MOLREP is an automated program for molecular replacement that utilizes a number of original approaches to rotational and translational search and data preparation. Since the first publication describing the program, MOLREP has acquired a variety of features that include weighting of the X-ray data and search models, multi-copy search, fitting the model into electron density, structural superposition of two models and rigid-body refinement. The program can run in a fully automatic mode using optimized parameters calculated from the input data.

The molecular-replacement method has been extended to a simultaneous search for multiple copies of the macromolecule in the unit cell. The central point of this approach is the construction of a multi-copy search model from the properly oriented monomers using a special translation function. The multi-copy search method has been implemented in the program MOLREP and successfully tested using experimental data.

D-amino acid oxidase is the prototype of the FAD-dependent oxidases. It catalyses the oxidation of Damino acids to the corresponding a-ketoacids. The reducing equivalents are transferred to molecular oxygen with production of hydrogen peroxide. We have solved the crystal structure of the complex of D-amino acid oxidase with benzoate, a competitive inhibitor of the substrate, by single isomorphous replacement and eightfold averaging. Each monomer is formed by two domains with an overall topology similar to that ofp-hydroxybenzoate hydroxylase. The benzoate molecule lays parallel to the flavin ring and is held in position by a salt bridge with Arg-283. Analysis of the active site shows that no side chains are properly positioned to act as the postulated base required for the catalytic carboanion mechanism. On the contrary, the benzoate binding mode suggests a direct transfer of the substrate a-hydrogen to the flavin during the enzyme reductive half-reaction. The active site of D-amino acid oxidase exhibits a striking similarity with that of flavocytochrome b2, a structurally unrelated FMN-dependent flavoenzyme. The active site groups of these two enzymes are in fact superimposable once the mirror-image of the flavocytochrome b2 active site is generated with respect to the flavin plane. Therefore, the catalytic sites of D-amino acid oxidase and flavocytochrome b2 appear to have converged to a highly similar but enantiomeric architecture in order to catalyze similar reactions (oxidation of a-amino acids or a-hydroxy acids), although with opposite stereochemistry.Since the description of D-amino acid oxidase (EC 1.4.3.3; DAAO) activity in mammalian tissues by Krebs in 1935 (1), DAAO has been the subject of a number of biochemical, spectroscopic, and kinetic investigations, becoming the prototype for the oxidase class of the flavin-containing enzymes [for a recent review, see ref. 2]. Its primary structure has been determined and its gene has been cloned (3, 4). Its kinetic and mechanistic properties have been studied in detail by a variety of techniques, while information on the topology of the active site and on its three-dimensional structure have only been derived from chemical modification studies and site-directed mutagenesis of selected residues. Based on these approaches, a catalytic mechanism for DAAO has been proposed, although definitive evidence against alternative mechanisms has not been found (refs. 2 and 5 and references therein).The enzyme catalyzes the oxidation of D-a-amino acids into the corresponding a-ketoacids. The reaction formally proceeds according to the following scheme:E-FADH2 + 02-*E-FAD + H202[2]The reductive half reaction (Eq. 1), in which the noncovalently bound FAD becomes reduced, is followed by the oxidative step in which FAD is reoxidized by molecular oxygen, with the release of hydrogen peroxide (Eq. 2). The imino acid product spontaneously hydrolyzes to the ketoacid in a nonenzymatic process (Eq. 3). DAAO displays a broad substrate specificity, with a preference for D-amin...

Protein aggregation is of great concern to pharmaceutical formulations and has been implicated in several diseases. We engineered an anti-IL-13 monoclonal antibody CNTO607 for improved solubility. Three structure-based engineering approaches were employed in this study: (i) modifying the isoelectric point (pI), (ii) decreasing the overall surface hydrophobicity and (iii) re-introducing an N-linked carbohydrate moiety within a complementarity-determining region (CDR) sequence. A mutant was identified with a modified pI that had a 2-fold improvement in solubility while retaining the binding affinity to IL-13. Several mutants with decreased overall surface hydrophobicity also showed moderately improved solubility while maintaining a similar antigen affinity. Structural studies combined with mutagenesis data identified an aggregation 'hot spot' in heavy-chain CDR3 (H-CDR3) that contains three residues ((99)FHW(100a)). The same residues, however, were found to be essential for high affinity binding to IL-13. On the basis of the spatial proximity and germline sequence, we reintroduced the consensus N-glycosylation site in H-CDR2 which was found in the original antibody, anticipating that the carbohydrate moiety would shield the aggregation 'hot spot' in H-CDR3 while not interfering with antigen binding. Peptide mapping and mass spectrometric analysis revealed that the N-glycosylation site was generally occupied. This variant showed greatly improved solubility and bound to IL-13 with affinity similar to CNTO607 without the N-linked carbohydrate. All three engineering approaches led to improved solubility and adding an N-linked carbohydrate to the CDR was the most effective route for enhancing the solubility of CNTO607.

sugar transport ͉ phosphorylation ͉ x-ray crystallography T he phosphoenolpyruvate (PEP):sugar phosphotransferase system (PTS) (1) catalyzes the synchronized uptake and phosphorylation of a number of carbohydrates in eubacteria (group translocation) (2, 3). With some variations, the PTS comprises three proteins. In the cytoplasm, PEP phosphorylates enzyme I (EI), which then transfers the phosphoryl group to the histidine phosphocarrier protein, HPr. From HPr, the phosphoryl group is transferred to various sugar-specific membrane associated transporters [enzyme II (EII)], each comprising two cytoplasmic domains, EIIA and EIIB, and an integral membrane domain EIIC. Within EII, EIIA accepts the phosphoryl group from HPr and donates it to EIIB, whereupon EIIC mediates sugar translocation. In addition to controlling sugar translocation, the phosphorylation state of PTS proteins is associated with regulation of metabolic pathways and signaling in bacterial cells (4-8).The Ϸ64-kDa EI is a homodimer, which is more tightly associated at the phosphorylated state than the unphosphorylated state (9-14). The phosphorylation by PEP requires Mg 2ϩ and targets the N atom of His-189 (numbering scheme of EI from Escherichia coli) (15). The dimer association rate constant is two to three orders of magnitude slower than typical rates measured for other dimeric proteins, suggesting that oligomerization is accompanied by major conformational rearrangements (13,16,17). The monomer-dimer equilibrium has been studied in vitro by various methods (18-21), and it has been proposed that the transition plays a regulatory role in the PEP:sugar phosphotransferase system. Yet, transient kinetic studies indicated that the EI dimer phosphorylates HPr without dissociating into monomers (17).Proteolytic cleavage of EI produces two domains (22, 23). The EI N-terminal domain (EIN, residues 1-230) contains the residue that transfers the phosphoryl group, 24) and the HPr-binding domain, whereas the EI C-terminal domain (EIC, residues 261-575) binds PEP in the presence of Mg 2ϩ (the PEP-binding domain) (22,25) and mediates dimerization (26,27). Site-directed mutagenesis showed that Cys-502, located on EIC, is essential for phosphorylation of His-189 by PEP (28). The structure of EIN from E. coli has been determined by x-ray crystallography (29) and NMR spectroscopy (30).

To assess the state of the art in antibody 3D modeling, 11 unpublished high-resolution x-ray Fab crystal structures from diverse species and covering a wide range of antigen-binding site conformations were used as a benchmark to compare Fv models generated by seven structure prediction methodologies. The participants included: Accerlys Inc, Chemical Computer Group (CCG), Schrodinger, Jeff Gray's lab at John Hopkins University, Macromoltek, Astellas Pharma/Osaka University and Prediction of ImmunoGlobulin Structure (PIGS). The sequences of benchmark structures were submitted to the modelers and PIGS, and a set of models were generated for each structure. We provide here an overview of the organization, participants and main results of this second antibody modeling assessment (AMA-II). Also, we compare the results with the first antibody assessment published in this journal (Almagro et al., 2011;79:3050).

No abstract

The wealth of kinetic and structural information makes inorganic pyrophosphatases (PPases) a good model system to study the details of enzymatic phosphoryl transfer. The enzyme accelerates metal-complexed phosphoryl transfer 10 10 -fold: but how? Our structures of the yeast PPase product complex at 1.15 Å and fluoride-inhibited complex at 1.9 Å visualize the active site in three different states: substrate-bound, immediate product bound, and relaxed product bound. These span the steps around chemical catalysis and provide strong evidence that a water molecule (O nu) directly attacks PPi with a pK a vastly lowered by coordination to two metal ions and D117. They also suggest that a low-barrier hydrogen bond (LBHB) forms between D117 and O nu, in part because of steric crowding by W100 and N116. Direct visualization of the double bonds on the phosphates appears possible. The flexible side chains at the top of the active site absorb the motion involved in the reaction, which may help accelerate catalysis. Relaxation of the product allows a new nucleophile to be generated and creates symmetry in the elementary catalytic steps on the enzyme. We are thus moving closer to understanding phosphoryl transfer in PPases at the quantum mechanical level. Ultra-high resolution structures can thus tease out overlapping complexes and so are as relevant to discussion of enzyme mechanism as structures produced by time-resolved crystallography. Inorganic pyrophosphatases (PPases) catalyze one of the oldest and most common reactions in cells and provide a good system for detailed analysis of enzymatic phosphoryl transfer from polyphosphate to water. The kinetics are well characterized (1, 2) and high-resolution structures are available along the reaction pathway (3). The enzyme accelerates hydrolysis of metal complexed inorganic pyrophosphate by 10 10 compared with the uncatalyzed reaction (1)-but for PPases, as for enzymes in general, the exact source of rate enhancement remains unclear.The original model of catalysis suggested that the mechanism proceeded in four steps with all steps after substrate binding partially rate-determining (1). The nucleophile, which is generated by coordinating a water molecule (O nu ) to two metal ions and which is further strengthened by donating a hydrogen bond to D117, is one key to pyrophosphate hydrolysis in PPases. In addition, the substrate pK a is adjusted by extensive coordination to charged atoms (positively charged side chains and M 2ϩ ; ref.3).Our most recent solution studies (P. Halonen, unpublished data; refs. 2 and 4) indicate that the enzyme-substrate complex (EM 2 :MPPi or EM 2 :M 2 PPi) undergoes isomerization during the catalytic cycle (Scheme 1; ref. 4). In addition, fluoride inhibition studies (4) are consistent with structural studies (3,5) suggesting that the nucleophile is coordinated to D117.We earlier determined the structure of complexes A and E (Scheme 1), but now have direct structural information on the mechanistically key intermediates C and D, as well as much higher res...