ABSTRACT
Here we report the crystal structure of a stablilized plasminogen activator inhibitor-1 variant (PAI-1-N150H-K154T-Q301P-Q319L-M354I (PAI-1-stab)) that shows a cleavage within the reactive centre loop. The new structure is of superior quality compared to the previously determined structure of the cleaved PAI-1-A335P mutant. We present a detailed comparison of the two structures and also compare them with the structure of the active PAI-1-stab. The structural data give important insights into the working mechanism of PAI-1 and also explain the role of various stabilizing mutations.
Subject(s)
Plasminogen Activator Inhibitor 1/genetics , Plasminogen Activator Inhibitor 1/physiology , Catalytic Domain , Crystallography, X-Ray , Humans , Models, Molecular , Molecular Conformation , Molecular Structure , Mutation , Protein Binding , Protein Conformation , Protein Structure, Tertiary , Serpins/chemistrySubject(s)
Endo-1,4-beta Xylanases/antagonists & inhibitors , Endo-1,4-beta Xylanases/metabolism , Enzyme Inhibitors/pharmacology , Triticum/enzymology , Bacillus subtilis/enzymology , Binding Sites , Edible Grain/enzymology , Endo-1,4-beta Xylanases/chemistry , Endo-1,4-beta Xylanases/genetics , Genetic Engineering , Kinetics , Mutagenesis, Site-Directed , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/chemistry , Recombinant Proteins/metabolismABSTRACT
The structure of a catalytically inactive RNase-related protein from Calystegia sepium (CalsepRRP) has been resolved by protein crystallography at a resolution of 2.05 A and an R factor of 20.74%. Although the protein is completely devoid of ribonuclease activity, it adopts the typical alpha + beta structure of non-base-specific RNases. Analysis of the structure revealed that two amino-acid substitutions in the 'active' P1 site, in combination with the less hydrophobic/aromatic character of the B1 base-recognition site and a completely disrupted B2 base-recognition site, might account for this complete lack of activity.
Subject(s)
Plant Proteins/chemistry , Amino Acid Sequence , Binding Sites , Crystallography, X-Ray , Models, Molecular , Molecular Sequence Data , Protein Structure, Secondary , Sequence AlignmentABSTRACT
The structure of the bark lectin RPbAI (isoform A4) from Robinia pseudoacacia has been determined by protein crystallography both in the free form and complexed with N-acetylgalactosamine. The free form is refined at 1.80 A resolution to an R-factor of 18.9% whereas the complexed structure has an R-factor of 19.7% at 2.05 A resolution. Both structures are compared to each other and to other available legume lectin structures. The polypeptide chains of the two structures exhibit the characteristic legume lectin tertiary fold. The quaternary structure resembles that of the Phaseolus vulgaris lectin, the soybean agglutinin, and the Dolichos biflorus lectin, but displays some unique features leading to the extreme stability of this lectin.
Subject(s)
Acacia/chemistry , Acetylglucosamine/metabolism , Lectins/chemistry , Lectins/metabolism , Acetylglucosamine/chemistry , Binding Sites , Crystallography, X-Ray , Hydrogen Bonding , Models, Molecular , Plant Lectins , Protein Binding , Protein Conformation , Static Electricity , Water/chemistry , Water/metabolismABSTRACT
The vitamin D binding protein binds globular actin with high affinity and is involved in the clearance of actin from the blood circulation. A complex of the human vitamin D binding protein and rabbit muscle actin was subjected to purification steps. The pure complex was crystallized using the hanging-drop vapour-diffusion procedure. The best obtained crystals belong to the monoclinic space group P2(1), with unit-cell parameters a = 74.44, b = 74.90, c = 88.02 A, beta = 110.19 degrees. A complete data set to 2.4 A was collected from a single crystal using synchrotron radiation at DESY, Hamburg, Germany.
Subject(s)
Actins/chemistry , Muscles/chemistry , Vitamin D-Binding Protein/chemistry , Actins/isolation & purification , Animals , Crystallization , Crystallography, X-Ray , Electrophoresis, Polyacrylamide Gel , Humans , Protein Conformation , Rabbits , Vitamin D-Binding Protein/isolation & purificationABSTRACT
MornigaM, a lectin from Morus nigra, belongs to the mannose-binding subgroup of the family of jacalin-related plant lectins. It was crystallized in the P6(5) space group, with unit-cell parameters a = b = 110.74, c = 159.28 A. The partially merohedrally twinned crystals could be detwinned and a subsequent molecular-replacement solution could be found using the coordinates of jacalin. Preliminary analysis clearly shows the tetrameric assembly of this protein. Furthermore, data from MornigaM crystals soaked in a mannose solution were collected.
Subject(s)
Lectins/chemistry , Magnoliopsida/chemistry , Mannose/metabolism , Crystallization , Interferon Inducers/chemistry , Lectins/metabolism , Plant Lectins , Protein Structure, Quaternary , Structure-Activity Relationship , Substrate Specificity , X-Ray DiffractionABSTRACT
The A(4) isoform of the bark lectin RPbAI from Robinia pseudoacacia has been crystallized in two different crystal forms. Crystal form I grows in the P2(1) space group with two tetramers in the asymmetric unit, whereas crystal form II grows in the I222 space group with a monomer in the asymmetric unit. Data sets were collected for both crystal forms to resolution limits of 2.55 and 1.81 A, respectively, which will allow successful structure determinations.
Subject(s)
Fabaceae/chemistry , Lectins/chemistry , Plants, Medicinal , Crystallization , Lectins/isolation & purification , Plant Lectins , Polymers/chemistry , Protein Conformation , X-Ray DiffractionABSTRACT
Because of its intrinsic lability, wild-type plasminogen activator inhibitor 1 (PAI-1) cannot be crystallized in its active conformation. Therefore, a stable variant of PAI-1 was used to retain the active conformation during crystallization. Four different crystallization conditions were evaluated in detail and two major types of crystals were detected. Whereas solutions consisting of either (i) cacodylate and sodium acetate, (ii) lithium sulfate and polyethylene glycol 4K, or (iii) imidazole, sodium chloride and sodium potassium phosphate buffer revealed thin platelet crystals, a solution (iv) containing ammonium acetate, citrate and polyethylene glycol 4K appeared to enhance the formation of clustered brush-like crystals. Crystals grown under condition (iii) were found to be suitable for X-ray data collection and consequent structural investigation. Data collection was 79.8% complete with a maximum resolution of 2.92 A. Importantly, PAI-1 retained its functional properties under all conditions.
Subject(s)
Plasminogen Activator Inhibitor 1/chemistry , Crystallization , Crystallography, X-Ray , Electrophoresis, Polyacrylamide Gel , Mutagenesis, Site-Directed , Plasminogen Activator Inhibitor 1/genetics , Protein Conformation , Recombinant Proteins/chemistry , Recombinant Proteins/geneticsABSTRACT
Two non-isomorphous monoclinic types of diffraction-quality crystals of Delta10Sak, a fully functional staphylokinase derivative lacking the first ten amino acids, have been grown by the hanging-drop vapour-diffusion technique. Type I crystals grow from a solution containing Zn(OAc)2, Tris buffer pH 7.5 and PEG 8000, while type II crystals can be obtained from a solution containing MgCl2, Tris buffer pH 8.5 and PEG 4000. Both crystal types were suitable for data collection (to 2.4 and 2.6 A resolution, respectively) and further structural investigation.
Subject(s)
Metalloendopeptidases/chemistry , Base Sequence , Crystallization , DNA Primers , Metalloendopeptidases/genetics , Protein Conformation , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Sequence DeletionABSTRACT
The three-dimensional structure of staphylokinase has been determined at 1.8 A. The puntative site of interaction with plasminogen was identified and epitopes were mapped.
Subject(s)
Metalloendopeptidases/chemistry , Plasminogen Activators/chemistry , Crystallography, X-Ray , Immunodominant Epitopes/analysis , Models, Molecular , Recombinant Proteins/chemistry , Staphylococcus aureus/enzymologyABSTRACT
Diffraction quality crystals of recombinant staphylokinase (STAR) have been grown by the hanging drop vapor diffusion technique from a solution containing MgCl2, Tris buffer (pH 8.5), and polyethylene glycol 4000. The crystals belong to the monoclinic space group C2 with unit cell dimensions alpha = 60.6 A, b = 43.7 A, c = 54.3 A, and beta = 115.6 degrees. A complete native data set to 1.8 A resolution has been collected using synchrotron radiation.
