Discovery of a Ni2+-dependent guanidine hydrolase in bacteria.

Nitrogen availability is a growth-limiting factor in many habitats1, and the global nitrogen cycle involves prokaryotes and eukaryotes competing for this precious resource. Only some bacteria and archaea can fix elementary nitrogen; all other organisms depend on the assimilation of mineral or organic nitrogen. The nitrogen-rich compound guanidine occurs widely in nature2-4, but its utilization is impeded by pronounced resonance stabilization5, and enzymes catalysing hydrolysis of free guanidine have not been identified. Here we describe the arginase family protein GdmH (Sll1077) from Synechocystis sp. PCC 6803 as a Ni2+-dependent guanidine hydrolase. GdmH is highly specific for free guanidine. Its activity depends on two accessory proteins that load Ni2+ instead of the typical Mn2+ ions into the active site. Crystal structures of GdmH show coordination of the dinuclear metal cluster in a geometry typical for arginase family enzymes and allow modelling of the bound substrate. A unique amino-terminal extension and a tryptophan residue narrow the substrate-binding pocket and identify homologous proteins in further cyanobacteria, several other bacterial taxa and heterokont algae as probable guanidine hydrolases. This broad distribution suggests notable ecological relevance of guanidine hydrolysis in aquatic habitats.

The importance of chain context in assessing small nucleotide variants in titin: in silico case study of the I10-I11 tandem and its arrhythmic right ventricular cardiomyopathy linked position T2580.

Non-synonymous small nucleotide variations (nsSNVs) in the giant muscle protein, titin, have key roles in the development of several myopathologies. Although there is considerable motive to screen at-risk individuals for nsSNVs, to identify patients in early disease stages while therapeutic intervention is still possible, the clinical significance of most titin variations remains unclear. Therefore, there is a growing need to establish methods to classify nsSNVs in a simple, economic and rapid manner. Due to its strong correlation to arrhythmogenic right ventricular cardiomyopathy (ARVC), one particular mutation in titin-T2580I, located in the I10 immunoglobulin domain-has received considerable attention. Here, we use the I10-I11 tandem as a case study to explore the possible benefits of considering the titin chain context-i.e. domain interfaces-in the assessment of titin nsSNVs. Specifically, we investigate which exchanges mimic the conformational molecular phenotype of the T2580I mutation at the I10-I11 domain interface. Then, we computed a residue stability landscape for domains alone and in tandem to define a Domain Interface Score (DIS) which identifies several hotspot residues. Our findings suggest that the T2580 position is highly sensitive to exchange and that any variant found in this position should be considered with care. Furthermore, we conclude that the consideration of the higher order structure of the titin chain is important to gain accurate insights into the vulnerability of positions in linker regions and that titin nsSNV prediction benefits from a contextual analysis. Communicated by Ramaswamy H. Sarma.

Structural evidence for a possible role of reversible disulphide bridge formation in the elasticity of the muscle protein titin.

BACKGROUND: The giant muscle protein titin contributes to the filament system in skeletal and cardiac muscle cells by connecting the Z disk and the central M line of the sarcomere. One of the physiological functions of titin is to act as a passive spring in the sarcomere, which is achieved by the elastic properties of its central I band region. Titin contains about 300 domains of which more than half are folded as immunoglobulin-like (Ig) domains. Ig domain segments of the I band of titin have been extensively used as templates to investigate the molecular basis of protein elasticity. RESULTS: The structure of the Ig domain I1 from the I band of titin has been determined to 2.1 A resolution. It reveals a novel, reversible disulphide bridge, which is neither required for correct folding nor changes the chemical stability of I1, but it is predicted to contribute mechanically to the elastic properties of titin in active sarcomeres. From the 92 Ig domains in the longest isoform of titin, at least 40 domains have a potential for disulphide bridge formation. CONCLUSIONS: We propose a model where the formation of disulphide bridges under oxidative stress conditions could regulate the elasticity of the I band in titin by increasing sarcomeric resistance. In this model, the formation of the disulphide bridge could refrain a possible directed motion of the two beta sheets or other mechanically stable entities of the I1 Ig domain with respect to each other when exposed to mechanical forces.

Purification, characterization and crystallization of thermostable anthranilate phosphoribosyltransferase from Sulfolobus solfataricus.

Anthranilate phosphoribosyltransferase (TrpD; EC 2.4.2.18) from the hyperthermophilic archaeon Sulfolobus solfataricus (ssTrpD) was expressed in Escherichia coli, purified and crystallized. Analytical gel permeation chromatography revealed a homodimeric composition of the enzyme. The steady-state kinetic characteristics suggest tight binding of the substrate anthranilic acid and efficient catalysis at the physiological growth temperature of S. solfataricus. Crystals of ssTrpD diffract to better than 2.6 A resolution and preliminary X-ray characterization was carried out. The crystals are suitable for structure determination.

Three-dimensional structure of Erwinia chrysanthemi pectin methylesterase reveals a novel esterase active site.

Most structures of neutral lipases and esterases have been found to adopt the common alpha/beta hydrolase fold and contain a catalytic Ser-His-Asp triad. Some variation occurs in both the overall protein fold and in the location of the catalytic triad, and in some enzymes the role of the aspartate residue is replaced by a main-chain carbonyl oxygen atom. Here, we report the crystal structure of pectin methylesterase that has neither the common alpha/beta hydrolase fold nor the common catalytic triad. The structure of the Erwinia chrysanthemi enzyme was solved by multiple isomorphous replacement and refined at 2.4 A to a conventional crystallographic R-factor of 17.9 % (R(free) 21.1 %). This is the first structure of a pectin methylesterase and reveals the enzyme to comprise a right-handed parallel beta-helix as seen in the pectinolytic enzymes pectate lyase, pectin lyase, polygalacturonase and rhamnogalacturonase, and unlike the alpha/beta hydrolase fold of rhamnogalacturonan acetylesterase with which it shares esterase activity. Pectin methylesterase has no significant sequence similarity with any protein of known structure. Sequence conservation among the pectin methylesterases has been mapped onto the structure and reveals that the active site comprises two aspartate residues and an arginine residue. These proposed catalytic residues, located on the solvent-accessible surface of the parallel beta-helix and in a cleft formed by external loops, are at a location similar to that of the active site and substrate-binding cleft of pectate lyase. The structure of pectin methylesterase is an example of a new family of esterases.

Activation of calcium/calmodulin regulated kinases.

Among numerous protein kinases found in mammalian cell systems there is a distinct subfamily of serine/threonine kinases that are regulated by calmodulin or other related activators in a calcium concentration dependent manner. Members of this family are involved in various cellular processes like cell proliferation and death, cell motility and metabolic pathways. In this contribution we shall review the available structural biology data on five members of this kinase family (calcium/calmodulin dependent kinase, twitchin kinase, titin kinase, phosphorylase kinase, myosin light chain kinase). As a common element, all these kinases contain a regulatory tail, which is C-terminal to their catalytic domain. The available 3D structures of two members, the serine/threonine kinases of the giant muscle proteins twitchin and titin in the autoinhibited conformation, show how this regulatory tail blocks their active sites. The structures suggest that activation of these kinases requires unblocking the active site from the C-terminal extension and conformational rearrangement of the active site loops. Small angle scattering data for myosin light chain kinase indicate a complete release of the C-terminal extension upon calcium/calmodulin binding. In addition, members of this family are regulated by diverse add-on mechanisms, including phosphorylation of residues within the activation segment or the P+1 loop as well as by additional regulatory subunits. The available structural data lead to the hypothesis of two different activation mechanisms upon binding to calcium sensitive proteins. In one model, the regulatory tail is entirely released ("fall-apart"). The alternative model ("looping-out") proposes a two-anchored release mechanism.

Structural basis for activation of the titin kinase domain during myofibrillogenesis.

The giant muscle protein titin (connectin) is essential in the temporal and spatial control of the assembly of the highly ordered sarcomeres (contractile units) of striated muscle. Here we present the crystal structure of titin's only catalytic domain, an autoregulated serine kinase (titin kinase). The structure shows how the active site is inhibited by a tyrosine of the kinase domain. We describe a dual mechanism of activation of titin kinase that consists of phosphorylation of this tyrosine and binding of calcium/calmodulin to the regulatory tail. The serine kinase domain of titin is the first known non-arginine-aspartate kinase to be activated by phosphorylation. The phosphorylated tyrosine is not located in the activation segment, as in other kinases, but in the P + 1 loop, indicating that this tyrosine is a binding partner of the titin kinase substrate. Titin kinase phosphorylates the muscle protein telethonin in early differentiating myocytes, indicating that this kinase may act in myofibrillogenesis.

Crystallization and preliminary X-ray analysis of a member of a new family of pectate lyases, PeIL from Erwinia chrysanthemi.

PeIL, a pectate lyase (E.C. 4.2.2.9) from E. chrysanthemi 3937 that is not homologous to the lyases with known structures, has been purified and crystallized by the hanging-drop method using a variety of organic solvents as precipitant. Elongated lathes grown from poly(ethylene glycol) plus isopropanol belong to the space group P212121 with cell dimensions a = 55.5, b = 58.2, c = 16.4 A with a single molecule in the asymmetric unit. Although complete data sets have been collected to 2.3 A resolution, these crystals diffract to at least 1.9 A resolution and are suitable for structure determination. Chunky plates grown using other organic solvents as the precipitant diffracted to 3 A resolution and were partially characterized as a second orthorhombic crystal form with space group P21212 and cell dimensions a = 119.1, b = 140.5 and c = 105.4 A, suggesting four molecules in the asymmetric unit.

Structure and evolution of parallel beta-helix proteins.

Three bacterial pectate lyases, a pectin lyase from Aspergillus niger, the structures of rhamnogalacturonase A from Aspergillus aculeatus, RGase A, and the P22-phage tailspike protein, TSP, display the right-handed parallel beta-helix architecture first seen in pectate lyase. The lyases have 7 complete coils while RGase A and TSP have 11 and 12, respectively. Each coil contains three beta-strands and three turn regions named PB1, T1, PB2, T2, PB3, and T3 in their order of occurrence. The lyases have homologous sequences but RGase A and TSP do not show obvious sequence homology either to the lyases or to each other. However, the structural similarities between all these molecules are so extensive that divergence from a common ancestor is much more probable than convergence to the same fold. The region PB2-T2-PB3 is the best conserved region in the lyases and shows the clearest structural similarity. Not only is the pleating and the direction of the hydrogen bonding in the sheets conserved, but so is the unusual alphaL-conformation turn between the two sheets. However, the overall shape, the position of long loops, a conserved alpha-helix that covers the amino-terminal end of the parallel beta-helix and stacks of residues in alphaR-conformation at the start of PB1 all suggest a common ancestor. The functional similarity, that the enzymes all bind alpha-galactose containing polymers at an equivalent site involving PB1 and its two flanking turn regions, further supports divergent evolution. We suggest that the stacking of the coils and the unusual near perpendicular junction of PB2 and PB3 make the parallel beta-helix fold especially likely to maintain similar main chain conformations during divergent evolution even after all vestige of similarity in primary structure has vanished.

Two crystal structures of pectin lyase A from Aspergillus reveal a pH driven conformational change and striking divergence in the substrate-binding clefts of pectin and pectate lyases.

BACKGROUND: Microbial pectin and pectate lyases are virulence factors that degrade the pectic components of the plant cell wall. The homogalacturan backbone of pectin varies in its degree of methylation from the highly methylated and relatively hydrophobic form known as pectin, to the fully demethylated and highly charged form known as pectate. Methylated and demethylated regions of pectin are cleaved by pectin lyase and calcium-dependent pectate lyases, respectively. Protein engineering of lyases specific for particular patterns of methylation, will yield modified pectins of high value to the food and pharmaceutical industries. RESULTS: The crystal structures of pectin lyase A from two strains of Aspergillus niger, N400 and 4M-147, have been determined at pH 6.5 (2.4 A resolution) and pH 8.5 (1.93 A resolution), respectively. The structures were determined by a combination of molecular replacement, multiple isomorphous replacement and intercrystal averaging. Pectin lyase A folds into a parallel beta helix and shares many of the structural features of pectate lyases, despite no more than 17% sequence identity after pairwise structure-based alignment. These shared structural features include amino acid stacks and the asparagine ladder. However, the differences in the substrate-binding clefts of these two enzymes are striking. In pectin lyase A, the cleft is dominated by aromatic residues and is enveloped by negative electrostatic potential. In pectate lyases, this cleft is rich in charged residues and contains an elongated ribbon of positive potential when Ca2+ is bound. The major difference between the two pectin lyase A structures from the two strains is in the conformation of the loop formed by residues 182-187. These observed differences are due to the different pH values of crystallization. CONCLUSIONS: The substrate-binding clefts and catalytic machinery of pectin and pectate lyases have diverged significantly. Specificity is dictated by both the nature of the protein-carbohydrate interaction and long-range electrostatic forces. Three potential catalytic residues have been identified in pectin lyase, two of these are common to pectate lyases. Pectin lyase A does not bind Ca2+ but an arginine residue is found in an equivalent position to the Ca2+ ion in pectate lyase, suggesting a similar role in catalysis. The activity of pectin lyase A is pH -dependent with an optimum activity at pH 5.5. The activity drops above pH 7.0 due to a conformational change at the binding cleft, triggered by the proximity of two buried aspartate residues.

Crystallization and preliminary X-ray analysis of pectin lyase A from Aspergillus niger.

The major secreted pectin lyase (E.C. 4.2.2.10) from Aspergillus niger, strain 4M-147, has been purified and crystallized by the hanging-drop method using polyethylene glycol as precipitant. The crystals belong to the space group P2(1)2(1)2(1) with cell dimensions a = 45.2, b = 83.2, c = 93.1 A (1 A = 0.1 nm) and a single molecule in the asymmetric unit. The crystals diffract to at least 2.0 A resolution and are suitable for structure determination.