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PubMed2004

Mechanism of ammonia transport by Amt/MEP/Rh: structure of AmtB at 1.35 A.

The first structure of an ammonia channel from the Amt/MEP/Rh protein superfamily, determined to 1.35 angstrom resolution, shows it to be a channel that spans the membrane 11 times. Two structurally similar halves span the membrane with opposite polarity. Structures with and without ammonia or methyl ammonia show a vestibule that recruits NH4+/NH3, a binding site for NH4+, and a 20 angstrom-long hydrophobic channel that lowers the NH4+ pKa to below 6 and conducts NH3. Favorable interactions for NH3 are seen within the channel and use conserved histidines. Reconstitution of AmtB into vesicles shows that AmtB conducts uncharged NH3.

Amino Acid Sequence↗
PubMed2001

Structure of TrwB, a gatekeeper in bacterial conjugation.

Bacterial conjugation implies a trans-membrane passage of DNA, mediated by proteins encoded in conjugative plasmids. This results in a spread of genetic information, including antibiotic resistance acquisition by pathogens. Special cases of conjugation are trans-kingdom gene transfer from bacteria to plants or fungi, and even bacterial sporulation and cell division. One of the main actors in this process is an integral inner membrane DNA-binding protein, called TrwB in the E. coli R388 conjugative system. It is responsible for coupling the single-strand DNA to be transferred from the donor to the acceptor cell in its complex with other proteins, with a type IV secretion system making up the mating apparatus. The TrwB protomer consists of two domains: a nucleotide-binding domain of alpha/beta topology, similar to RecA and DNA ring helicases, and an all-alpha domain. The quaternary structure reveals an almost spherical homohexamer, strikingly similar to F(1)-ATPase. A central 20 A wide channel traverses the hexamer, thus connecting cytoplasm with periplasm.

Bacterial Proteins↗
PubMed2005

Glycoprotein tertiary and quaternary structures are monitored by the same quality control mechanism.

Folding of glycoproteins entering the secretory pathway is strictly surveyed in the endoplasmic reticulum by a quality control system. Folding intermediates and proteins irreparably misfolded are marked via glucosylation by the UDPglucose:glycoprotein glucosyltransferase, an enzyme that acts as a folding sensor by exclusively labeling glycoproteins not displaying their native structures. Here we show that this sensing mechanism also applies to the oligomerization of protein complexes, as the glucosyltransferase appeared to be able to glucosylate folded complex subunits lacking the full complement of oligomer components.

Glycoproteins↗
PubMed2006

Identification of novel quaternary domain interactions in the Hsp90 chaperone, GRP94.

The structural basis for the coupling of ATP binding and hydrolysis to chaperone activity remains a central question in Hsp90 biology. By analogy to MutL, ATP binding to Hsp90 is thought to promote intramolecular N-terminal dimerization, yielding a molecular clamp functioning in substrate protein activation. Though observed in studies with recombinant domains, whether such quaternary states are present in native Hsp90s is unknown. In this study, native subunit interactions in GRP94, the endoplasmic reticulum Hsp90, were analyzed using chemical cross-linking in conjunction with tandem mass spectrometry. We report the identification of two distinct intermolecular interaction sites. Consistent with previous studies, one site comprises the C-terminal dimerization domain. The remaining site represents a novel intermolecular contact between the N-terminal and middle (M) domains of opposing subunits. This N+M domain interaction was present in the nucleotide-empty, ADP-, ATP-, or geldanamycin-bound states and could be selectively disrupted upon addition of synthetic geldanamycin dimers. These results identify a compact, intertwined quaternary conformation of native GRP94 and suggest that intersubunit N+M interactions are integral to the structural biology of Hsp90.

Adenosine Diphosphate↗
PubMed2005

Intersubunit and domain interactions of the meprin B metalloproteinase. Disulfide bonds and protein-protein interactions in the MAM and TRAF domains.

Meprins, multimeric metalloproteases expressed in kidney and intestinal epithelial cells as well as in certain leukocytes and cancer cells, have the ability to hydrolyze a variety of growth factors, vasoactive peptides, cytokines, and extracellular matrix proteins. The meprin B isoform exists primarily as a cell-surface homooligomer composed of disulfide-linked, multidomain beta-subunits. To gain insight into how the tertiary and quaternary structure of meprin B affects function, the disulfide-bonding pattern and sites of domain-domain interactions were investigated using sedimentation equilibrium ultracentrifugation, cross-linking, and mass spectrometry techniques. Three symmetrical intersubunit disulfide bonds were identified in the noncatalytic interaction domains; two in the MAM (meprin, A-5 protein, protein-tyrosine phosphatase mu) domain and one in the TRAF (tumor necrosis factor receptor-associated factor) domain. These disulfide bridges are unique for the known homophilic interactions of these domains. Mutation of any of the intersubunit cysteine residues resulted in the inability of meprin B to form disulfide-linked dimers. The four cysteines of the protease domain formed intradomain disulfide bonds. The MAM domain also had one intradomain disulfide bond and one free cysteine. Cross-linking studies of the meprin B dimer with the amine-reactive cross-linker disuccinimidyl suberate revealed inter- and intradomain contacts within the protein, including prosequence-prosequence, protease-TRAF, protease-epidermal growth factor, and TRAF-TRAF interactions. From these observations, a model of the meprin B dimer structure is proposed that provides insight into the relationship between structure and function of this isoform.

Animals↗
PubMed2006

Limulus polyphemus hemocyanin: 10 A cryo-EM structure, sequence analysis, molecular modelling and rigid-body fitting reveal the interfaces between the eight hexamers.

The blue copper protein hemocyanin from the horseshoe crab Limulus polyphemus is among the largest respiratory proteins found in nature (3.5 MDa) and exhibits a highly cooperative oxygen binding. Its 48 subunits are arranged as eight hexamers (1x6mers) that form the native 8x6mer in a nested hierarchy of 2x6mers and 4x6mers. This quaternary structure is established by eight subunit types (termed I, IIA, II, IIIA, IIIB, IV, V, and VI), of which only type II has been sequenced. Crystal structures of the 1x6mer are available, but for the 8x6mer only a 40 A 3D reconstruction exists. Consequently, the structural parameters of the 8x6mer are not firmly established, and the molecular interfaces between the eight hexamers are still to be defined. This, however, is crucial for understanding how allosteric transitions are mediated between the different levels of hierarchy. Here, we show the 10 A structure (FSC(1/2-bit) criterion) of the oxygenated 8x6mer from cryo-electron microscopy (cryo-EM) and single-particle analysis. Moreover, we show its molecular model as obtained by DNA sequencing of subunits II, IIIA, IV and VI, and molecular modelling and rigid-body fitting of all subunit types. Remarkably, the latter enabled us to improve the resolution of the cryo-EM structure from 11 A to the final 10 A. The 10 A structure allows firm assessment of various structural parameters of the 8x6mer, the 4x6mer and the 2x6mer, and reveals a total of 46 inter-hexamer bridges. These group as 11 types of interface: four at the 2x6mer level (II-II, II-IV, V-VI, IV-VI), three form the 4x6mer (V-V, V-VI, VI-IIIB/IV/V), and four are required to assemble the 8x6mer (IIIA-IIIA, IIIA-IIIB, II-IV, IV-IV). The molecular model shows the amino acid residues involved, and reveals that several of the interfaces are intriguingly histidine-rich and likely to transfer allosteric signals between the different levels of the nested hierarchy.

Amino Acid Sequence↗
PubMed2005

Crystallization and preliminary X-ray diffraction analysis of myotoxin I, a Lys49-phospholipase A2 from Bothrops moojeni.

A new myotoxic Lys49-phospholipase A2 isolated from Bothrops moojeni snake venom has been crystallized. The crystals diffracted to 2.18 A resolution using a synchrotron-radiation source and belong to space group C2. The unit-cell parameters are a = 56.8, b = 125.0, c = 64.7 A, beta = 105.5 degrees. Preliminary analysis indicates the presence of four molecules in the asymmetric unit. This may suggest a new quaternary structure for this Lys49-phospholipase A2 in contrast to the dimeric and monomeric structures solved so far for this class of proteins.

Animals↗
PubMed2004

Characterization of polynucleotide kinase/phosphatase enzymes from Mycobacteriophages omega and Cjw1 and vibriophage KVP40.

Coliphage T4 Pnkp is a bifunctional polynucleotide 5'-kinase/3'-phosphatase that catalyzes the end-healing steps of a RNA repair pathway. Here we show that mycobacteriophages Omega and Cjw1 and vibriophage KVP40 also encode bifunctional Pnkp enzymes consisting of a proximal 5'-kinase module with an essential P-loop motif, GXGK(S/T), and a distal 3'-phosphatase module with an essential acyl-phosphatase motif, DX- DGT. Biochemical characterization of the viral Pnkp proteins reveals several shared features, including an alkaline pH optimum for the kinase component, an intrinsic RNA kinase activity, and a homotetrameric or homodimeric quaternary structure, that distinguish them from the monomeric DNA-specific phosphatase/kinase enzymes found in mammals and fission yeast. Whereas the phage 5'-kinases differ from each other in their preferences for phosphorylation of 5' overhangs, blunt ends, or recessed ends, none of them displays the preference for recessed ends reported for mammalian DNA kinase. We hypothesize that Pnkp provides phages that have it with a means to evade an RNA-damaging antiviral host response. Genetic complementation of the essential end-healing steps of yeast tRNA splicing by the Omega and Cjw1 Pnkp enzymes establishes their capacity to perform RNA repair reactions in vivo. A supportive correlation is that Omega and Cjw1, which are distinguished from other mycobacteriophages by their possession of a Pnkp enzyme, are also unique among the mycobacteriophages in their specification of putative RNA ligases.

Adenosine Triphosphate↗
PubMed2000

Chromophore-apoprotein interactions in Synechocystis sp. PCC6803 phytochrome Cph1.

The secondary, tertiary, and quaternary structures of the Synechocystis Cph1 phytochrome were investigated by absorption and circular dichroism spectroscopy, size exclusion chromatography, and limited proteolysis. The Cph1 protein was coexpressed with a bacterial thioredoxin in Escherichia coli, reconstituted in vitro with tetrapyrrole chromophores, and purified by chitin affinity chromatography. The resultant Cph1 holoproteins were essentially pure and had the specific absorbance ratio (SAR) of 0.8-0.9. Circular dichroism spectroscopy and limited proteolysis showed that the chromophore binding induced marked conformational changes in the Cph1 protein. The alpha-helical content increased to 42-44% in the holoproteins from 37% in the apoprotein. However, no significant difference in the secondary structure was detected between the Pr and Pfr forms. The tertiary structure of the Cph1 apoprotein appeared to be relatively flexible but became more compact and resistant to tryptic digestion upon chromophore binding. Interestingly, a small chromopeptide of about 30 kDa was still predominant even after longer tryptic digestion. The N-terminal location of this chromopeptide was confirmed by expression in E. coli and in vitro reconstitution with chromophores of the 32.5 kDa N-terminal fragment of the Cph1 protein. This chromopeptide was fully photoreversible with the spectral characteristic similar to that of the full-size Cph1 protein. The Cph1 protein forms dimers through the C-terminal region. These results suggest that the prokaryotic Cph1 phytochrome shares the structural and conformational characteristics of plant phytochromes, such as the two-domain structure consisting of the relatively compact N-terminal and the relatively flexible C-terminal regions, in addition to the chromophore-induced conformational changes.

Apoproteins↗
PubMed2003

GrpE, a nucleotide exchange factor for DnaK.

The cochaperone GrpE functions as a nucleotide exchange factor to promote dissociation of adenosine 5'-diphosphate (ADP) from the nucleotide-binding cleft of DnaK. GrpE and the DnaJ cochaperone act in concert to control the flux of unfolded polypeptides into and out of the substrate-binding domain of DnaK by regulating the nucleotide-bound state of DnaK. DnaJ stimulates nucleotide hydrolysis, and GrpE promotes the exchange of ADP for adenosine triphosphate (ATP) and also augments peptide release from the DnaK substrate-binding domain in an ATP-independent manner. The eukaryotic cytosol does not contain GrpE per se because GrpE-like function is provided by the BAG1 protein, which acts as a nucleotide exchange factor for cytosolic Hsp70s. GrpE, which plays a prominent role in mitochondria, chloroplasts, and bacterial cytoplasms, is a fascinating molecule with an unusual quaternary structure. The long alpha-helices of GrpE have been hypothesized to act as a thermosensor and to be involved in the decrease in GrpE-dependent nucleotide exchange that is observed in vitro at temperatures relevant to heat shock. This review describes the molecular biology of GrpE and focuses on the structural and kinetic aspects of nucleotide exchange, peptide release, and the thermosensor hypothesis.

Animals↗
PubMed2004

Targeting, import, and dimerization of a mammalian mitochondrial ATP binding cassette (ABC) transporter, ABCB10 (ABC-me).

ATP binding cassette (ABC) transporters are a diverse superfamily of energy-dependent membrane translocases. Although responsible for the majority of transmembrane transport in bacteria, they are relatively uncommon in eukaryotic mitochondria. Organellar trafficking and import, in addition to quaternary structure assembly, of mitochondrial ABC transporters is poorly understood and may offer explanations for the paucity of their diversity. Here we examine these processes in ABCB10 (ABC-me), a mitochondrial inner membrane erythroid transporter involved in heme biosynthesis. We report that ABCB10 possesses an unusually long 105-amino acid mitochondrial targeting presequence (mTP). The central subdomain of the mTP (amino acids (aa) 36-70) is sufficient for mitochondrial import of enhanced green fluorescent protein. The N-terminal subdomain (aa 1-35) of the mTP, although not necessary for the trafficking of ABCB10 to mitochondria, participates in the proper import of the molecule into the inner membrane. We performed a series of amino acid mutations aimed at changing specific properties of the mTP. The mTP requires neither arginine residues nor predictable alpha-helices for efficient mitochondrial targeting. Disruption of its hydrophobic character by the mutation L46Q/I47Q, however, greatly diminishes its efficacy. This mutation can be rescued by cryptic downstream (aa 106-715) mitochondrial targeting signals, highlighting the redundancy of this protein's targeting qualities. Mass spectrometry analysis of chemically cross-linked, immunoprecipitated ABCB10 indicates that ABCB10 embedded in the mitochondrial inner membrane homodimerizes and homo-oligomerizes. A deletion mutant of ABCB10 that lacks its mTP efficiently targets to the endoplasmic reticulum. Quaternary structure assembly of ABCB10 in the ER appears to be similar to that in the mitochondria.

ATP-Binding Cassette Transporters↗
PubMed2003

Active-site copper and zinc ions modulate the quaternary structure of prokaryotic Cu,Zn superoxide dismutase.

The influence of the constitutive metal ions on the equilibrium properties of dimeric Photobacterium leiognathi Cu,Zn superoxide dismutase has been studied for the wild-type and for two mutant protein forms bearing a negative charge in the amino acid clusters at the dimer association interface. Depletion of copper and zinc dissociates the two mutant proteins into monomers, which reassemble toward the dimeric state upon addition of stoichiometric amounts of zinc. Pressure-dependent dissociation is observed for the copper-depleted wild-type and mutated enzymes, as monitored by the fluorescence shift of a unique tryptophan residue located at the subunit association interface. The spectral shift occurs slowly, reaching a plateau after 15-20 minutes, and is fully reversible. The recovery of the original fluorescence properties, after decompression, is fast (less than four minutes), suggesting that the isolated subunit has a relatively stable structure, and excluding the presence of stable intermediates during the dimer-monomer transition. The dimer dissociation process is still incomplete at 6.5 kbar for the copper-depleted wild-type and mutated enzymes, at variance with what is generally observed for oligomeric proteins that dissociate below 3 kbar. Measurement of the degree of dissociation, at two different protein concentrations, allows us to calculate the standard volume variation upon association, Delta V, and the dissociation constant K(d0), at atmospheric pressure, (25 ml/mol and 3 x 10(-7)M, respectively). The holoprotein is fully dimeric even at 6.5 kbar, which allows us to evaluate a lower Delta G degrees limit of 11.5 kcal/mol, corresponding to a dissociation constant K(d0)<10(-9)M.

Binding Sites↗
PubMed2005

Structure of unliganded GRP94, the endoplasmic reticulum Hsp90. Basis for nucleotide-induced conformational change.

GRP94, the endoplasmic reticulum paralog of Hsp90, is regulated by adenosine nucleotides that bind to its N-terminal regulatory domain. Because of its weak affinity for nucleotides, the functionally relevant transition in GRP94 is likely to be between the unliganded and nucleotide-bound states. We have determined the structure of the unliganded GRP94 N-domain. The helix 1-4-5 subdomain of the unliganded protein adopts the closed conformation seen in the structure of the protein in complex with inhibitors. This conformation is distinct from the open conformation of the subdomain seen when the protein is bound to ATP or ADP. ADP soaked into crystals of the unliganded protein reveals an intermediate conformation midway between the open and closed states and demonstrates that in GRP94 the conversion between the open and closed states is driven by ligand binding. The direction of the observed movement in GRP94 shows that nucleotides act to open the subdomain elements rather than close them, which is contrary to the motion proposed for Hsp90. These observations support a model where ATP binding dictates the conformation of the N-domain and regulates its ability to form quaternary structural interactions.

Adenosine Diphosphate↗
PubMed2005

Roles of the N-terminal domain on the function and quaternary structure of the ionotropic glutamate receptor.

The alpha-amino-3-hydroxyl-5-methyl-4-isoxazolepropionic acid (AMPA) subtype of ionotropic glutamate receptors (iGluRs) mediates fast excitatory neurotransmission in the mammalian brain. Although the most N-terminal leucine/isoleucine/valine-binding protein (LIVBP) domain is suggested to play a role in the initial assembly of iGluR subunits, it is unclear how this domain is arranged and functions in intact iGluRs. Similarly, although recent crystallographic analyses indicate that the isolated ligand-binding lysine/arginine/ornithine-binding protein domain forms a 2-fold symmetric dimer, the subunit stoichiometry of intact iGluRs remains elusive. Here, we developed a new approach to address these issues. The LIVBP domain of the GluR1 subunit of AMPA receptors was replaced by leucine-zipper peptides designed to form stable symmetric dimers, trimers, tetramers, or pentamers. All these mutant GluR1s were expressed in human embryonic kidney 293 cells and were transported to the cell surface as well as wild type GluR1. Functional and biochemical analyses indicated that these oligomerizing peptides specifically controlled the formation of the expected number of subunits in a channel complex. However, the channel function was only restored by the tetramer-forming peptide. Although the purified LIVBP domain of GluR1 formed a dimmer in solution, a dimer-forming peptide could not restore the function of GluR1. Moreover, a cross-linking assay indicated that four LIVBP domains are located in proximity to each other. These results suggest that the function of the LIVBP domain is not simply to form initial dimers but to adopt a conformation compatible with the overall tetrameric arrangement of subunits in intact AMPA receptors.

Amino Acid Sequence↗
PubMed2000

Quaternary organization of the Staphylothermus marinus phosphoenolpyruvate synthase: angular reconstitution from cryoelectron micrographs with molecular modeling.

Digital electron images of frozen-hydrated preparations of the 2.25-MDa Staphylothermus marinus phosphoenolpyruvate synthase (EC 2.7.9.2) have been analyzed by single-particle classification and averaging and iterative quaternion-based angular reconstitution. Contrast transfer function correction of micrographs obtained at different defocus values was used to improve the informational quality of the projection averages. Three-dimensional reconstructions were obtained to roughly 3-nm spatial resolution, in which the 24 identical subunits were arranged to form an octahedral complex, although the amino-terminal nucleotide-binding domain was not resolved. An atomic model of the subunit was generated by homology modeling using as the reference the known X-ray crystallographic structure of the related enzyme pyruvate orthophosphate dikinase (EC 2.7.9.1) from Clostridium symbiosum (Protein Data Bank entry 1DIK). The S. marinus protein could be arranged into an assembly of 12 homodimers to match the three-dimensional reconstruction in terms of shape and size of the homodimers, as well as overall shape and size of the complex. The quaternary model indicated that active sites of three monomers were localized around cavities (or putative channels) centered at the threefold axes of rotational symmetry and that carboxyl-terminal alpha-helical segments of four monomers were localized at the fourfold axes of rotational symmetry where they could facilitate interdimer interaction. The quaternary arrangement also indicated numerous potential hydrophobic and electrostatic interactions at the interdimer interfaces that could contribute further to structural stability.

Bacterial Proteins↗
PubMed2003

Crystal structure of a full-length LysR-type transcriptional regulator, CbnR: unusual combination of two subunit forms and molecular bases for causing and changing DNA bend.

The LysR-type transcriptional regulator (LTTR) proteins are one of the most common transcriptional regulators in prokaryotes. Here we report the crystal structure of CbnR, which is one of the LTTRs derived from Ralstonia eutropha NH9. This is the first crystal structure of a full-length LTTR. CbnR was found to form a homo-tetramer, which seems to be a biologically active form. Surprisingly, the tetramer can be regarded as a dimer of dimers, whereby each dimer is composed of two subunits in different conformations. In the CbnR tetramer, the DNA-binding domains are located at the V-shaped bottom of the main body of the tetramer, and seem to be suitable to interact with a long stretch of the promoter DNA, which is approximately 60bp. Interaction between the four DNA-binding domains and the two binding sites on the target DNA is likely to bend the target DNA along the V-shaped bottom of the CbnR tetramer. The relaxation of the bent DNA, which occurs upon inducer binding to CbnR, seems to be associated with a quaternary structure change of the tetramer.

Amino Acid Sequence↗
PubMed2006

The crystal and solution structures of glyceraldehyde-3-phosphate dehydrogenase reveal different quaternary structures.

The presence of an isoform of glyceraldehyde-3-phosphate dehydrogenase (kmGAPDH1p) associated with the cell wall of a flocculent strain of Kluyveromyces marxianus was the first report of a non-cytosolic localization of a glycolytic enzyme, but the mechanism by which the protein is transported to the cell surface is not known. To identify structural features that could account for the multiple localizations of the protein, the three-dimensional structure of kmGAPDH1p was determined by x-ray crystallography and small angle x-ray scattering. The x-ray crystallographic structure of kmGAPDH1p revealed a dimer, although all GAPDH homologs studied thus far have a tetrameric structure with 222 symmetry. Interestingly, the structure of kmGAPDH1p in solution revealed a tetramer with a 70 degrees tilt angle between the dimers. Moreover, the separation between the centers of the dimers composing the kmGAPDH1p tetramer diminished from 34 to 30 A upon NAD(+) binding, this latter value being similar to the observed in the crystallographic models of GAPDH homologs. The less compact structure of apo-kmGAPDH1p could already be the first image of the transition intermediate between the tetramer observed in solution and the dimeric form found in the crystal structure, which we postulate to exist in vivo because of the protein's multiple subcellular localizations in this yeast species.

Amino Acid Sequence↗