Additional Secondary Structural Elements Research Articles

NMR resonance assignment and dynamics of profilin from Heimdallarchaeota

The origin of the eukaryotic cell is an unsettled scientific question. The Asgard superphylum has emerged as a compelling target for studying eukaryogenesis due to the previously unseen diversity of eukaryotic signature proteins. However, our knowledge about these proteins is still relegated to metagenomic data and very little is known about their structural properties. Additionally, it is still unclear if these proteins are functionally homologous to their eukaryotic counterparts. Here, we expressed, purified and structurally characterized profilin from Heimdallarchaeota in the Asgard superphylum. The structural analysis shows that while this profilin possesses similar secondary structural elements as eukaryotic profilin, it contains additional secondary structural elements that could be critical for its function and an indication of divergent evolution.

Molecular envelope and atomic model of an anti-terminated glyQS T-box regulator in complex with tRNAGly.

A T-box regulator or riboswitch actively monitors the levels of charged/uncharged tRNA and participates in amino acid homeostasis by regulating genes involved in their utilization or biosynthesis. It has an aptamer domain for cognate tRNA recognition and an expression platform to sense the charge state and modulate gene expression. These two conserved domains are connected by a variable linker that harbors additional secondary structural elements, such as Stem III. The structural basis for specific tRNA binding is known, but the structural basis for charge sensing and the role of other elements remains elusive. To gain new structural insights on the T-box mechanism, a molecular envelope was calculated from small angle X-ray scattering data for the Bacillus subtilis glyQS T-box riboswitch in complex with an uncharged tRNAGly. A structural model of an anti-terminated glyQS T-box in complex with its cognate tRNAGly was derived based on the molecular envelope. It shows the location and relative orientation of various secondary structural elements. The model was validated by comparing the envelopes of the wild-type complex and two variants. The structural model suggests that in addition to a possible regulatory role, Stem III could aid in preferential stabilization of the T-box anti-terminated state allowing read-through of regulated genes.

Ligand Binding Reduces Conformational Flexibility in the Active Site of Tyrosine Phosphatase Related to Biofilm Formation A (TpbA) from Pseudomonasaeruginosa

Tyrosine phosphatase related to biofilm formation A (TpbA) is a periplasmic dual-specificity phosphatase (DUSP) that controls biofilm formation in the pathogenic bacterium Pseudomonas aeruginosa. While DUSPs are known to regulate important cellular functions in both prokaryotes and eukaryotes, very few structures of bacterial DUSPs are available. Here, we present the solution structure of TpbA in the ligand-free open conformation, along with an analysis of the structural and dynamic changes that accompany ligand/phosphate binding. While TpbA adopts a typical DUSP fold, it also possesses distinct structural features that distinguish it from eukaryotic DUSPs. These include additional secondary structural elements, β0 and α6, and unique conformations of the variable insert, the α4–α5 loop and helix α5 that impart TpbA with a flat active-site surface. In the absence of ligand, the protein tyrosine phosphatase loop is disordered and the general acid loop adopts an open conformation, placing the catalytic aspartate, Asp105, more than 11Å away from the active site. Furthermore, the loops surrounding the active site experience motions on multiple timescales, consistent with a combination of conformational heterogeneity and fast (picosecond to nanosecond) timescale dynamics, which are significantly reduced upon ligand binding. Taken together, these data structurally distinguish TpbA and possibly other bacterial DUSPs from eukaryotic DUSPs and provide a rich picture of active-site dynamics in the ligand-free state that are lost upon ligand binding.

Homology swapping of intrinsic secondary structural elements between cellulosomal types I and II cohesins and their effect on dockerin binding

The high-affinity cohesin–dockerin interaction dictates the suprastructural assembly of the multienzyme cellulosome complex. The interaction between these two complementary families of protein modules was found to be species-specific and type-dependent. The structure of the type II cohesin module possesses additional intrinsic secondary structural elements absent in the type I cohesin, i.e., an α-helix and two singular "β-flaps". The role of these extra secondary structures in dockerin recognition was studied in this work using gene swapping, in which corresponding homologous stretches of types I and II cohesins were interchanged. The specificity of binding of the resultant chimaeric cohesins was determined by enzyme-linked affinity assay. Several chimaeric cohesins retained dockerin recognition properties. Hence, these cohesins may undergo manipulations (insertion/deletion of peptide segments) without altering their affinity toward their counterpart dockerin, although type-dependent binding specificity cannot be converted by swapping of the additional secondary structural elements between the two cohesin types. The results further emphasize the strong affinity and plasticity between the cohesin and dockerin pair and are consistent with the known findings on specificity of the types I and II interactions. These studies provide insight into the structural and functional resilience of the cohesins and thus have direct bearing on their potential use in biotechnological applications.

Structural Insights into the Specificity of Xyn10B from Paenibacillus barcinonensis and Its Improved Stability by Forced Protein Evolution

Paenibacillus barcinonensis is a soil bacterium bearing a complex set of enzymes for xylan degradation, including several secreted enzymes and Xyn10B, one of the few intracellular xylanases reported to date. The crystal structure of Xyn10B has been determined by x-ray analysis. The enzyme folds into the typical (beta/alpha)(8) barrel of family 10 glycosyl hydrolases (GH10), with additional secondary structure elements within the beta/alpha motifs. One of these loops -L7- located at the beta7 C terminus, was essential for xylanase activity as its partial deletion yielded an inactive enzyme. The loop contains residues His(249)-Glu(250), which shape a pocket opened to solvent in close proximity to the +2 subsite, which has not been described in other GH10 enzymes. This wide cavity at the +2 subsite, where methyl-2,4-pentanediol from the crystallization medium was found, is a noteworthy feature of Xyn10B, as compared with the narrow crevice described for other GH10 xylanases. Docking analysis showed that this open cavity can accommodate glucuronic acid decorations of xylo-oligosaccharides. Co-crystallization experiments with conduramine derivative inhibitors supported the importance of this open cavity at the +2 subsite for Xyn10B activity. Several mutant derivatives of Xyn10B with improved thermal stability were obtained by forced evolution. Among them, mutant xylanases S15L and M93V showed increased half-life, whereas the double mutant S15L/M93V exhibited a further increase in stability, showing a 20-fold higher heat resistance than the wild type xylanase. All the mutations obtained were located on the surface of Xyn10B. Replacement of a Ser by a Leu residue in mutant xylanase S15L can increase hydrophobic packing efficiency and fill a superficial indentation of the protein, giving rise to a more compact structure of the enzyme.

3D architecture of DNA Pol α reveals the functional core of multi-subunit replicative polymerases

Eukaryotic DNA replication requires the coordinated activity of the multi-subunit DNA polymerases: Pol α, Pol δ and Pol ɛ. The conserved catalytic and regulatory B subunits associate in a constitutive heterodimer that represents the functional core of all three replicative polymerases. Here, we combine X-ray crystallography and electron microscopy (EM) to describe subunit interaction and 3D architecture of heterodimeric yeast Pol α. The crystal structure of the C-terminal domain (CTD) of the catalytic subunit bound to the B subunit illustrates a conserved mechanism of accessory factor recruitment by replicative polymerases. The EM reconstructions of Pol α reveal a bilobal shape with separate catalytic and regulatory modules. Docking of the B–CTD complex in the EM reconstruction shows that the B subunit is tethered to the polymerase domain through a structured but flexible linker. Our combined findings provide a structural template for the common functional architecture of the three major replicative DNA polymerases.

The Structure and Binding Behavior of the Bacterial Cell Surface Layer Protein SbsC

Surface layers (S-layers) comprise the outermost cell envelope component of most archaea and many bacteria. Here we present the structure of the bacterial S-layer protein SbsC from Geobacillus stearothermophilus, showing a very elongated and flexible molecule, with strong and specific binding to the secondary cell wall polymer (SCWP). The crystal structure of rSbsC((31-844)) revealed a novel fold, consisting of six separate domains, which are connected by short flexible linkers. The N-terminal domain exhibits positively charged residues regularly spaced along the putative ligand binding site matching the distance of the negative charges on the extended SCWP. Upon SCWP binding, a considerable stabilization of the N-terminal domain occurs. These findings provide insight into the processes of S-layer attachment to the underlying cell wall and self-assembly, and also accommodate the observed mechanical strength, the polarity of the S-layer, and the pronounced requirement for surface flexibility inherent to cell growth and division.

The carbohydrate recognition domain of Langerin reveals high structural similarity with the one of DC-SIGN but an additional, calcium-independent sugar-binding site

Langerin is a type II transmembrane oligosaccharide receptor on Langerhans cells (LCs), a prominent subclass of dendritic cells (DCs) that mediate immune responses in epithelia and play a role in HIV degradation. Its extracellular moiety comprises a neck region with several heptad repeats and an exposed carboxy-terminal calcium-type carbohydrate-recognition domain (CRD). The CRD of human Langerin, which was expressed as a soluble protein in the periplasm of E. coli, was crystallized both alone and in the presence of two sugars, followed by X-ray analyses to resolutions of 2.5 Å for apo-Langerin and to 1.6 Å and 2.1 Å for the complexes with mannose and maltose, respectively. The fold of the Langerin CRD (dubbed LangA) resembles that of other typical C-type lectins such as DC-SIGN. However, especially in the long loop region (LLR), which is responsible for carbohydrate-binding, two additional secondary structure elements are present: a 3 10 helix and a small β-sheet arising from the extended β-strand 2, which enters into a hairpin and a new strand β2′. Unexpectedly, the crystal structures in the presence of maltose and mannose reveal two sugar-binding sites. One is calcium-dependent and structurally conserved in the C-type lectin family whereas the second one represents a novel, calcium-independent type. Based on these data, a model for the binding of mannan, a component of many endogenous as well as viral glycoproteins, is proposed and the differences in binding behavior between Langerin and DC-SIGN with respect to the Lewis X carbohydrate antigen and its derivatives can be explained. Therefore, the crystal structure of LangA should be helpful for the development of new marker reagents selective for LCs and also of therapeutic compounds that may enhance the inhibitory role of Langerin towards HIV infection.

Crystal Structure of the Non-heme Iron Dioxygenase PtlH in Pentalenolactone Biosynthesis

The non-heme iron dioxygenase PtlH from the soil organism Streptomyces avermitilis is a member of the iron(II)/alpha-ketoglutarate-dependent dioxygenase superfamily and catalyzes an essential reaction in the biosynthesis of the sesquiterpenoid antibiotic pentalenolactone. To investigate the structural basis for substrate recognition and catalysis, we have determined the x-ray crystal structure of PtlH in several complexes with the cofactors iron, alpha-ketoglutarate, and the non-reactive enantiomer of the substrate, ent-1-deoxypentalenic acid, in four different crystal forms to up to 1.31 A resolution. The overall structure of PtlH forms a double-stranded barrel helix fold, and the cofactor-binding site for iron and alpha-ketoglutarate is similar to other double-stranded barrel helix fold enzymes. Additional secondary structure elements that contribute to the substrate-binding site in PtlH are not conserved in other double-stranded barrel helix fold enzymes. Binding of the substrate enantiomer induces a reorganization of the monoclinic crystal lattice leading to a disorder-order transition of a C-terminal alpha-helix. The newly formed helix blocks the major access to the active site and effectively traps the bound substrate. Kinetic analysis of wild type and site-directed mutant proteins confirms a critical function of two arginine residues in substrate binding, while simulated docking of the enzymatic reaction product reveals the likely orientation of bound substrate.

GntR family of regulators in Mycobacterium smegmatis: a sequence and structure based characterization

BackgroundMycobacterium smegmatis is fast growing non-pathogenic mycobacteria. This organism has been widely used as a model organism to study the biology of other virulent and extremely slow growing species like Mycobacterium tuberculosis. Based on the homology of the N-terminal DNA binding domain, the recently sequenced genome of M. smegmatis has been shown to possess several putative GntR regulators. A striking characteristic feature of this family of regulators is that they possess a conserved N-terminal DNA binding domain and a diverse C-terminal domain involved in the effector binding and/or oligomerization. Since the physiological role of these regulators is critically dependent upon effector binding and operator sites, we have analysed and classified these regulators into their specific subfamilies and identified their potential binding sites.ResultsThe sequence analysis of M. smegmatis putative GntRs has revealed that FadR, HutC, MocR and the YtrA-like regulators are encoded by 45, 8, 8 and 1 genes respectively. Further out of 45 FadR-like regulators, 19 were classified into the FadR group and 26 into the VanR group. All these proteins showed similar secondary structural elements specific to their respective subfamilies except MSMEG_3959, which showed additional secondary structural elements. Using the reciprocal BLAST searches, we further identified the orthologs of these regulators in Bacillus subtilis and other mycobacteria. Since the expression of many regulators is auto-regulatory, we have identified potential operator sites for a number of these GntR regulators by analyzing the upstream sequences.ConclusionThis study helps in extending the annotation of M. smegmatis GntR proteins. It identifies the GntR regulators of M. smegmatis that could serve as a model for studying orthologous regulators from virulent as well as other saprophytic mycobacteria. This study also sheds some light on the nucleotide preferences in the target-motifs of GntRs thus providing important leads for initiating the experimental characterization of these proteins, construction of the gene regulatory network for these regulators and an understanding of the influence of these proteins on the physiology of the mycobacteria.

NMR structure of the three quasi RNA recognition motifs (qRRMs) of human hnRNP F and interaction studies with Bcl-x G-tract RNA: a novel mode of RNA recognition.

The heterogeneous nuclear ribonucleoprotein (hnRNP) F belongs to the hnRNP H family involved in the regulation of alternative splicing and polyadenylation and specifically recognizes poly(G) sequences (G-tracts). In particular, hnRNP F binds a G-tract of the Bcl-x RNA and regulates its alternative splicing, leading to two isoforms, Bcl-xS and Bcl-xL, with antagonist functions. In order to gain insight into G-tract recognition by hnRNP H members, we initiated an NMR study of human hnRNP F. We present the solution structure of the three quasi RNA recognition motifs (qRRMs) of hnRNP F and identify the residues that are important for the interaction with the Bcl-x RNA by NMR chemical shift perturbation and mutagenesis experiments. The three qRRMs exhibit the canonical βαββαβ RRM fold but additional secondary structure elements are present in the two N-terminal qRRMs of hnRNP F. We show that qRRM1 and qRRM2 but not qRRM3 are responsible for G-tract recognition and that the residues of qRRM1 and qRRM2 involved in G-tract interaction are not on the β-sheet surface as observed for the classical RRM but are part of a short β-hairpin and two adjacent loops. These regions define a novel interaction surface for RNA recognition by RRMs.

The NMR and X-ray Structures of the Saccharomyces cerevisiae Vts1 SAM Domain Define a Surface for the Recognition of RNA Hairpins

The SAM domain of the Saccharomyces cerevisiae post-transcriptional regulator Vts1 has a high affinity towards RNA hairpins containing a CUGGC pentaloop. We present the 1.6 Angstroms X-ray crystal structure of the Vts1 SAM domain in its unliganded state, and the NMR solution structure of this domain in its RNA-bound state. Both structures reveal a canonical five helix SAM domain flanked by additional secondary structural elements at the N and C termini. The two structures are essentially identical, implying that no major structural rearrangements occur upon RNA binding. Amide chemical shift changes map the RNA-binding site to a shallow, basic patch at the junction of helix alpha5 and the loop connecting helices alpha1 and alpha2.

Escherichia coli FolC Structure Reveals an Unexpected Dihydrofolate Binding Site Providing an Attractive Target for Anti-microbial Therapy

In some bacteria, such as Escherichia coli, the addition of L-glutamate to dihydropteroate (dihydrofolate synthetase activity) and the subsequent additions of L-glutamate to tetrahydrofolate (folylpolyglutamate synthetase (FPGS) activity) are catalyzed by the same enzyme, FolC. The crystal structure of E. coli FolC is described in this paper. It showed strong similarities to that of the FPGS enzyme of Lactobacillus casei within the ATP binding site and the catalytic site, as do all other members of the Mur synthethase superfamily. FolC structure revealed an unexpected dihydropteroate binding site very different from the folate site identified previously in the FPGS structure. The relevance of this site is exemplified by the presence of phosphorylated dihydropteroate, a reaction intermediate in the DHFS reaction. L. casei FPGS is considered a relevant model for human FPGS. As such, the presence of a folate binding site in E. coli FolC, which is different from the one seen in FPGS enzymes, provides avenues for the design of specific inhibitors of this enzyme in antimicrobial therapy.

Crystal Structure of a Type-II Cohesin Module from the Bacteroides cellulosolvens Cellulosome Reveals Novel and Distinctive Secondary Structural Elements

The incorporation of enzymes into the multi-enzyme cellulosome complex and its anchoring to the bacterial cell surface are dictated by a set of binding interactions between two complementary protein modules: the cohesin and the dockerin. In this work, the X-ray crystal structure of a type-II cohesin from scaffoldin A of Bacteroides cellulosolvens has been determined to a resolution of 1.6 angstroms using molecular replacement. The type-II B. cellulosolvens cohesin (Bc-cohesin-II) is the first detailed description of a crystal structure for a type-II cohesin, and its features were compared with the known type-I cohesins from Clostridium thermocellum and Clostridium cellulolyticum (Ct-cohesin-I and Cc-cohesin-I, respectively). The overall jelly-roll topology of the type-II Bc-cohesin is very similar to that observed for the type-I cohesins with three additional secondary structures: an alpha-helix and two "beta-flaps" that disrupt the normal course of a beta-strand. In addition, beta-strand 5 is elevated by approximately 4 angstroms on the surface of the molecule, relative to the type-I Ct and Cc-cohesins. Like its type-I analogue, the hydrophobic/aromatic core of Bc-cohesin-II comprises an upper and lower core, but an additional aromatic patch and conserved tryptophan at the crown of the molecule serves to stabilize the alpha-helix of the type-II cohesin. Comparison of Bc-cohesin-II with the known type-I cohesin-dockerin heterodimer suggests that each of the additional secondary structural elements assumes a flanking position relative to the putative dockerin-binding surface. The raised ridge formed by beta-strand 5 confers additional distinctive topographic features to the proposed binding interface that collectively distinguish between the type-II and type-I cohesins.

Molecular Dynamics Simulation of Dimeric and Monomeric Forms of Human Prion Protein: Insight into Dynamics and Properties

A central theme in prion protein research is the detection of the process that underlies the conformational transition from the normal cellular prion form (PrP C) to its pathogenic isoform (PrP Sc). Although the three-dimensional structures of monomeric and dimeric human prion protein (HuPrP) have been revealed by NMR spectroscopy and x-ray crystallography, the process underlying the conformational change from PrP C to PrP Sc and the dynamics and functions of PrP C remain unknown. The dimeric form is thought to play an important role in the conformational transition. In this study, we performed molecular dynamics (MD) simulations on monomeric and dimeric HuPrP at 300 K and 500 K for 10 ns to investigate the differences in the properties of the monomer and the dimer from the perspective of dynamic and structural behaviors. Simulations were also undertaken with Asp178Asn and acidic pH, which is known as a disease-associated factor. Our results indicate that the dynamics of the dimer and monomer were similar (e.g., denaturation of helices and elongation of the β-sheet). However, additional secondary structure elements formed in the dimer might result in showing the differences in dynamics and properties between the monomer and dimer (e.g., the greater retention of dimeric than monomeric tertiary structure).

Crystal Structure of Decameric Fructose-6-Phosphate Aldolase from Escherichia coli Reveals Inter-subunit Helix Swapping as a Structural Basis for Assembly Differences in the Transaldolase Family

Fructose-6-phosphate aldolase from Escherichia coli is a member of a small enzyme subfamily (MipB/TalC family) that belongs to the class I aldolases. The three-dimensional structure of this enzyme has been determined at 1.93 Å resolution by single isomorphous replacement and tenfold non-crystallographic symmetry averaging and refined to an R-factor of 19.9% ( R free 21.3%). The subunit folds into an α/β barrel, with the catalytic lysine residue on barrel strand β4. It is very similar in overall structure to that of bacterial and mammalian transaldolases, although more compact due to extensive deletions of additional secondary structural elements. The enzyme forms a decamer of identical subunits with point group symmetry 52. Five subunits are arranged as a pentamer, and two ring-like pentamers pack like a doughnut to form the decamer. A major interaction within the pentamer is through the C-terminal helix from one monomer, which runs across the active site of the neighbouring subunit. In classical transaldolases, this helix folds back and covers the active site of the same subunit and is involved in dimer formation. The inter-subunit helix swapping appears to be a major determinant for the formation of pentamers rather than dimers while at the same time preserving importing interactions of this helix with the active site of the enzyme. The active site lysine residue is covalently modified, by forming a carbinolamine with glyceraldehyde from the crystallisation mixture. The catalytic machinery is very similar to that of transaldolase, which together with the overall structural similarity suggests that enzymes of the MipB/TALC subfamily are evolutionary related to the transaldolase family.

High-resolution structure of a polyomavirus VP1-oligosaccharide complex: implications for assembly and receptor binding.

The crystal structure of a recombinant polyomavirus VP1 pentamer (residues 32-320) in complex with a branched disialylated hexasaccharide receptor fragment has been determined at 1.9 A resolution. The result extends our understanding of oligosaccharide receptor recognition. It also suggests a mechanism for enhancing the fidelity of virus assembly. We have previously described the structure of the complete polyomavirus particle complexed with this receptor fragment at 3.65 A. The model presented here offers a much more refined view of the interactions that determine carbohydrate recognition and allows us to assign additional specific contacts, in particular those involving the (alpha2,6)-linked, branching sialic acid. The structure of the unliganded VP1 pentamer, determined independently, shows that the oligosaccharide fits into a preformed groove and induces no measurable structural rearrangements. A comparison with assembled VP1 in the virus capsid reveals a rearrangement of residues 32-45 at the base of the pentamer. This segment may help prevent the formation of incorrectly assembled particles by reducing the likelihood that the C-terminal arm will fold back into its pentamer of origin.

The structure of Pneumocystis carinii dihydrofolate reductase to 1.9 A resolution.

The fungal pathogen Pneumocystis carinii causes a pneumonia which is an opportunistic infection of AIDS patients. Current therapy includes the dihydrofolate reductase (DHFR) inhibitor trimethoprim which is selective but only a relatively weak inhibitor of the enzyme for P. carinii. Determination of the three-dimensional structure of the enzyme should form the basis for design of more potent and selective therapeutic agents for treatment of the disease. The structure of P. carinii DHFR in complex with reduced nicotinamide adenine dinucleotide phosphate and trimethoprim has accordingly been solved by X-ray crystallography. The structure of the ternary complex has been refined at 1.86 A resolution (R = 0.181). A similar ternary complex with piritrexim (which is a tighter binding, but less selective inhibitor) has also been solved, as has the binary complex holoenzyme, both at 2.5 A resolution. These structures show how two drugs interact with a fungal DHFR. A comparison of the three-dimensional structure of this relatively large DHFR with bacterial or mammalian enzyme-inhibitor complexes determined previously highlights some additional secondary structure elements in this particular enzyme species. These comparisons provide further insight into the principles governing DHFR-inhibitor interaction, in which the volume of the active site appears to determine the strength of inhibitor binding.