Chain flexibility and configurational dimensions of left handed Z- DNA and right handed B-DNA helices

The configurational behaviour of flexible helices of right handed B- and left handed Z-types have been analysed using statistical mechanical procedures. The configuration-dependent parameter, most importantly, the persistence length has been computed, using the heminucleotide scheme of treating polynucleotide chains under the approximation that perturbations in the backbone torsions produce sufficient flexibility in these helices. The values of persistence lengths obtained for Z-helices are very much higher than that of B-helices indicating that former is less flexible compared to the latter. These are in accordance with the results obtained recently on B- and Z-forms of poly(dG-dC). (dG-dC) using light scattering studies. Also the persistence lengths of B_II-DNA helices characterised by a skew 3'-hemiucleotide (ε-270°), and also when they coexist with B-DNA have been computed and the values lie within the range of experimentally reported values on B-helices. It is argued that the decrease in the persistence length values of B-DNA at higher salt concentration is due to additional small fluctuations in sugar residue torsions induced due to neutralisation of electrostatic repulsions between adjacent phosphates of the nucleotide. Noteworthy is that these are correlated to winding angle variations and the consequent bending of the helix. Contribution No. 659.     

Crystal structures and small-angle x-ray scattering analysis of UDP-galactopyranose mutase from the pathogenic fungus Aspergillus fumigatus

UDP-galactopyranose mutase (UGM) is a flavoenzyme that catalyzes the conversion of UDP-galactopyranose to UDP-galactofuranose, which is a central reaction in galactofuranose biosynthesis. Galactofuranose has never been found in humans but is an essential building block of the cell wall and extracellular matrix of many bacteria, fungi, and protozoa. The importance of UGM for the viability of many pathogens and its absence in humans make UGM a potential drug target. Here we report the first crystal structures and small-angle x-ray scattering data for UGM from the fungus Aspergillus fumigatus, the causative agent of aspergillosis. The structures reveal that Aspergillus UGM has several extra secondary and tertiary structural elements that are not found in bacterial UGMs yet are important for substrate recognition and oligomerization. Small-angle x-ray scattering data show that Aspergillus UGM forms a tetramer in solution, which is unprecedented for UGMs. The binding of UDP or the substrate induces profound conformational changes in the enzyme. Two loops on opposite sides of the active site move toward each other by over 10 Å to cover the substrate and create a closed active site. The degree of substrate-induced conformational change exceeds that of bacterial UGMs and is a direct consequence of the unique quaternary structure of Aspergillus UGM. Galactopyranose binds at the re face of the FAD isoalloxazine with the anomeric carbon atom poised for nucleophilic attack by the FAD N5 atom. The structural data provide new insight into substrate recognition and the catalytic mechanism and thus will aid inhibitor design.     

Modeling microdomains: the surface area of globin helices.

The accessible surface areas of 53 high-resolution globin helices are correlated with molecular weight. The linear fit is assessed for statistical accuracy using a boot-strap analysis, and by comparison to the areas of 13 ideal polyalanine alpha-helices. The accessible area of the unfolded helices is compared with the folded values before helix-helix packing. An analytical physical model is presented to explain the correlation, and to provide an analytical value for the surface area parameter in the diffusion-collision model of protein folding.     

Crystal structures explain functional differences in the two actin depolymerization factors of the malaria parasite

Apicomplexan parasites, such as the malaria-causing Plasmodium, utilize an actin-based motor for motility and host cell invasion. The actin filaments of these parasites are unusually short, and actin polymerization is under strict control of a small set of regulatory proteins, which are poorly conserved with their mammalian orthologs. Actin depolymerization factors (ADFs) are among the most important actin regulators, affecting the rates of filament turnover in a multifaceted manner. Plasmodium has two ADFs that display low sequence homology with each other and with the higher eukaryotic family members. Here, we show that ADF2, like canonical ADF proteins but unlike ADF1, binds to both globular and filamentous actin, severing filaments and inducing nucleotide exchange on the actin monomer. The crystal structure of Plasmodium ADF1 shows major differences from the ADF consensus, explaining the lack of F-actin binding. Plasmodium ADF2 structurally resembles the canonical members of the ADF/cofilin family.     

Additional criterion for the determination of the handedness of 21 helices in crystals of bile acids: Crystal structure of a tert‐butylphenyl derivative of cholic acid

[3β,5β,7α,12α]‐3‐(4‐t‐Butylbenzoilamine)‐7,12‐dihydroxycholan‐24‐oic acid was synthesized and recrystallized from chlorobenzene and acetone. Orthorhombic P2₁2₁2₁ and monoclinic P2₁ crystals were obtained, respectively, and both crystals include solvent and water molecules with a 1:1:1 stoichiometry. In the second case, there are two nonequivalent molecules of the steroid in the unit cell. In both crystals, a crossing structure results for the molecular packing, stabilized by hydrogen bonds between the steroid molecules. In the second crystal, water links steroid molecules of the same type, which are not directly connected through hydrogen bonds. The steroid molecules define helicoidal assemblies along 2₁ screw axes. The handedness in the crystal in chlorobenzene is unambiguously defined by following the criterion proposed by Miyata et al., as the steroids are in a belly‐to‐belly disposition around the axis. This motif does not appear in the crystal in acetone, and other criterion is required. On the basis of the fact that a staircase and its banister have the same handedness and using the crystal in chlorobenzene as reference, the additional criterion has been established. According to it, in the absence of a belly‐to‐belly orientation, the handedness must be defined by keeping the bile acid with its right side oriented toward the axis. Pitch angles of the helices and tilt angles of the molecules are also determined. Chirality, 2011. © 2011 Wiley‐Liss, Inc.     

Differentiation between transmembrane helices and peripheral helices by the deconvolution of circular dichroism spectra of membrane proteins.

The interpretation of the circular dichroism (CD) spectra of proteins to date requires additional secondary structural information of the proteins to be analyzed, such as X-ray or NMR data. Therefore, these methods are inappropriate for a CD database whose secondary structures are unknown, as in the case of the membrane proteins. The convex constraint analysis algorithm (Perczel, A., Hollósi, M., Tusnády, G., & Fasman, G. D., 1991, Protein Eng. 4, 669-679), on the other hand, operates only on a collection of spectral data to extract the common spectral components with their spectral weights. The linear combinations of these derived "pure" CD curves can reconstruct the original data set with great accuracy. For a membrane protein data set, the five-component spectra so obtained from the deconvolution consisted of two different types of alpha helices (the alpha helix in the soluble domain and the alpha T helix, for the transmembrane alpha helix), a beta-pleated sheet, a class C-like spectrum related to beta turns, and a spectrum correlated with the unordered conformation. The deconvoluted CD spectrum for the alpha T helix was characterized by a positive red-shifted band in the range 195-200 nm (+95,000 deg cm2 dmol-1), with the intensity of the negative band at 208 nm being slightly less negative than that of the 222-nm band (-50,000 and -60,000 deg cm2 dmol-1, respectively) in comparison with the regular alpha helix, with a positive band at 190 nm and two negative bands at 208 and 222 nm with magnitudes of +70,000, -30,000, and -30,000 deg cm2 dmol-1, respectively.     

Optimal bundling of transmembrane helices using sparse distance constraints

We present a two-step approach to modeling the transmembrane spanning helical bundles of integral membrane proteins using only sparse distance constraints, such as those derived from chemical cross-linking, dipolar EPR and FRET experiments. In Step 1, using an algorithm, we developed, the conformational space of membrane protein folds matching a set of distance constraints is explored to provide initial structures for local conformational searches. In Step 2, these structures refined against a custom penalty function that incorporates both measures derived from statistical analysis of solved membrane protein structures and distance constraints obtained from experiments. We begin by describing the statistical analysis of the solved membrane protein structures from which the theoretical portion of the penalty function was derived. We then describe the penalty function, and, using a set of six test cases, demonstrate that it is capable of distinguishing helical bundles that are close to the native bundle from those that are far from the native bundle. Finally, using a set of only 27 distance constraints extracted from the literature, we show that our method successfully recovers the structure of dark-adapted rhodopsin to within 3.2 A of the crystal structure.     

Assembly of transmembrane helices of simple polytopic membrane proteins from sequence conservation patterns

The transmembrane (TM) domains of most membrane proteins consist of helix bundles. The seemingly simple task of TM helix bundle assembly has turned out to be extremely difficult. This is true even for simple TM helix bundle proteins, i.e., those that have the simple form of compact TM helix bundles. Herein, we present a computational method that is capable of generating native-like structural models for simple TM helix bundle proteins having modest numbers of TM helices based on sequence conservation patterns. Thus, the only requirement for our method is the presence of more than 30 homologous sequences for an accurate extraction of sequence conservation patterns. The prediction method first computes a number of representative well-packed conformations for each pair of contacting TM helices, and then a library of tertiary folds is generated by overlaying overlapping TM helices of the representative conformations. This library is scored using sequence conservation patterns, and a subsequent clustering analysis yields five final models. Assuming that neighboring TM helices in the sequence contact each other (but not that TM helices A and G contact each other), the method produced structural models of Calpha atom root-mean-square deviation (CA RMSD) of 3-5 A from corresponding crystal structures for bacteriorhodopsin, halorhodopsin, sensory rhodopsin II, and rhodopsin. In blind predictions, this type of contact knowledge is not available. Mimicking this, predictions were made for the rotor of the V-type Na(+)-adenosine triphosphatase without such knowledge. The CA RMSD between the best model and its crystal structure is only 3.4 A, and its contact accuracy reaches 55%. Furthermore, the model correctly identifies the binding pocket for sodium ion. These results demonstrate that the method can be readily applied to ab initio structure prediction of simple TM helix bundle proteins having modest numbers of TM helices.     

Crystal Structures Capture Three States in the Catalytic Cycle of a Pyridoxal Phosphate (PLP) Synthase

PLP synthase (PLPS) is a remarkable single-enzyme biosynthetic pathway that produces pyridoxal 5′-phosphate (PLP) from glutamine, ribose 5-phosphate, and glyceraldehyde 3-phosphate. The intact enzyme includes 12 synthase and 12 glutaminase subunits. PLP synthesis occurs in the synthase active site by a complicated mechanism involving at least two covalent intermediates at a catalytic lysine. The first intermediate forms with ribose 5-phosphate. The glutaminase subunit is a glutamine amidotransferase that hydrolyzes glutamine and channels ammonia to the synthase active site. Ammonia attack on the first covalent intermediate forms the second intermediate. Glyceraldehyde 3-phosphate reacts with the second intermediate to form PLP. To investigate the mechanism of the synthase subunit, crystal structures were obtained for three intermediate states of the Geobacillus stearothermophilus intact PLPS or its synthase subunit. The structures capture the synthase active site at three distinct steps in its complicated catalytic cycle, provide insights into the elusive mechanism, and illustrate the coordinated motions within the synthase subunit that separate the catalytic states. In the intact PLPS with a Michaelis-like intermediate in the glutaminase active site, the first covalent intermediate of the synthase is fully sequestered within the enzyme by the ordering of a generally disordered 20-residue C-terminal tail. Following addition of ammonia, the synthase active site opens and admits the Lys-149 side chain, which participates in formation of the second intermediate and PLP. Roles are identified for conserved Asp-24 in the formation of the first intermediate and for conserved Arg-147 in the conversion of the first to the second intermediate.     

Crystal structure of the yeast metacaspase yca1

Yca1, the only metacaspase in Saccharomyces cerevisiae, is thought to be a clan CD cysteine protease that includes the caspase subfamily. Although yeast is a single cell eukaryote, it can undergo a cell death process reminiscent of apoptosis. Yca1 has been reported to play an important role in the regulation of such apoptotic process. However, the structure and functional mechanism of Yca1 remain largely enigmatic. In this study, we report the crystal structure of the Yca1 metacaspase at 1.7 Å resolution, confirming a caspase-like fold. In sharp contrast to canonical caspases, however, Yca1 exists as a monomer both in solution and in the crystals. Canonical caspase contains six β-strands, with strand β6 pairing up with β6 of another caspase molecule to form a homodimerization interface. In Yca1, an extra pair of antiparallel β-strands forms a continuous β-sheet with the six caspase-common β-strands, blocking potential dimerization. Yca1 was reported to undergo autocatalytic processing in yeast; overexpression in bacteria also led to autoprocessing of Yca1 into two fragments. Unexpectedly, we found that both the autocatalytic processing and the proteolytic activity of Yca1 are greatly facilitated by the presence of calcium (Ca²⁺), but not other divalent cations. Our structural and biochemical characterization identifies Yca1 as a Ca²⁺-activated cysteine protease that may cleave specific substrates during stress response in yeast.     

TM-MOTIF: an alignment viewer to annotate predicted transmembrane helices and conserved motifs in aligned set of sequences

Multiple sequence alignments become biologically meaningful only if conserved and functionally important residues and secondary structural elements preserved can be identified at equivalent positions. This is particularly important for transmembrane proteins like G-protein coupled receptors (GPCRs) with seven transmembrane helices. TM-MOTIF is a software package and an effective alignment viewer to identify and display conserved motifs and amino acid substitutions (AAS) at each position of the aligned set of homologous sequences of GPCRs. The key feature of the package is to display the predicted membrane topology for seven transmembrane helices in seven colours (VIBGYOR colouring scheme) and to map the identified motifs on its respective helices /loop regions. It is an interactive package which provides options to the user to submit query or pre-aligned set of GPCR sequences to align with a reference sequence, like rhodopsin, whose structure has been solved experimentally. It also provides the possibility to identify the nearest homologue from the available inbuilt GPCR or Olfactory Receptor cluster dataset whose association is already known for its receptor type. AVAILABILITY: The database is available for free at mini@ncbs.res.in.     

Crystal structure of a novel cysteinless plant Kunitz-type protease inhibitor

Bauhinia bauhinioides Cruzipain Inhibitor (BbCI) is a cysteine protease inhibitor highly homologous to plant Kunitz-type inhibitors. However, in contrast to classical Kunitz family inhibitors it lacks cysteine residues and therefore disulfide bridges. BbCI is also distinct in the ability to inactivate enzymes belonging to two different classes, cysteine and serine proteases. Besides inhibiting the cysteine protease cruzipain, BbCI also inhibits cathepsin L and the serine proteases HNE (human neutrophil elastase) and PPE (porcine pancreatic elastase). Monoclinic crystals of the recombinant inhibitor that diffract to 1.7 Å resolution were obtained using hanging drop method by vapor diffusion at 18 °C. The refined structure shows the conservative β-trefoil fold features of the Kunitz inhibitors. In BbCI, one of the two characteristic S-S bonds is replaced by the water-mediated interaction between Tyr125 and Gly132. In this work we explore the structural differences between Kunitz-type inhibitors and analyze the essential interactions that maintain the protein structural stability preserving its biological function.     

Crystal structure of a Josephin-ubiquitin complex: evolutionary restraints on ataxin-3 deubiquitinating activity

The Josephin domain is a conserved cysteine protease domain found in four human deubiquitinating enzymes: ataxin-3, the ataxin-3-like protein (ATXN3L), Josephin-1, and Josephin-2. Josephin domains from these four proteins were purified and assayed for their ability to cleave ubiquitin substrates. Reaction rates differed markedly both among the different proteins and for different substrates with a given protein. The ATXN3L Josephin domain is a significantly more efficient enzyme than the ataxin-3 domain despite their sharing 85% sequence identity. To understand the structural basis of this difference, the 2.6 Å x-ray crystal structure of the ATXN3L Josephin domain in complex with ubiquitin was determined. Although ataxin-3 and ATXN3L adopt similar folds, they bind ubiquitin in different, overlapping sites. Mutations were made in ataxin-3 at selected positions, introducing the corresponding ATXN3L residue. Only three such mutations are sufficient to increase the catalytic activity of the ataxin-3 domain to levels comparable with that of ATXN3L, suggesting that ataxin-3 has been subject to evolutionary restraints that keep its deubiquitinating activity in check.     

Ferrocenylboronates: Crystal structures and electrochemical properties

The electrochemical behavior of a series of eight previously reported monoferrocenyl- and diferrocenyl-boronates derived from tridentate ligands has been studied. Even if most mononuclear and all the dinuclear complexes examined showed to undergo slow decomposition in nonaqueous solution (releasing free ferrocenyl boronic acid), their redox activity has been investigated. It has been proved that the oxidation of the two ferrocenyl subunits in the dimeric species proceeds simultaneously, indicating that no mutual electronic interaction exists between them. Additionally, the solid-state molecular structures of two diferrocenyl complexes (2a and 2b) were studied by X-ray diffraction. The analysis confirms the formation of a [5.4.0] heterobicycle with the presence of two different boron atoms, one in a tetrahedral geometry and the other one in a trigonal geometry.     

Crystal Structure of the cis-Dimer of Nectin-1: implications for the architecture of cell-cell junctions

In multicellular organisms, cells are interconnected by cell adhesion molecules. Nectins are immunoglobulin (Ig)-like cell adhesion molecules that mediate homotypic and heterotypic cell-cell adhesion, playing key roles in tissue organization. To mediate cell-cell adhesion, nectin molecules dimerize in cis on the surface of the same cell, followed by trans-dimerization of the cis-dimers between the neighboring cells. Previous cell biological studies deduced that the first Ig-like domain of nectin and the second Ig-like domain are involved in trans-dimerization and cis-dimerization, respectively. However, to understand better the steps involved in nectin adhesion, the structural basis for the dimerization of nectin must be determined. In this study, we determined the first crystal structure of the entire extracellular region of nectin-1. In the crystal, nectin-1 formed a V-shaped homophilic dimer through the first Ig-like domain. Structure-based site-directed mutagenesis of the first Ig-like domain identified four essential residues that are involved in the homophilic dimerization. Upon mutating the four residues, nectin-1 significantly decreased cis-dimerization on the surface of cultured cells and abolished the homophilic and heterophilic adhesion activities. These results indicate that, in contrast with the previous notion, our structure represents a cis-dimer. Thus, our findings clearly reveal the structural basis for the cis-dimerization of nectins through the first Ig-like domains.     

The Crystal Structure of Human Transketolase and New Insights into Its Mode of Action

The crystal structure of human transketolase (TKT), a thiamine diphosphate (ThDP) and Ca²⁺-dependent enzyme that catalyzes the interketol transfer between ketoses and aldoses as part of the pentose phosphate pathway, has been determined to 1.75 Å resolution. The recombinantly produced protein crystallized in space group C2 containing one monomer in the asymmetric unit. Two monomers form the homodimeric biological assembly with two identical active sites at the dimer interface. Although the protomer exhibits the typical three (α/β)-domain structure and topology reported for TKTs from other species, structural differences are observed for several loop regions and the linker that connects the PP and Pyr domain. The cofactor and substrate binding sites of human TKT bear high resemblance to those of other TKTs but also feature unique properties, including two lysines and a serine that interact with the β-phosphate of ThDP. Furthermore, Gln¹⁸⁹ spans over the thiazolium moiety of ThDP and replaces an isoleucine found in most non-mammalian TKTs. The side chain of Gln⁴²⁸ forms a hydrogen bond with the 4′-amino group of ThDP and replaces a histidine that is invariant in all non-mammalian TKTs. All other amino acids involved in substrate binding and catalysis are strictly conserved. Besides a steady-state kinetic analysis, microscopic equilibria of the donor half-reaction were characterized by an NMR-based intermediate analysis. These studies reveal that formation of the central 1,2-dihydroxyethyl-ThDP carbanion-enamine intermediate is thermodynamically favored with increasing carbon chain length of the donor ketose substrate. Based on the structure of human transketolase and sequence alignments, putative functional properties of the related transketolase-like proteins TKTL1 and -2 are discussed in light of recent findings suggesting that TKTL1 plays a role in cancerogenesis.     

Crystal structures of saposins A and C

Saposins A and C are sphingolipid activator proteins required for the lysosomal breakdown of galactosylceramide and glucosylceramide, respectively. The saposins interact with lipids, leading to an enhanced accessibility of the lipid headgroups to their cognate hydrolases. We have determined the crystal structures of human saposins A and C to 2.0 Angstroms and 2.4 Angstroms, respectively, and both reveal the compact, monomeric saposin fold. We confirmed that these two proteins were monomeric in solution at pH 7.0 by analytical centrifugation. However, at pH 4.8, in the presence of the detergent C(8)E(5), saposin A assembled into dimers, while saposin C formed trimers. Saposin B was dimeric under all conditions tested. The self-association of the saposins is likely to be relevant to how these small proteins interact with lipids, membranes, and hydrolase enzymes.