The transactivation region of the fis protein that controls site-specific DNA inversion contains extended mobile beta-hairpin arms

The Fis protein regulates site-specific DNA inversion catalyzed by a family of DNA invertases when bound to a cis-acting recombinational enhancer. As is often found for transactivation domains, previous crystal structures have failed to resolve the conformation of the N-terminal inversion activation region within the Fis dimer. A new crystal form of a mutant Fis protein now reveals that the activation region contains two beta-hairpin arms that protrude over 20 A from the protein core. Saturation mutagenesis identified the regulatory and structurally important amino acids. The most critical activating residues are located near the tips of the beta-arms. Disulfide cross-linking between the beta-arms demonstrated that they are highly flexible in solution and that efficient inversion activation can occur when the beta-arms are covalently linked together. The emerging picture for this regulatory motif is that contacts with the recombinase at the tip of the mobile beta-arms activate the DNA invertase in the context of an invertasome complex.     

Differential activity of the G protein beta5 gamma2 subunit at receptors and effectors

The G protein beta5 subunit differs substantially in amino acid sequence from the other known beta subunits suggesting that beta gamma dimers containing this protein may play specialized roles in cell signaling. To examine the functional properties of the beta5 subunit, recombinant beta5 gamma2 dimers were purified from baculovirus-infected Sf9 insect cells using a strategy based on two affinity tags (hexahistidine and FLAG) engineered into the N terminus of the gamma2 subunit (gamma2HF). The function of the pure beta5 gamma2HF dimers was examined in three assays: activation of pure phospholipase C-beta in lipid vesicles; activation of recombinant, type II adenylyl cyclase expressed in Sf9 cell membranes; and coupling of alpha subunits to the endothelin B (ETB) and M1 muscarinic receptors. In each case, the efficacy of the beta5 gamma2HF dimer was compared with that of the beta1 gamma2HF dimer, which has demonstrated activity in these assays. The beta5 gamma2HF dimer activated phospholipase C-beta with a potency and efficacy similar to that of beta1 gamma2 or beta1 gamma2HF; however, it was markedly less effective than the beta1 gamma2HF or beta1 gamma2 dimer in its ability to activate type II adenylyl cyclase (EC50 of approximately 700 nM versus 25 nM). Both the beta5 gamma2HF and the beta1 gamma2HF dimers supported coupling of M1 muscarinic receptors to the Gq alpha subunit. The ETB receptor coupled effectively to both the Gi and Gq alpha subunits in the presence of the beta1 gamma2HF dimer. In contrast, the beta5 gamma2HF dimer only supported coupling of the Gq alpha subunits to the ETB receptor and did not support coupling of the Gi alpha subunit. These results suggest that the beta5 gamma2HF dimer binds selectively to Gq alpha subunits and does not activate the same set of effectors as dimers containing the beta1 subunit. Overall, the data support a specialized role for the beta5 subunit in cell signaling.     

In vivo identification of intermediate stages of the DNA inversion reaction catalyzed by the Salmonella Hin recombinase

The Hin recombinase catalyzes a site-specific recombination reaction that results in the reversible inversion of a 1-kbp segment of the Salmonella chromosome. The DNA inversion reaction catalyzed by the Salmonella Hin recombinase is a dynamic process proceeding through many intermediate stages, requiring multiple DNA sites and the Fis accessory protein. Biochemical analysis of this reaction has identified intermediate steps in the inversion reaction but has not yet revealed the process by which transition from one step to another occurs. Because transition from one reaction step to another proceeds through interactions between specific amino acids, and between amino acids and DNA bases, it is possible to study these transitions through mutational analysis of the proteins involved. We isolated a large number of mutants in the Hin recombinase that failed to carry out the DNA exchange reaction. We generated genetic tools that allowed the assignment of these mutants to specific transition steps in the recombination reaction. This genetic analysis, combined with further biochemical analysis, allowed us to define contributions by specific amino acids to individual steps in the DNA inversion reaction. Evidence is also presented in support of a model that Fis protein enhances the binding of Hin to the hixR recombination site. These studies identified regions within the Hin recombinase involved in specific transition steps of the reaction and provided new insights into the molecular details of the reaction mechanism.     

Hin-mediated inversion on positively supercoiled DNA

Hin recombinase requires negatively supercoiled DNA for an efficient inversion. We have generated positively supercoiled plasmid DNA using reverse gyrase from Sulfolobus shibatae and subjected it to the Hin-mediated inversion reaction. Both Hin and Fis showed the same DNA binding activity regardless of the superhelical handedness of the substrate plasmid. However, inversion activity on positively supercoiled DNA was less than 1% of negatively supercoiled DNA. Assays designed to probe steps in inversion, showed that on positively supercoiled DNA, Hin was able to cleave the recombination sites with the same efficiency shown on negatively supercoiled DNA but was not able to exchange the cleaved DNA. Based on the theoretical differences between positive and negative supercoiling, our data may suggest that unwinding of the double helix at recombination sites is needed after DNA cleavage for strand exchange to occur.     

High resolution crystal structure of pyruvate decarboxylase from Zymomonas mobilis. Implications for substrate activation in pyruvate decarboxylases

The crystal structure of tetrameric pyruvate decarboxylase from Zymomonas mobilis has been determined at 1.9 A resolution and refined to a crystallographic R-factor of 16.2% and Rfree of 19.7%. The subunit consists of three domains, all of the alpha/beta type. Two of the subunits form a tight dimer with an extensive interface area. The thiamin diphosphate binding site is located at the subunit-subunit interface, and the cofactor, bound in the V conformation, interacts with residues from the N-terminal domain of one subunit and the C-terminal domain of the second subunit. The 2-fold symmetry generates the second thiamin diphosphate binding site in the dimer. Two of the dimers form a tightly packed tetramer with pseudo 222 symmetry. The interface area between the dimers is much larger in pyruvate decarboxylase from Z. mobilis than in the yeast enzyme, and structural differences in these parts result in a completely different packing of the subunits in the two enzymes. In contrast to other pyruvate decarboxylases, the enzyme from Z. mobilis is not subject to allosteric activation by the substrate. The tight packing of the dimers in the tetramer prevents large rearrangements in the quaternary structure as seen in the yeast enzyme and locks the enzyme in an activated conformation. The architecture of the cofactor binding site and the active site is similar in the two enzymes. However, the x-ray analysis reveals subtle but significant structural differences in the active site that might be responsible for variations in the biochemical properties in these enzymes.     

The crystal structure of a 3D domain-swapped dimer of RNase A at a 2.1-A resolution

The dimer of bovine pancreatic ribonuclease A (RNase A) discovered by Crestfield, Stein, and Moore in 1962 has been crystallized and its structure determined and refined to a 2.1-A resolution. The dimer is 3D domain-swapped. The N-terminal helix (residues 1-15) of each subunit is swapped into the major domain (residues 23-124) of the other subunit. The dimer of bull seminal ribonuclease (BS-RNase) is also known to be domain-swapped, but the relationship of the subunits within the two dimers is strikingly different. In the RNase A dimer, the 3-stranded beta sheets of the two subunits are hydrogen-bonded at their edges to form a continuous 6-stranded sheet across the dimer interface; in the BS-RNase dimer, it is instead the two helices that abut. Whereas the BS-RNase dimer has 2-fold molecular symmetry, the two subunits of the RNase A dimer are related by a rotation of approximately 160 degrees. Taken together, these structures show that intersubunit adhesion comes mainly from the swapped helical domain binding to the other subunit in the "closed interface" but that the overall architecture of the domain-swapped oligomer depends on the interactions in the second type of interface, the "open interface." The RNase A dimer crystals take up the dye Congo Red, but the structure of a Congo Red-stained crystal reveals no bound dye molecule. Dimer formation is inhibited by excess amounts of the swapped helical domain. The possible implications for amyloid formation are discussed.     

Trypsin cleavage of human cystathionine beta-synthase into an evolutionarily conserved active core: structural and functional consequences

Cystathionine beta-synthase (CBS) catalyzes the condensation of homocysteine and serine to cystathionine-an irreversible step in the eukaryotic transsulfuration pathway. The native enzyme is a homotetramer or multimer of 63-kDa (551 amino acids) subunits and is activated by S-adenosyl-l-methionine (AdoMet) or by partial cleavage with trypsin. Amino-terminal analysis of the early products of trypsinolysis demonstrated that the first cleavages occur at Lys 30, 36, and 39. The enzyme still retains the subunit organization as a tetramer or multimer composed of 58-kDa subunits. Analysis by electrospray ionization mass spectrometry showed that further trypsin treatment cleaves CBS in its COOH-terminal region at Arg 413 to yield 45-kDa subunits. This 45-kDa active core is the portion of CBS most conserved with the evolutionarily related enzymes isolated from plants, yeast, and bacteria. The active core of CBS forms a dimer of approximately 85 kDa. The dimer is about twice as active as the tetramer. It binds both pyridoxal 5'-phosphate and heme cofactors but is no longer activated by AdoMet. Further analysis suggests that the dissociation of CBS to dimers causes a decrease in enzyme thermostability and a threefold increase in affinity toward the sulfhydryl-containing substrate-homocysteine. We found that the COOH-terminal region, residues 414-551, is essential for maintaining the tetrameric structure and AdoMet activation of the enzyme. The inability of the active core to form multimeric aggregates has facilitated its crystallization and X-ray diffraction studies.     

Intra- vs intersubunit communication in the homodimeric restriction enzyme EcoRV: Thr 37 and Lys 38 involved in indirect readout are only important for the catalytic activity of their own subunit

EcoRV is a dimer of two identical subunits which together form one binding site for the double-stranded DNA substrate. Concerted cleavage of both strands of the duplex requires intersubunit communication to synchronize the two catalytic centers of EcoRV. Here we address the question of how contacts to the DNA backbone trigger conformational changes which lead to the activation of both catalytic centers. The structure of the specific EcoRV-DNA complex shows that a region including amino acids Thr 37 and Lys 38 is involved in interactions with the DNA backbone and is a candidate for intersubunit communication. Homodimeric EcoRV T37A and K38A variants have a 1000-fold reduced catalytic activity. To examine whether Thr 37 and Lys 38 of one subunit affect the catalytic center in the same subunit and/or in the other subunit, we have produced heterodimeric variants containing a Thr 37 --> Ala or Lys 38 --> Ala substitution in one subunit combined with a wild type (wt) subunit (wt/T37A and wt/K38A) or with a subunit which contains an amino acid substitution (Asp 90 --> Ala) in the active site (D90A/T37A and D90A/K38A). Cleavage experiments with supercoiled pAT153 show that wt/T37A and wt/K38A preferentially nick the DNA. A steady-state kinetic analysis of the cleavage of an oligodeoxynucleotide substrate shows that the activity of wt/T37A and wt/K38A is half of that of wild type EcoRV, whereas D90A/T37A and D90A/K38A are almost inactive. These results demonstrate that Thr 37 and Lys 38 affect primarily the catalytic center in their own subunit and that both subunits of EcoRV can be activated independently of each other. We suggest that Thr 37 and Lys 38 control the catalytic activity of the active site in their own subunit by positioning alpha-helix B.     

The 2.0 A structure of a cross-linked complex between snowdrop lectin and a branched mannopentaose: evidence for two unique binding modes

BACKGROUND: Galanthus nivalis agglutinin (GNA), a mannose-specific lectin from snowdrop bulbs, is a tetrameric member of the family of Amaryllidaceae lectins that exhibit antiviral activity towards HIV. Its subunits are composed of three pseudo-symmetrically related beta sheet domains, each with a conserved mannose-binding site. Crystal structures of monosaccharide and disaccharide complexes of GNA have revealed that all 12 binding sites of the tetramer are functional, and that the degree of occupancy is dependent on the availability of subsidiary interactions from neighboring subunits. The complex of GNA with a branched mannopentaose ((Manalpha1,6-(alpha1, 3-Man)Man-alpha1,6-(alpha1,3-Man)Man) described here simulates a more biologically relevant complex. RESULTS: Two unique mannopentaose binding modes co-exist in the tetragonal structure (1 subunit/asymmetric unit) of the complex. In one, the conserved monosaccharide-binding pocket in domain 1 (CRD 1) is utilized for cross-linkage of twofold related GNA dimers by the outer 3,6 tri-Man arm, which alternates between two orientations consistent with crystal symmetry. Inter-linked dimers assemble helically along the 41 crystal axis forming a pore-like structure. In the second binding mode, the complete 3,6 tri-Man arm binds to an extended binding region in domain 3 (CRD 3) with subsites for each terminal Man and the internal Man positioned in the conserved monosaccharide pocket. The two remaining mannose residues are not visible in either binding mode. CONCLUSIONS: This structure provides insights into possible mechanisms of the cross-linkage that is known to occur when lectins interact with specific multivalent cell surface receptors during events such as agglutination and mitogenic stimulation. By virtue of the large number of sites available for mannose binding, GNA has multiple possibilities of forming unique lattice structures. The two distinctly different binding modes observed in this study confirm that high affinity mannose binding occurs only at the two domain sites located near dimer interfaces.     

Role of the prenyl group on the G protein gamma subunit in coupling trimeric G proteins to A1 adenosine receptors

The coupling of receptors to heterotrimeric G proteins is determined by interactions between the receptor and the G protein alpha subunits and by the composition of the betagamma dimers. To determine the role of the gamma subunit prenyl modification in this interaction, the CaaX motifs in the gamma1 and gamma2 subunits were altered to direct modification with different prenyl groups, recombinant betagamma dimers expressed in the baculovirus/Sf9 insect cell system, and the dimers purified. The activity of the betagamma dimers was compared in two assays: formation of the high affinity agonist binding conformation of the A1 adenosine receptor and receptor-catalyzed exchange of GDP for GTP on the alpha subunit. The beta1gamma1 dimer (modified with farnesyl) was significantly less effective than beta1gamma2 (modified with geranylgeranyl) in either assay. The beta1gamma1-S74L dimer (modified with geranylgeranyl) was nearly as effective as beta1gamma2 in either assay. The beta1gamma2-L71S dimer (modified with farnesyl) was significantly less active than beta1gamma2. Using 125I-labeled betagamma subunits, it was determined that native and altered betagamma dimers reconstituted equally well into Sf9 membranes containing A1 adenosine receptors. These data suggest that the prenyl group on the gamma subunit is an important determinant of the interaction between receptors and G protein gamma subunits.     

Hin recombinase bound to DNA: the origin of specificity in major and minor groove interactions

The structure of the 52-amino acid DNA-binding domain of the prokaryotic Hin recombinase, complexed with a DNA recombination half-site, has been solved by x-ray crystallography at 2.3 angstrom resolution. The Hin domain consists of a three-alpha-helix bundle, with the carboxyl-terminal helix inserted into the major groove of DNA, and two flanking extended polypeptide chains that contact bases in the minor groove. The overall structure displays features resembling both a prototypical bacterial helix-turn-helix and the eukaryotic homeodomain, and in many respects is an intermediate between these two DNA-binding motifs. In addition, a new structural motif is seen: the six-amino acid carboxyl-terminal peptide of the Hin domain runs along the minor groove at the edge of the recombination site, with the peptide backbone facing the floor of the groove and side chains extending away toward the exterior. The x-ray structure provides an almost complete explanation for DNA mutant binding studies in the Hin system and for DNA specificity observed in the Hin-related family of DNA invertases.     

Structure-function relationships and flexible tetramer assembly in pyruvate decarboxylase revealed by analysis of crystal structures

The crystal structures of pyruvate decarboxylase from the yeast Saccharomyces uvarum and Saccharomyces cerevisiae have been determined at 2.4 and 2.3 A resolution, respectively. These structures provide details about the protein fold and domain assembly within subunits, about subunit assembly to form dimers and about dimer assembly to form tetramers. They also provide a clear picture of the active site centered on the thiamin diphosphate cofactor, and have allowed amino acids critical for catalysis and involved in stabilization of the unusual cofactor conformation to be identified. The structural information has enabled identification of the site of allosteric activation to be centered on Cys-221, and suggests that a six residue segment leading from the regulatory site to the catalytic site may be involved in transmission of a binding signal. The importance of several amino acids within this segment in the regulatory process, as well as some involved in stabilizing and activating the cofactor has been confirmed by analyzing the behavior of recombinant enzymes with single point mutations introduced at these sites. Additional structures have been determined for pyruvate decarboxylase in multiple crystal forms, some of which were obtained from crystals grown with known allosteric activators present in the media. Currently four distinct types of tetramers have been observed, with each showing a different mode of association of dimers to form the tetramers. In some of the cases involving the presence of allosteric activators drastic changes in the mode of dimer assembly to form tetramers is seen.     

ATP hydrolysis catalyzed by human replication factor C requires participation of multiple subunits

Human replication factor C (hRFC) is a five-subunit protein complex (p140, p40, p38, p37, and p36) that acts to catalytically load proliferating cell nuclear antigen onto DNA, where it recruits DNA polymerase delta or epsilon to the primer terminus at the expense of ATP, leading to processive DNA synthesis. We have previously shown that a subcomplex of hRFC consisting of three subunits (p40, p37, and p36) contained DNA-dependent ATPase activity. However, it is not clear which subunit(s) hydrolyzes ATP, as all five subunits include potential ATP binding sites. In this report, we introduced point mutations in the putative ATP-binding sequences of each hRFC subunit and examined the properties of the resulting mutant hRFC complex and the ATPase activity of the hRFC or the p40.p37.p36 complex. A mutation in any one of the ATP binding sites of the p36, p37, p40, or p140 subunits markedly reduced replication activity of the hRFC complex and the ATPase activity of the hRFC or the p40.p37.p36 complex. A mutation in the ATP binding site of the p38 subunit did not alter the replication activity of hRFC. These findings indicate that the replication activity of hRFC is dependent on efficient ATP hydrolysis contributed to by the action of four hRFC subunits.     

Fatty acid synthase dimers containing catalytically active beta-ketoacyl synthase or malonyl/acetyltransferase domains in only one subunit can support fatty acid synthesis at the acyl carrier protein domains of both subunits

A double-tagging, dual affinity chromatographic procedure, which permits isolation of dimers independently mutated in each subunit, has been exploited to probe the functional topology of the animal fatty acid synthase. Dimers were engineered in which the chain-terminating thioesterase reaction was compromised by mutation of the (active-site) serine residue in both subunits; these dimers assembled two long-chain fatty acyl moieties, which remained covalently linked to the 4'-phosphopantetheine residues of the two acyl carrier protein domains. Significantly, dimers that contained an additional mutation that compromised the activity of either the beta-ketoacyl synthase or malonyl/acetyltransferase activity in only one subunit also assembled two long-chain acyl moieties. In contrast, in a control experiment, introduction of an additional mutation that compromised the function of the acyl carrier protein domain in only one subunit resulted in the assembly of only one long-chain acyl moiety per dimer. Because the beta-ketoacyl synthase and malonyl/acetyltransferase domains are located near the amino terminus of the polypeptide and the acyl carrier protein domain near the carboxyl terminus, these results support a modified model for the animal fatty acid synthase in which head-to-tail functional contacts are possible both within as well as between subunits.     

Conversion of a beta-strand to an alpha-helix induced by a single-site mutation observed in the crystal structure of Fis mutant Pro26Ala

The conversion from an alpha-helix to a beta-strand has received extensive attention since this structural change may induce many amyloidogenic proteins to self-assemble into fibrils and cause fatal diseases. Here we report the conversion of a peptide segment from a beta-strand to an alpha-helix by a single-site mutation as observed in the crystal structure of Fis mutant Pro26Ala determined at 2.0 A resolution. Pro26 in Fis occurs at the point where a flexible extended beta-hairpin arm leaves the core structure. Thus it can be classified as a "hinge proline" located at the C-terminal end of the beta2-strand and the N-terminal cap of the A alpha-helix. The replacement of Pro26 to alanine extends the A alpha-helix for two additional turns in one of the dimeric subunits; therefore, the structure of the peptide from residues 22 to 26 is converted from a beta-strand to an alpha-helix. This result confirms the structural importance of the proline residue located at the hinge region and may explain the mutant's reduced ability to activate Hin-catalyzed DNA inversion. The peptide (residues 20 to 26) in the second monomer subunit presumably retains its beta-strand conformation in the crystal; therefore, this peptide shows a "chameleon-like" character since it can adopt either an alpha-helix or a beta-strand structure in different environments. The structure of Pro26Ala provides an additional example where not only the protein sequence, but also non-local interactions determine the secondary structure of proteins.     

Comparison of the sequence-selective DNA binding by peptide dimers with covalent and noncovalent dimerization domains

Sequence-specific DNA binding proteins generally consist of more than two DNA-contacting regions to ensure the selectivity of recognition. The multiple DNA binding modules are connected either through the covalent linker or through the noncovalent dimerization domain. We have compared the DNA binding of peptide dimers with covalent and noncovalent dimerization domains to explore the potential advantage of each linkage on the sequence-specific DNA binding. Three sets of head-to-tail peptide dimers were synthesized by using the same basic region peptide to target the same DNA sequence; one dimer was assembled with a bridged biphenyl derivative as a covalent dimerization domain, and two other dimers were assembled with the cyclodextrin guest noncovalent dimerization domains. One of the noncovalent dimers was a heterodimer that consisted of cyclodextrin and guest peptides, while the other was a homodimer that consisted of peptides bearing both cyclodextrin and the guest molecule within the same chain. Both noncovalent dimers formed the specific DNA complexes within narrower ranges of peptide concentrations and showed higher sequence selectivity than the covalent dimer did. Among the three dimers, the noncovalent homodimer that can form an intramolecular inclusion complex showed the highest sequence selectivity. Because the noncovalent homodimer with the higher stability of the circular intramolecular inclusion complex exhibited the higher sequence selectivity, it was concluded that an equilibrium involving a conformational transition of a monomeric peptide effectively reduced the stability of its nonspecific binding complex, hence increasing the efficacy of cooperative dimer formation at the specific DNA sequence.     

Single chain dimers of MASH-1 bind DNA with enhanced affinity

By designing recombinant genes containing tandem copies of the coding region of the BHLH domain of MASH-1 (MASH-BHLH) with intervening DNA sequences encoding linker sequences of 8 or 17 amino acids, the two subunits of the MASH dimer have been connected to form the single chain dimers MM8 and MM17. Despite the long and flexible linkers which connect the C-terminus of the first BHLH subunit to the N-terminus of the second, a distance of approximately 55 A, the single chain dimers could be produced in Escherichia coli at high levels. MM8 and MM17 were monomeric and no 'cross-folding' of the subunits was observed. CD spectroscopy revealed that, like wild-type MASH-BHLH, MM8 and MM17 adopt only partly folded structures in the absence of DNA, but undergo a folding transition to a mainly alpha-helical conformation on DNA binding. Titrations by electrophoretic mobility shift assays revealed that the affinity of the single chain dimers for E box-containing DNA sequences was increased approximately 10-fold when compared with wild-type MASH-BHLH. On the other hand, the affinity for heterologous DNA sequences was increased only 5-fold. Therefore, the introduction of the peptide linker led to a 4-fold increase in DNA binding specificity from -0.14 to -0.57 kcal/mol.