Three-dimensional Structure of L-2-Haloacid Dehalogenase from Xanthobacter autotrophicus GJ10 Complexed with the Substrate-analogue Formate

The L-2-haloacid dehalogenase from the 1,2-dichloroethane degrading bacterium Xanthobacter autotrophicus GJ10 catalyzes the hydrolytic dehalogenation of small L-2-haloalkanoic acids to yield the corresponding D-2-hydroxyalkanoic acids. Its crystal structure was solved by the method of multiple isomorphous replacement with incorporation of anomalous scattering information and solvent flattening, and was refined at 1.95-Å resolution to an R factor of 21.3%. The three-dimensional structure is similar to that of the homologous L-2-haloacid dehalogenase from Pseudomonas sp. YL (1), but the X. autotrophicus enzyme has an extra dimerization domain, an active site cavity that is completely shielded from the solvent, and a different orientation of several catalytically important amino acid residues. Moreover, under the conditions used, a formate ion is bound in the active site. The position of this substrate-analogue provides valuable information on the reaction mechanism and explains the limited substrate specificity of the Xanthobacter L-2-haloacid dehalogenase.

Equilibrium Sampling From Nonequilibrium Dynamics

We present some applications of an Interacting Particle System (IPS) methodology to the field of Molecular Dynamics. This IPS method allows several simulations of a switched random process to keep closer to equilibrium at each time, thanks to a selection mechanism based on the relative virtual work induced on the system. It is therefore an efficient improvement of usual non-equilibrium simulations, which can be used to compute canonical averages, free energy differences, and typical transitions paths

QMCube (QM3): An all‐purpose suite for multiscale QM/MM calculations

QMCube (QM3) is a suite written in the Python programming language, initially focused on multiscale QM/MM simulations of biological systems, but open enough to address other kinds of problems. It allows the user to combine highly efficient QM and MM programs, providing unified access to a wide range of computational methods. The suite also supplies additional modules with extra functionalities. These modules facilitate common tasks such as performing the setup of the models or process the data generated during the simulations. The design of QM3 has been carried out considering the least number of external dependencies (only an algebra library, already included in the distribution), which makes it extremely portable. Also, the modular structure of the suite should help to expand and develop new computational methods

High-resolution NMR studies of structure and dynamics of human ERp27 indicate extensive interdomain flexibility

ERp27 (endoplasmic reticulum protein 27.7 kDa) is a homologue of PDI (protein disulfide-isomerase) localized to the endoplasmic reticulum. ERp27 is predicted to consist of two thioredoxinfold domains homologous with the non-catalytic b and b domains of PDI. The structure in solution of the N-terminal blike domain of ERp27 was solved using high-resolution NMR data. The structure confirms that it has the thioredoxin fold and that ERp27 is a member of the PDI family. 15N-NMR relaxation data were obtained and ModelFree analysis highlighted limited exchange contributions and slow internal motions, and indicated that the domain has an average order parameter S 2 of 0.79. Comparison of the single-domain structure determined in the present study with the equivalent domain within fulllength ERp27, determined independently by X-ray diffraction, indicated very close agreement. The domain interface inferred from NMR data in solution was much more extensive than that observed in the X-ray structure, suggesting that the domains flex independently and that crystallization selects one specific interdomain orientation. This led us to apply a new rapid method to simulate the flexibility of the full-length protein, establishing that the domains show considerable freedom to flex (tilt and twist) about the interdomain linker, consistent with the NMR data

An initial event in insect innate immune response: structural and biological studies of interactions between β-1,3-glucan and the N-terminal domain of β-1,3-glucan recognition protein

In response to invading microorganisms, insect β-1,3-glucan recognition protein (βGRP), a soluble receptor in the hemolymph, binds to the surfaces of bacteria and fungi and activates serine protease cascades that promote destruction of pathogens by means of melanization or expression of antimicrobial peptides. Here we report on the NMR solution structure of the N-terminal domain of βGRP (N-βGRP) from Indian meal moth (Plodia interpunctella), which is sufficient to activate the prophenoloxidase (proPO) pathway resulting in melanin formation. NMR and isothermal calorimetric titrations of N-βGRP with laminarihexaose, a glucose hexamer containing β-1,3 links, suggest a weak binding of the ligand. However, addition of laminarin, a glucose polysaccharide (~ 6 kDa) containing β-1,3 and β-1,6 links that activates the proPO pathway, to N-βGRP results in the loss of NMR cross-peaks from the backbone 15N-1H groups of the protein, suggesting the formation of a large complex. Analytical ultra centrifugation (AUC) studies of formation of N-βGRP:laminarin complex show that ligand-binding induces sel-fassociation of the protein:carbohydrate complex into a macro structure, likely containing six protein and three laminarin molecules (~ 102 kDa). The macro complex is quite stable, as it does not undergo dissociation upon dilution to sub-micromolar concentrations. The structural model thus derived from the present studies for N-βGRP:laminarin complex in solution differs from the one in which a single N-βGRP molecule has been proposed to bind to a triple helical form of laminarin on the basis of an X-ray crystallographic structure of N-βGRP:laminarihexaose complex [Kanagawa, M., Satoh, T., Ikeda, A., Adachi, Y., Ohno, N., and Yamaguchi, Y. (2011) J. Biol. Chem. 286, 29158-29165]. AUC studies and phenoloxidase activation measurements carried out with the designed mutants of N-βGRP indicate that electrostatic interactions involving Asp45, Arg54, and Asp68 between the ligand-bound protein molecules contribute in part to the stability of N-βGRP:laminarin macro complex and that a decreased stability is accompanied by a reduced activation of the proPO pathway. Increased β-1,6 branching in laminarin also results in destabilization of the macro complex. These novel findings suggest that ligand-induced self-association of βGRP:β-1,3-glucan complex may form a platform on a microbial surface for recruitment of downstream proteases, as a means of amplification of the initial signal of pathogen recognition for the activation of the proPO pathway

Protein folding using contact maps

We present the development of the idea to use dynamics in the space of contact maps as a computational approach to the protein folding problem. We first introduce two important technical ingredients, the reconstruction of a three dimensional conformation from a contact map and the Monte Carlo dynamics in contact map space. We then discuss two approximations to the free energy of the contact maps and a method to derive energy parameters based on perceptron learning. Finally we present results, first for predictions based on threading and then for energy minimization of crambin and of a set of 6 immunoglobulins. The main result is that we proved that the two simple approximations we studied for the free energy are not suitable for protein folding. Perspectives are discussed in the last section.Comment: 29 pages, 10 figure

Structure and mechanism of human DNA polymerase η

The variant form of the human syndrome xeroderma pigmentosum (XPV) is caused by a deficiency in DNA polymerase eta (Pol eta), a DNA polymerase that enables replication through ultraviolet-induced pyrimidine dimers. Here we report high-resolution crystal structures of human Pol eta at four consecutive steps during DNA synthesis through cis-syn cyclobutane thymine dimers. Pol eta acts like a 'molecular splint' to stabilize damaged DNA in a normal B-form conformation. An enlarged active site accommodates the thymine dimer with excellent stereochemistry for two-metal ion catalysis. Two residues conserved among Pol eta orthologues form specific hydrogen bonds with the lesion and the incoming nucleotide to assist translesion synthesis. On the basis of the structures, eight Pol eta missense mutations causing XPV can be rationalized as undermining the molecular splint or perturbing the active-site alignment. The structures also provide an insight into the role of Pol eta in replicating through D loop and DNA fragile sites

Ribosomal oxygenases are structurally conserved from prokaryotes to humans

2-Oxoglutarate (2OG)-dependent oxygenases have important roles in the regulation of gene expression via demethylation of N-methylated chromatin components1,2 and in the hydroxylation of transcription factors3 and splicing factor proteins4. Recently, 2OG-dependent oxygenases that catalyse hydroxylation of transfer RNA5,6,7 and ribosomal proteins8 have been shown to be important in translation relating to cellular growth, TH17-cell differentiation and translational accuracy9,10,11,12. The finding that ribosomal oxygenases (ROXs) occur in organisms ranging from prokaryotes to humans8 raises questions as to their structural and evolutionary relationships. In Escherichia coli, YcfD catalyses arginine hydroxylation in the ribosomal protein L16; in humans, MYC-induced nuclear antigen (MINA53; also known as MINA) and nucleolar protein 66 (NO66) catalyse histidine hydroxylation in the ribosomal proteins RPL27A and RPL8, respectively. The functional assignments of ROXs open therapeutic possibilities via either ROX inhibition or targeting of differentially modified ribosomes. Despite differences in the residue and protein selectivities of prokaryotic and eukaryotic ROXs, comparison of the crystal structures of E. coli YcfD and Rhodothermus marinus YcfD with those of human MINA53 and NO66 reveals highly conserved folds and novel dimerization modes defining a new structural subfamily of 2OG-dependent oxygenases. ROX structures with and without their substrates support their functional assignments as hydroxylases but not demethylases, and reveal how the subfamily has evolved to catalyse the hydroxylation of different residue side chains of ribosomal proteins. Comparison of ROX crystal structures with those of other JmjC-domain-containing hydroxylases, including the hypoxia-inducible factor asparaginyl hydroxylase FIH and histone Nε-methyl lysine demethylases, identifies branch points in 2OG-dependent oxygenase evolution and distinguishes between JmjC-containing hydroxylases and demethylases catalysing modifications of translational and transcriptional machinery. The structures reveal that new protein hydroxylation activities can evolve by changing the coordination position from which the iron-bound substrate-oxidizing species reacts. This coordination flexibility has probably contributed to the evolution of the wide range of reactions catalysed by oxygenases

Towards automated crystallographic structure refinement with phenix.refine

phenix.refine is a program within the PHENIX package that supports crystallographic structure refinement against experimental data with a wide range of upper resolution limits using a large repertoire of model parameterizations. This paper presents an overview of the major phenix.refine features, with extensive literature references for readers interested in more detailed discussions of the methods

phenix.model_vs_data: a high-level tool for the calculation of crystallographic model and data statistics

Application of phenix.model_vs_data to the contents of the Protein Data Bank shows that the vast majority of deposited structures can be automatically analyzed to reproduce the reported quality statistics. However, the small fraction of structures that elude automated re-analysis highlight areas where new software developments can help retain valuable information for future analysis