Exploiting tertiary structure through local folds for crystallographic phasing.

We describe an algorithm for phasing protein crystal X-ray diffraction data that identifies, retrieves, refines and exploits general tertiary structural information from small fragments available in the Protein Data Bank. The algorithm successfully phased, through unspecific molecular replacement combined with density modification, all-helical, mixed alpha-beta, and all-beta protein structures. The method is available as a software implementation: Borges.

Combining phase information in reciprocal space for molecular replacement with partial models.

ARCIMBOLDO allows ab initio phasing of macromolecular structures below atomic resolution by exploiting the location of small model fragments combined with density modification in a multisolution frame. The model fragments can be either secondary-structure elements predicted from the sequence or tertiary-structure fragments. The latter can be derived from libraries of typical local folds or from related structures, such as a low-homology model that is unsuccessful in molecular replacement. In all ARCIMBOLDO applications, fragments are searched for sequentially. Correct partial solutions obtained after each fragment-search stage but lacking the necessary phasing power can, if combined, succeed. Here, an analysis is presented of the clustering of partial solutions in reciprocal space and of its application to a set of different cases. In practice, the task of combining model fragments from an ARCIMBOLDO run requires their referral to a common origin and is complicated by the presence of correct and incorrect solutions as well as by their not being independent. The F-weighted mean phase difference has been used as a figure of merit. Clustering perfect, non-overlapping fragments dismembered from test structures in polar and nonpolar space groups shows that density modification before determining the relative origin shift enhances its discrimination. In the case of nonpolar space groups, clustering of ARCIMBOLDO solutions from secondary-structure models is feasible. The use of partially overlapping search fragments provides a more favourable circumstance and was assessed on a test case. Applying the devised strategy, a previously unknown structure was solved from clustered correct partial solutions.

Unexpected tautomeric equilibria of the carbanion-enamine intermediate in pyruvate oxidase highlight unrecognized chemical versatility of thiamin.

Thiamin diphosphate, the vitamin B1 coenzyme, plays critical roles in fundamental metabolic pathways that require acyl carbanion equivalents. Studies on chemical models and enzymes had suggested that these carbanions are resonance-stabilized as enamines. A crystal structure of this intermediate in pyruvate oxidase at 1.1 Å resolution now challenges this paradigm by revealing that the enamine does not accumulate. Instead, the intermediate samples between the ketone and the carbanion both interlocked in a tautomeric equilibrium. Formation of the keto tautomer is associated with a loss of aromaticity of the cofactor. The alternate confinement of electrons to neighboring atoms rather than π-conjugation seems to be of importance for the enzyme-catalyzed, redox-coupled acyl transfer to phosphate, which requires a dramatic inversion of polarity of the reacting substrate carbon in two subsequent catalytic steps. The ability to oscillate between a nucleophilic (carbanion) and an electrophilic (ketone) substrate center highlights a hitherto unrecognized versatility of the thiamin cofactor. It remains to be studied whether formation of the keto tautomer is a general feature of all thiamin enzymes, as it could provide for stable storage of the carbanion state, or whether this feature represents a specific trait of thiamin oxidases. In addition, the protonation state of the two-electron reduced flavin cofactor can be fully assigned, demonstrating the power of high-resolution cryocrystallography for elucidation of enzymatic mechanisms.

Structure solution of DNA-binding proteins and complexes with ARCIMBOLDO libraries.

Protein-DNA interactions play a major role in all aspects of genetic activity within an organism, such as transcription, packaging, rearrangement, replication and repair. The molecular detail of protein-DNA interactions can be best visualized through crystallography, and structures emphasizing insight into the principles of binding and base-sequence recognition are essential to understanding the subtleties of the underlying mechanisms. An increasing number of high-quality DNA-binding protein structure determinations have been witnessed despite the fact that the crystallographic particularities of nucleic acids tend to pose specific challenges to methods primarily developed for proteins. Crystallographic structure solution of protein-DNA complexes therefore remains a challenging area that is in need of optimized experimental and computational methods. The potential of the structure-solution program ARCIMBOLDO for the solution of protein-DNA complexes has therefore been assessed. The method is based on the combination of locating small, very accurate fragments using the program Phaser and density modification with the program SHELXE. Whereas for typical proteins main-chain α-helices provide the ideal, almost ubiquitous, small fragments to start searches, in the case of DNA complexes the binding motifs and DNA double helix constitute suitable search fragments. The aim of this work is to provide an effective library of search fragments as well as to determine the optimal ARCIMBOLDO strategy for the solution of this class of structures.

Structure of sulfamidase provides insight into the molecular pathology of mucopolysaccharidosis IIIA.

Mucopolysaccharidosis type IIIA (Sanfilippo A syndrome), a fatal childhood-onset neurodegenerative disease with mild facial, visceral and skeletal abnormalities, is caused by an inherited deficiency of the enzyme N-sulfoglucosamine sulfohydrolase (SGSH; sulfamidase). More than 100 mutations in the SGSH gene have been found to reduce or eliminate its enzymatic activity. However, the molecular understanding of the effect of these mutations has been confined by a lack of structural data for this enzyme. Here, the crystal structure of glycosylated SGSH is presented at 2 Å resolution. Despite the low sequence identity between this unique N-sulfatase and the group of O-sulfatases, they share a similar overall fold and active-site architecture, including a catalytic formylglycine, a divalent metal-binding site and a sulfate-binding site. However, a highly conserved lysine in O-sulfatases is replaced in SGSH by an arginine (Arg282) that is positioned to bind the N-linked sulfate substrate. The structure also provides insight into the diverse effects of pathogenic mutations on SGSH function in mucopolysaccharidosis type IIIA and convincing evidence for the molecular consequences of many missense mutations. Further, the molecular characterization of SGSH mutations will lay the groundwork for the development of structure-based drug design for this devastating neurodegenerative disorder.

Structure and activity of the only human RNase T2.

Mutations in the gene of human RNase T2 are associated with white matter disease of the human brain. Although brain abnormalities (bilateral temporal lobe cysts and multifocal white matter lesions) and clinical symptoms (psychomotor impairments, spasticity and epilepsy) are well characterized, the pathomechanism of RNase T2 deficiency remains unclear. RNase T2 is the only member of the Rh/T2/S family of acidic hydrolases in humans. In recent years, new functions such as tumor suppressing properties of RNase T2 have been reported that are independent of its catalytic activity. We determined the X-ray structure of human RNase T2 at 1.6 Å resolution. The α+ß core fold shows high similarity to those of known T2 RNase structures from plants, while, in contrast, the external loop regions show distinct structural differences. The catalytic features of RNase T2 in presence of bivalent cations were analyzed and the structural consequences of known clinical mutations were investigated. Our data provide further insight into the function of human RNase T2 and may prove useful in understanding its mode of action independent of its enzymatic activity.

Molecular architecture with carbohydrate functionalized ß-peptides adopting 314-helical conformation.

Carbohydrate recognition is essential in cellular interactions and biological processes. It is characterized by structural diversity, multivalency and cooperative effects. To evaluate carbohydrate interaction and recognition, the structurally defined attachment of sugar units to a rigid template is highly desired. ß-Peptide helices offer conformationally stable templates for the linear presentation of sugar units in defined distances. The synthesis and ß-peptide incorporation of sugar-ß-amino acids are described providing the saccharide units as amino acid side chain. The respective sugar-ß-amino acids are accessible by Michael addition of ammonia to sugar units derivatized as α,ß-unsaturated esters. Three sugar units were incorporated in ß-peptide oligomers varying the sugar (glucose, galactose, xylose) and sugar protecting groups. The influence of sugar units and the configuration of sugar-ß-amino acids on ß-peptide secondary structure were investigated by CD spectroscopy.

Modes and model building in SHELXE.

Density modification is a standard step to provide a route for routine structure solution by any experimental phasing method, with single-wavelength or multi-wavelength anomalous diffraction being the most popular methods, as well as to extend fragments or incomplete models into a full solution. The effect of density modification on the starting maps from either source is illustrated in the case of SHELXE. The different modes in which the program can run are reviewed; these include less well known uses such as reading external phase values and weights or phase distributions encoded in Hendrickson-Lattman coefficients. Typically in SHELXE, initial phases are calculated from experimental data, from a partial model or map, or from a combination of both sources. The initial phase set is improved and extended by density modification and, if the resolution of the data and the type of structure permits, polyalanine tracing. As a feature to systematically eliminate model bias from phases derived from predicted models, the trace can be set to exclude the area occupied by the starting model. The trace now includes an extension into the gamma position or hydrophobic and aromatic side chains if a sequence is provided, which is performed in every tracing cycle. Once a correlation coefficient of over 30% between the structure factors calculated from such a trace and the native data indicates that the structure has been solved, the sequence is docked in all model-building cycles and side chains are fitted if the map supports it. The extensions to the tracing algorithm brought in to provide a complete model are discussed. The improvement in phasing performance is assessed using a set of tests.

Extending molecular-replacement solutions with SHELXE.

Although the program SHELXE was originally intended for the experimental phasing of macromolecules, it can also prove useful for expanding a small protein fragment to an almost complete polyalanine trace of the structure, given a favourable combination of native data resolution (better than about 2.1 Å) and solvent content. A correlation coefficient (CC) of more than 25% between the native structure factors and those calculated from the polyalanine trace appears to be a reliable indicator of success and has already been exploited in a number of pipelines. Here, a more detailed account of this usage of SHELXE for molecular-replacement solutions is given.

Continuous ß-turn fold of an alternating alanyl/homoalanyl peptide nucleic acid.

The crystal structure of the PNA (peptide nucleic acid) oligomer H-Lys-HalG-AlaG-HalC-AlaG-HalC-AlaC-Lys-NH(2) (PNA1, amino acids with D-configuration are underlined, Ala = alanyl, Hal = homoalanyl) has been determined by ab initio direct methods and refined against 1.0 Å data. The asymmetric unit consists of a tetrameric cage with almost ideal Watson-Crick C-G base pairing of all the guanine and cytosine side-chain substituents. Each PNA strand has a 90° ß-turn every second residue, stabilized by three hydrogen bonds between the backbone amides. The first, second, fifth and sixth bases stack on one side of the monomer and pair with the corresponding complementary bases of a second monomer to form a dimer. The two remaining bases on each side of the resulting dimer form Watson-Crick pairs with the complementary bases of a second dimer, leading to a unique cage structure. The extra methylene groups in the homoalanyl residues enable stacking of the bases with an optimal distance between base-planes but also with an appreciable lateral displacement (slide).

Practical structure solution with ARCIMBOLDO.

Since its release in September 2009, the structure-solution program ARCIMBOLDO, based on the combination of locating small model fragments such as polyalanine α-helices with density modification with the program SHELXE in a multisolution frame, has evolved to incorporate other sources of stereochemical or experimental information. Fragments that are more sophisticated than the ubiquitous main-chain α-helix can be proposed by modelling side chains onto the main chain or extracted from low-homology models, as locally their structure may be similar enough to the unknown one even if the conventional molecular-replacement approach has been unsuccessful. In such cases, the program may test a set of alternative models in parallel against a specified figure of merit and proceed with the selected one(s). Experimental information can be incorporated in three ways: searching within ARCIMBOLDO for an anomalous fragment against anomalous differences or MAD data or finding model fragments when an anomalous substructure has been determined with another program such as SHELXD or is subsequently located in the anomalous Fourier map calculated from the partial fragment phases. Both sources of information may be combined in the expansion process. In all these cases the key is to control the workflow to maximize the chances of success whilst avoiding the creation of an intractable number of parallel processes. A GUI has been implemented to aid the setup of suitable strategies within the various typical scenarios. In the present work, the practical application of ARCIMBOLDO within each of these scenarios is described through the distributed test cases.

Crystallographic ab initio protein structure solution below atomic resolution.

Ab initio macromolecular phasing has been so far limited to small proteins diffracting at atomic resolution (beyond 1.2 A) unless heavy atoms are present. We describe a general ab initio phasing method for 2 A data, based on combination of localizing model fragments such as small á-helices with Phaser and density modification with SHELXE. We implemented this approach in the program Arcimboldo to solve a 222-amino-acid structure at 1.95 A.

Cln5 represents a new type of cysteine-based S-depalmitoylase linked to neurodegeneration.

Genetic CLN5 variants are associated with childhood neurodegeneration and Alzheimer's disease; however, the molecular function of ceroid lipofuscinosis neuronal protein 5 (Cln5) is unknown. We solved the Cln5 crystal structure and identified a region homologous to the catalytic domain of members of the N1pC/P60 superfamily of papain-like enzymes. However, we observed no protease activity for Cln5; and instead, we discovered that Cln5 and structurally related PPPDE1 and PPPDE2 have efficient cysteine palmitoyl thioesterase (S-depalmitoylation) activity using fluorescent substrates. Mutational analysis revealed that the predicted catalytic residues histidine-166 and cysteine-280 are critical for Cln5 thioesterase activity, uncovering a new cysteine-based catalytic mechanism for S-depalmitoylation enzymes. Last, we found that Cln5-deficient neuronal progenitor cells showed reduced thioesterase activity, confirming live cell function of Cln5 in setting S-depalmitoylation levels. Our results provide new insight into the function of Cln5, emphasize the importance of S-depalmitoylation in neuronal homeostasis, and disclose a new, unexpected enzymatic function for the N1pC/P60 superfamily of proteins.

Structure of a highly NADP+-specific isocitrate dehydrogenase.

Isocitrate dehydrogenase catalyzes the first oxidative and decarboxylation steps in the citric acid cycle. It also lies at a crucial bifurcation point between CO2-generating steps in the cycle and carbon-conserving steps in the glyoxylate bypass. Hence, the enzyme is a focus of regulation. The bacterial enzyme is typically dependent on the coenzyme nicotinamide adenine dinucleotide phosphate. The monomeric enzyme from Corynebacterium glutamicum is highly specific towards this coenzyme and the substrate isocitrate while retaining a high overall efficiency. Here, a 1.9 Šresolution crystal structure of the enzyme in complex with its coenzyme and the cofactor Mg2+ is reported. Coenzyme specificity is mediated by interactions with the negatively charged 2'-phosphate group, which is surrounded by the side chains of two arginines, one histidine and, via a water, one lysine residue, forming ion pairs and hydrogen bonds. Comparison with a previous apoenzyme structure indicates that the binding site is essentially preconfigured for coenzyme binding. In a second enzyme molecule in the asymmetric unit negatively charged aspartate and glutamate residues from a symmetry-related enzyme molecule interact with the positively charged arginines, abolishing coenzyme binding. The holoenzyme from C. glutamicum displays a 36° interdomain hinge-opening movement relative to the only previous holoenzyme structure of the monomeric enzyme: that from Azotobacter vinelandii. As a result, the active site is not blocked by the bound coenzyme as in the closed conformation of the latter, but is accessible to the substrate isocitrate. However, the substrate-binding site is disrupted in the open conformation. Hinge points could be pinpointed for the two molecules in the same crystal, which show a 13° hinge-bending movement relative to each other. One of the two pairs of hinge residues is intimately flanked on both sides by the isocitrate-binding site. This suggests that binding of a relatively small substrate (or its competitive inhibitors) in tight proximity to a hinge point could lead to large conformational changes leading to a closed, presumably catalytically active (or inactive), conformation. It is possible that the small-molecule concerted inhibitors glyoxylate and oxaloacetate similarly bind close to the hinge, leading to an inactive conformation of the enzyme.

Experimental phasing with SHELXC/D/E: combining chain tracing with density modification.

The programs SHELXC, SHELXD and SHELXE are designed to provide simple, robust and efficient experimental phasing of macromolecules by the SAD, MAD, SIR, SIRAS and RIP methods and are particularly suitable for use in automated structure-solution pipelines. This paper gives a general account of experimental phasing using these programs and describes the extension of iterative density modification in SHELXE by the inclusion of automated protein main-chain tracing. This gives a good indication as to whether the structure has been solved and enables interpretable maps to be obtained from poorer starting phases. The autotracing algorithm starts with the location of possible seven-residue alpha-helices and common tripeptides. After extension of these fragments in both directions, various criteria are used to decide whether to accept or reject the resulting poly-Ala traces. Noncrystallographic symmetry (NCS) is applied to the traced fragments, not to the density. Further features are the use of a 'no-go' map to prevent the traces from passing through heavy atoms or symmetry elements and a splicing technique to combine the best parts of traces (including those generated by NCS) that partly overlap.

The magic triangle goes MAD: experimental phasing with a bromine derivative.

Experimental phasing is an essential technique for the solution of macromolecular structures. Since many heavy-atom ion soaks suffer from nonspecific binding, a novel class of compounds has been developed that combines heavy atoms with functional groups for binding to proteins. The phasing tool 5-amino-2,4,6-tribromoisophthalic acid (B3C) contains three functional groups (two carboxylate groups and one amino group) that interact with proteins via hydrogen bonds. Three Br atoms suitable for anomalous dispersion phasing are arranged in an equilateral triangle and are thus readily identified in the heavy-atom substructure. B3C was incorporated into proteinase K and a multiwavelength anomalous dispersion (MAD) experiment at the Br K edge was successfully carried out. Radiation damage to the bromine-carbon bond was investigated. A comparison with the phasing tool I3C that contains three I atoms for single-wavelength anomalous dispersion (SAD) phasing was also carried out.

Structure analysis of endosialidase NF at 0.98 A resolution.

Endosialidase NF (endoNF) is a bacteriophage-derived endosialidase that specifically degrades alpha-2,8-linked polysialic acid. The structure of a new crystal form of endoNF in complex with sialic acid has been refined at 0.98 A resolution. The 210 kDa homotrimeric multi-domain enzyme displays outstanding stability and resistance to SDS. Even at atomic resolution, only a minor fraction of side chains possess alternative conformations. However, multiple conformations of an active-site residue imply that it has an important catalytic function in the cleavage mechanism of polysialic acid.

The crystal structure of non-modified and bipyridine-modified PNA duplexes.

Peptide nucleic acid (PNA) is a synthetic analogue of DNA that commonly has an N-aminoethyl glycine backbone. The crystal structures of two PNA duplexes, one containing eight standard nucleobase pairs (GGCATGCC)(2), and the other containing the same nucleobase pairs and a central pair of bipyridine ligands, have been solved with a resolution of 1.22 and 1.10 Å, respectively. The non-modified PNA duplex adopts a P-type helical structure similar to that of previously characterized PNAs. The atomic-level resolution of the structures allowed us to observe for the first time specific modes of interaction between the terminal lysines of the PNA and the backbone and the nucleobases situated in the vicinity of the lysines, which are considered an important factor in the induction of a preferred handedness in PNA duplexes. Our results support the notion that whereas PNA typically adopts a P-type helical structure, its flexibility is relatively high. For example, the base-pair rise in the bipyridine-containing PNA is the largest measured to date in a PNA homoduplex. The two bipyridines bulge out of the duplex and are aligned parallel to the major groove of the PNA. In addition, two bipyridines from adjacent PNA duplexes form a π-stacked pair that relates the duplexes within the crystal. The bulging out of the bipyridines causes bending of the PNA duplex, which is in contrast to the structure previously reported for biphenyl-modified DNA duplexes in solution, where the biphenyls are π stacked with adjacent nucleobase pairs and adopt an intrahelical geometry. This difference shows that relatively small perturbations can significantly impact the relative position of nucleobase analogues in nucleic acid duplexes.

Crystal structure of trioxacarcin A covalently bound to DNA.

We report a crystal structure that shows an antibiotic that extracts a nucleobase from a DNA molecule 'caught in the act' after forming a covalent bond but before departing with the base. The structure of trioxacarcin A covalently bound to double-stranded d(AACCGGTT) was determined to 1.78 A resolution by MAD phasing employing brominated oligonucleotides. The DNA-drug complex has a unique structure that combines alkylation (at the N7 position of a guanine), intercalation (on the 3'-side of the alkylated guanine), and base flip-out. An antibiotic-induced flipping-out of a single, nonterminal nucleobase from a DNA duplex was observed for the first time in a crystal structure.

The 2A resolution crystal structure of HetL, a pentapeptide repeat protein involved in regulation of heterocyst differentiation in the cyanobacterium Nostoc sp. strain PCC 7120.

The hetL gene from the cyanobacterium Nostoc sp. PCC 7120 encodes a 237 amino acid protein (25.6kDa) containing 40 predicted tandem pentapeptide repeats. Nostoc sp. PCC 7120 is a filamentous cyanobacterium that forms heterocysts, specialized cells capable of fixing atmospheric N(2) during nitrogen starvation in its aqueous environment. Under these conditions, heterocysts occur in a regular pattern of approximately one out of every 10-15 vegetative cells. Heterocyst differentiation is highly regulated involving hundreds of genes, one of which encodes PatS, thought to be an intercellular peptide signal made by developing heterocysts to inhibit heterocyst differentiation in neighboring vegetative cells, thus contributing to pattern formation and spacing of heterocysts along the filament. While overexpression of PatS suppresses heterocyst differentiation in Nostoc sp. PCC 7120, overexpression of HetL produces a multiple contiguous heterocyst phenotype with loss of the wild type heterocyst pattern, and strains containing extra copies of hetL allow heterocyst formation even in cells overexpressing PatS. Thus, HetL appears to interfere with heterocyst differentiation inhibition by PatS, however, the mechanism for HetL function remains unknown. As a first step towards exploring the mechanism for its biochemical function, the crystal structure of HetL has been solved at 2.0A resolution using sulfur anomalous scattering.