. NMR chemical shift mapping using the MCP-1 mutants showed that the sulfated N-terminal region of CCR2 binds to the same region (N-loop and β3-strand) of both monomeric and dimeric MCP-1 but that binding to the dimeric form also influences the environment of chemokine N-terminal residues, which are involved in dimer formation. We conclude that interaction with the sulfated N terminus of CCR2 destabilizes the dimerization interface of inactive dimeric MCP-1, thus inducing dissociation to the active monomeric state.

Structure and Assembly of a Trans-Periplasmic Channel for Type IV Pili in Neisseria meningitidis

Type IV pili are polymeric fibers which protrude from the cell surface and play a critical role in adhesion and invasion by pathogenic bacteria. The secretion of pili across the periplasm and outer membrane is mediated by a specialized secretin protein, PilQ, but the way in which this large channel is formed is unknown. Using NMR, we derived the structures of the periplasmic domains from N. meningitidis PilQ: the N-terminus is shown to consist of two β-domains, which are unique to the type IV pilus-dependent secretins. The structure of the second β-domain revealed an eight-stranded β-sandwich structure which is a novel variant of the HSP20-like fold. The central part of PilQ consists of two α/β fold domains: the structure of the first of these is similar to domains from other secretins, but with an additional α-helix which links it to the second α/β domain. We also determined the structure of the entire PilQ dodecamer by cryoelectron microscopy: it forms a cage-like structure, enclosing a cavity which is approximately 55 Å in internal diameter at its largest extent. Specific regions were identified in the density map which corresponded to the individual PilQ domains: this allowed us to dock them into the cryoelectron microscopy density map, and hence reconstruct the entire PilQ assembly which spans the periplasm. We also show that the C-terminal domain from the lipoprotein PilP, which is essential for pilus assembly, binds specifically to the first α/β domain in PilQ and use NMR chemical shift mapping to generate a model for the PilP:PilQ complex. We conclude that passage of the pilus fiber requires disassembly of both the membrane-spanning and the β-domain regions in PilQ, and that PilP plays an important role in stabilising the PilQ assembly during secretion, through its anchorage in the inner membrane.

Mapping of the CD23 Binding Site on Immunoglobulin E (IgE) and Allosteric Control of the IgE-FcϵRI Interaction

IgE, the antibody that mediates allergic responses, acts as part of a self-regulating protein network. Its unique effector functions are controlled through interactions of its Fc region with two cellular receptors, FcεRI on mast cells and basophils and CD23 on B cells. IgE cross-linked by allergen triggers mast cell activation via FcεRI, whereas IgE-CD23 interactions control IgE expression levels. We have determined the CD23 binding site on IgE, using a combination of NMR chemical shift mapping and site-directed mutagenesis. We show that the CD23 and FcεRI interaction sites are at opposite ends of the Cε3 domain of IgE, but that receptor binding is mutually inhibitory, mediated by an allosteric mechanism. This prevents CD23-mediated cross-linking of IgE bound to FcεRI on mast cells and resulting antigen-independent anaphylaxis. The mutually inhibitory nature of receptor binding provides a degree of autonomy for the individual activities mediated by IgE-FcεRI and IgE-CD23 interactions.

Characterization of Chromoshadow Domain-mediated Binding of Heterochromatin Protein 1α (HP1α) to Histone H3

The chromoshadow domain (CSD) of heterochromatin protein 1 (HP1) was recently shown to contribute to chromatin binding and transcriptional regulation through interaction with histone H3. Here, we demonstrate the structural basis of this interaction for the CSD of HP1α. This mode of H3 binding is dependent on dimerization of the CSD and recognition of a PxVxL-like motif, as for other CSD partners. NMR chemical shift mapping showed that the H3 residues that mediate the CSD interaction occur in and adjacent to the αN helix just within the nucleosome core. Access to the binding region would require some degree of unwrapping of the DNA near the nucleosomal DNA entry/exit site.

ZBP1 KH34 consensus RNA‐binding site identifies posttranscriptional regulatory networks

ZBP1 is a multifunctional regulator of RNA metabolism that has been shown to control aspects of localization, stability and translation for many mRNAs. To reveal the basis for ZBP1 recognition of its RNA targets, we have biochemically characterized the interaction between ZBP1 and the β‐actin zipcode. We have determined that ZBP1 KH34 recognizes a bipartite RNA element located within the first 28 nucleotides of the zipcode. The 5′ sequence of the bipartite element was determined to be CGGAC while the 3′ sequence was C/A‐CA‐C/U. The spacing between these RNA sequences is consistent with the structure of KH34 that we solved by X‐ray crystallography where the tandem KH domains are arranged in an anti‐parallel pseudodimer conformation. NMR chemical shift mapping of KH34 in complex with zipcode RNA fragments identified that KH4 recognizes the 5′ region of the bipartite element and KH3 binds to the 3′ region. In order to identify other mRNA targets of ZBP1, the KH34 consensus sequence was used to search the 3′ UTRs of human and mouse and the bipartite element was conserved in 114 mRNAs. Many of the predicted mRNA targets were found to coimmunoprecipitate with ZBP1 from mouse embryonic brain lysate. Gene ontology analysis of the ZBP1 target mRNAs indicated that these mRNAs were significantly enriched in categories involved in cellular development, assembly and motility, which correspond to the known cellular functions of ZBP1.