Critical points for an accurate human genome analysis.

Next-generation sequencing is radically changing how DNA diagnostic laboratories operate. What started as a single-gene profession is now developing into gene panel sequencing and whole-exome and whole-genome sequencing (WES/WGS) analyses. With further advances in sequencing technology and concomitant price reductions, WGS will soon become the standard and be routinely offered. Here, we focus on the critical steps involved in performing WGS, with a particular emphasis on points where WGS differs from WES, the important variables that should be taken into account, and the quality control measures that can be taken to monitor the process. The points discussed here, combined with recent publications on guidelines for reporting variants, will facilitate the routine implementation of WGS into a diagnostic setting.

Involvement of Tyr108 in the enzyme mechanism of the small laccase from Streptomyces coelicolor.

The enzyme mechanism of the multicopper oxidase (MCO) SLAC from Streptomyces coelicolor was investigated by structural (XRD), spectroscopic (optical, EPR), and kinetics (stopped-flow) experiments on variants in which residue Tyr108 had been replaced by Phe or Ala through site-directed mutagenesis. Contrary to the more common three-domain MCOs, a tyrosine in the two-domain SLAC is found to participate in the enzyme mechanism by providing an electron during oxygen reduction, giving rise to the temporary appearance of a tyrosyl radical. The relatively low k(cat)/K(M) of SLAC and the involvement of Y108 in the enzyme mechanism may reflect an adaptation to a milieu in which there is an imbalance between the available reducing and oxidizing co-substrates. The purported evolutionary relationship between the two-domain MCOs and human ceruloplasmin appears to extend not only to the 3D structure and the mode of binding of the Cu's in the trinuclear center, as noted before, but also to the enzyme mechanism.

Structure of a post-translationally processed heterodimeric double-headed Kunitz-type serine protease inhibitor from potato.

Potato serine protease inhibitor (PSPI) constitutes about 22% of the total amount of proteins in potato tubers (cv. Elkana), making it the most abundant protease inhibitor in the plant. PSPI is a heterodimeric double-headed Kunitz-type serine protease inhibitor that can tightly and simultaneously bind two serine proteases by mimicking the substrate of the enzyme with its reactive-site loops. Here, the crystal structure of PSPI is reported, representing the first heterodimeric double-headed Kunitz-type serine protease inhibitor structure to be determined. PSPI has a ß-trefoil fold and, based on the structure, two reactive-site loops bearing residues Phe75 and Lys95 were identified.

Crystal structure of chlorite dismutase, a detoxifying enzyme producing molecular oxygen.

Chlorite dismutase (Cld) is a key enzyme of perchlorate and chlorate respiration. This heme-based protein reduces the toxic compound chlorite into the innocuous chloride anion in a very efficient way while producing molecular oxygen. A sequence comparison between Cld homologues shows a highly conserved family. The crystal structure of Azospira oryzae strain GR-1 Cld is reported to 2.1 A resolution. The structure reveals a hexameric organization of the Cld, while each monomer exhibits a ferredoxin-like fold. The six subunits are organized in a ring structure with a maximal diameter of 9 nm and an inner diameter of 2 nm. The heme active-site pocket is solvent accessible both from the inside and the outside of the ring. Moreover, a second anion binding site that could accommodate the assumed reaction intermediate ClO(-) for further transformation has been identified near the active site. The environment of the heme cofactor was investigated with electron paramagnetic resonance spectroscopy. Apart from the high-spin ferric signal of the five-coordinate resting-state enzyme, two low-spin signals were found corresponding to six-coordinate species. The current crystal structure confirms and complements a recently proposed catalytic mechanism that proceeds via a ferryl species and a ClO(-) anion. Our structural data exclude cooperativity between the iron centers.

Involvement of a carboxylated lysine in UV damage endonuclease.

UV damage endonuclease is a DNA repair enzyme that can both recognize damage such as UV lesions and introduce a nick directly 5' to them. Recently, the crystal structure of the enzyme from Thermus thermophilus was solved. In the electron density map of this structure, unexplained density near the active site was observed at the tip of Lys229. Based on this finding, it was proposed that Lys229 is post-translationally modified. In this article, we give evidence that this modification is a carboxyl group. By combining activity assays and X-ray crystallography on several point mutants, we show that the carboxyl group assists in metal binding required for catalysis by donating negative charge to the metal-coordinating residue His231. Moreover, functional and structural analysis of the K229R mutant reveals that if His231 shifts away, an increased activity results on both damaged and undamaged DNA. Taken together, the results show that T. thermophilus ultraviolet damage endonuclease is carboxylated and the modified lysine is required for proper catalysis and preventing increased incision of undamaged DNA.

Characterization of a diagnostic Fab fragment binding trimeric Lewis X.

Lewis X trisaccharides normally function as essential cell-cell interaction mediators. However, oligomers of Lewis X trisaccharides expressed by the parasite Schistosoma mansoni seem to be related to its evasion of the immune response of its human host. Here we show that monoclonal antibody 54-5C10-A, which is used to diagnose schistosomiasis in humans, interacts with oligomers of at least three Lewis X trisaccharides, but not with monomeric Lewis X. We describe the sequence and the 2.5 A crystal structure of its Fab fragment and infer a possible mode of binding of the polymeric Lewis X from docking studies. Our studies indicate a radically different mode of binding compared to Fab 291-2G3-A, which is specific for monomeric Lewis X, thus providing a structural explanation of the diagnostic success of 54-5C10-A.

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of chlorite dismutase: a detoxifying enzyme producing molecular oxygen.

Chlorite dismutase, a homotetrameric haem-based protein, is one of the key enzymes of (per)chlorate-reducing bacteria. It is highly active (>2 kU mg(-1)) in reducing the toxic compound chlorite to the innocuous chloride anion and molecular oxygen. Chlorite itself is produced as the intermediate product of (per)chlorate reduction. The chlorite dismutase gene in Azospira oryzae strain GR-1 employing degenerate primers has been identified and the active enzyme was subsequently overexpressed in Escherichia coli. Chlorite dismutase was purified, proven to be active and crystallized using sitting drops with PEG 2000 MME, KSCN and ammonium sulfate as precipitants. The crystals belonged to space group P2(1)2(1)2 and were most likely to contain six subunits in the asymmetric unit. The refined unit-cell parameters were a = 164.46, b = 169.34, c = 60.79 A. The crystals diffracted X-rays to 2.1 A resolution on a synchrotron-radiation source and a three-wavelength MAD data set has been collected. Determination of the chlorite dismutase structure will provide insights into the active site of the enzyme, for which no structures are currently available.

Crystal structure of the DNA repair enzyme ultraviolet damage endonuclease.

The ultraviolet damage endonuclease (UVDE) performs the initial step in an alternative excision repair pathway of UV-induced DNA damage, nicking immediately adjacent to the 5' phosphate of the damaged nucleotides. Unique for a single-protein DNA repair endonuclease, it can detect different types of damage. Here we show that Thermus thermophilus UVDE shares some essential structural features with Endo IV, an enzyme from the base excision repair pathway that exclusively nicks at abasic sites. A comparison between the structures indicates how DNA is bound by UVDE, how UVDE may recognize damage, and which of its residues are involved in catalysis. Furthermore, the comparison suggests an elegant explanation of UVDE's potential to recognize different types of damage. Incision assays including point mutants of UVDE confirmed the relevance of these conclusions.

Protonation of a histidine copper ligand in fern plastocyanin.

Plastocyanin is a small blue copper protein that shuttles electrons as part of the photosynthetic redox chain. Its redox behavior is changed at low pH as a result of protonation of the solvent-exposed copper-coordinating histidine. Protonation and subsequent redox inactivation could have a role in the down regulation of photosynthesis. As opposed to plastocyanin from other sources, in fern plastocyanin His90 protonation at low pH has been reported not to occur. Two possible reasons for that have been proposed: pi-pi stacking between Phe12 and His90 and lack of a hydrogen bond with the backbone oxygen of Gly36. We have produced this fern plastocyanin recombinantly and examined the properties of wild-type protein and mutants Phe12Leu, Gly36Pro, and the double mutant with NMR spectroscopy, X-ray crystallography, and cyclic voltammetry. The results demonstrate that, contrary to earlier reports, protonation of His90 in the wild-type protein does occur in solution with a pKa of 4.4 (+/-0.1). Neither the single mutants nor the double mutant exhibit a change in protonation behavior, indicating that the suggested interactions have no influence. The crystal structure at low pH of the Gly36Pro variant does not show His90 protonation, similar to what was found for the wild-type protein. The structure suggests that movement of the imidazole ring is hindered by crystal contacts. This study illustrates a significant difference between results obtained in solution by NMR and by crystallography.

The protein structure of recombinant human lactoferrin produced in the milk of transgenic cows closely matches the structure of human milk-derived lactoferrin.

Human lactoferrin (hLF) is an iron-binding glycoprotein involved in the host defence against infection and excessive inflammation. As the availability of (human milk-derived) natural hLF is limited, alternative means of production of this biopharmaceutical are extensively researched. Here we report the crystal structure of recombinant hLF (rhLF) expressed in the milk of transgenic cows at a resolution of 2.4 A. To our knowledge, the first reported structure of a recombinant protein produced in milk of transgenic livestock. Even though rhLF contains oligomannose- and hybrid-type N-linked glycans next to complex-type glycans, which are the only glycans found on natural hLF, the structures are identical within the experimental error (r.m.s. deviation of only 0.28 A for the main-chain atoms). Of the differences in polymorphic amino acids between the natural and rhLF variant used, only the side-chain of Asp561 could be modeled into the rhLF electron density map. Taken together, the results confirm the structural integrity of the rhLF variant used in this study. It also confirms the validity of the transgenic cow mammary gland as a vehicle to produce recombinant human proteins.

Crystallization and preliminary X-ray crystallographic studies on a Kunitz-type potato serine protease inhibitor.

Interest in protease inhibitors has been renewed because of their potent activity in preventing carcinogenesis in a wide variety of in vivo and in vitro model systems. Potato tubers contain a wide range of such protease inhibitors. In cv. Elkana potato tubers, protease inhibitors represent about 50% of the total amount of soluble protein. Potato serine protease inhibitor (PSPI), one of the isoforms of the most abundant group of protease inhibitors, is a dimeric double-headed Kunitz-type inhibitor. No high-resolution structural information on this type of inhibitor has so far been obtained, as all currently known structures are of the monomeric single-headed or monomeric double-headed types. Crystals were grown in 0.1 M HEPES pH 7.5, 10% PEG 8000 and 8% ethylene glycol complemented with 9 mM 1-s-octyl-beta-D-thioglucoside or 0.1 M glycine. Data were collected from a single crystal under cryoconditions to 1.8 A resolution. The protein crystallized in space group P2(1), with unit-cell parameters a = 54.82, b = 93.92, c = 55.44 A, beta = 100.7 degrees; the scaling Rsym is 0.044 for 45,456 unique reflections.

The structure of the receptor-binding domain of the bacteriophage T4 short tail fibre reveals a knitted trimeric metal-binding fold.

Adsorption of T4 bacteriophage to the Escherichia coli host cell is mediated by six long and six short tail fibres. After at least three long tail fibres have bound, short tail fibres extend and bind irreversibly to the core region of the host cell lipo-polysaccharide (LPS), serving as inextensible stays during penetration of the cell envelope by the tail tube. The short tail fibres consist of a parallel, in-register, trimer of gene product 12 (gp12).X-ray crystallography at 1.5A resolution of a protease-stable fragment of gp12 generated in the presence of zinc chloride reveals the structure of the C-terminal receptor-binding domain. It has a novel "knitted" fold, consisting of three extensively intertwined monomers. It reveals a metal-binding site, containing a zinc ion coordinated by six histidine residues in an octahedral conformation. We also suggest an LPS-binding region.