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At least 127 records · Page 7Linked to original sources

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↗

PubMed2001

The lack of chaperonelike activity of Caenorhabditis elegans Hsp12.2 cannot be restored by domain swapping with human alphaB-crystallin.

The small heat shock proteins Hsp12.2 and alphaB-crystallin differ in that the former occurs as tetramers, without chaperonelike activity, whereas the latter forms multimers and is a good chaperone. To investigate whether the lack of chaperone activity of Hsp12.2 is primarily due to its tetrameric structure or rather to intrinsic sequence features, we engineered chimeric proteins by swapping the N-terminal, C-terminal, and tail regions of Hsp12.2 and alphaB-crystallin, designated as n-c-t and N-C-T, respectively. Three of the chimeric sHsps, namely N-c-T, n-c-T, and N-C-t, showed nativelike secondary and quaternary structures as measured by circular dichroism and gel permeation chromatography. Combining the conserved alpha-crystallin domain of Hsp12.2 with the N-terminal and tail regions of alphaB-crystallin (N-c-T) resulted in multimeric complexes, but did not restore chaperonelike activity. Replacing the tail region of Hsp12.2 with that of alphaB-crystallin (n-c-T) did not alter the tetrameric structure and lack of chaperone activity. Similarly, providing alphaB-crystallin with the tail of Hsp12.2 (N-C-t) did not substantially influence the multimeric complex size, but it reduced the chaperoning ability, especially for small substrates. These results suggest that the conserved alpha-crystallin domain of Hsp12.2 is intrinsically unsuitable to confer chaperonelike activity and confirms that the tail region in alphaB-crystallin modulates chaperonelike capacity in a substrate-dependent manner.

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↗

PubMed2006

The Brichos domain-containing C-terminal part of pro-surfactant protein C binds to an unfolded poly-val transmembrane segment.

Native lung surfactant protein C (SP-C) is a 4.2-kDa acylpeptide that associates with alveolar surfactant phospholipids via a transmembrane alpha-helix. This helix contains mainly Val, although poly-Val is inefficient in helix formation, and helical SP-C can spontaneously convert to beta-sheet aggregates and amyloid-like fibrils. SP-C is cleaved out from a 21-kDa integral membrane protein, proSP-C, in the alveolar type II cell. Recently several mutations localized in the endoplasmic reticulum-lumenal (C-terminal) part of proSP-C (CTproSP-C) have been associated with intracellular accumulation of toxic forms of proSP-C, low levels of mature SP-C, and development of interstitial lung disease. CTproSP-C contains a approximately 100-residue Brichos domain of unknown function that is also found in other membrane proteins associated with amyloid formation, dementia, and cancer. Here we find that recombinant CTproSP-C binds lipid-associated SP-C, which is in beta-strand conformation, and that this interaction results in an increased helical content. In contrast, CTproSP-C does not bind alpha-helical SP-C. Recombinant CTproSP-C(L188Q), a mutation associated with interstitial lung disease, shows secondary and quaternary structures similar to those of wild type CTproSP-C but is unable to bind lipid-associated beta-strand SP-C. Transfection of CTproSP-C into HEK293 cells that express proSP-C(L188Q) increases the amount of proSP-C protein, whereas no effect is seen on cells expressing wild type proSP-C. These findings suggest that CTproSP-C binds nonhelical SP-C and thereby prevents beta-sheet aggregation and that mutations in CTproSP-C can interfere with this function.

Amino Acid Sequence↗

PubMedDate not supplied

[Effect of hydrostatic pressure on quaternary structure on the chaperone function of alpha-crystallin].

High hydrostatic pressure-induced changes in bovine lens alpha-crystallin oligomers size and chaperone-like function were studied by a static light scattering. Under pressure 1.5 kbar, alpha-crystallin oligomers size is almost unaffected. Increase of the size was observed during several hours of incubation at 3 kbar. Such high-pressure effect on association has been previously revealed for detergent micelles, whereas the "typical" protein oligomers are known to dissociate under high pressure. Our results about pressure influence on alpha-crystallin association supports the previously proposed "protein micelle" model of the protein quaternary structure. Chaperone-like activity of alpha-crystallin is shown to increase after incubation at 3 kbar. After the end of the incubation this activity is slowly decreasing during several hours.

Animals↗

PubMed2005

Kinetic stabilization of the native state by protein engineering: implications for inhibition of transthyretin amyloidogenesis.

The amyloidogenic homotetrameric protein transthyretin (TTR) must undergo rate-limiting dissociation to partially denatured monomers in order to aggregate. TTR contains two distinct quaternary interfaces, one of which defines the binding sites for thyroxine and small-molecule amyloidogenesis inhibitors. Kinetic stabilization of the tetramer can be accomplished either by the binding of amyloidogenesis inhibitors selectively to the native state over the dissociative transition state or by the introduction of trans-suppressor subunits (T119M) into heterotetramers to destabilize the dissociative transition state. In each case, increasing the dissociation activation barrier prevents tetramer dissociation. Herein, we demonstrate that tethering two subunits whose quaternary interface defines the thyroxine binding site also dramatically increases the barrier for tetramer dissociation, apparently by destabilization of the dissociative transition state. The tethered construct (TTR-L-TTR)2 is structurally and functionally equivalent to wild-type TTR. Urea is unable to denature (TTR-L-TTR)2, yet it is able to maintain the denatured state once denaturation is achieved by GdnHCl treatment, suggesting that (TTR-L-TTR)2 is kinetically rather than thermodynamically stabilized, consistent with the identical wild-type TTR and (TTR-L-TTR)2 GdnHCl denaturation curves. Studies focused on a construct containing a single TTR-L-TTR chain and two normal monomer subunits establish that alteration of only one quaternary structural interface is sufficient to impose kinetic stabilization on the entire quaternary structure.

Amyloid↗

PubMed2000

Divergence in macromolecular assembly: X-ray crystallographic structure analysis of lumazine synthase from Brucella abortus.

We have determined the three-dimensional structure of 6, 7-dimethyl-8-ribityllumazine synthase (lumazine synthase) from Brucella abortus, the infectious organism of the disease brucellosis in animals. This enzyme catalyses the formation of 6, 7-dimethyl-8-ribityllumazine, the penultimate product in the synthesis of riboflavin. The three-dimensional X-ray crystal structure of the enzyme from B. abortus has been solved and refined at 2.7 A resolution to a final R-value of 0.18 (R(free)=0.23). The macromolecular assembly of the enzyme differs from that of the enzyme from Bacillus subtilis, the only other lumazine synthase structure known. While the protein from B. subtilis assembles into a 60 subunit icosahedral capsid built from 12 pentameric units, the enzyme from B. abortus is pentameric in its crystalline form. Nonetheless, the active sites of the two enzymes are virtually identical indicating inhibitors to theses enzymes could be effective pharmaceuticals across a broad species range. Furthermore, we compare the structures of the enzyme from B. subtilis and B. abortus and describe the C teminus structure which accounts for the differences in quaternary structure.

Bacillus subtilis↗

PubMed2005

The pathway by which the tetrameric protein transthyretin dissociates.

The homotetrameric protein transthyretin (TTR) must undergo rate-limiting dissociation to its constituent monomers in order to enable partial denaturation that allows the process of amyloidogenesis associated with human pathology to ensue. The TTR quaternary structure contains two distinct dimer interfaces, one of which creates the two binding sites for the natural ligand thyroxine. Tetramer dissociation could proceed through three distinct pathways; scission into dimers along either of the two unique quaternary interfaces followed by dimer dissociation represents two possibilities. Alternatively, the tetramer could lose monomers sequentially. To elucidate the TTR dissociation pathway, we employed two different TTR constructs, each featuring covalent attachment of proximal subunits. We demonstrate that tethering the A and B subunits of TTR with a disulfide bond (as well as the symmetrically disposed C and D subunits) allows urea-mediated dissociation of the resulting (TTR-S-S-TTR)(2) construct, affording (TTR-S-S-TTR)(1) retaining a stable 16-stranded beta-sheet structure that is equivalent to the dimer not possessing a thyroid binding site. In contrast, linking the A and C subunits employing a peptide tether (TTR-L-TTR)(2) affords a kinetically stable quaternary structure that does not dissociate or denature in urea. Both tethered constructs and wild-type TTR exhibit analogous stability based on guanidine hydrochloride denaturation curves. The latter denaturant can denature the tetramer, unlike urea, which can only denature monomeric TTR; hence urea requires dissociation to monomers to function. Under native conditions, the (TTR-S-S-TTR)(2) construct is able to dissociate and incorporate subunits from labeled WT TTR homotetramers at a rate equivalent to that exhibited by WT TTR. In contrast, the (TTR-L-TTR)(2) construct is unable to exchange any subunits, even after 180 h. All of the data presented herein and elsewhere demonstrate that the pathway of TTR tetramer dissociation occurs by scission of the tetramer along the crystallographic C(2) axis affording AB and CD dimers that rapidly dissociate into monomers. Determination of the mechanism of dissociation provides an explanation for why small molecules that bind at the AB/CD dimer-dimer interface impose kinetic stabilization upon TTR and disease-associated variants thereof.

Dimerization↗

PubMed2004

Effect of glycosylation on the structure of Erythrina corallodendron lectin.

The three-dimensional structure of the recombinant form of Erythrina corallodendron lectin, complexed with lactose, has been elucidated by X-ray crystallography at 2.55 A resolution. Comparison of this non-glycosylated structure with that of the native glycosylated lectin reveals that the tertiary and quaternary structures are identical in the two forms, with local changes observed at one of the glycosylation sites (Asn17). These changes take place in such a way that hydrogen bonds with the neighboring protein molecules in rECorL compensate those made by the glycan with the protein in ECorL. Contrary to an earlier report, this study demonstrates that the glycan attached to the lectin does not influence the oligomeric state of the lectin. Identical interactions between the lectin and the non-covalently bound lactose in the two forms indicate, in line with earlier reports, that glycosylation does not affect the carbohydrate specificity of the lectin. The present study, the first of its kind involving a glycosylated protein with a well-defined glycan and the corresponding deglycosylated form, provides insights into the structural aspects of protein glycosylation.

Binding Sites↗

PubMed2005

Unusual effect of salts on the homodimeric structure of NADH oxidase from Thermus thermophilus in acidic pH.

The unusual salt-dependent behavior of the homodimeric flavoenzyme NADH oxidase from Thermus thermophilus in acidic pH has been studied using circular dichroism (CD) and sedimentation velocity. The native-like secondary and quaternary structures in acidic low ionic strength conditions were significantly perturbed by the addition of salts. The peptide region of the CD spectra showed a major salt-induced conformational change in the protein secondary structure. Sedimentation velocity experiments showed dissociation of the homodimeric structure of NADH oxidase in the presence of salt (>1 M). The new acidic conformation of the protein was stabilized by high ionic strength as indicated by a salt-induced increase in the melting temperature of the protein, and by a shift in the apparent pK(a) values of the conformational transition to a less acidic pH. Distortion of the dominant alpha-helical signal was expressed as the disappearance of the parallel polarized Moffitt exciton band at 208 nm without an accompanying loss of amplitude of n-->pi* electronic transitions at 222 nm. The unusual CD spectra correlated qualitatively with the theoretically calculated CD spectra of short alpha-helical structures and/or twisted beta-sheets. Differences between the experimentally obtained CD spectra and theoretical calculations (AGADIR) of the alpha-helical content of NADH oxidase indicate a role for non-local interactions in the protein conformation at high ionic strength and low pH. These findings indicate the importance of the homodimeric interface and electrostatic interactions for maintaining the structural integrity of this thermophilic protein.

Dimerization↗

PubMed2001

A correlation between differential structural features and the degree of endopeptidase activity of type A botulinum neurotoxin in aqueous solution.

Botulinum neurotoxin type A is one of the most toxic substances known to man (LD(50) for mouse 0.1 ng/kg). It is also an effective therapeutic drug against involuntary muscle disorders and for pain management. BoNT/A is a Zn(2+) endopeptidase which selectively cleaves SNAP-25 (synaptosomal-associated protein of 25 kDa), a critical component of the exocytotic machinery. Based on nucleotide sequence, BoNT/A is a 145 kDa protein, which appears as a 145 kDa protein band on sodium dodecyl sulfate--polyacrylamide gel electrophoresis. We have examined the structure of BoNT/A in aqueous solution, and found the structure in aqueous solution differs dramatically from that resolved by X-ray crystallography, both at secondary and at quaternary levels. In terms of secondary structure, BoNT/A in aqueous solution has about 47% beta-sheet structure as revealed by infrared spectroscopy, while X-ray crystallography revealed only 17% beta-sheet structure. In terms of quaternary structure, the estimated molecular mass of the native BoNT/A in aqueous solution ranged between 230 and 314 kDa, based on results from different chemical and biophysical techniques (native gel electrophoresis, chemical cross-linking, size exclusion chromatography, and fluorescence anisotropy). These results indicate that BoNT/A exists as a dimer in aqueous solution, which contrasts with the reported monomeric structure of BoNT/A based on X-ray crystallography. The dimeric form of BoNT/A can self-dissociate into the monomeric form at a concentration lower than 50 nM. This concentration-dependent structural change has a significant impact on the endopeptidase activity of BoNT/A: the catalytic efficiency of the monomeric BoNT/A is about 4-fold higher than that of its dimeric form. This difference implies a sterically restricted catalytic site of BoNT/A in the dimeric form of BoNT/A.

Botulinum Toxins, Type A↗

PubMed2003

Quaternary structure dependence of kinetic hole burning and conformational substates interconversion in hemoglobin.

Using a sol-gel encapsulation technique, we have prepared samples of CO saturated human adult hemoglobin locked in the R or T quaternary conformation. We report time-resolved spectra of these samples in the Soret region following flash photolysis, in the time interval ranging from 250 ns to 200 ms and in the temperature interval of 100-170 K. A suitable analysis of the measured difference spectra enables us to obtain the spectral contribution of deoxyHb and HbCO molecules as a function of time and/or of the fraction N(t) of deoxyHb molecules. In our experimental time window geminate CO rebinding to hemoglobin in the T quaternary conformation is about 2 orders of magnitude slower than to hemoglobin in the R conformation: this suggests that the barrier distribution for the CO rebinding, g(H), depends strongly on the protein quaternary structure. In our temperature interval, spectral shifts due to kinetic hole burning (KHB) are present: for HbCO the KHB effect is large in the R conformation and small in the T conformation. For deoxyHb the opposite is true. We attribute the observed behavior to the effect of interconversion between the relevant substates. This effect is stronger for HbCO molecules in the T conformation and for deoxyHb molecules in the R conformation; it confirms the quaternary structure dependence of the hemoglobin energy landscape and suggests enhanced dynamics of ligation intermediate species such as T-state HbCO or R-state deoxyHb.

Hemoglobins↗

PubMed2006

Retention of core catalytic functions by a conserved minimal ribonuclease E peptide that lacks the domain required for tetramer formation.

Ribonuclease E (RNase E) is a multifunctional endoribonuclease that has been evolutionarily conserved in both Gram-positive and Gram-negative bacteria. X-ray crystallography and biochemical studies have concluded that the Escherichia coli RNase E protein functions as a homotetramer formed by Zn linkage of dimers within a region extending from amino acid residues 416 through 529 of the 116-kDa protein. Using fragments of RNase E proteins from E. coli and Haemophilus influenzae, we show here that RNase E derivatives that are as short as 395 amino acid residues and that lack the Zn-link region shown previously to be essential for tetramer formation (i.e. amino acid residues 400-415) are catalytically active enzymes that retain the 5' to 3' scanning ability and cleavage site specificity characteristic of full-length RNase E and that also confer colony forming ability on rne null mutant bacteria. Further truncation leads to loss of these properties. Our results, which identify a minimal catalytically active RNase E sequence, indicate that contrary to current models, a tetrameric quaternary structure is not required for RNase E to carry out its core enzymatic functions.

Catalytic Domain↗

PubMed2002

Crystal structure of the Bse634I restriction endonuclease: comparison of two enzymes recognizing the same DNA sequence.

Crystal structures of Type II restriction endonucleases demonstrate a conserved common core and active site residues but diverse structural elements involved in DNA sequence discrimination. Comparative structural analysis of restriction enzymes recognizing the same nucleotide sequence might therefore contribute to our understanding of the structural diversity of specificity determinants within restriction enzymes. We have solved the crystal structure of the Bacillus stearothermophilus restriction endonuclease Bse634I by the multiple isomorphous replacement technique to 2.17 A resolution. Bse634I is an isoschisomer of the Cfr10I restriction enzyme whose crystal structure has been reported previously. Comparative structural analysis of the first pair of isoschisomeric enzymes revealed conserved structural determinants of sequence recognition and catalysis. However, conformations of the N-terminal subdomains differed between Bse634I/Cfr10I, suggesting a rigid body movement that might couple DNA recognition and catalysis. Structural similarities extend to the quaternary structure level: crystal contacts suggest that Bse634I similarly to Cfr10I is arranged as a tetramer. Kinetic analysis reveals that Bse634I is able to interact simultaneously with two recognition sites supporting the tetrameric architecture of the protein. Thus, restriction enzymes Bse634I, Cfr10I and NgoMIV, recognizing overlapping nucleotide sequences, exhibit a conserved tetrameric architecture that is of functional importance.

Base Sequence↗

PubMed2001

pH-induced structural changes regulate histidine decarboxylase activity in Lactobacillus 30a.

Histidine decarboxylase (HDC) from Lactobacillus 30a produces histamine that is essential to counter waste acids, and to optimize cell growth. The HDC trimer is active at low pH and inactive at neutral to alkaline pH. We have solved the X-ray structure of HDC at pH 8 and revealed the novel mechanism of pH regulation. At high pH helix B is unwound, destroying the substrate binding pocket. At acid pH the helix is stabilized, partly through protonation of Asp198 and Asp53 on either side of the molecular interface, acting as a proton trap. In contrast to hemoglobin regulation, pH has a large effect on the tertiary structure of HDC monomers and relatively little or no effect on quaternary structure.

Binding Sites↗

PubMed2006

High-level expression and purification of human thymidine kinase 1: quaternary structure, stability, and kinetics.

Human cytosolic thymidine kinase (hTK1) is the key enzyme of the pyrimidine salvage pathway and phosphorylates thymidine to thymidine monophosphate, a precursor building block of the DNA. Wild-type hTK1 (hTK1W) as well as a truncated form of the enzyme (hTK1M) carrying deletions at the N- and C-terminal regions were cloned as His(6)-tagged fusion proteins. Expression, isolation, and purification protocols have been established, leading to high yields of soluble and active wild type (approximately 35 mg) and truncated hTK1 (approximately 23 mg) per liter of culture. The protein was purified to near homogeneity. The chaperone DnaK was identified to be the major contaminant that could be removed by applying an additional ATP-MgCl(2) incubation and washing step. hTK1W was a permanent tetramer in solution, whereas the truncated construct hTK1M appears to be a dimer in absence and presence of substrates. Both hTK1W and hTK1M exhibit pronounced thermal stability with transition temperatures (T(m)) of 71.7 and 73.4 degrees C, respectively, when measured without adding substrates. The presence of substrates stabilized both hTK1W (DeltaT(m) ranging from 5.6 to 12.5 degrees C) and hTK1M (DeltaT(m) ranging from 0.8 to 5.3 degrees C). Both enzymes show high activity over a broad range of pH, temperature, and ionic strength. Kinetic studies determined a K(M) of 0.51 microM and a k(cat) of 0.28 s(-1) for wild-type hTK1. The truncated hTK1M has a K(M) of 0.87 microM and k(cat) of 1.65 s(-1), thus exhibiting increased catalytic efficiency. The availability of recombinant human TK1 will facilitate further biochemical and crystallographic studies.

Amino Acid Sequence↗

PubMed2003

Quaternary structure of alpha-crustacyanin from lobster as seen by small-angle X-ray scattering.

The structure of alpha-crustacyanin, the blue carotenoprotein of lobster (Homarus gammarus) carapace, has been investigated for the first time using small-angle X-ray scattering. In this paper, we have determined the dimensions of this protein composed of eight heterodimeric subunits of beta-crustacyanin. Analysis of the scattering spectra and estimation of the shape of alpha-crustacyanin show that the protein fits into a cylinder with an axial length of 238 A and a radius of 47.5 A, in which the eight beta-crustacyanin molecules are probably arranged in a helical manner.

Animals↗

PubMed2006

Structure of thiocyanate hydrolase: a new nitrile hydratase family protein with a novel five-coordinate cobalt(III) center.

Thiocyanate hydrolase (SCNase) of Thiobacillus thioparus THI115 is a cobalt(III)-containing enzyme catalyzing the degradation of thiocyanate to carbonyl sulfide and ammonia. We determined the crystal structures of the apo- and native SCNases at a resolution of 2.0 A. SCNases in both forms had a conserved hetero-dodecameric structure, (alphabetagamma)(4). Four alphabetagamma hetero-trimers were structurally equivalent. One alphabetagamma hetero-trimer was composed of the core domain and the betaN domain, which was located at the center of the molecule and linked the hetero-trimers with novel quaternary interfaces. In both the apo- and native SCNases, the core domain was structurally conserved between those of iron and cobalt-types of nitrile hydratase (NHase). Native SCNase possessed the post-translationally modified cysteine ligands, gammaCys131-SO(2)H and gammaCys133-SOH like NHases. However, the low-spin cobalt(III) was found to be in the distorted square-pyramidal geometry, which had not been reported before in any protein. The size as well as the electrostatic properties of the substrate-binding pocket was totally different from NHases with respect to the charge distribution and the substrate accessibility, which rationally explains the differences in the substrate preference between SCNase and NHase.

Amino Acid Motifs↗