The refined structure of canavalin from jack bean in two crystal forms at 2.1 and 2.0 Å resolution

The structure of canavalin was refined to 2.1 and 2.0 A resolution in cubic and hexagonal crystals of space group P2(1)3 and P6(3), respectively. The threefold molecular symmetry is expressed in the symmetry of both crystals, where each identical subunit is an asymmetric unit. The canavalin subunit consists of two very similar domains, each comprised of a core subdomain having Swiss-roll topology with a loop subdomain that contains helices. The refined canavalin models resolved the discrepancy in amino-acid registers of the secondary-structural elements compared with phaseolin. The presence of strand Z in both domains of canavalin was confirmed and a new helix in the loop between strands A and B of each domain was observed. The models were analyzed in terms of the duplicated vicilin domains. Three strictly conserved residues, two glycines and a proline, were identified. The similarity between entire vicilin molecules is greater than that between separate domains of canavalin and phaseolin. Homology modeling of the sucrose-binding protein (SBP) from soybean showed a plausible trimeric assembly of subunits similar to that of vicilins

X-ray diffraction and atomic force microscopy analysis of twinned crystals: rhombohedral canavalin

The structure of canavalin, the vicilin-class storage protein from jack bean, was refined to 1.7 A resolution in a highly twinned rhombohedral crystal of space group R3 and unit-cell parameters a = b = c = 83.0 A, alpha = beta = gamma = 111.1 degrees. The resulting R and R(free) were 0.176 and 0.245, respectively. The orthorhombic crystal structure (space group C222(1), unit-cell parameters a = 136.5, b = 150.3, c = 133.4 A) was also refined with threefold non-crystallographic symmetry restraints. R and R(free) were 0.181 and 0.226, respectively, for 2.6 A resolution data. No significant difference in the protein structure was seen between these two crystal forms, nor between these two and the hexagonal and cubic crystal forms reported elsewhere [Ko et al. (1993), Acta Cryst. D49, 478-489; Ko et al. (1993), Plant Physiol. 101, 729-744]. A phosphate ion was identified in the lumen of the C-terminal beta-barrel. Lattice interactions showed that the trimeric molecule could be well accommodated in both 'top-up' and 'bottom-up' orientations in a rhombohedral unit cell of the R3 crystal and explained the presence of a high twin fraction. The large inter-trimer stacking interface of the C222(1) crystal may account for its relative stability. Atomic force microscopy (AFM) investigations of the growth of three crystal forms of canavalin indicate the rhombohedral form to be unique. Unlike the other two crystal forms, it contains at least an order of magnitude more screw dislocations and stacking faults than any other macromolecular crystal yet studied, and it alone grows principally by generation of steps from the screw dislocations. The unusually high occurrence of the screw dislocations and stacking faults is attributed to mechanical stress produced by the alternate molecular orientations in the rhombohedral crystals and their organization into discrete domains or blocks. At boundaries of alternate domains, lattice strain is relieved by the formation of the screw dislocations

The crystal structure of the DNase domain of colicin E7 in complex with its inhibitor Im7 protein

AbstractBackground: Colicin E7 (ColE7) is one of the bacterial toxins classified as a DNase-type E-group colicin. The cytotoxic activity of a colicin in a colicin-producing cell can be counteracted by binding of the colicin to a highly specific immunity protein. This biological event is a good model system for the investigation of protein recognition.Results: The crystal structure of a one-to-one complex between the DNase domain of colicin E7 and its cognate immunity protein Im7 has been determined at 2.3 Å resolution. Im7 in the complex is a varied four-helix bundle that is identical to the structure previously determined for uncomplexed Im7. The structure of the DNase domain of ColE7 displays a novel α/β fold and contains a Zn2+ ion bound to three histidine residues and one water molecule in a distorted tetrahedron geometry. Im7 has a V-shaped structure, extending two arms to clamp the DNase domain of ColE7. One arm (α1∗–loop12–α2∗; where ∗ represents helices in Im7) is located in the region that displays the greatest sequence variation among members of the immunity proteins in the same subfamily. This arm mainly uses acidic sidechains to interact with the basic sidechains in the DNase domain of ColE7. The other arm (loop 23–α3∗–loop 34) is more conserved and it interacts not only with the sidechain but also with the mainchain atoms of the DNase domain of ColE7.Conclusions: The protein interfaces between the DNase domain of ColE7 and Im7 are charge-complementary and charge interactions contribute significantly to the tight and specific binding between the two proteins. The more variable arm in Im7 dominates the binding specificity of the immunity protein to its cognate colicin. Biological and structural data suggest that the DNase active site for ColE7 is probably near the metal-binding site

Structure of orthorhombic crystals of beef liver catalase

The growth mechanisms and physical properties of the orthorhombic crystal form of beef liver catalase were investigated using in situ atomic force microscopy (AFM). It was observed that the crystals grow in the <001> direction by an unusual progression of sequential two-dimensional nuclei of half unit-cell layers corresponding to the 'bottoms' and 'tops' of unit cells. These were easily discriminated by their alternating asymmetric shapes and their strong growth-rate anisotropy. This pattern has not previously been observed with other macromolecular crystals. Orthorhombic beef liver catalase crystals exhibit an extremely high defect density and incorporate great numbers of misoriented microcrystals, revealed intact by etching experiments, which may explain their marginal diffraction properties. To facilitate interpretation of AFM results in terms of intermolecular interactions, the structure of the orthorhombic crystals, having an entire tetramer of the enzyme as the asymmetric unit, was solved by molecular replacement using a model derived from a trigonal crystal form. It was subsequently refined by conventional techniques. Although the packing of molecules in the two unit cells was substantially different, with very few exceptions no significant differences in the molecular structures were observed. In addition, no statistically significant deviation from ideal 222 molecular symmetry appeared within the tetramer. The packing of molecules in the crystal revealed by X-ray analysis explained in a satisfying way the process of crystal growth revealed by AFM

Probing the DNA kink structure induced by the hyperthermophilic chromosomal protein Sac7d

Sac7d, a small, abundant, sequence-general DNA-binding protein from the hyperthermophilic archaeon Sulfolobus acidocaldarius, causes a single-step sharp kink in DNA (∼60°) via the intercalation of both Val26 and Met29. These two amino acids were systematically changed in size to probe their effects on DNA kinking. Eight crystal structures of five Sac7d mutant–DNA complexes have been analyzed. The DNA-binding pattern of the V26A and M29A single mutants is similar to that of the wild-type, whereas the V26A/M29A protein binds DNA without side chain intercalation, resulting in a smaller overall bending (∼50°). The M29F mutant inserts the Phe29 side chain orthogonally to the C2pG3 step without stacking with base pairs, inducing a sharp kink (∼80°). In the V26F/M29F-GCGATCGC complex, Phe26 intercalates deeply into DNA bases by stacking with the G3 base, whereas Phe29 is stacked on the G15 deoxyribose, in a way similar to those used by the TATA box-binding proteins. All mutants have reduced DNA-stabilizing ability, as indicated by their lower T(m) values. The DNA kink patterns caused by different combinations of hydrophobic side chains may be relevant in understanding the manner by which other minor groove-binding proteins interact with DNA

Crystal structure and substrate specificity of plant adenylate isopentenyltransferase from Humulus lupulus: distinctive binding affinity for purine and pyrimidine nucleotides

Cytokinins are important plant hormones, and their biosynthesis most begins with the transfer of isopentenyl group from dimethylallyl diphosphate (DMAPP) to the N6-amino group of adenine by either adenylate isopentenyltransferase (AIPT) or tRNA–IPT. Plant AIPTs use ATP/ADP as an isopentenyl acceptor and bacterial AIPTs prefer AMP, whereas tRNA–IPTs act on specific sites of tRNA. Here, we present the crystal structure of an AIPT–ATP complex from Humulus lupulus (HlAIPT), which is similar to the previous structures of Agrobacterium AIPT and yeast tRNA–IPT. The enzyme is structurally homologous to the NTP-binding kinase family of proteins but forms a solvent-accessible channel that binds to the donor substrate DMAPP, which is directed toward the acceptor substrate ATP/ADP. When measured with isothermal titration calorimetry, some nucleotides displayed different binding affinities to HlAIPT with an order of ATP > dATP ∼ ADP > GTP > CTP > UTP. Two basic residues Lys275 and Lys220 in HlAIPT interact with the β and γ-phosphate of ATP. By contrast, the interactions are absent in Agrobacterium AIPT because they are replaced by the acidic residues Asp221 and Asp171. Despite its structural similarity to the yeast tRNA–IPT, HlAIPT has evolved with a different binding strategy for adenylate

Crystal structure of the left-handed archaeal RadA helical filament: identification of a functional motif for controlling quaternary structures and enzymatic functions of RecA family proteins

The RecA family of proteins mediates homologous recombination, an evolutionarily conserved pathway that maintains genomic stability by protecting against DNA double strand breaks. RecA proteins are thought to facilitate DNA strand exchange reactions as closed-rings or as right-handed helical filaments. Here, we report the crystal structure of a left-handed Sulfolobus solfataricus RadA helical filament. Each protomer in this left-handed filament is linked to its neighbour via interactions of a β-strand polymerization motif with the neighbouring ATPase domain. Immediately following the polymerization motif, we identified an evolutionarily conserved hinge region (a subunit rotation motif) in which a 360° clockwise axial rotation accompanies stepwise structural transitions from a closed ring to the AMP–PNP right-handed filament, then to an overwound right-handed filament and finally to the left-handed filament. Additional structural and functional analyses of wild-type and mutant proteins confirmed that the subunit rotation motif is crucial for enzymatic functions of RecA family proteins. These observations support the hypothesis that RecA family protein filaments may function as rotary motors

Crystal structure of IcaR, a repressor of the TetR family implicated in biofilm formation in Staphylococcus epidermidis

Expression of the gene cluster icaADBC is necessary for biofilm production in Staphylococcus epidermidis. The ica operon is negatively controlled by the repressor IcaR. Here, the crystal structure of IcaR was determined and the refined structure revealed a homodimer comprising entirely α-helices, typical of the tetracycline repressor protein family for gene regulations. The N-terminal domain contains a conserved helix-turn-helix DNA-binding motif with some conformational variations, indicating flexibility in this region. The C-terminal domain shows a complementary surface charge distribution about the dyad axis, ideal for efficient and specific dimer formation. The results of the electrophoretic mobility shift assay and isothermal titration calorimetry suggested that a 28 bp core segment of the ica operator is implicated in the cooperative binding of two IcaR dimers on opposite sides of the duplex DNA. Computer modeling based on the known DNA-complex structure of QacR and site-specific mutagenesis experiments showed that direct protein–DNA interactions are mostly conserved, but with slight variations for recognizing the different sequences. By interfering with the binding of IcaR to DNA, aminoglycoside gentamicin and other antibiotics may activate the icaADBC genes and elicit biofilm production in S. epidermidis, and likely S. aureus, as a defense mechanism

Structural and Functional Analyses of Five Conserved Positively Charged Residues in the L1 and N-Terminal DNA Binding Motifs of Archaeal RadA Protein

RecA family proteins engage in an ATP-dependent DNA strand exchange reaction that includes a ssDNA nucleoprotein helical filament and a homologous dsDNA sequence. In spite of more than 20 years of efforts, the molecular mechanism of homology pairing and strand exchange is still not fully understood. Here we report a crystal structure of Sulfolobus solfataricus RadA overwound right-handed filament with three monomers per helical pitch. This structure reveals conformational details of the first ssDNA binding disordered loop (denoted L1 motif) and the dsDNA binding N-terminal domain (NTD). L1 and NTD together form an outwardly open palm structure on the outer surface of the helical filament. Inside this palm structure, five conserved basic amino acid residues (K27, K60, R117, R223 and R229) surround a 25 Å pocket that is wide enough to accommodate anionic ssDNA, dsDNA or both. Biochemical analyses demonstrate that these five positively charged residues are essential for DNA binding and for RadA-catalyzed D-loop formation. We suggest that the overwound right-handed RadA filament represents a functional conformation in the homology search and pairing reaction. A new structural model is proposed for the homologous interactions between a RadA-ssDNA nucleoprotein filament and its dsDNA target