Eila Cedergren-Zeppezauer

Homotrimeric dUTPases; structural solutions for specific recognition and hydrolysis of dUTP.

Prevention of incorporation of dUTP into DNA is essential for maintenance of the genetic information. Prompt and specific removal of dUTP from the nucleotide pool, as expedited by the ubiquitous enzyme dUTPase, is therefore required for full viability in most biological systems. Conserved structural features perpetuate specificity in choice of substrate, which is crucial as hydrolysis of the structurally closely related nucleotides dTTP, dCTP and UTP would debilitate DNA and RNA synthesis. The most common family of dUTPases is the homotrimeric variety where X-ray structures are available for one bacterial, one mammalian and two retroviral dUTPases. These four enzymes have similar overall structural layouts, but the interactions that stabilise the trimer vary markedly, ranging from exclusively hydrophobic to water-mediated interactions. Trimeric dUTPases contain five conserved sequence motifs, positioned at the subunit interfaces where they contribute to the formation of the active sites. Each of the three identical active sites per trimer is built of residues contributed by all three subunits. One subunit provides residues involved in base and sugar recognition, where a beta-hairpin acts to maintain exquisite selectivity, while a second subunit contributes residues for phosphate interactions. The third subunit supplies a glycine-rich consensus motif located in the flexible C-terminal part of the subunit, known from crystallographic studies to cover the active site in the presence of substrate and certain substrate analogues. All dUTPases studied require the presence of a divalent metal ion, preferably Mg(2+), for optimal activity. The putative position of the essential metal ion has been identified in the structure of one retroviral dUTPase. Structure-function studies are essential if the properties of dUTPases are to be understood fully in relation to their biological role. In this review the structural arrangement of the homotrimeric dUTPases is discussed in the context of active site geometry, achievement of specificity and subunit interactions.

Atomic resolution structure of Escherichia coli dUTPase determined ab initio

Cryocooled crystals of a mercury complex of Escherichia coli dUTPase diffract to atomic resolution. Data to 1.05 A resolution were collected from a derivative crystal and the structure model was derived from a Fourier map with phases calculated from the coordinates of the Hg atom (one site per subunit of the trimeric enzyme) using the program ARP/wARP. After refinement with anisotropic temperature factors a highly accurate model of the bacterial dUTPase was obtained. Data to 1.45 A from a native crystal were also collected and the 100 K structures were compared. Inspection of the refined models reveals that a large part of the dUTPase remains rather mobile upon freezing, with 14% of the main chain being totally disordered and with numerous side chains containing disordered atoms in multiple discrete conformations. A large number of those residues surround the active-site cavity. Two glycerol molecules (the cryosolvent) occupy the deoxyribose-binding site. Comparison between the native enzyme and the mercury complex shows that the active site is not adversely affected by the binding of mercury. An unexpected effect seems to be a stabilization of the crystal lattice by means of long-range interactions, making derivatization a potentially useful tool for further studies of inhibitor-substrate-analogue complexes of this protein at very high resolution.

REFINED STRUCTURE OF CU-SUBSTITUTED ALCOHOL DEHYDROGENASE AT 2.1 A RESOLUTION

Liver alcohol dehydrogenase (LADH) is a Zn(II)-dependent dimeric enzyme. LADH with the active-site Zn(II) substituted by Cu(II) resembles blue (type I) copper proteins by its spectroscopic characteristics. In this work we present the X-ray structure of the active site Cu(II)-substituted LADH complex with NADH and dimethyl sulfoxide (DMSO). The structure was solved by molecular replacement. The space group is P2(1) with cell dimensions a = 44.4, b = 180.6, c = 50.8 A and beta = 108 degrees. There is one dimer of the enzyme in the asymmetric unit. The refinement was carried out to a crystallographic R-factor of 16.1% for 41 119 unique reflections in the resolution range 12.0 to 2.1 A. The coordination geometry of Cu(II) in LADH is compared with the active-site metal coordination in the Zn-LADH-NADH-DMSO complex and blue-copper proteins. The distances from the metal to the protein ligands (Cys46, His67 and Cys174) are similar for the Zn(II) and Cu(II) ions. The distances of the O atom of the inhibitor DMSO to the Cu(II) ion in the two subunits of the dimer are 3.19 and 3.45 A. These are considerably longer than the corresponding distances for the Zn(II) enzyme, 2.19 and 2.15 A. The Cu(II) ion is positioned nearly in the plane of the three protein ligands (NS(2)) with a geometry similar to the trigonal arrangement of the three strongly bound ligands (N(2)S) in blue-copper proteins. This coordination probably accounts for the similarity of the spectral characteristics of Cu(II)-LADH and type I copper proteins.

A variety of electrostatic interactions and adducts can activate NAD(P) cofactors for hydride transfer.

In NAD(P)-dependent enzymes the coenzyme gives or takes a hydride ion, but how the nicotinamide ring is activated to form the transition state for hydride transfer is not clear. On the basis of ultra-high resolution X-ray crystal structures of liver alcohol dehydrogenase (LADH) in complex with NADH and a number of substrate analogues we proposed that the activation of NADH is an integral part of the enzyme mechanism of aldehyde reduction [R. Meijers, R.J. Morris, H.W. Adolph, A. Merli, V.S. Lamzin, E.S. Cedergren-Zeppezauer, On the enzymatic activation of NADH, The Journal of Biological Chemistry 276(12) (2001) 9316-9321, %U http://www.ncbi.nlm.nih.gov/pubmed/11134046; R. Meijers, H.-W. Adolph, Z. Dauter, K.S. Wilson, V.S. Lamzin, E.S. Cedergren-Zeppezauer, Structural evidence for a ligand coordination switch in liver alcohol dehydrogenase, Biochemistry 46(18) (2007) 5446-5454, %U http://www.ncbi.nlm.nih.gov/pubmed/17429946]. We observed a nicotinamide with a severely distorted pyridine ring and a water molecule in close proximity to the ring. Quantum chemical calculations indicated that (de)protonation of the water molecule can be directly coupled to activation of NADH for hydride transfer. A systematic search of the Protein Data Bank (PDB) for atoms that come within van der Waals distance of the pyridine ring of the nicotinamide reveals that a large number of NAD(P)-containing protein complexes are involved in electrostatic interactions with the enzymatic environment. Using the deposited diffraction data to analyze the cofactor and its surroundings, we observe several adducts between protein atoms and the pyridine ring that were not previously reported. This further indicates that the enzymatic activation of NAD(P) induced by electrostatic interactions is an essential part of the hydride transfer mechanism.

Crystallization and preliminary X-ray diffraction of Trypanosoma cruzi dUTPase

Crystals of Trypanosoma cruzi dUTPase have been grown. Two different morphologies are observed, depending on the molecular weight of the PEG used as precipitating agent in the mother liquor, both having a hexagonal unit cell with similar dimensions. Complete X-ray diffraction data have been collected to low resolution for one of the forms. The space group is P6322, with unit-cell dimensions a = 134.15, c = 147.05 A. Peaks in the self-rotation function and the solvent content are consistent with two molecules of dUTPase per asymmetric unit.

Airborn Chemistry Levitated Protein Droplets as a Novel Analytical Tool for Nucleation Screening in Macromolecular Crystallisation

A system of novel analytical tools for fast screening of protein nucleation conditions have been developed, based on acoustic levitation and piezo-electric flow-through micro-dispensing and volume measurement in nano-volumes. The system has been used for study different proteins ability to precipitate by testing different crystallization agents. From this data precipitation diagrams are created and “normal” protein-crystallization procedure can then be performed. Described methodology consumes only minute amounts of the precious protein