Minh-Hoa Dao-Thi

THE CRYSTALLOGRAPHIC STRUCTURE OF PHYTOHEMAGGLUTININ-L

The structure of phytohemagglutinin-L (PHA-L), a leucoagglutinating seed lectin from Phaseolus vulgaris, has been solved with molecular replacement using the coordinates of lentil lectin as model, and refined at a resolution of 2.8 Å. The final R-factor of the structure is 20.0%. The quaternary structure of the PHA-L tetramer differs from the structures of the concanavalin A and peanut lectin tetramers, but resembles the structure of the soybean agglutinin tetramer. PHA-L consists of two canonical legume lectin dimers that pack together through the formation of a close contact between two β-strands. Of the two covalently bound oligosaccharides per monomer, only one GlcNAc residue per monomer is visible in the electron density. In this article we describe the structure of PHA-L, and we discuss the putative position of the high affinity adenine-binding site present in a number of legume lectins. A comparison with transthyretin, a protein that shows a remarkable resemblance to PHA-L, gives further ground to our proposal.

NMR, molecular modeling, and crystallographic studies of lentil lectin-sucrose interaction.

The conformational features of sucrose in the combining site of lentil lectin have been characterized through elucidation of a crystalline complex at 1.9-Å resolution, transferred nuclear Overhauser effect experiments performed at 600 Mhz, and molecular modeling. In the crystal, the lentil lectin dimer binds one sucrose molecule per monomer. The locations of 229 water molecules have been identified. NMR experiments have provided 11 transferred NOEs. In parallel, the docking study and conformational analysis of sucrose in the combining site of lentil lectin indicate that three different conformations can be accommodated. Of these, the orientation with lowest energy is identical with the one observed in the crystalline complex and provides good agreement with the observed transferred NOEs. These structural investigations indicate that the bound sucrose has a unique conformation for the glycosidic linkage, close to the one observed in crystalline sucrose, whereas the fructofuranose ring remains relatively flexible and does not exhibit any strong interaction with the protein. Major differences in the hydrogen bonding network of sucrose are found. None of the two inter-residue hydrogen bonds in crystalline sucrose are conserved in the complex with the lectin. Instead, a water molecule bridges hydroxyl groups O2-g and O3-f of sucrose.

The monosaccharide binding site of lentil lectin: an X-ray and molecular modelling study.

The X-ray crystal structure of lentil lectin in complex with α-d-glucopyranose has been determined by molecular replacement and refined to anR-value of 0.20 at 3.0 Å resolution. The glucose interacts with the protein in a manner similar to that found in the mannose complexes of concanavalin A, pea lectin and isolectin I fromLathyrus ochrus. The complex is stabilized by a network of hydrogen bonds involving the carbohydrate oxygens O6, O4, O3 and O5. In addition, the α-d-glucopyranose residue makes van der Waals contacts with the protein, involving the phenyl ring of Phe123β. The overall structure of lentil lectin, at this resolution, does not differ significantly from the highly refined structures of the uncomplexed lectin.Molecular docking studies were performed with mannose and its 2-O and 3-O-m-nitro-benzyl derivatives to explain their high affinity binding. The interactions of the modelled mannose with lentil lectin agree well with those observed experimentally for the protein-carbohydrate complex. The highly flexible Me-2-O-(m-nitro-benzyl)-α-d-mannopyranoside and Me-3-O-(m-nitro-benzyl)-α-d-mannopyranoside become conformationally restricted upon binding to lentil lectin. For best orientations of the two substrates in the combining site, the loss of entropy is accompanied by the formation of a strong hydrogen bond between the nitro group and one amino acid, Gly97β and Asn125β, respectively, along with the establishment of van der Waals interactions between the benzyl group and the aromatic amino acids Tyr100β and Trp128β.

Crystallization of CcdB in complex with a GyrA fragment.

Plasmid addiction systems consist of a plasmid-encoded toxin-antidote pair that serves to stabilize low-copy-number plasmids in bacterial populations. CcdB, the toxin from the ccd system on the Escherichia coli F plasmid, acts as a gyrase poison. A 14 kDa fragment of gyrase, GyrA14, was found to bind to the toxin CcdB with an affinity of 1.75 x 10(-8) M. Crystals of the (GyrA14)(2) dimer in its free state belong to space group P4(3)2(1)2, with unit-cell parameters a = 86.4, c = 89.4 angstroms, and diffract to 2.4 angstroms. Crystals of the (GyrA14)(2)-(CcdB)(2) complex belong to space group P2(1)2(1)2(1), with a = 52.1, b = 83.3, c = 110.9 angstroms, and diffract to 2.8 angstroms resolution.

Crystallization of Doc and the Phd–Doc toxin–antitoxin complex

The phd/doc addiction system is responsible for the stable inheritance of lysogenic bacteriophage P1 in its plasmidic form in Escherichia coli and is the archetype of a family of bacterial toxin-antitoxin modules. The His66Tyr mutant of Doc (Doc(H66Y)) was crystallized in space group P2(1), with unit-cell parameters a = 53.1, b = 198.0, c = 54.1 A, beta = 93.0 degrees . These crystals diffracted to 2.5 A resolution and probably contained four dimers of Doc in the asymmetric unit. Doc(H66Y) in complex with a 22-amino-acid C-terminal peptide of Phd (Phd(52-73Se)) was crystallized in space group C2, with unit-cell parameters a = 111.1, b = 38.6, c = 63.3 A, beta = 99.3 degrees , and diffracted to 1.9 A resolution. Crystals of the complete wild-type Phd-Doc complex belonged to space group P3(1)21 or P3(2)21, had an elongated unit cell with dimensions a = b = 48.9, c = 354.9 A and diffracted to 2.4 A resolution using synchrotron radiation.

Untangle, a tool for filtering overlapping diffraction patterns from multicrystals.

Standard crystallographic data-processing protocols are based on single-crystal models; data from aggregates of multiple crystals with different orientations are difficult to process. In certain cases, it is possible to separately index the diffraction patterns from the dominant crystals in the aggregate. Untangle is a program designed to identify and eliminate overlapping spots from such patterns in order to improve data quality. The program has a Python core with a simple and highly portable graphical user interface, permitting visual verification of the process and interactive modification of the overlap threshold. The software is available under an open-source license.

Crystallization of glycosylated and nonglycosylated phytohemagglutinin-L.

In the seeds of legume plants a class of sugar‐binding proteins can be found, generally called legume lectins. In this paper we present the crystallization of phytohemagglutinin‐L (PHA‐L), a glycosylated lectin from the seeds of the common bean (Phaseolus vulgaris). Single PHA‐L crystals were grown by vapor diffusion, using PEG as precipitant. The protein crystallizes in the monoclinic space group C2, and diffracts to a resolution of 2.7 Å. The unit cell parameters are a = 106.3 Å, b = 121.2 Å, c = 90.8 Å, and β = 93.7°. The asymmetric unit probably contains one PHA‐L tetramer. Crystals of a recombinant nonglycosylated form of PHA‐L, grown under identical conditions, and crystals of the native PHA‐L, grown in the presence of isopropanol, did not survive the mounting process.

Crystallization of the C-terminal domain of the addiction antidote CcdA in complex with its toxin CcdB

CcdA and CcdB are the antidote and toxin of the ccd addiction module of Escherichia coli plasmid F. The CcdA C-terminal domain (CcdAC36; 36 amino acids) was crystallized in complex with CcdB (dimer of 2 x 101 amino acids) in three different crystal forms, two of which diffract to high resolution. Form II belongs to space group P2(1)2(1)2(1), with unit-cell parameters a = 37.6, b = 60.5, c = 83.8 A and diffracts to 1.8 A resolution. Form III belongs to space group P2(1), with unit-cell parameters a = 41.0, b = 37.9, c = 69.6 A, beta = 96.9 degrees, and diffracts to 1.9 A resolution.

Quaternary Structure of UEA-II, the Chitobiose Specific Lectin from Gorse

The chitobiose specific Ulex europaeus lectin II crystallizes in space group P3221 with unit-cell dimensions a = b = 105.54, c = 176.26 A. The asymmetric unit contains a complete lectin tetramer. The crystals were shown to diffract to 4.5 A on a rotating-anode source and to 2.7 A at the Daresbury synchrotron source. Molecular replacement and subsequent rigid-body refinement using data to 4.5 A yielded a solution corresponding to a tetramer very similar to that of phytohemagglutinin-L and soybean agglutinin. The monomers in the Ulex lectin tetramer are rotated approximately 5 degrees compared with the phytohemagglutinin-L and soybean agglutinin structures.

CRYSTALLIZATION OF CCDB

CcdB is a small dimeric protein that poisons DNA-topoisomerase II complexes. Its crystallization properties in terms of precipitant type, precipitant concentration, pH and protein concentration have been investigated leading to a novel crystal form which, in contrast to previously reported crystals, is suitable for structure determination using the multiple isomorphous replacement (MIR) method. The space group of this new form is C2, with unit-cell parameters a = 74.94, b = 36.24, c = 35.77 A, beta = 115.27 degrees. The asymmetric unit contains a single monomer. Flash-frozen crystals diffract to at least 1.5 A resolution, while room-temperature diffraction can be observed up to 1.6 A. The double mutant S74C/G77Q, which acts as a super-killer, crystallizes in space group I222 (or I212121) with unit-cell dimensions a = 105.58, b = 105.80, c = 91.90 A. These crystals diffract to 2.5 A resolution.