Locking in alternate conformations of the integrin αLβ2 I domain with disulfide bonds reveals functional relationships among integrin domains

We used integrin αLβ2 heterodimers containing I domains locked open (active) or closed (inactive) with disulfide bonds to investigate regulatory interactions among domains in integrins. mAbs to the αL I domain and β2 I-like domain inhibit adhesion of wild-type αLβ2 to intercellular adhesion molecule-1. However, with αLβ2 containing a locked open I domain, mAbs to the I domain were subdivided into subsets (i) that did not inhibit, and thus appear to inhibit by favoring the closed conformation, and (ii) that did inhibit, and thus appear to bind to the ligand binding site. Furthermore, αLβ2 containing a locked open I domain was completely resistant to inhibition by mAbs to the β2 I-like domain, but became fully susceptible to inhibition after disulfide reduction with DTT. This finding suggests that the I-like domain indirectly contributes to ligand binding by regulating opening of the I domain in wild-type αLβ2. Conversely, locking the I domain closed partially restrained conformational change of the I-like domain by Mn2+, as measured with mAb m24, which we map here to the β2 I-like domain. By contrast, locking the I domain closed or open did not affect constitutive or Mn2+-induced exposure of the KIM127 epitope in the β2 stalk region. Furthermore, locked open I domains, in αLβ2 complexes or expressed in isolation on the cell surface, bound to intercellular adhesion molecule-1 equivalently in Mg2+ and Mn2+. These results suggest that Mn2+ activates αLβ2 by binding to a site other than the I domain, most likely the I-like domain of β2.

Genomic and genetic dissection of an archaeal regulon

The extremely halophilic archaeon Halobacterium sp. NRC-1 can grow phototrophically by means of light-driven proton pumping by bacteriorhodopsin in the purple membrane. Here, we show by genetic analysis of the wild type, and insertion and double-frame shift mutants of Bat that this transcriptional regulator coordinates synthesis of a structural protein and a chromophore for purple membrane biogenesis in response to both light and oxygen. Analysis of the complete Halobacterium sp. NRC-1 genome sequence showed that the regulatory site, upstream activator sequence (UAS), the putative binding site for Bat upstream of the bacterio-opsin gene (bop), is also present upstream to the other Bat-regulated genes. The transcription regulator Bat contains a photoresponsive cGMP-binding (GAF) domain, and a bacterial AraC type helix–turn–helix DNA binding motif. We also provide evidence for involvement of the PAS/PAC domain of Bat in redox-sensing activity by genetic analysis of a purple membrane overproducer. Five additional Bat-like putative regulatory genes were found, which together are likely to be responsible for orchestrating the complex response of this archaeon to light and oxygen. Similarities of the bop-like UAS and transcription factors in diverse organisms, including a plant and a γ-proteobacterium, suggest an ancient origin for this regulon capable of coordinating light and oxygen responses in the three major branches of the evolutionary tree of life. Finally, sensitivity of four of five regulon genes to DNA supercoiling is demonstrated and correlated to presence of alternating purine–pyrimidine sequences (RY boxes) near the regulated promoters.

Evaluation of the role of heterogeneous nuclear ribonucleoprotein A1 as a host factor in murine coronavirus discontinuous transcription and genome replication

Viruses with RNA genomes often capture and redirect host cell components to assist in mechanisms particular to RNA-dependent RNA synthesis. The nidoviruses are an order of positive-stranded RNA viruses, comprising coronaviruses and arteriviruses, that employ a unique strategy of discontinuous transcription, producing a series of subgenomic mRNAs linking a 5′ leader to distal portions of the genome. For the prototype coronavirus mouse hepatitis virus (MHV), heterogeneous nuclear ribonucleoprotein (hnRNP) A1 has been shown to be able to bind in vitro to the negative strand of the intergenic sequence, a cis-acting element found in the leader RNA and preceding each downstream ORF in the genome. hnRNP A1 thus has been proposed as a host factor in MHV transcription. To test this hypothesis genetically, we initially constructed MHV mutants with a very high-affinity hnRNP A1 binding site inserted in place of, or adjacent to, an intergenic sequence in the MHV genome. This inserted hnRNP A1 binding site was not able to functionally replace, or enhance transcription from, the intergenic sequence. This finding led us to test more directly the role of hnRNP A1 by analysis of MHV replication and RNA synthesis in a murine cell line that does not express this protein. The cellular absence of hnRNP A1 had no detectable effect on the production of infectious virus, the synthesis of genomic RNA, or the quantity or quality of subgenomic mRNAs. These results strongly suggest that hnRNP A1 is not a required host factor for MHV discontinuous transcription or genome replication.

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-Å resolution. The α/β structure is strikingly similar to the globular domain of linker histone H5 and the DNA-binding domain of hepatocyte nuclear factor 3γ (HNF-3γ), 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-3γ and RFX1) and from the winged-helix domains found within the RAP30 subunit of TFIIF and the β 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 method for soluble overexpression of the α7 nicotinic acetylcholine receptor extracellular domain

We describe the construction of a soluble protein carrying the N-terminal extracellular domain (ECD) of the α7 subunit of the nicotinic acetylcholine receptor. The approach was to fuse the α7 ECD at the C and N termini of several monomeric and pentameric soluble carrier proteins and to investigate the soluble expression of the product in Escherichia coli. An initial screening of six carrier proteins resulted in the selection of a fusion protein comprising, from the N to the C terminus, the maltose binding protein, a 17-aa linker containing an enterokinase binding site, and the α7 ECD. This protein is soluble upon expression in bacteria and is purified by affinity chromatography. It binds the competitive nicotinic antagonist α-bungarotoxin with 2.5 μM affinity and displays a CD spectrum corresponding to a folded protein. The method might be suitable to produce large quantities of protein for crystallization and immunochemical experiments.

Competitive regulation of CaT-like-mediated Ca2+ entry by protein kinase C and calmodulin

A finely tuned Ca2+ signaling system is essential for cells to transduce extracellular stimuli, to regulate growth, and to differentiate. We have recently cloned CaT-like (CaT-L), a highly selective Ca2+ channel closely related to the epithelial calcium channels (ECaC) and the calcium transport protein CaT1. CaT-L is expressed in selected exocrine tissues, and its expression also strikingly correlates with the malignancy of prostate cancer. The expression pattern and selective Ca2+ permeation properties suggest an important function in Ca2+ uptake and a role in tumor progression, but not much is known about the regulation of this subfamily of ion channels. We now demonstrate a biochemical and functional mechanism by which cells can control CaT-L activity. CaT-L is regulated by means of a unique calmodulin binding site, which, at the same time, is a target for protein kinase C-dependent phosphorylation. We show that Ca2+-dependent calmodulin binding to CaT-L, which facilitates channel inactivation, can be counteracted by protein kinase C-mediated phosphorylation of the calmodulin binding site.

Crystal structure of the ectodomain of Methuselah, a Drosophila G protein-coupled receptor associated with extended lifespan

The Drosophila mutant methuselah (mth) was identified from a screen for single gene mutations that extended average lifespan. Mth mutants have a 35% increase in average lifespan and increased resistance to several forms of stress, including heat, starvation, and oxidative damage. The protein affected by this mutation is related to G protein-coupled receptors of the secretin receptor family. Mth, like secretin receptor family members, has a large N-terminal ectodomain, which may constitute the ligand binding site. Here we report the 2.3-Å resolution crystal structure of the Mth extracellular region, revealing a folding topology in which three primarily β-structure-containing domains meet to form a shallow interdomain groove containing a solvent-exposed tryptophan that may represent a ligand binding site. The Mth structure is analyzed in relation to predicted Mth homologs and potential ligand binding features.

Crystal structure of cis-prenyl chain elongating enzyme, undecaprenyl diphosphate synthase

Undecaprenyl diphosphate synthase (UPS) catalyzes the cis-prenyl chain elongation onto trans, trans-farnesyl diphosphate (FPP) to produce undecaprenyl diphosphate (UPP), which is indispensable for the biosynthesis of bacterial cell walls. We report here the crystal structure of UPS as the only three-dimensional structure among cis-prenyl chain elongating enzymes. The structure is classified into a protein fold family and is completely different from the so-called “isoprenoid synthase fold” that is believed to be a common structure for the enzymes relating to isoprenoid biosynthesis. Conserved amino acid residues among cis-prenyl chain elongating enzymes are located around a large hydrophobic cleft in the UPS structure. A structural P-loop motif, which frequently appears in the various kinds of phosphate binding site, is found at the entrance of this cleft. The catalytic site is determined on the basis of these structural features, from which a possible reaction mechanism is proposed.

Overexpression of Arabidopsis Phytochrome B Inhibits Phytochrome A Function in the Presence of Sucrose1

Overexpression of phytochrome B (phyB) in Arabidopsis has previously been demonstrated to result in dominant negative interference of phytochrome A (phyA)-mediated hypocotyl growth inhibition in far-red (FR) light. This phenomenon has been examined further in this study and has been found to be dependent on the FR fluence rate and on the availability of metabolizable sugars in the growth medium. Poorly metabolized sugars capable of activating the putative hexokinase sensory function were not effective in eliciting the phytochrome interference response. Overexpressed phyB lacking the chromophore-binding site was also effective at inhibiting the phyA response, especially at higher fluence rates of FR. Overexpressed phyB produces the dominant negative phenotype without any apparent effect on phyA abundance or degradation. It is possible that phyA and phyB interact with a common reaction partner but that either the energy state of the cell or a separate sugar-signaling mechanism modulates the phytochrome-signaling interactions.

Molecular Analysis of (R)-(+)-Mandelonitrile Lyase Microheterogeneity in Black Cherry1

The flavoprotein (R)-(+)-mandelonitrile lyase (MDL; EC 4.1.2.10), which plays a key role in cyanogenesis in rosaceous stone fruits, occurs in black cherry (Prunus serotina Ehrh.) homogenates as several closely related isoforms. Biochemical and molecular biological methods were used to investigate MDL microheterogeneity and function in this species. Three novel MDL cDNAs of high sequence identity (designated MDL2, MDL4, and MDL5) were isolated. Like MDL1 and MDL3 cDNAs (Z. Hu, J.E. Poulton [1997] Plant Physiol 115: 1359–1369), they had open reading frames that predicted a flavin adenine dinucleotide-binding site, multiple N-glycosylation sites, and an N-terminal signal sequence. The N terminus of an MDL isoform purified from seedlings matched the derived amino acid sequence of the MDL4 cDNA. Genomic sequences corresponding to the MDL1, MDL2, and MDL4 cDNAs were obtained by polymerase chain reaction amplification of genomic DNA. Like the previously reported mdl3 gene, these genes are interrupted at identical positions by three short, conserved introns. Given their overall similarity, we conclude that the genes mdl1, mdl2, mdl3, mdl4, and mdl5 are derived from a common ancestral gene and constitute members of a gene family. Genomic Southern-blot analysis showed that this family has approximately eight members. Northern-blot analysis using gene-specific probes revealed differential expression of the genes mdl1, mdl2, mdl3, mdl4, and mdl5.

GTPase Activity and Biochemical Characterization of a Recombinant Cotton Fiber Annexin1

A cDNA encoding annexin was isolated from a cotton (Gossypium hirsutum) fiber cDNA library. The cDNA was expressed in Escherichia coli, and the resultant recombinant protein was purified. We then investigated some biochemical properties of the recombinant annexin based on the current understanding of plant annexins. An “add-back experiment” was performed to study the effect of the recombinant annexin on β-glucan synthase activity, but no effect was found. However, it was found that the recombinant annexin could display ATPase/GTPase activities. The recombinant annexin showed much higher GTPase than ATPase activity. Mg2+ was essential for these activities, whereas a high concentration of Ca2+ was inhibitory. A photolabeling assay showed that this annexin could bind GTP more specifically than ATP. The GTP-binding site on the annexin was mapped into the carboxyl-terminal fourth repeat of annexin from the photolabeling experiment using domain-deletion mutants of this annexin. Northern-blot analysis showed that the annexin gene was highly expressed in the elongation stages of cotton fiber differentiation, suggesting a role of this annexin in cell elongation.

Layilin, a Novel Integral Membrane Protein, Is a Hyaluronan Receptor

The actin cytoskeleton plays a significant role in changes of cell shape and motility, and interactions between the actin filaments and the cell membrane are crucial for a variety of cellular processes. Several adaptor proteins, including talin, maintain the cytoskeleton-membrane linkage by binding to integral membrane proteins and to the cytoskeleton. Layilin, a recently characterized transmembrane protein with homology to C-type lectins, is a membrane-binding site for talin in peripheral ruffles of spreading cells. To facilitate studies of layilin's function, we have generated a layilin-Fc fusion protein comprising the extracellular part of layilin joined to human immunoglobulin G heavy chain and used this chimera to identify layilin ligands. Here, we demonstrate that layilin-Fc fusion protein binds to hyaluronan immobilized to Sepharose. Microtiter plate-binding assays, coprecipitation experiments, and staining of sections predigested with different glycosaminoglycan-degrading enzymes and cell adhesion assays all revealed that layilin binds specifically to hyaluronan but not to other tested glycosaminoglycans. Layilin's ability to bind hyaluronan, a ubiquitous extracellular matrix component, reveals an interesting parallel between layilin and CD44, because both can bind to cytoskeleton-membrane linker proteins through their cytoplasmic domains and to hyaluronan through their extracellular domains. This parallelism suggests a role for layilin in cell adhesion and motility.

The crystal structure of a heptameric archaeal Sm protein: Implications for the eukaryotic snRNP core

Sm proteins form the core of small nuclear ribonucleoprotein particles (snRNPs), making them key components of several mRNA-processing assemblies, including the spliceosome. We report the 1.75-Å crystal structure of SmAP, an Sm-like archaeal protein that forms a heptameric ring perforated by a cationic pore. In addition to providing direct evidence for such an assembly in eukaryotic snRNPs, this structure (i) shows that SmAP homodimers are structurally similar to human Sm heterodimers, (ii) supports a gene duplication model of Sm protein evolution, and (iii) offers a model of SmAP bound to single-stranded RNA (ssRNA) that explains Sm binding-site specificity. The pronounced electrostatic asymmetry of the SmAP surface imparts directionality to putative SmAP–RNA interactions.

Arg-302 facilitates deprotonation of Glu-325 in the transport mechanism of the lactose permease from Escherichia coli

A mechanistic model for lactose/H+ symport via the lactose permease of Escherichia coli proposed recently indicates that the permease must be protonated to bind ligand with high affinity. Moreover, in the ground state, the symported H+ is shared between His-322 (helix X) and Glu-269 (helix VIII), whereas Glu-325 (helix X) is charge-paired with Arg-302 (helix IX). Substrate binding at the outer surface induces a conformational change that leads to transfer of the H+ to Glu-325 and reorientation of the binding site to the inner surface. After release of the substrate, Glu-325 is deprotonated on the inside because of rejuxtapositioning with Arg-302. To test the role of Arg-302 in the mechanism, the catalytic properties of mutants Arg-302→Ala and Arg-302→Ser were studied. Both mutants are severely defective in active lactose transport, as well as in efflux or influx down a concentration gradient, translocation modes that involve net H+ movement. In marked contrast, the mutants catalyze equilibrium exchange of lactose and bind ligand with high affinity. These characteristics are remarkably analogous to those of permease mutants with neutral replacements for Glu-325, a residue that plays a direct role in H+ translocation. These observations lend strong support for the argument that Arg-302 interacts with Glu-325 to facilitate deprotonation of the carboxylic acid during turnover.

An isoleucine/leucine residue in the carboxyltransferase domain of acetyl-CoA carboxylase is critical for interaction with aryloxyphenoxypropionate and cyclohexanedione inhibitors

cDNA fragments encoding the carboxyltransferase domain of the multidomain plastid acetyl-CoA carboxylase (ACCase) from herbicide-resistant maize and from herbicide-sensitive and herbicide-resistant Lolium rigidum were cloned and sequenced. A Leu residue was found in ACCases from herbicide-resistant plants at a position occupied by Ile in all ACCases from sensitive grasses studied so far. Leu is present at the equivalent position in herbicide-resistant ACCases from other eukaryotes. Chimeric ACCases containing a 1000-aa fragment of two ACCase isozymes found in a herbicide-resistant maize were expressed in a yeast ACC1 null mutant to test herbicide sensitivity of the enzyme in vivo and in vitro. One of the enzymes was resistant/tolerant, and one was sensitive to haloxyfop and sethoxydim, rendering the gene-replacement yeast strains resistant and sensitive to these compounds, respectively. The sensitive enzyme has an Ile residue, and the resistant one has a Leu residue at the putative herbicide-binding site. Additionally, a single Ile to Leu replacement at an equivalent position changes the wheat plastid ACCase from sensitive to resistant. The effect of the opposite substitution, Leu to Ile, makes Toxoplasma gondii apicoplast ACCase resistant to haloxyfop and clodinafop. In this case, inhibition of the carboxyltransferase activity of ACCase (second half-reaction) of a large fragment of the Toxoplasma enzyme expressed in Escherichia coli was tested. The critical amino acid residue is located close to a highly conserved motif of the carboxyltransferase domain, which is probably a part of the enzyme active site, providing the basis for the activity of fop and dim herbicides.