Crystal structure of the C-terminal three-helix bundle subdomain of C. elegans Hsp70

Hsp70 chaperones are composed of two domains; the 40 kDa N-terminal nucleotide-binding domain (NDB) and the 30 kDa C-terminal substrate-binding domain (SBD). Structures of the SBD from Escherichia coli homologues DnaK and HscA show it can be further divided into an 18 kDa beta-sandwich subdomain, which forms the hydrophobic binding pocket, and a 10 kDa C-terminal three-helix bundle that forms a lid over the binding pocket. Across prokaryotes and eukaryotes, the NBD and beta-sandwich subdomain are well conserved in both sequence and structure. The C-terminal subdomain is, however, more evolutionary variable and the only eukaryotic structure from rat Hsc70 revealed a diverged helix-loop-helix fold. We have solved the crystal structure of the C-terminal 10 kDa subdomain from Caenorhabditis elegans Hsp70 which forms a helical-bundle similar to the prokaryotic homologues. This provides the first confirmation of the structural conservation of this subdomain in eukaryotes. Comparison with the rat structure reveals a domain-swap dimerisation mechanism; however, the C. elegans subdomain exists exclusively as a monomer in solution in agreement with the hypothesis that regions out with the C-terminal subdomain are necessary for Hsp70 self-association.     

Chitobiose phosphorylase from Vibrio proteolyticus, a member of glycosyl transferase family 36, has a clan GH-L-like (alpha/alpha)(6) barrel fold

Vibrio proteolyticus chitobiose phosphorylase (ChBP) belongs to glycosyl transferase family 36 (GT-36), and catalyzes the reversible phosphorolysis of chitobiose into alpha-GlcNAc-1-phosphate and GlcNAc with inversion of the anomeric configuration. As the first known structures of a GT-36 enzyme, we determined the crystal structure of ChBP in a ternary complex with GlcNAc and SO(4). It is also the first structures of an inverting phosphorolytic enzyme in a complex with a sugar and a sulfate ion, and reveals a pseudo-ternary complex structure of enzyme-sugar-phosphate. ChBP comprises a beta sandwich domain and an (alpha/alpha)(6) barrel domain, constituting a distinctive structure among GT families. Instead, it shows significant structural similarity with glycoside hydrolase (GH) enzymes, glucoamylases (GH-15), and maltose phosphorylase (GH-65) in clan GH-L. The structural similarity reported here, together with distant sequence similarities between ChBP and GHs, led to the reclassification of family GT-36 into a novel GH family, namely GH-94.     

Solution structure of human initiation factor eIF2alpha reveals homology to the elongation factor eEF1B

The GTP-bound form of the trimeric eukaryotic translation initiation factor 2 (eIF2) transfers aminoacylated initiator methionyl tRNA onto the 40S ribosome. We have solved with solution NMR the structure of the alpha subunit of human eIF2 (heIF2alpha). The protein consists of two domains that are mobile relative to each other. The N-terminal domain has an S1-type oligonucleotide/oligosaccharide binding-fold subdomain and an alpha-helical subdomain. The C-terminal domain adopts an alphabeta-fold very similar to the C-terminal domain of elongation factor (eEF) 1Balpha, the guanine-nucleotide exchange factor for eEF1A. The structural and functional similarities found between eIF2alpha/eIF2gamma and eEF1Balpha/eEF1A suggest a model for the interaction of eIF2alpha with eIF2gamma, and eIF2 with Met-tRNAiMet. It further indicates a previously unrecognized evolutionary lineage of eIF2alpha/gamma from the functionally related elongation factor eEF1Balpha/eEF1A complex.     

Structural and functional analysis of a glycoside hydrolase family 97 enzyme from Bacteroides thetaiotaomicron

SusB, an 84-kDa alpha-glucoside hydrolase involved in the starch utilization system (sus) of Bacteroides thetaiotaomicron, belongs to glycoside hydrolase (GH) family 97. We have determined the enzymatic characteristics and the crystal structures in free and acarbose-bound form at 1.6A resolution. SusB hydrolyzes the alpha-glucosidic linkage, with inversion of anomeric configuration liberating the beta-anomer of glucose as the reaction product. The substrate specificity of SusB, hydrolyzing not only alpha-1,4-glucosidic linkages but also alpha-1,6-, alpha-1,3-, and alpha-1,2-glucosidic linkages, is clearly different from other well known glucoamylases belonging to GH15. The structure of SusB was solved by the single-wavelength anomalous diffraction method with sulfur atoms as anomalous scatterers using an in-house x-ray source. SusB includes three domains as follows: the N-terminal, catalytic, and C-terminal domains. The structure of the SusB-acarbose complex shows a constellation of carboxyl groups at the catalytic center; Glu532 is positioned to provide protonic assistance to leaving group departure, with Glu439 and Glu508 both positioned to provide base-catalyzed assistance for inverting nucleophilic attack by water. A structural comparison with other glycoside hydrolases revealed significant similarity between the catalytic domain of SusB and those of alpha-retaining glycoside hydrolases belonging to GH27, -36, and -31 despite the differences in catalytic mechanism. SusB and the other retaining enzymes appear to have diverged from a common ancestor and individually acquired the functional carboxyl groups during the process of evolution. Furthermore, sequence comparison of the active site based on the structure of SusB indicated that GH97 included both retaining and inverting enzymes.     

Glucoamylase: structure/function relationships, and protein engineering

Glucoamylases are inverting exo-acting starch hydrolases releasing beta-glucose from the non-reducing ends of starch and related substrates. The majority of glucoamylases are multidomain enzymes consisting of a catalytic domain connected to a starch-binding domain by an O-glycosylated linker region. Three-dimensional structures have been determined of free and inhibitor complexed glucoamylases from Aspergillus awamori var. X100, Aspergillus niger, and Saccharomycopsis fibuligera. The catalytic domain folds as a twisted (alpha/alpha)(6)-barrel with a central funnel-shaped active site, while the starch-binding domain folds as an antiparallel beta-barrel and has two binding sites for starch or beta-cyclodextrin. Certain glucoamylases are widely applied industrially in the manufacture of glucose and fructose syrups. For more than a decade mutational investigations of glucoamylase have addressed fundamental structure/function relationships in the binding and catalytic mechanisms. In parallel, issues of relevance for application have been pursued using protein engineering to improve the industrial properties. The present review focuses on recent findings on the catalytic site, mechanism of action, substrate recognition, the linker region, the multidomain architecture, the engineering of specificity and stability, and roles of individual substrate binding subsites.     

GAT (GGA and Tom1) domain responsible for ubiquitin binding and ubiquitination

GGAs (Golgi-localizing, gamma-adaptin ear domain homology, ADP-ribosylation factor (ARF)-binding proteins) are a family of monomeric adaptor proteins involved in membrane trafficking from the trans-Golgi network to endosomes. The GAT (GGA and Tom1) domains of GGAs have previously been shown to interact with GTP-bound ARF and to be crucial for membrane recruitment of GGAs. Here we show that the C-terminal subdomain of the GAT domain, which is distinct from the N-terminal GAT subdomain responsible for ARF binding, can bind ubiquitin. The binding is mediated by interactions between residues on one side of the alpha3 helix of the GAT domain and those on the so-called Ile-44 surface patch of ubiquitin. The binding of the GAT domain to ubiquitin can be enhanced by the presence of a GTP-bound form of ARF. Furthermore, GGA itself is ubiquitinated in a manner dependent on the GAT-ubiquitin interaction. These results delineate the molecular basis for the interaction between ubiquitin and GAT and suggest that GGA-mediated trafficking is regulated by the ubiquitin system as endosomal trafficking mediated by other ubiquitin-binding proteins.     

Molecular cloning and 3D structure prediction of the first raw-starch-degrading glucoamylase without a separate starch-binding domain

Raw-starch-degrading glucoamylases have been known as multidomain enzymes consisting of a catalytic domain connected to a starch-binding domain (SBD) by an O-glycosylated linker region. A molecular genetics approach has been chosen to find structural differences between two related glucoamylases, raw-starch-degrading Glm and nondegrading Glu, from the yeasts Saccharomycopsis fibuligera IFO 0111 and HUT 7212, respectively. We have found that Glm and Glu show a high primary (77%) and tertiary structure similarity. Glm, although possessing a good ability for raw starch degradation, did not show consensus amino acid residues to any SBD found in glucoamylases or other amylolytic enzymes. Raw starch binding and digestion by Glm must thus depend on the existence of a site(s) lying within the intact protein which lacks a separate SBD. The enzyme represents a structurally new type of raw-starch-degrading glucoamylase.     

Structure of the eukaryotic initiation factor (eIF) 5 reveals a fold common to several translation factors

Eukaryotic initiation factor 5 (eIF5) plays multiple roles in translation initiation. Its N-terminal domain functions as a GTPase-activator protein (GAP) for GTP bound to eIF2, while its C-terminal region nucleates the interactions between multiple translation factors, including eIF1, which acts to inhibit GTP hydrolysis or P(i) release, and the beta subunit of eIF2. These proteins and the events in which they participate are critical for the accurate recognition of the correct start codon during translation initiation. Here, we report the three-dimensional solution structure of the N-terminal domain of human eIF5, comprising two subdomains, both reminiscent of nucleic-acid-binding modules. The N-terminal subdomain contains the "arginine finger" motif that is essential for GAP function but which, unusually, resides in a partially disordered region of the molecule. This implies that a conformational reordering of this portion of eIF5 is likely to occur upon formation of a competent complex for GTP hydrolysis, following the appropriate activation signal. Interestingly, the N-terminal subdomain of eIF5 reveals an alpha/beta fold structurally similar to both the archaeal orthologue of the beta subunit of eIF2 and, unexpectedly, to eIF1. These results reveal a novel protein fold common to several factors involved in related steps of translation initiation. The implications of these observations are discussed in terms of the mechanism of translation initiation.     

Partial identification of carbohydrate-binding sites of a Galalpha1,3Galbeta1,4GlcNAc-specific lectin from the mushroom Marasmius oreades by site-directed mutagenesis

The Galalpha1,3Galbeta1,4GlcNAc-specific lectin from the mushroom Marasmius oreades (MOA) contains a ricin B chain-like (QXW)(3) domain at its N-terminus that is composed of three identical subdomains (alpha, beta, and gamma) and a C-terminal domain of unknown function. Here, we investigate the structure-function relationship of MOA to define the number and location of its carbohydrate-binding sites. Based on the sequence alignment of MOA to the ricin B-chain lactose-binding sites, we systematically constructed mutants by site-directed mutagenesis. We have used precipitation and hemagglutination assay for the primary analyses, and surface plasmon resonance for the kinetic analysis. Among amino acid residues at the putative carbohydrate-binding sites, Gln(46) in the alpha subdomain and Trp(138) in the gamma subdomain have been identified to be important amino acid residues directly or indirectly involved in carbohydrate recognition. By surface plasmon resonance, Q46A and W138A were 2.4- and 4.3-fold less active than that of the wild-type MOA (K(a) = 2 x 10(7)), respectively. A double-site mutant (Q46A/W138A) had activity similar to W138A. The C-terminal deletion mutant MOADeltaC showed hemagglutination and precipitation activity, although its binding constant was 12.5-fold less active (K(a) = 1.6 x 10(6)) than that of the wild-type MOA. A C-terminal deletion mutant with mutations at both Gln(46) and Trp(138) (MOADeltaC-Q46A/W138A) was 12,500-fold less active (K(a) = 1.6 x 10(3)) than that of the wild-type MOA. On the basis of this observation, we conclude that both alpha and gamma subdomains are most probably involved in carbohydrate binding, but the beta subdomain appears to be inactive.     

Glucoamylase from Thermoanaerobacterium thermosaccharolyticum: Sequence studies and analysis of the macromolecular architecture of the enzyme

A chromosomal DNA fragment with a length of 2,025 bp, carrying the structural gene coding for glucoamylase in Thermoanaerobacterium thermosaccharolyticum, was cloned and sequenced. It coded for 695 amino acids, representing a polypeptide with a predicted molecular mass of 77.5 kDa. The deduced amino acid sequence exhibited high homologies with the glucoamylase sequence of another bacterial glucoamylase (Clostridium sp. G0005) and with fungal glucoamylases. The catalytic domain (amino acids 271 to 695) of the T. thermosaccharolyticum enzyme shared a high degree of similarity (five conserved regions) with the catalytic domain of Aspergillus awamori glucoamylase. By comparing the secondary structure of the sequence of the catalytic domain of the T. thermosaccharolyticum enzyme with that of glucoamylase from A. awamori, and on the basis of X-ray crystallographic data available for the A. awamori enzyme, it turned out that, most probably, both enzymes have a catalytic domain organized into an "(alpha/alpha)(6)-barrel" and an overall size and shape that is very similar. These findings confirm and extend our working model for the macromolecular architecture of the T. thermosaccharolyticum glucoamylase obtained, in earlier experiments, by electron microscopy of negatively stained isolated enzyme molecules. Antibodies for an enzyme-specific peptide located near the active site were successfully applied for inhibition studies of enzyme activity and for electron microscopic epitope mapping. A study comparing the site of attachment of this kind of antibody to the T. thermosaccharolyticum glucoamylase molecule with the expected attachment site as deduced from the A. awamori enzyme structure confirmed the close similarity of both glucoamylases regarding the macromolecular architecture of that part of the enzyme carrying the catalytic center, though helices H9, H10, and H11 in peripheral parts of the A. awamori enzyme are missing in the T. thermosaccharolyticum enzyme.     

Insights into DNA replication: the crystal structure of DNA polymerase B1 from the archaeon Sulfolobus solfataricus

To minimize the large number of mispairs during genome duplication owing to the large amount of DNA to be synthesized, many replicative polymerases have accessory domains with complementary functions. We describe the crystal structure of replicative DNA polymerase B1 from the archaeon Sulfolobus solfataricus. Comparison between other known structures indicates that although the protein is folded into the typical N-terminal, editing 3'-5'exonuclease, and C-terminal right-handed polymerase domains, it is characterized by the unusual presence of two extra alpha helices in the N-terminal domain interacting with the fingers helices to form an extended fingers subdomain, a structural feature that can account for some functional features of the protein. We explore the structural basis of specific lesion recognition, the initial step in DNA repair, describing how the N-terminal subdomain pocket of archaeal DNA polymerases could allow specific recognition of deaminated bases such as uracil and hypoxanthine in addition to the typical DNA bases.     

Competition between intradomain and interdomain interactions: a buried salt bridge is essential for villin headpiece folding and actin binding

Villin-type headpiece domains are ～70 residue motifs that reside at the C-terminus of a variety of actin-associated proteins. Villin headpiece (HP67) is a commonly used model system for both experimental and computational studies of protein folding. HP67 is made up of two subdomains that form a tightly packed interface. The isolated C-terminal subdomain of HP67 (HP35) is one of the smallest autonomously folding proteins known. The N-terminal subdomain requires the presence of the C-terminal subdomain to fold. In the structure of HP67, a conserved salt bridge connects N- and C-terminal subdomains. This buried salt bridge between residues E39 and K70 is unusual in a small protein domain. We used mutational analysis, monitored by CD and NMR, and functional assays to determine the role of this buried salt bridge. First, the two residues in the salt bridge were replaced with strictly hydrophobic amino acids, E39M/K70M. Second, the two residues in the salt bridge were swapped, E39K/K70E. Any change from the wild-type salt bridge residues results in unfolding of the N-terminal subdomain, even when the mutations were made in a stabilized variant of HP67. The C-terminal subdomain remains folded in all mutants and is stabilized by some of the mutations. Using actin sedimentation assays, we find that a folded N-terminal domain is essential for specific actin binding. Therefore, the buried salt bridge is required for the specific folding of the N-terminal domain which confers actin-binding activity to villin-type headpiece domains, even though the residues required for this specific interaction destabilize the C-terminal subdomain.     

Structural insights into substrate specificity and function of glucodextranase

A glucodextranase (iGDase) from Arthrobacter globiformis I42 hydrolyzes alpha-1,6-glucosidic linkages of dextran from the non-reducing end to produce beta-D-glucose via an inverting reaction mechanism and classified into the glycoside hydrolase family 15 (GH15). Here we cloned the iGDase gene and determined the crystal structures of iGDase of the unliganded form and the complex with acarbose at 2.42-A resolution. The structure of iGDase is composed of four domains N, A, B, and C. Domain A forms an (alpha/alpha)(6)-barrel structure and domain N consists of 17 antiparallel beta-strands, and both domains are conserved in bacterial glucoamylases (GAs) and appear to be mainly concerned with catalytic activity. The structure of iGDase complexed with acarbose revealed that the positions and orientations of the residues at subsites -1 and +1 are nearly identical between iGDase and GA; however, the residues corresponding to subsite 3, which form the entrance of the substrate binding pocket, and the position of the open space and constriction of iGDase are different from those of GAs. On the other hand, domains B and C are not found in the bacterial GAs. The primary structure of domain C is homologous with a surface layer homology domain of pullulanases, and the three-dimensional structure of domain C resembles the carbohydrate-binding domain of some glycohydrolases.     

Crystal structure of avian carboxypeptidase D domain II: a prototype for the regulatory metallocarboxypeptidase subfamily.

The crystal structure of domain II of duck carboxypeptidase D, a prohormone/propeptide processing enzyme integrated in a three repeat tandem in the natural system, has been solved, constituting a prototype for members of the regulatory metallocarboxypeptidase subfamily. It displays a 300 residue N-terminal alpha/beta-hydrolase subdomain with overall topological similarity to and general coincidence of the key catalytic residues with the archetypal pancreatic carboxypeptidase A. However, numerous significant insertions/deletions in segments forming the funnel-like access to the active site explain differences in specificity towards larger protein substrates or inhibitors. This alpha/beta-hydrolase subdomain is followed by a C-terminal 80 residue beta-sandwich subdomain, unique for these regulatory metalloenzymes and topologically related to transthyretin and sugar-binding proteins. The structure described here establishes the fundamentals for a better understanding of the mechanism ruling events such as prohormone processing and will enable modelling of regulatory carboxypeptidases as well as a more rational design of inhibitors of carboxypeptidase D.     

Crystal structure of α-galactosidase from Lactobacillus acidophilus NCFM: insight into tetramer formation and substrate binding

Lactobacillus acidophilus NCFM is a probiotic bacterium known for its beneficial effects on human health. The importance of α-galactosidases (α-Gals) for growth of probiotic organisms on oligosaccharides of the raffinose family present in many foods is increasingly recognized. Here, the crystal structure of α-Gal from L. acidophilus NCFM (LaMel36A) of glycoside hydrolase (GH) family 36 (GH36) is determined by single-wavelength anomalous dispersion. In addition, a 1.58-Å-resolution crystallographic complex with α-d-galactose at substrate binding subsite − 1 was determined. LaMel36A has a large N-terminal twisted β-sandwich domain, connected by a long α-helix to the catalytic (β/α)₈-barrel domain, and a C-terminal β-sheet domain. Four identical monomers form a tightly packed tetramer where three monomers contribute to the structural integrity of the active site in each monomer. Structural comparison of LaMel36A with the monomeric Thermotoga maritima α-Gal (TmGal36A) reveals that O2 of α-d-galactose in LaMel36A interacts with a backbone nitrogen in a glycine-rich loop of the catalytic domain, whereas the corresponding atom in TmGal36A is from a tryptophan side chain belonging to the N-terminal domain. Thus, two distinctly different structural motifs participate in substrate recognition. The tetrameric LaMel36A furthermore has a much deeper active site than the monomeric TmGal36A, which possibly modulates substrate specificity. Sequence analysis of GH36, inspired by the observed structural differences, results in four distinct subgroups having clearly different active-site sequence motifs. This novel subdivision incorporates functional and architectural features and may aid further biochemical and structural analyses within GH36.     

Growth hormone receptor targeting to lipid rafts requires extracellular subdomain 2

GH receptor (GHR) is a single membrane-spanning glycoprotein dimer that binds GH in its extracellular domain (ECD). GH activates the GHR intracellular domain (ICD)-associated tyrosine kinase, JAK2, which causes intracellular signaling. We previously found that plasma membrane (PM)-associated GHR was dramatically enriched in the lipid raft (LR) component of the membrane and that localization of GHR within PM regions may regulate GH signaling by influencing the profile of pathway activation. In this study, we examined determinants of LR localization of the GHR using a reconstitution system which lacks endogenous JAK2 and GHR. By non-detergent extraction and multistep fractionation, we found that GHR was highly enriched in the LR fraction independent of JAK2 expression. Various GHR mutants were examined in transfectants harboring JAK2. LR concentration was observed for a GHR in which the native transmembrane domain (TMD) is replaced by that of the unrelated LDL receptor and for a GHR that lacks its ICD. Thus, LR association requires neither the TMD nor the ICD. Similarly, a GHR that lacks the ECD, except for the membrane-proximal ECD stem region, was only minimally LR-concentrated. Mutants with internal stem deletions in the context of the full-length receptor were LR-concentrated similar to the wild-type. A GHR lacking ECD subdomain 1 reached the PM and was LR-concentrated, while one lacking ECD subdomain 2, also reached the PM, but was not LR-concentrated. These data suggest LR targeting resides in ECD subdomain 2, a region relatively uninvolved in GH binding.     

The N-terminal N5 subdomain of the alpha 3(VI) chain is important for collagen VI microfibril formation

Collagen VI assembly is unique within the collagen superfamily in that the alpha 1(VI), alpha 2(VI), and alpha 3(VI) chains associate intracellularly to form triple helical monomers, and then dimers and tetramers, which are secreted from the cell. Secreted tetramers associate end-to-end to form the distinctive extracellular microfibrils that are found in virtually all connective tissues. Although the precise protein interactions involved in this process are unknown, the N-terminal globular regions, which are composed of multiple copies of von Willebrand factor type A-like domains, are likely to play a critical role in microfibril formation, because they are exposed at both ends of the tetramers. To explore the role of these subdomains in collagen VI intracellular and extracellular assembly, alpha 3(VI) cDNA expression constructs with sequential N-terminal deletions were stably transfected into SaOS-2 cells, producing cell lines that express alpha 3(VI) chains with N-terminal globular domains containing modules N9-N1, N6-N1, N5-N1, N4-N1, N3-N1, or N1, as well as the complete triple helix and C-terminal globular domain (C1-C5). All of these transfected alpha 3(VI) chains were able to associate with endogenous alpha 1(VI) and alpha 2(VI) to form collagen VI monomers, dimers, and tetramers, which were secreted. Importantly, cells that expressed alpha 3(VI) chains containing the N5 subdomain, alpha 3(VI) N9-C5, N6-C5, and N5-C5, formed microfibrils and deposited a collagen VI matrix. In contrast, cells that expressed the shorter alpha 3(VI) chains, N4-C5, N3-C5, and N1-C5, were severely compromised in their ability to form end-to-end tetramer assemblies and failed to deposit a collagen VI matrix. These data demonstrate that the alpha 3(VI) N5 module is critical for microfibril formation, thus identifying a functional role for a specific type A subdomain in collagen VI assembly.