Periplasmic domains of Pseudomonas aeruginosa PilN and PilO form a stable heterodimeric complex.

Type IV pili (T4P) are bacterial virulence factors responsible for attachment to surfaces and for twitching motility, a motion that involves a succession of pilus extension and retraction cycles. In the opportunistic pathogen Pseudomonas aeruginosa, the PilM/N/O/P proteins are essential for T4P biogenesis, and genetic and biochemical analyses strongly suggest that they form an inner-membrane complex. Here, we show through co-expression and biochemical analysis that the periplasmic domains of PilN and PilO interact to form a heterodimer. The structure of residues 69-201 of the periplasmic domain of PilO was determined to 2.2 A resolution and reveals the presence of a homodimer in the asymmetric unit. Each monomer consists of two N-terminal coiled coils and a C-terminal ferredoxin-like domain. This structure was used to generate homology models of PilN and the PilN/O heterodimer. Our structural analysis suggests that in vivo PilN/O heterodimerization would require changes in the orientation of the first N-terminal coiled coil, which leads to two alternative models for the role of the transmembrane domains in the PilN/O interaction. Analysis of PilN/O orthologues in the type II secretion system EpsL/M revealed significant similarities in their secondary structures and the tertiary structures of PilO and EpsM, although the way these proteins interact to form inner-membrane complexes appears to be different in T4P and type II secretion. Our analysis suggests that PilN interacts directly, via its N-terminal tail, with the cytoplasmic protein PilM. This work shows a direct interaction between the periplasmic domains of PilN and PilO, with PilO playing a key role in the proper folding of PilN. Our results suggest that PilN/O heterodimers form the foundation of the inner-membrane PilM/N/O/P complex, which is critical for the assembly of a functional T4P complex.

Structural aspects of interactions within the Myc/Max/Mad network.

Recently determined structures of a number of Myc family proteins have provided significant insights into the molecular nature of complex assembly and DNA binding. These structures illuminate the details of specific interactions that govern the assembly of nucleoprotein complexes and, in doing so, raise more questions regarding Myc biology. In this review, we focus on the lessons provided by these structures toward understanding (1) interactions that govern transcriptional repression by Mad via the Sin3 pathway, (2) homodimerization of Max, (3) heterodimerization of Myc-Max and Mad-Max, and (4) DNA recognition by each of the Max-Max, Myc-Max, and Mad-Max dimers.

Deposit3D: a tool for automating structure depositions to the Protein Data Bank.

Almost all successful protein structure-determination projects in the public sector culminate in a structure deposition to the Protein Data Bank (PDB). In order to expedite the deposition process, Deposit3D has been developed. This command-line script calculates or gathers all the required structure-deposition information and outputs this data into a mmCIF file for subsequent upload through the RCSB PDB ADIT interface. Deposit3D might be particularly useful for structural genomics pipeline projects because it allows workers involved with various stages of a structure-determination project to pool their different categories of annotation information before starting a deposition session.

Engaging the ribosome: universal IFs of translation.

Eukaryotic initiation factor 1A (eIF1A) and the GTPase IF2/eIF5B are the only universally conserved translation initiation factors. Recent structural, biochemical and genetic data indicate that these two factors form an evolutionarily conserved structural and functional unit in translation initiation. Based on insights gathered from studies of the translation elongation factor GTPases, we propose that these factors occupy the aminoacyl-tRNA site (A site) on the ribosome, and promote initiator tRNA binding and ribosomal subunit joining. These processes yield a translationally competent ribosome with Met-tRNA in the ribosomal peptidyl-tRNA site (P site), base-paired to the AUG start codon of a mRNA.

Structural genomics of enzymes involved in sterol/isoprenoid biosynthesis.

X-ray structures of two enzymes in the sterol/isoprenoid biosynthesis pathway have been determined in a structural genomics pilot study. Mevalonate-5-diphosphate decarboxylase (MDD) is a single-domain alpha/beta protein that catalyzes the last of three sequential ATP-dependent reactions which convert mevalonate to isopentenyl diphosphate. Isopentenyl disphosphate isomerase (IDI) is an alpha/beta metalloenzyme that catalyzes interconversion of isopentenyl diphosphate and dimethylallyl diphosphate, which condense in the next step toward synthesis of sterols and a host of natural products. Homology modeling of related proteins and comparisons of the MDD and IDI structures with two other experimentally determined structures have shown that MDD is a member of the GHMP superfamily of small-molecule kinases and IDI is similar to the nudix hydrolases, which act on nucleotide diphosphatecontaining substrates. Structural models were produced for 379 proteins, encompassing a substantial fraction of both protein superfamilies. All three enzymes responsible for synthesis of isopentenyl diphosphate from mevalonate (mevalonate kinase, phosphomevalonate kinase, and MDD) share the same fold, catalyze phosphorylation of chemically similar substrates (MDD decarboxylation involves phosphorylation of mevalonate diphosphate), and seem to have evolved from a common ancestor. These structures and the structural models derived from them provide a framework for interpreting biochemical function and evolutionary relationships.

Hierarchical phosphorylation of the translation inhibitor 4E-BP1.

In most instances, translation is regulated at the initiation phase, when a ribosome is recruited to the 5' end of an mRNA. The eIF4E-binding proteins (4E-BPs) interdict translation initiation by binding to the translation factor eIF4E, and preventing recruitment of the translation machinery to mRNA. The 4E-BPs inhibit translation in a reversible manner. Hypophosphorylated 4E-BPs interact avidly with eIF4E, whereas 4E-BP hyperphosphorylation, elicited by stimulation of cells with hormones, cytokines, or growth factors, results in an abrogation of eIF4E-binding activity. We reported previously that phosphorylation of 4E-BP1 on Thr 37 and Thr 46 is relatively insensitive to serum deprivation and rapamycin treatment, and that phosphorylation of these residues is required for the subsequent phosphorylation of a set of unidentified serum-responsive sites. Here, using mass spectrometry, we identify the serum-responsive, rapamycin-sensitive sites as Ser 65 and Thr 70. Utilizing a novel combination of two-dimensional isoelectric focusing/SDS-PAGE and Western blotting with phosphospecific antibodies, we also establish the order of 4E-BP1 phosphorylation in vivo; phosphorylation of Thr 37/Thr 46 is followed by Thr 70 phosphorylation, and Ser 65 is phosphorylated last. Finally, we show that phosphorylation of Ser 65 and Thr 70 alone is insufficient to block binding to eIF4E, indicating that a combination of phosphorylation events is necessary to dissociate 4E-BP1 from eIF4E.

A novel peptide recognition mode revealed by the X-ray structure of a core U2AF35/U2AF65 heterodimer.

U2 auxiliary factor (U2AF) is an essential splicing factor that recognizes the 3' splice site and recruits the U2 snRNP to the branch point. The X-ray structure of the human core U2AF heterodimer, consisting of the U2AF35 central domain and a proline-rich region of U2AF65, has been determined at 2.2 A resolution. The structure reveals a novel protein-protein recognition strategy, in which an atypical RNA recognition motif (RRM) of U2AF35 and the U2AF65 polyproline segment interact via reciprocal "tongue-in-groove" tryptophan residues. Complementary biochemical experiments demonstrate that the core U2AF heterodimer binds RNA, and that the interacting tryptophan side chains are essential for U2AF dimerization. Atypical RRMs in other splicing factors may serve as protein-protein interaction motifs elsewhere during spliceosome assembly.

Crystal structure of negative cofactor 2 recognizing the TBP-DNA transcription complex.

The X-ray structure of a ternary complex of Negative Cofactor 2 (NC2), the TATA box binding protein (TBP), and DNA has been determined at 2.6 A resolution. The N termini of NC2 alpha and beta resemble histones H2A and H2B, respectively, and form a heterodimer that binds to the bent DNA double helix on the underside of the preformed TBP-DNA complex via electrostatic interactions. NC2beta contributes to inhibition of TATA-dependent transcription through interactions of its C-terminal alpha helix with a conserved hydrophobic feature on the upper surface of TBP, which in turn positions the penultimate alpha helix of NC2beta to block recognition of the TBP-DNA complex by transcription factor IIB. Further regulatory implications of the NC2 heterodimer structure are discussed.

X-ray structure of the human hyperplastic discs protein: an ortholog of the C-terminal domain of poly(A)-binding protein.

The poly(A)-binding protein (PABP) recognizes the 3' mRNA poly(A) tail and plays an essential role in eukaryotic translation initiation and mRNA stabilization/degradation. PABP is a modular protein, with four N-terminal RNA-binding domains and an extensive C terminus. The C-terminal region of PABP is essential for normal growth in yeast and has been implicated in mediating PABP homo-oligomerization and protein-protein interactions. A small, proteolytically stable, highly conserved domain has been identified within this C-terminal segment. Remarkably, this domain is also present in the hyperplastic discs protein (HYD) family of ubiquitin ligases. To better understand the function of this conserved region, an x-ray structure of the PABP-like segment of the human HYD protein has been determined at 1.04-A resolution. The conserved domain adopts a novel fold resembling a right-handed supercoil of four alpha-helices. Sequence profile searches and comparative protein structure modeling identified a small ORF from the Arabidopsis thaliana genome that encodes a structurally similar but distantly related PABP/HYD domain. Phylogenetic analysis of the experimentally determined (HYD) and homology modeled (PABP) protein surfaces revealed a conserved feature that may be responsible for binding to a PABP interacting protein, Paip1, and other shared interaction partners.

Thermodynamic analysis of the interaction between YY1 and the AAV P5 promoter initiator element.

BACKGROUND: We previously determined the co-crystal structure of the zinc finger region of transcription factor YY1 (YY1Delta) bound to the initiator element (Inr) of the adenoassociated virus (AAV) P5 gene promoter [Houbaviy, H.B. et al. (1996) Proc. Natl. Acad. Sci. USA 93, 13577-13582]. Our structure explained both binding specificity and the ability of YY1 to support specific, unidirectional transcription initiation. RESULTS: To further understand Inr recognition by YY1, we analyzed the YY1Delta-Inr interaction by isothermal titration calorimetry (ITC) and used limited proteolysis, DNase I footprinting and missing nucleoside experiments to show that YY1Delta and full-length YY1 (YY1WT) have indistinguishable DNA binding properties. CONCLUSIONS: YY1 binding occurs at an equilibrium dissociation constant (K(d)) of about 1 microM, and exhibits a large negative heat capacity change (DeltaC(p)). We analyzed the thermodynamic behavior of YY1Delta in terms of buried solvent-accessible surface area resulting from interaction of two rigid bodies, which could not explain our measured value of DeltaC(p). We must, therefore, postulate conformational changes in YY1 and/or the Inr or question the validity of current DeltaC(p) analysis methods for protein-DNA interactions.

Crystal structure of the C-terminal domain of the RAP74 subunit of human transcription factor IIF.

The x-ray structure of a C-terminal fragment of the RAP74 subunit of human transcription factor (TF) IIF has been determined at 1.02-A resolution. The alpha/beta structure is strikingly similar to the globular domain of linker histone H5 and the DNA-binding domain of hepatocyte nuclear factor 3gamma (HNF-3gamma), making it a winged-helix protein. The surface electrostatic properties of this compact domain differ significantly from those of bona fide winged-helix transcription factors (HNF-3gamma and RFX1) and from the winged-helix domains found within the RAP30 subunit of TFIIF and the beta subunit of TFIIE. RAP74 has been shown to interact with the TFIIF-associated C-terminal domain phosphatase FCP1, and a putative phosphatase binding site has been identified within the RAP74 winged-helix domain.

A conserved HEAT domain within eIF4G directs assembly of the translation initiation machinery.

The X-ray structure of the phylogenetically conserved middle portion of human eukaryotic initiation factor (eIF) 4GII has been determined at 2.4 A resolution, revealing a crescent-shaped domain consisting of ten alpha helices arranged as five HEAT repeats. Together with the ATP-dependent RNA helicase eIF4A, this HEAT domain suffices for 48S ribosomal complex formation with a picornaviral RNA internal ribosome entry site (IRES). Structure-based site-directed mutagenesis was used to identify two adjacent features on the surface of this essential component of the translation initiation machinery that, respectively, bind eIF4A and a picornaviral IRES. The structural and biochemical results provide mechanistic insights into both cap-dependent and cap-independent translation initiation.

Translational repression by a novel partner of human poly(A) binding protein, Paip2.

The eukaryotic mRNA 3' poly(A) tail acts synergistically with the 5' cap structure to enhance translation. This effect is mediated by a bridging complex, composed of the poly(A) binding protein (PABP), eIF4G, and the cap binding protein, eIF4E. PABP-interacting protein 1 (Paip1) is another factor that interacts with PABP to coactivate translation. Here, we describe a novel human PABP-interacting protein (Paip2), which acts as a repressor of translation both in vitro and in vivo. Paip2 preferentially inhibits translation of a poly(A)-containing mRNA, but has no effect on the translation of hepatitis C virus mRNA, which is cap- and eIF4G-independent. Paip2 decreases the affinity of PABP for polyadenylate RNA, and disrupts the repeating structure of poly(A) ribonucleoprotein. Furthermore, Paip2 competes with Paip1 for PABP binding. Thus, Paip2 inhibits translation by interdicting PABP function.

Crystal structures of ribosome anti-association factor IF6.

Ribosome anti-association factor eIF6 (originally named according to translation initiation terminology as eukaryotic initiation factor 6) binds to the large ribosomal subunit, thereby preventing inappropriate interactions with the small subunit during initiation of protein synthesis. We have determined the X-ray structures of two IF6 homologs, Methanococcus jannaschii archaeal aIF6 and Sacchromyces cerevisiae eIF6, revealing a phylogenetically conserved 25 kDa protein consisting of five quasi identical alpha/beta subdomains arrayed about a five-fold axis of pseudosymmetry. Yeast eIF6 prevents ribosomal subunit association. Comparative protein structure modeling with other known archaeal and eukaryotic homologs demonstrated the presence of two conserved surface regions, one or both of which may bind the large ribosomal subunit.

X-Ray structures of the universal translation initiation factor IF2/eIF5B: conformational changes on GDP and GTP binding.

X-ray structures of the universal translation initiation factor IF2/eIF5B have been determined in three states: free enzyme, inactive IF2/eIF5B.GDP, and active IF2/eIF5B.GTP. The "chalice-shaped" enzyme is a GTPase that facilitates ribosomal subunit joining and Met-tRNA(i) binding to ribosomes in all three kingdoms of life. The conserved core of IF2/eIF5B consists of an N-terminal G domain (I) plus an EF-Tu-type beta barrel (II), followed by a novel alpha/beta/alpha-sandwich (III) connected via an alpha helix to a second EF-Tu-type beta barrel (IV). Structural comparisons reveal a molecular lever, which amplifies a modest conformational change in the Switch 2 region of the G domain induced by Mg(2+)/GTP binding over a distance of 90 A from the G domain active center to domain IV. Mechanisms of GTPase function and ribosome binding are discussed.

An overview of structural genomics.

With access to sequences of entire human genomes plus those of various model organisms and many important microbial pathogens, structural biology is on the verge of a dramatic transformation. Our newfound wealth of sequence information will serve as the foundation for an important initiative in structural genomics. We are poised to embark on a systematic program of high-throughput X-ray crystallography and NMR spectroscopy aimed at developing a comprehensive view of the protein structure universe. Structural genomics will yield a large number of experimental protein structures (tens of thousands) and an even larger number of calculated comparative protein structure models (millions). This enormous body of structural data will be freely available, and promises to accelerate scientific discovery in all areas of biological science, including biodiversity and evolution in natural ecosystems, agricultural plant genetics, breeding of farm and domestic animals, and human health and disease.

Physical and functional interaction between the eukaryotic orthologs of prokaryotic translation initiation factors IF1 and IF2.

To initiate protein synthesis, a ribosome with bound initiator methionyl-tRNA must be assembled at the start codon of an mRNA. This process requires the coordinated activities of three translation initiation factors (IF) in prokaryotes and at least 12 translation initiation factors in eukaryotes (eIF). The factors eIF1A and eIF5B from eukaryotes show extensive amino acid sequence similarity to the factors IF1 and IF2 from prokaryotes. By a combination of two-hybrid, coimmunoprecipitation, and in vitro binding assays eIF1A and eIF5B were found to interact directly, and the eIF1A binding site was mapped to the C-terminal region of eIF5B. This portion of eIF5B was found to be critical for growth in vivo and for translation in vitro. Overexpression of eIF1A exacerbated the slow-growth phenotype of yeast strains expressing C-terminally truncated eIF5B. These findings indicate that the physical interaction between the evolutionarily conserved factors eIF1A and eIF5B plays an important role in translation initiation, perhaps to direct or stabilize the binding of methionyl-tRNA to the ribosomal P site.

The tetranucleotide UCAY directs the specific recognition of RNA by the Nova K-homology 3 domain.

The Nova family of proteins are target antigens in the autoimmune disorder paraneoplastic opsoclonus-myoclonus ataxia and contain K-homology (KH)-type RNA binding domains. The Nova-1 protein has recently been shown to regulate alternative splicing of the alpha2 glycine receptor subunit pre-mRNA by binding to an intronic element containing repeats of the tetranucleotide UCAU. Here, we have used selection-amplification to demonstrate that the KH3 domain of Nova recognizes a single UCAY element in the context of a 20-base hairpin RNA; the UCAY tetranucleotide is optimally presented as a loop element of the hairpin scaffold and requires protein residues C-terminal to the previously defined KH domain. These results suggest that KH domains in general recognize tetranucleotide motifs and that biological RNA targets of KH domains may use either RNA secondary structure or repeated sequence elements to achieve high affinity and specificity of protein binding.