Sandwich Fold Research Articles

NMR structure of YcgL, a conserved protein from Escherichia coli representing the DUF709 family, with a novel α/β/α sandwich fold

NMR structure of YcgL, a conserved protein from <i>Escherichia coli</i> representing the DUF709 family, with a novel α/β/α sandwich fold

A Novel Two-domain Architecture Within the Amino Acid Kinase Enzyme Family Revealed by the Crystal Structure of Escherichia coli Glutamate 5-kinase

Glutamate 5-kinase (G5K) makes the highly unstable product glutamyl 5-phosphate (G5P) in the initial, controlling step of proline/ornithine synthesis, being feedback-inhibited by proline or ornithine, and causing, when defective, clinical hyperammonaemia. We determined two crystal structures of G5K from Escherichia coli, at 2.9 Å and 2.5 Å resolution, complexed with glutamate and sulphate, or with G5P, sulphate and the proline analogue 5-oxoproline. E. coli G5K presents a novel tetrameric (dimer of dimers) architecture. Each subunit contains a 257 residue AAK domain, typical of acylphosphate-forming enzymes, with characteristic α 3β 8α 4 sandwich topology. This domain is responsible for catalysis and proline inhibition, and has a crater on the β sheet C-edge that hosts the active centre and bound 5-oxoproline. Each subunit contains a 93 residue C-terminal PUA domain, typical of RNA-modifying enzymes, which presents the characteristic β 5β 4 sandwich fold and three α helices. The AAK and PUA domains of one subunit associate non-canonically in the dimer with the same domains of the other subunit, leaving a negatively charged hole between them that hosts two Mg ions in one crystal, in line with the G5K requirement for free Mg. The tetramer, formed by two dimers interacting exclusively through their AAK domains, is flat and elongated, and has in each face, pericentrically, two exposed active centres in alternate subunits. This would permit the close apposition of two active centres of bacterial glutamate-5-phosphate reductase (the next enzyme in the proline/ornithine-synthesising route), supporting the postulated channelling of G5P. The structures clarify substrate binding and catalysis, justify the high glutamate specificity, explain the effects of known point mutations, and support the binding of proline near glutamate. Proline binding may trigger the movement of a loop that encircles glutamate, and which participates in a hydrogen bond network connecting active centres, which is possibly involved in the cooperativity for glutamate.

Identification by Mn2+ rescue of two residues essential for the proton transfer of tRNase Z catalysis

Thermotoga maritima tRNase Z cleaves pre-tRNAs containing the 74CCA76 sequence precisely after the A76 residue to create the mature 3′ termini. Its crystal structure has revealed a four-layer αβ/βα sandwich fold that is typically found in the metallo-β-lactamase superfamily. The well-conserved six histidine and two aspartate residues together with metal ions are assumed to form the tRNase Z catalytic center. Here, we examined tRNase Z variants containing single amino acid substitutions in the catalytic center for pre-tRNA cleavage. Cleavage by each variant in the presence of Mg2+ was hardly detected, although it is bound to pre-tRNA. Surprisingly, however, Mn2+ ions restored the lost Mg2+-dependent activity with two exceptions of the Asp52Ala and His222Ala substitutions, which abolished the activity almost completely. These results provide a piece of evidence that Asp-52 and His-222 directly contribute the proton transfer for the catalysis.

Multiple Distinct Assemblies Reveal Conformational Flexibility in the Small Heat Shock Protein Hsp26

Small heat shock proteins are a superfamily of molecular chaperones that suppress protein aggregation and provide protection from cell stress. A key issue for understanding their action is to define the interactions of subunit domains in these oligomeric assemblies. Cryo-electron microscopy of yeast Hsp26 reveals two distinct forms, each comprising 24 subunits arranged in a porous shell with tetrahedral symmetry. The subunits form elongated, asymmetric dimers that assemble via trimeric contacts. Modifications of both termini cause rearrangements that yield a further four assemblies. Each subunit contains an N-terminal region, a globular middle domain, the alpha-crystallin domain, and a C-terminal tail. Twelve of the C termini form 3-fold assembly contacts which are inserted into the interior of the shell, while the other 12 C termini form contacts on the surface. Hinge points between the domains allow a variety of assembly contacts, providing the flexibility required for formation of supercomplexes with non-native proteins.

Solution Structure of the NEAT (NEAr Transporter) Domain from IsdH/HarA: the Human Hemoglobin Receptor in Staphylococcus aureus

During infections the pathogen Staphylococcus aureus procures the essential nutrient iron from its host using iron-regulated surface determinant (Isd) proteins, which scavenge heme bound iron from host hemoproteins. Four Isd proteins are displayed in the cell wall, where they function as receptors for host proteins and heme. Each of the receptors contains one or more copies of a recently discovered domain called NEAT (NEAr Transporter) that has been shown to mediate protein binding. Here we report the three-dimensional solution structure of the NEAT domain from the IsdH/HarA protein, which is the hemoglobin receptor in the Isd system. This is the first structure of a NEAT domain and reveals that they adopt a beta sandwich fold that consists of two five-stranded antiparallel beta sheets. Although unrelated at the primary sequence level, our results indicate that NEAT domains belong to the immunoglobulin superfamily. Binding studies indicate that two IsdH/HarA NEAT domains bind a single molecule of methemoglobin, while the distantly related NEAT domain from the S. aureus IsdC protein binds only heme. A comparison of their primary sequences in light of the new structure is used to predict the hemoglobin and heme binding surfaces on NEAT domains.

Crystal Structure of Human Vacuolar Protein Sorting Protein 29 Reveals a Phosphodiesterase/Nuclease-like Fold and Two Protein-Protein Interaction Sites

Vacuolar protein sorting protein 29 (Vps29p), which is involved in retrograde trafficking from prevacuolar endosomes to the trans-Golgi network, performs its biological functions by participating in the formation of a "retromer complex." In human cells, this complex comprises four conserved proteins: hVps35p, hVps29p, hVps26p, and sorting nexin 1 protein (SNX1). Here, we report the crystal structure of hVps29p at 2.1 Angstroms resolution, the first three-dimensional structure of the retromer subunits. This novel structure adopts a four-layered alpha-beta-beta-alpha sandwich fold. hVps29p contains a metal-binding site that is very similar to the active sites of some proteins of the phosphodiesterase/nuclease protein family, indicating that hVps29p may carry out chemically similar functions. Structure and sequence conservation analysis suggests that hVps29p contains two protein-protein interaction sites. One site, which potentially serves as the interface between hVps29p and hVps35p, comprises 5 conserved hydrophobic and 8 hydrophilic residues. The other site is relatively more hydrophilic and may serve as a binding interface with hVps26p, SNX1, or other target proteins.

Crystal Structure of the tRNA 3′ Processing Endoribonuclease tRNase Z from Thermotoga maritima

The maturation of the tRNA 3' end is catalyzed by a tRNA 3' processing endoribonuclease named tRNase Z (RNase Z or 3'-tRNase) in eukaryotes, Archaea, and some bacteria. The tRNase Z generally cuts the 3' extra sequence from the precursor tRNA after the discriminator nucleotide. In contrast, Thermotoga maritima tRNase Z cleaves the precursor tRNA precisely after the CCA sequence. In this study, we determined the crystal structure of T. maritima tRNase Z at 2.6-A resolution. The tRNase Z has a four-layer alphabeta/betaalpha sandwich fold, which is classified as a metallo-beta-lactamase fold, and forms a dimer. The active site is located at one edge of the beta-sandwich and is composed of conserved motifs. Based on the structure, we constructed a docking model with the tRNAs that suggests how tRNase Z may recognize the substrate tRNAs.

Crystal Structure of a Hyperthermophilic Archaeal Acylphosphatase from Pyrococcus horikoshiiStructural Insights into Enzymatic Catalysis, Thermostability, and Dimerization,

Acylphosphatases catalyze the hydrolysis of the carboxyl-phosphate bond in acyl phosphates. Although acylphosphatase-like sequences are found in all three domains of life, no structure of acylphosphatase has been reported for bacteria and archaea so far. Here, we report the characterization of enzymatic activities and crystal structure of an archaeal acylphosphatase. A putative acylphosphatase gene (PhAcP) was cloned from the genomic DNA of Pyrococcus horikoshii and was expressed in Escherichia coli. Enzymatic parameters of the recombinant PhAcP were measured using benzoyl phosphate as the substrate. Our data suggest that, while PhAcP is less efficient than other mammalian homologues at 25 degrees C, the thermophilic enzyme is fully active at the optimal growth temperature (98 degrees C) of P. horikoshii. PhAcP is extremely stable; its apparent melting temperature was 111.5 degrees C and free energy of unfolding at 25 degrees C was 54 kJ mol(-)(1). The 1.5 A crystal structure of PhAcP adopts an alpha/beta sandwich fold that is common to other acylphosphatases. PhAcP forms a dimer in the crystal structure via antiparallel association of strand 4. Structural comparison to mesophilic acylphosphatases reveals significant differences in the conformation of the L5 loop connecting strands 4 and 5. The extreme thermostability of PhAcP can be attributed to an extensive ion-pair network consisting of 13 charge residues on the beta sheet of the protein. The reduced catalytic efficiency of PhAcP at 25 degrees C may be due to a less flexible active-site residue, Arg20, which forms a salt bridge to the C-terminal carboxyl group. New insights into catalysis were gained by docking acetyl phosphate to the active site of PhAcP.

Crystal Structure of Chorismate Synthase: A Novel FMN-binding Protein Fold and Functional Insights

Chorismate synthase catalyzes the conversion of 5-enolpyruvylshikimate 3-phosphate to chorismate in the shikimate pathway, which represents an attractive target for discovering antimicrobial agents and herbicides. Chorismate serves as a common precursor for the synthesis of aromatic amino acids and many aromatic compounds in microorganisms and plants. Chorismate synthase requires reduced FMN as a cofactor but the catalyzed reaction involves no net redox change. Here, we have determined the crystal structure of chorismate synthase from Helicobacter pylori in both FMN-bound and FMN-free forms. It is a tetrameric enzyme, with each monomer possessing a novel “β-α-β sandwich fold”. Highly conserved regions, including several flexible loops, cluster together around the bound FMN to form the active site. The unique FMN-binding site is formed largely by a single subunit, with a small contribution from a neighboring subunit. The isoalloxazine ring of the bound FMN is significantly non-planar. Our structure illuminates the essential functional roles played by the cofactor.

X-ray crystal structure of CutA from Thermotoga maritima at 1.4 A resolution.

The structure of the CutA protein from Thermotoga maritima (tmCutA) was determined at 1.4 A resolution using the Se-Met multiwavelength anomalous diffraction (MAD) technique. This protein (TIGR annotation - TM1056, DNA bases 1,069,580–1,069,885) is conserved in numerous bacteria, archaea and eucarya, including plants and mammals (COG1324, Fig. 1). The CutA Escherichia coli homolog—CutA1 (35% ID) is involved in divalent cation homeostasis,1 while the mammalian homolog—mCutA (40% ID) was found to be associated with cell surface acetylcholinesterase.2 However, the biological function of the CutA proteins is yet to be determined. Fig. 1 Multiple sequence alignment of CutA from Thermotoga maritima (Thema) against other periplasmic divalent cation tolerance proteins from bacteria (Aquae = Aquifex aeolicus, Deira = Deinococcus radiodurans) and archaea (Pyrho = Pyrococcus horikoshii, Arcfu ... The tmCutA monomer assumes a two-layer α/β α/β sandwich fold (β1β2α1β3β4α3β5β6α5) shared by a large number of proteins (CATH number 3.30.70.120). Two short (β2β5 and two long (β3β4) β-strands arranged in an antiparallel fashion β5↑β2↓β3↑β4↓ form a curved sheet. Three α-helices cover one side of the molecule (Fig. 2a). Each monomer is elongated with overall dimensions approximately 25 × 30× 60 A (Fig. 2a). The monomers are assembled into a trimer with dimensions 55 × 55 × 35 A through interaction of the edges of three β-strands (β3 on the top and β5β6 on the bottom) (Fig. 2b). In the trimer, the subunits are related by crystallographic three fold axes. The same oligomeric state was observed for tmCutA in solution by size exclusion experiments (see Methods) and appears to be functionally relevant. In the trimer, there is a cavity that is accessible from outside via three solvent accessible channels. The inner surface of the cavity is lined with several conserved residues including Cys35, which may serve as a potential metal binding site. Fig. 2 Ribbon representations of the tmCutA monomer (a), trimer (b) and superimposed structures of tmCutA and GlnK’ complex with ATP (c). a: The α-helixes and β-strains are colored in red and blue, respectively. b: Each monomer in the ... Structural homologues of tmCutA were identified using Dali search3 and SSAP structure comparison program 4. The closest structural matches, with a Dali score of 9.4 Z, were the E. coli signal transducer proteins GlnB (PII)5 and GlnK6 (Fig. 2c). These two proteins are 67% identical to each other but have insignificant sequence similarity (less than 15% ID) to tmCutA. GlnB and GlnK, which are also trimeric, are involved in maintaining nitrogen homeostasis7–9 and have been shown to cooperatively bind ATP and α-ketoglutarate.6,10 The ATP binding sites of GlnB and GlnK are located in clefts between the momoners,11 but most of the conserved amino acids that mediate ATP binding in PII proteins are not found in tmCutA. The cavity on the side of tmCutA is formed by a number of conserved aromatic (Tyr45, Trp47, Tyr81, and Trp101) and charged residues (Asp54, Glu56, Glu82) (Figs. 1 and ​and2c),2c), which makes this cleft a strong candidate for a conserved function. In PII-type proteins, the two central β-strains β3 and β4 are joined by a flexible loop called the T-loop, which is composed of 17 amino acids. The T-loop plays a key role in protein-protein interactions with downstream effector proteins.12 The β3 and β4 strands in tmCutA, while longer than the corresponding strands in the PII proteins (12 residues in the β3 strand of tmCutA compared with 9 in the β2 strand of PII proteins), are joined by only a two-amino acid (Lys48Gly49) turn and form a hairpin. Although the residues in the C-terminal extension of β3-strand in tmCutA proteins (Tyr45, Trp46, and Trp47, Fig. 1) are conserved among tmCutA homologs, the structure of this region in tmCutA is different than in PII proteins and thus this region of tmCutA may not participate in protein-protein interactions. CutA is annotated as being involved in metal homeostasis. Several other structural homologs of tmCutA, including the PII proteins, metallochaperone Atx1 (5.9 Z score) and the metal-binding domain of the Menkes copper-transporting ATPase (4.5 Z score) have metal-binding properties.13,14 These proteins coordinate the metal through a CXXC sequence motif,15,16 but this motif is not found in CutA. Therefore, tmCutA may represent a new branch of this family that differs from PII and metal binding proteins. The biochemical experiments are under way to define possible ligands and functional partners of CutA proteins.

3D Domain Swapping Modulates the Stability of Members of an Icosahedral Virus Group

Background: Rice yellow mottle virus (RYMV) is a major pathogen that dramatically reduces rice production in many African countries. RYMV belongs to the genus sobemovirus, one group of plant viruses with icosahedral capsids and single-stranded, positive-sense RNA genomes. Results: The structure of RYMV was determined and refined to 2.8 Å resolution by X-ray crystallography. The capsid contains 180 copies of the coat protein subunit arranged with T = 3 icosahedral symmetry. Each subunit adopts a jelly-roll β sandwich fold. The RYMV capsid structure is similar to those of other sobemoviruses. When compared with these viruses, however, the βA arm of the RYMV C subunit, which is a molecular switch that regulates quasi-equivalent subunit interactions, is swapped with the 2-fold-related βA arm to a similar, noncovalent bonding environment. This exchange of identical structural elements across a symmetry axis is categorized as 3D domain swapping and produces long-range interactions throughout the icosahedral surface lattice. Biochemical analysis supports the notion that 3D domain swapping increases the stability of RYMV. Conclusions: The quasi-equivalent interactions between the RYMV proteins are regulated by the N-terminal ordered residues of the βA arm, which functions as a molecular switch. Comparative analysis suggests that this molecular switch can also modulate the stability of the viral capsids.

Structure and function of laminin LG modules.

Laminin G domain-like (LG) modules of approximately 180-200 residues are found in a number of extracellular and receptor proteins and often are present in tandem arrays. LG modules are implicated in interactions with cellular receptors (integrins, alpha-dystroglycan), sulfated carbohydrates and other extracellular ligands. The recently determined crystal structures of LG modules of the laminin alpha2 chain reveal a compact beta sandwich fold and identify a novel calcium binding site. Binding epitopes for heparin, sulfatides and alpha-dystroglycan have been mapped by site-directed mutagenesis and show considerable overlap. The epitopes are located in surface loops around the calcium site, which in other proteins (agrin, neurexins) are modified by alternative splicing. Efficient ligand binding often requires LG modules to be present in tandem. The close proximity of the N- and C-termini in the LG module, as well as a unique link region between laminin LG3 and LG4, impose certain constraints on the arrangement of LG tandems. Further modifications may be introduced by proteolytic processing of laminin G domains, which is known to occur in the alpha2, alpha3 and alpha4 chains.

A novel two-over-two alpha-helical sandwich fold is characteristic of the truncated hemoglobin family

Small hemoproteins displaying amino acid sequences 20-40 residues shorter than (non-)vertebrate hemoglobins (Hbs) have recently been identified in several pathogenic and non-pathogenic unicellular organisms, and named 'truncated hemoglobins' (trHbs). They have been proposed to be involved not only in oxygen transport but also in other biological functions, such as protection against reactive nitrogen species, photosynthesis or to act as terminal oxidases. Crystal structures of trHbs from the ciliated protozoan Paramecium caudatum and the green unicellular alga Chlamydomonas eugametos show that the tertiary structure of both proteins is based on a 'two-over-two' alpha-helical sandwich, reflecting an unprecedented editing of the classical 'three-over-three' alpha-helical globin fold. Based on specific Gly-Gly motifs the tertiary structure accommodates the deletion of the N-terminal A-helix and replacement of the crucial heme-binding F-helix with an extended polypeptide loop. Additionally, concerted structural modifications allow burying of the heme group and define the distal site, which hosts a TyrB10, GlnE7 residue pair. A set of structural and amino acid sequence consensus rules for stabilizing the fold and the bound heme in the trHbs homology subfamily is deduced.

Three-dimensional structure of nucleoside diphosphate kinase.

Three-dimensional structures are known from X-ray studies of the nucleoside diphosphate (NDP) kinase of many organisms from bacteria to human. All NDP kinases have subunits of about 150 residues with a very similar fold based on the alphabeta sandwich or ferredoxin fold. This fold is found in many nucleotide or polynucleotide-binding proteins with no sequence relationship to NDP kinase. This common fold is augmented here with specific features: a surface alpha-helix hairpin, the Kpn loop, and the C-terminal extension. The alpha-helix hairpin and Kpn loop make up the nucleotide binding site, which is unique to NDP kinase and different from that of other kinases or ATPases. The Kpn loop and the C-terminal extension are also involved in the quaternary structure. Whereas all known eukaryotic NDP kinases, including mitochondral enzymes, are hexamers, some bacterial enzymes are tetramers. However, hexameric and tetrameric NDP kinases are built from the same dimer. The structural environment of the active histidine is identical in all. The nucleotide binding site is also fully conserved, except for a feature implicating C-terminal residues in the hexamer, but not in the tetramer. Structural data on the native and phosphorylated enzyme, complexes with substrates, inhibitor, and a transition state analog, give a solid basis to a mechanism of phosphate transfer in which the largest contributors to catalysis are the 3'-OH of the sugar and the bound Mg2+ in the nucleotide substrate. In contrast, we still lack structural data relating to DNA binding and other functions of NDP kinases.