Dimer-dimer Interface Research Articles

Crystal Structures of Nonoxidative Zinc-dependent 2,6-Dihydroxybenzoate (γ-Resorcylate) Decarboxylase from Rhizobium sp. Strain MTP-10005

Reversible 2,6-dihydroxybenzoate decarboxylase from Rhizobium sp. strain MTP-10005 belongs to a nonoxidative decarboxylase family. We have determined the structures of the following three forms of the enzyme: the native form, the complex with the true substrate (2,6-dihydroxybenzoate), and the complex with 2,3-dihydroxybenzaldehyde at 1.7-, 1.9-, and 1.7-A resolution, respectively. The enzyme exists as a tetramer, and the subunit consists of one (alphabeta)8 triose-phosphate isomerase-barrel domain with three functional linkers and one C-terminal tail. The native enzyme possesses one Zn2+ ion liganded by Glu8, His10, His164, Asp287, and a water molecule at the active site center, although the enzyme has been reported to require no cofactor for its catalysis. The substrate carboxylate takes the place of the water molecule and is coordinated to the Zn2+ ion. The 2-hydroxy group of the substrate is hydrogen-bonded to Asp287, which forms a triad together with His218 and Glu221 and is assumed to be the catalytic base. On the basis of the geometrical consideration, substrate specificity is uncovered, and the catalytic mechanism is proposed for the novel Zn2+-dependent decarboxylation.

Engineering a novel, stable dimeric streptavidin with lower isoelectric point

We have engineered a soluble, stable two-chain dimeric streptavidin (TCD) in Escherchia coli. Examination of the three-dimensional structure of streptavidin aided by empirical binding free-energy calculations helped us to select mutations at subunit interfaces that dissociate the native tetramer and stabilize the desired dimer. We chose positions W120, L124, V125 and H127 and mutated them to 120D/124D/125D/127D (TCD-1); 120D/124N/125S/127D (TCD-2); and 120D/124D/125S/127D (TCD-3). The H127D mutation creates electrostatic repulsion that disrupts the dimer-dimer interface, but leaves it very hydrophobic. Therefore, W120, L124 and V125 were mutated to hydrophilic residues to increase dimer solubility. Among the three candidates, TCD-2 gave the best result: a stable, active dimer with K(d) for biotin of approximately 1x10(-7)M after purification by gel-filtration chromatography. The experimental results confirm the possibility of rational engineering of low-pI dimeric streptavidins. Reduced-size streptavidin mutants with a net negative charge may be more suitable than antibodies or wild-type streptavidin for the targeting step in radioimmunotherapy because they should clear faster from the bloodstream and the kidney.

Effects of Protein-Ligand Associations on the Subunit Interactions of Phosphofructokinase from B. stearothermophilus

Differences between the crystal structures of inhibitor-bound and uninhibited forms of phosphofructokinase (PFK) from B. stearothermophilus have led to a structural model for allosteric inhibition by phosphoenolpyruvate (PEP) wherein a dimer-dimer interface within the tetrameric enzyme undergoes a quaternary shift. We have developed a labeling and hybridization technique to generate a tetramer with subunits simultaneously containing two different extrinsic fluorophores in known subunit orientations. This construct has been utilized in the examination of the effects of allosteric ligand and substrate binding on the subunit affinities of tetrameric PFK using several biophysical and spectroscopic techniques including 2-photon, dual-channel fluorescence correlation spectroscopy (FCS). We demonstrate that PEP-binding at the allosteric site is sufficient to reduce the affinity of the active site interface from beyond the limits of experimental detection to nanomolar affinity, while conversely strengthening the interface at which it is bound. The reduced interface affinity is specific to inhibitor binding because binding the activator ADP at the same allosteric site causes no reduction in subunit affinity. With inhibitor bound, the weakened subunit affinity has allowed the kinetics of dimer association to be elucidated.

Allosteric Communication Network in the Tetrameric Restriction Endonuclease Bse634I

Restriction endonuclease Bse634I is a homotetramer arranged as a dimer of two primary dimers. Bse634I displays its maximum catalytic efficiency upon binding of two copies of cognate DNA, one per each primary dimer. The catalytic activity of Bse634I on a single DNA copy is down-regulated due to the cross-talking interactions between the primary dimers. The mechanism of signal propagation between the individual active sites of Bse634I remains unclear. To identify communication pathways involved in the catalytic activity regulation of Bse634I tetramer we mutated a selected set of amino acid residues at the dimer–dimer interface and analysed the oligomeric state and catalytic properties of the mutant proteins. We demonstrate that alanine replacement of N262 and V263 residues located in the loop at the tetramerisation interface did not inhibit tetramer assembly but dramatically altered the catalytic properties of Bse634I despite of the distal location from the active site. Kinetic analysis using cognate hairpin oligonucleotide and one and two-site plasmids as substrates allowed us to identify two types of communication signals propagated through the dimer-dimer interface in the Bse634I tetramer: the inhibitory, or “stopper” and the activating, or “sync” signal. We suggest that the interplay between the two signals determines the catalytic and regulatory properties of the Bse634I and mutant proteins.

Mutational Analysis of the Flagellar Rotor Protein FliN: Identification of Surfaces Important for Flagellar Assembly and Switching

FliN is a component of the flagellar switch complex in many bacterial species. The crystal structure is known for most of FliN, and a targeted cross-linking study (K. Paul and D. F. Blair, J. Bacteriol. 188:2502-2511, 2006) showed that it is organized in ring-shaped tetramers at the bottom of the basal body C ring. FliN is essential for flagellar assembly and direction switching, but its precise functions have not been defined. Here, we identify functionally important regions on FliN by systematic mutagenesis. Nonconservative mutations were made at positions sampling the surface of the protein, and the effects on flagellar assembly and function were measured. Flagellar assembly was disrupted by mutations in a conserved hydrophobic patch centered on the dimer twofold axis or by mutations on the surface that forms the dimer-dimer interface in the tetramer. The assembly defect in hydrophobic-patch mutants was partially rescued by overexpression of the flagellar export proteins FliH and FliI, and coprecipitation assays demonstrated a binding interaction between FliN and FliH that was weakened by mutations in the hydrophobic patch. Thus, FliN might contribute to export by providing binding sites for FliH or FliH-containing complexes. The region around the hydrophobic patch is also important for switching; certain mutations in or near the patch caused a smooth-swimming chemotaxis defect that in most cases could be partially rescued by overexpression of the clockwise-signaling protein CheY. The results indicate that FliN is more closely involved in switching than has been supposed, possibly contributing to the binding site for CheY on the switch.

Molecular and Structural Characterization of Hexameric β-d-Glucosidases in Wheat and Rye

The wheat (Triticum aestivum) and rye (Secale cereale) beta-D-glucosidases hydrolyze hydroxamic acid-glucose conjugates, exist as different types of isozyme, and function as oligomers. In this study, three cDNAs encoding beta-D-glucosidases (TaGlu1a, TaGlu1b, and TaGlu1c) were isolated from young wheat shoots. Although the TaGlu1s share very high sequence homology, the mRNA level of Taglu1c was much lower than the other two genes in 48- and 96-h-old wheat shoots. The expression ratio of each gene was different between two wheat cultivars. Recombinant TaGlu1b expressed in Escherichia coli was electrophoretically distinct fromTaGlu1a and TaGlu1c. Furthermore, coexpression of TaGlu1a and TaGlu1b gave seven bands on a native-PAGE gel, indicating the formation of both homo- and heterohexamers. One distinctive property of the wheat and rye glucosidases is that they function as hexamers but lose activity when dissociated into smaller oligomers or monomers. The crystal structure of hexameric TaGlu1b was determined at a resolution of 1.8 A. The N-terminal region was located at the dimer-dimer interface and plays a crucial role in hexamer formation. Mutational analyses revealed that the aromatic side chain at position 378, which is located at the entrance to the catalytic center, plays an important role in substrate binding. Additionally, serine-464 and leucine-465 of TaGlu1a were shown to be critical in the relative specificity for DIMBOA-glucose (2-O-beta-D-glucopyranosyl-4-hydroxy-7-methoxy-1,4-benzoxazin-3-one) over DIBOA-glucose (7-demethoxy-DIMBOA-glucose).

Characterization of a Thermostable NADPH:FMN Oxidoreductase from the Mesophilic Bacterium Bacillus subtilis

The gene yhdA from Bacillus subtilis encoding a putative flavin mononucleotide (FMN)-dependent oxidoreductase was cloned and heterologously expressed in Escherichia coli. The purified enzyme has a noncovalently bound FMN cofactor, which is preferentially reduced by NADPH, indicating that YhdA is a NADPH:FMN oxidoreductase. The rate of NADPH oxidation is enhanced by the addition of external FMN, and analysis of initial rate measurements reveals the occurrence of a ternary complex in a bi-bi reaction mechanism. YhdA has also been shown to reductively cleave the -N=N- bond in azo dyes at the expense of NADPH, and hence, it possesses azoreductase activity, however, at a rate 100 times slower than that found for FMN. Using Cibacron Marine as a model compound, we could demonstrate that the dye is a competitive inhibitor of NADPH and FMN. The utilization of NADPH and the absence of a flavin semiquinone radical distinguish YhdA from flavodoxins, which adopt the same structural fold, i.e., a five-stranded beta sheet sandwiched by five alpha helices. The native molecular-mass of YhdA was determined to be 76 kDa, suggesting that the protein occurs as a tetramer, whereas the YhdA homologue in Saccharomyces cerevisiae (YLR011wp) forms a dimer in solution. Interestingly, the different oligomerization of these homologous proteins correlates to their thermostability, with YhdA exhibiting a melting point of 86.5 degrees C, which is 26.3 degrees C higher than that for the yeast protein. This unusually high melting point is proposed to be the result of increased hydrophobic packing between dimers and the additional presence of four salt bridges stabilizing the dimer-dimer interface.

Structural Analysis of N-acetylglucosamine-6-phosphate Deacetylase Apoenzyme from Escherichia coli

We report the crystal structure of the apoenzyme of N-acetylglucosamine-6-phosphate (GlcNAc6P) deacetylase from Escherichia coli (EcNAGPase) and the spectrometric evidence of the presence of Zn2+ in the native protein. The GlcNAc6P deacetylase is an enzyme of the amino sugar catabolic pathway that catalyzes the conversion of the GlcNAc6P into glucosamine 6-phosphate (GlcN6P). The crystal structure was phased by the single isomorphous replacement with anomalous scattering (SIRAS) method using low-resolution (2.9 A) iodine anomalous scattering and it was refined against a native dataset up to 2.0 A resolution. The structure is similar to two other NAGPases whose structures are known from Thermotoga maritima (TmNAGPase) and Bacillus subtilis (BsNAGPase); however, it shows a phosphate ion bound at the metal-binding site. Compared to these previous structures, the apoenzyme shows extensive conformational changes in two loops adjacent to the active site. The E. coli enzyme is a tetramer and its dimer-dimer interface was analyzed. The tetrameric structure was confirmed in solution by small-angle X-ray scattering data. Although no metal ions were detected in the present structure, experiments of photon-induced X-ray emission (PIXE) spectra and of inductively coupled plasma emission spectroscopy (ICP-AES) with enzyme that was neither exposed to chelating agents nor metal ions during purification, revealed the presence of 1.4 atoms of Zn per polypeptide chain. Enzyme inactivation by metal-sequestering agents and subsequent reactivation by the addition of several divalent cations, demonstrate the role of metal ions in EcNAGPase structure and catalysis.

Structural and functional roles for β‐strand 7 in the α‐crystallin domain of p26, a polydisperse small heat shock protein from Artemia franciscana

Oviparous development in the extremophile crustacean, Artemia franciscana, generates encysted embryos which enter a profound state of dormancy, termed diapause. Encystment is marked by the synthesis of p26, a polydisperse small heat shock protein thought to protect embryos from stress. In order to elucidate structural/functional relationships within p26 and other polydisperse small heat shock proteins, and to better define the protein's role during diapause, amino acid substitutions R110G, F112R, R114A and Y116D were generated within the p26 alpha-crystallin domain by site-directed mutagenesis. These residues were chosen because they are highly conserved across species boundaries, and molecular modelling indicates that they are part of a key structural interface between dimers. The F112R mutation, which had the greatest impact on oligomerization, placed two charged residues at the p26 dimer-dimer interface, demonstrating the importance of beta-strand 7 in tetramer formation. All mutated versions of p26 were less able than wild-type p26 to confer thermotolerance on transformed bacteria and they exhibited diminished chaperone action in three in vitro assays; however, all variants retained protective activity. This apparent stability of p26 may, by prolonging effective chaperone life in vivo, enhance embryo stress resistance. All substitutions modified p26 intrinsic fluorescence, surface hydrophobicity and secondary structure, and the pronounced changes in variant R114A, as indicated by these physical measurements, correlated with the greatest loss of function. Although mutation R114A had the greatest effect on p26 chaperoning, it had the least on oligomerization. These results demonstrate that in contrast to many other small heat shock proteins, p26 effectiveness as a chaperone is independent of oligomerization. The results also reinforce the idea, occasioned by modelling, that R114 is removed slightly from dimer-dimer interfaces. Moreover, beta-strand 7 is shown to have an important role in oligomerization of p26, a function first proposed for this structural element upon crystallization of wheat Hsp16.9, a small heat shock protein with different quaternary structure.

Functionally Important Substructures of Circadian Clock Protein KaiB in a Unique Tetramer Complex

KaiB is a component of the circadian clock molecular machinery in cyanobacteria, which are the simplest organisms that exhibit circadian rhythms. Here we report the x-ray crystal structure of KaiB from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1. The KaiB crystal diffracts at a resolution of 2.6 A and includes four subunits organized as a dimer of dimers, each composed of two non-equivalent subunits. The overall shape of the tetramer is an elongated hexagonal plate, with a single positively charged cleft flanked by two negatively charged ridges whose surfaces includes several terminal chains. Site-directed mutagenesis of Synechococcus KaiB confirmed that alanine substitution of residues Lys-11 or Lys-43 in the cleft, or deletion of C-terminal residues 95-108, which forms part of the ridges, strongly weakens in vivo circadian rhythms. Characteristics of KaiB deduced from the x-ray crystal structure were also confirmed by physicochemical measurements of KaiB in solution. These data suggest that the positively charged cleft and flanking negatively charged ridges in KaiB are essential for the biological function of KaiB in the circadian molecular machinery in cyanobacteria.

Crystallographic Trapping of the Glutamyl-CoA Thioester Intermediate of Family I CoA Transferases

Coenzyme A transferases are involved in a broad range of biochemical processes in both prokaryotes and eukaryotes, and exhibit a diverse range of substrate specificities. The YdiF protein from Escherichia coli O157:H7 is an acyl-CoA transferase of unknown physiological function, and belongs to a large sequence family of CoA transferases, present in bacteria to humans, which utilize oxoacids as acceptors. In vitro measurements showed that YdiF displays enzymatic activity with short-chain acyl-CoAs. The crystal structures of YdiF and its complex with CoA, the first co-crystal structure for any Family I CoA transferase, have been determined and refined at 1.9 and 2.0 A resolution, respectively. YdiF is organized into tetramers, with each monomer having an open alpha/beta structure characteristic of Family I CoA transferases. Co-crystallization of YdiF with a variety of CoA thioesters in the absence of acceptor carboxylic acid resulted in trapping a covalent gamma-glutamyl-CoA thioester intermediate. The CoA binds within a well defined pocket at the N- and C-terminal domain interface, but makes contact only with the C-terminal domain. The structure of the YdiF complex provides a basis for understanding the different catalytic steps in the reaction of Family I CoA transferases.

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.

Crystal Structure of Tetrameric Homoisocitrate Dehydrogenase from an Extreme Thermophile, Thermus thermophilus : Involvement of Hydrophobic Dimer-Dimer Interaction in Extremely High Thermotolerance

The crystal structure of homoisocitrate dehydrogenase involved in lysine biosynthesis from Thermus thermophilus (TtHICDH) was determined at 1.85-A resolution. Arg85, which was shown to be a determinant for substrate specificity in our previous study, is positioned close to the putative substrate binding site and interacts with Glu122. Glu122 is highly conserved in the equivalent position in the primary sequence of ICDH and archaeal 3-isopropylmalate dehydrogenase (IPMDH) but interacts with main- and side-chain atoms in the same domain in those paralogs. In addition, a conserved Tyr residue (Tyr125 in TtHICDH) which extends its side chain toward a substrate and thus has a catalytic function in the related beta-decarboxylating dehydrogenases, is flipped out of the substrate-binding site. These results suggest the possibility that the conformation of the region containing Glu122-Tyr125 is changed upon substrate binding in TtHICDH. The crystal structure of TtHICDH also reveals that the arm region is involved in tetramer formation via hydrophobic interactions and might be responsible for the high thermotolerance. Mutation of Val135, located in the dimer-dimer interface and involved in the hydrophobic interaction, to Met alters the enzyme to a dimer (probably due to steric perturbation) and markedly decreases the thermal inactivation temperature. Both the crystal structure and the mutation analysis indicate that tetramer formation is involved in the extremely high thermotolerance of TtHICDH.

The crystal structures of native and (S)-lysine-bound dihydrodipicolinate synthase fromEscherichia coliwith improved resolution show new features of biological significance

Dihydrodipicolinate synthase (DHDPS) mediates the key first reaction common to the biosynthesis of (S)-lysine and meso-diaminopimelate. The activity of DHDPS is allosterically regulated by the feedback inhibitor (S)-lysine. The crystal structure of DHDPS from Escherichia coli has previously been published, but to only a resolution of 2.5 A, and the structure of the lysine-bound adduct was known to only 2.94 A resolution. Here, the crystal structures of native and (S)-lysine-bound dihydrodipicolinate synthase from E. coli are presented to 1.9 and 2.0 A, respectively, resolutions that allow, in particular, more accurate definition of the protein structure. The general architecture of the active site is found to be consistent with previously determined structures, but with some important differences. Arg138, which is situated at the entrance of the active site and is thought to be involved in substrate binding, has an altered conformation and is connected via a water molecule to Tyr133 of the active-site catalytic triad. This suggests a hitherto unknown function for Arg138 in the DHDPS mechanism. Additionally, a re-evaluation of the dimer-dimer interface reveals a more extensive network of interactions than first thought. Of particular interest is the higher resolution structure of DHDPS with (S)-lysine bound at the allosteric site, which is remote to the active site, although connected to it by a chain of conserved water molecules. (S)-Lysine has a slightly altered conformation from that originally determined and does not appear to alter the DHDPS structure as others have reported.

Large improvement in the thermal stability of a tetrameric malate dehydrogenase by single point mutations at the dimer-dimer interface.

The stability of tetrameric malate dehydrogenase from the green phototrophic bacterium Chloroflexus aurantiacus (CaMDH) is at least in part determined by electrostatic interactions at the dimer-dimer interface. Since previous studies had indicated that the thermal stability of CaMDH becomes lower with increasing pH, attempts were made to increase the stability by removal of (excess) negative charge at the dimer-dimer interface. Mutation of Glu165 to Gln or Lys yielded a dramatic increase in thermal stability at pH 7.5 (+23.6 -- + 23.9 degrees C increase in apparent t(m)) and a more moderate increase at pH 4.4 (+4.6 -- + 5.4 degrees C). The drastically increased stability at neutral pH was achieved without forfeiture of catalytic performance at low temperatures. The crystal structures of the two mutants showed only minor structural changes close to the mutated residues, and indicated that the observed stability effects are solely due to subtle changes in the complex network of electrostatic interactions in the dimer-dimer interface. Both mutations reduced the concentration dependency of thermal stability, suggesting that the oligomeric structure had been reinforced. Interestingly, the two mutations had similar effects on stability, despite the charge difference between the introduced side-chains. Together with the loss of concentration dependency, this may indicate that both E165Q and E165K stabilize CaMDH to such an extent that disruption of the inter-dimer electrostatic network around residue 165 no longer limits kinetic thermal stability.

Crystallographic and solution studies of N-lithocholyl insulin: a new generation of prolonged-acting human insulins.

The addition of specific bulky hydrophobic groups to the insulin molecule provides it with affinity for circulating serum albumin and enables it to form soluble macromolecular complexes at the site of subcutaneous injection, thereby securing slow absorption of the insulin analogue into the blood stream and prolonging its half-life once there. N-Lithocholic acid acylated insulin [Lys(B29)-lithocholyl des-(B30) human insulin] has been crystallized and the structure determined by X-ray crystallography at 1.6 A resolution to explore the molecular basis of its assembly. The unit cell in the crystal consists of an insulin hexamer containing two zinc ions, with two m-cresol molecules bound at each dimer-dimer interface stabilizing an R(6) conformation. Six covalently bound lithocholyl groups are arranged symmetrically around the outside of the hexamer. These form specific van der Waals and hydrogen-bonding interactions at the interfaces between neighboring hexamers, possibly representing the kinds of interactions which occur in the soluble aggregates at the site of injection. Comparison with an equivalent nonderivatized native insulin hexamer shows that the addition of the lithocholyl group disrupts neither the important conformational features of the insulin molecule nor its hexamer-forming ability. Indeed, binding studies show that the affinity of N-lithocholyl insulin for the human insulin receptor is not significantly diminished.

Cooperative Effect of Two Surface Amino Acid Mutations (Q252L and E170K) in Glucose Dehydrogenase from Bacillus megaterium IWG3 on Stabilization of Its Oligomeric State

A thermostable glucose dehydrogenase (GlcDH) mutant of Bacillus megaterium IWG3 harboring the Q252L substitution (Y. Makino, S. Negoro, I. Urabe, and H. Okada, J. Biol. Chem. 264:6381-6385, 1989) is stable at pH values above 9, but only in the presence of 2 M NaCl. Another GlcDH mutant exhibiting increased stability at an alkaline pH in the absence of NaCl has been isolated previously (S.-H. Baik, T. Ide, H. Yoshida, O. Kagami, and S. Harayama, Appl. Microbiol. Biotechnol. 61:329-335, 2003). This mutant had two amino acid substitutions, Q252L and E170K. In the present study, we characterized three GlcDH mutants harboring the substitutions Q252L, E170K, and Q252L/E170K under low-salt conditions. The GlcDH mutant harboring two substitutions, Q252L/E170K, was stable, but mutants harboring a single substitution, either Q252L or E170K, were unstable at an alkaline pH. Gel filtration chromatography analyses demonstrated that the oligomeric state of the Q252/E170K enzyme was stable, while the tetramers of the enzymes harboring a single substitution (Q252L or E170K) dissociated into dimers at an alkaline pH. These results indicated that the Q252L and E170K substitutions synergistically strengthened the interaction at the dimer-dimer interface. The crystal structure of the E170K/Q252L mutant, determined at 2.0-angstroms resolution, showed that residues 170 and 252 are located in a hydrophobic cavity at the subunit-subunit interface. We concluded that these residues in the wild-type enzyme have thermodynamically unfavorable effects, while the Q252L and E170K substitutions increase the subunit-subunit interactions by stabilizing the hydrophobic cavity.

Determination of the multimerization state of the hepatitis delta virus antigens in vivo.

Hepatitis delta virus expresses two essential proteins, the small and large delta antigens, and both are required for viral propagation. Proper function of each protein depends on the presence of a common amino-terminal multimerization domain. A crystal structure, solved using a peptide fragment that contained residues 12 to 60, depicts the formation of an octameric ring composed of antiparallel coiled-coil dimers. Because this crystal structure was solved for only a fragment of the delta antigens, it is unknown whether octamers actually form in vivo at physiological protein concentrations and in the context of either intact delta antigen. To test the relevance of the octameric structure, we developed a new method to probe coiled-coil structures in vivo. We generated a panel of mutants containing cysteine substitutions at strategic locations within the predicted monomer-monomer interface and the dimer-dimer interface. Since the small delta antigen contains no cysteine residues, treatment of cell extracts with a mild oxidizing reagent was expected to induce disulfide bond formation only when the appropriate pairs of cysteine substitution mutants were coexpressed. We indeed found that, in vivo, both the small and large delta antigens assembled as antiparallel coiled-coil dimers. Likewise, we found that both proteins could assume an octameric quaternary structure in vivo. Finally, during the course of these experiments, we found that unprenylated large delta antigen molecules could be disulfide cross-linked via the sole cysteine residue located within the carboxy terminus. Therefore, in vivo, the C terminus likely provides an additional site of protein-protein interaction for the large delta antigen.

Structure of a Sir2 Substrate, Alba, Reveals a Mechanism for Deacetylation-induced Enhancement of DNA Binding

The targeted acetylation status of histones and several other transcriptional regulatory proteins plays an important role in gene expression, although the mechanism for this is not well understood. As a model to understand how targeted acetylation may effect transcription, we determined the x-ray crystal structure of the chromatin protein Alba from Archaeoglobus fulgidus, a substrate for the Sir2 protein that deacetylates it at lysine 11 to promote DNA binding by Alba. The structure reveals a dimer of dimers in which the dimer-dimer interface is stabilized by several conserved hydrophobic residues as well as the lysine 11 target of Sir2. We show that, in solution, the mutation of these hydrophobic residues or lysine 11 disrupts dimer-dimer formation and decreases DNA-binding affinity. We propose that the in vivo deacetylation of lysine 11 of archaeal Alba by Sir2 promotes protein oligomerization for optimal DNA binding. Implications for the mechanism by which histone acetylation modulates gene expression are discussed.

The First Crystal Structure of Archaeal Aldolase: UNIQUE TETRAMERIC STRUCTURE of 2-DEOXY-d-RIBOSE-5-PHOSPHATE ALDOLASE FROM THE HYPERTHERMOPHILIC ARCHAEA Aeropyrum pernix

A gene encoding a 2-deoxy-d-ribose-5-phosphate aldolase (DERA) homolog was identified in the hyperthermophilic Archaea Aeropyrum pernix. The gene was overexpressed in Escherichia coli, and the produced enzyme was purified and characterized. The enzyme is an extremely thermostable DERA; its activity was not lost after incubation at 100 degrees C for 10 min. The enzyme has a molecular mass of approximately 93 kDa and consists of four subunits with an identical molecular mass of 24 kDa. This is the first report of the presence of tetrameric DERA. The three-dimensional structure of the enzyme was determined by x-ray analysis. The subunit folds into an alpha/beta-barrel. The asymmetric unit consists of two homologous subunits, and a crystallographic 2-fold axis generates the functional tetramer. The main chain coordinate of the monomer of the A. pernix enzyme is quite similar to that of the E. coli enzyme. There was no significant difference in hydrophobic interactions and the number of ion pairs between the monomeric structures of the two enzymes. However, a significant difference in the quaternary structure was observed. The area of the subunit-subunit interface in the dimer of the A. pernix enzyme is much larger compared with the E. coli enzyme. In addition, the A. pernix enzyme is 10 amino acids longer than the E. coli enzyme in the N-terminal region and has an additional N-terminal helix. The N-terminal helix produces a unique dimer-dimer interface. This promotes the formation of a functional tetramer of the A. pernix enzyme and strengthens the hydrophobic intersubunit interactions. These structural features are considered to be responsible for the extremely high stability of the A. pernix enzyme. This is the first description of the structure of hyperthermophilic DERA and of aldolase from the Archaea domain.