Crystal Structure of Human Collagen XVIII Trimerization Domain: A Novel Collagen Trimerization Fold

Collagens contain a unique triple-helical structure with a repeating sequence -G-X-Y-, where proline and hydroxyproline are major constituents in X and Y positions, respectively. Folding of the collagen triple helix requires trimerization domains. Once trimerized, collagen chains are correctly aligned and the folding of the triple helix proceeds in a zipper-like fashion. Here we report the isolation, characterization, and crystal structure of the trimerization domain of human type XVIII collagen, a member of the multiplexin family. This domain differs from all other known trimerization domains in other collagens and exhibits a high trimerization potential at picomolar concentrations. Strong chain association and high specificity of binding are needed for multiplexins, which are present at very low levels.     

Prediction of 5-HT3 receptor agonist-binding residues using homology modeling

5-HT(3) receptors demonstrate significant structural and functional homology to other members of the Cys-loop ligand-gated ion channel superfamily. The extracellular domains of these receptors share similar sequence homology (approximately 20%) with Limnaea acetylcholine binding protein, for which an x-ray crystal structure is available. We used this structure as a template for computer-based homology modeling of the 5-HT(3) receptor extracellular domain. AutoDock software was used to dock 5-HT into the putative 5-HT(3) receptor ligand-binding site, resulting in seven alternative energetically favorable models. Residues located no more than 5 A from the docked 5-HT were identified for each model; of these, 12 were found to be common to all seven models with five others present in only certain models. Some docking models reflected the cation-pi interaction previously demonstrated for W183, and data from these and other studies were used to define our preferred models.     

Molecular modeling of the complex between Torpedo acetylcholine receptor and anti-MIR Fab198

Myasthenia gravis is a neuromuscular disorder caused by an antibody-mediated autoimmune response to the muscle-type nicotinic acetylcholine receptor (AChR). The majority of monoclonal antibodies (mAbs) produced in rats immunized with intact AChR compete with each other for binding to an area of the alpha-subunit called the main immunogenic region (MIR). The availability of a complex between the AChR and Fab198 (Fab fragment of the anti-MIR mAb198) would help understand how the antigen and antibody interact and in designing improved antibody fragments that protect against the destructive activity of myasthenic antibodies. In the present study, we modeled the Torpedo AChR/Fab198 complex, based primarily on the recent 4A resolution structure of the Torpedo AChR. In order to computationally dock the two structures, we used the ZDOCK software. The total accessible surface area change of the complex compared to those of experimentally determined antigen-antibody complexes indicates an intermediate size contact surface. CDRs H3 and L3 seem to contribute most to the binding, while L2 seems to contribute least. These data suggest mutagenesis experiments aimed at validating the model and improving the binding affinity of Fab198 for the AChR.     

Capturing state-dependent dynamic events of GABAA-receptors: a microscopic look into the structural and functional insights

The γ-amino butyric acid type A receptors (GABA_A-Rs) are the key players in the mammalian brain that meditate fast inhibitory neurotransmission events. The structural integrity of these ligand-gated ion channel controls chloride ion permeability, which in turn monitors important pharmacological functions. Despite ample studies on GABA_A-Rs, there was a need for a study on full-length receptor structures, devoted to track structure–function correlations based on their dynamic behavior consideration. We have employed molecular dynamics simulations accompanied by other biophysical methods to shed light on sequential and unaddressed questions like How GABA_A-R structure facilitates the entry of GABA molecules at its two orthosteric binding sites? After entry, what structural features and changes monitor site-wise GABA binding differences? In the same context, what are the roles and responsibilities of loops such as C and F? On physiologically relevant time scales, how open to close state transition occurs? How salt bridges such as E155-R207 and E153-R207 maintain state-dependent C-loop structures? In an attempt, our simulation study unravels the complete course of GABA binding-unbinding pathway. This provides us with the relevant understanding of state-dependent dynamic events of GABA_A-Rs.     

Unusual Conformation of the SxN Motif in the Crystal Structure of Penicillin-Binding Protein A from Mycobacterium tuberculosis

PBPA from Mycobacterium tuberculosis is a class B-like penicillin-binding protein (PBP) that is not essential for cell growth in M. tuberculosis, but is important for proper cell division in Mycobacterium smegmatis. We have determined the crystal structure of PBPA at 2.05 Å resolution, the first published structure of a PBP from this important pathogen. Compared to other PBPs, PBPA has a relatively small N-terminal domain, and conservation of a cluster of charged residues within this domain suggests that PBPA is more related to class B PBPs than previously inferred from sequence analysis. The C-terminal domain is a typical transpeptidase fold and contains the three conserved active-site motifs characterisitic of penicillin-interacting enzymes. Whilst the arrangement of the SxxK and KTG motifs is similar to that observed in other PBPs, the SxN motif is markedly displaced away from the active site, such that its serine (Ser281) is not involved in hydrogen bonding with residues of the other two motifs. A disulfide bridge between Cys282 (the “x” of the SxN motif) and Cys266, which resides on an adjacent loop, may be responsible for this unusual conformation. Another interesting feature of the structure is a relatively long connection between β5 and α11, which restricts the space available in the active site of PBPA and suggests that conformational changes would be required to accommodate peptide substrate or β-lactam antibiotics during acylation. Finally, the structure shows that one of the two threonines postulated to be targets for phosphorylation is inaccessible (Thr362), whereas the other (Thr437) is well placed on a surface loop near the active site.     

Crystal Structure of the N-Terminal Domain of Nup358/RanBP2

Key steps in mRNA export are the nuclear assembly of messenger ribonucleoprotein particles (mRNPs), the translocation of mRNPs through the nuclear pore complex (NPC), and the mRNP remodeling events at the cytoplasmic side of the NPC. Nup358/RanBP2 is a constituent of the cytoplasmic filaments of the NPC specific to higher eukaryotes and provides a multitude of binding sites for the nucleocytoplasmic transport machinery. Here, we present the crystal structure of the Nup358 N-terminal domain (NTD) at 0.95 Å resolution. The structure reveals an α-helical domain that harbors three central tetratricopeptide repeats (TPRs), flanked on each side by an additional solvating amphipathic α helix. Overall, the NTD adopts an unusual extended conformation that lacks the characteristic peptide-binding groove observed in canonical TPR domains. Strikingly, the vast majority of the NTD surface exhibits an evolutionarily conserved, positive electrostatic potential, and we demonstrate that the NTD possesses the capability to bind single-stranded RNA in solution. Together, these data suggest that the NTD contributes to mRNP remodeling events at the cytoplasmic face of the NPC.     

Crystal Structure of the Electron Carrier Domain of the Reaction Center Cytochrome cz Subunit from Green Photosynthetic Bacterium Chlorobium tepidum

In green sulfur photosynthetic bacteria, the cytochrome c_z (cyt c_z) subunit in the reaction center complex mediates electron transfer mainly from menaquinol/cytochrome c oxidoreductase to the special pair (P840) of the reaction center. The cyt c_z subunit consists of an N-terminal transmembrane domain and a C-terminal soluble domain that binds a single heme group. The periplasmic soluble domain has been proposed to be highly mobile and to fluctuate between oxidoreductase and P840 during photosynthetic electron transfer. We have determined the crystal structure of the oxidized form of the C-terminal functional domain of the cyt c_z subunit (C-cyt c_z) from thermophilic green sulfur bacterium Chlorobium tepidum at 1.3-Å resolution. The overall fold of C-cyt c_z consists of four α-helices and is similar to that of class I cytochrome c proteins despite the low similarity in their amino acid sequences. The N-terminal structure of C-cyt c_z supports the swinging mechanism previously proposed in relation with electron transfer, and the surface properties provide useful information on possible interaction sites with its electron transfer partners. Several characteristic features are observed for the heme environment: These include orientation of the axial ligands with respect to the heme plane, surface-exposed area of the heme, positions of water molecules, and hydrogen-bond network involving heme propionate groups. These structural features are essential for elucidating the mechanism for regulating the redox state of cyt c_z.     

Structural insights into the substrate specificity and function of Escherichia coli K12 YgjK, a glucosidase belonging to the glycoside hydrolase family 63

Proteins belonging to the glycoside hydrolase family 63 (GH63) are found in bacteria, archaea, and eukaryotes. Eukaryotic GH63 proteins are processing α-glucosidase I enzymes that hydrolyze an oligosaccharide precursor of eukaryotic N-linked glycoproteins. In contrast, the functions of the bacterial and archaeal GH63 proteins are unclear. Here we determined the crystal structure of a bacterial GH63 enzyme, Escherichia coli K12 YgjK, at 1.78 Å resolution and investigated some properties of the enzyme. YgjK consists of the N-domain and the A-domain, joined by a linker region. The N-domain is composed of 18 antiparallel β-strands and is classified as a super-β-sandwich. The A-domain contains 16 α-helices, 12 of which form an (α/α)₆-barrel; the remaining 4 α-helices are found in an extra structural unit that we designated as the A′-region. YgjK, a member of the glycoside hydrolase clan GH-G, shares structural similarity with glucoamylase (GH15) and chitobiose phosphorylase (GH65), both of which belong to clan GH-L. In crystal structures of YgjK in complex with glucose, mannose, and galactose, all of the glucose, mannose, and galactose units were located in the catalytic cleft. YgjK showed the highest activity for the α-1,3-glucosidic linkage of nigerose, but also hydrolyzed trehalose, kojibiose, and maltooligosaccharides from maltose to maltoheptaose, although the activities were low. These findings suggest that YgjK is a glucosidase with relaxed specificity for sugars.     

Crystal structure of Escherichia coli L-arabinose isomerase (ECAI), the putative target of biological tagatose production

Escherichia coli L-arabinose isomerase (ECAI; EC 5.3.1.4) catalyzes the isomerization of L-arabinose to L-ribulose in vivo. This enzyme is also of commercial interest as it catalyzes the conversion of D-galactose to D-tagatose in vitro. The crystal structure of ECAI was solved and refined at 2.6 A resolution. The subunit structure of ECAI is organised into three domains: an N-terminal, a central and a C-terminal domain. It forms a crystallographic trimeric architecture in the asymmetric unit. Packing within the crystal suggests the idea that ECAI can form a hexameric assembly. Previous electron microscopic and biochemical studies supports that ECAI is hexameric in solution. A comparison with other known structures reveals that ECAI adopts a protein fold most similar to E. coli fucose isomerase (ECFI) despite very low sequence identity 9.7%. The structural similarity between ECAI and ECFI with regard to number of domains, overall fold, biological assembly, and active site architecture strongly suggests that the enzymes have functional similarities. Further, the crystal structure of ECAI forms a basis for identifying molecular determinants responsible for isomerization of arabinose to ribulose in vivo and galactose to tagatose in vitro.     

Crystal Structure of the HA3 Subcomponent of Clostridium botulinum Type C Progenitor Toxin

The Clostridium botulinum type C 16S progenitor toxin contains a neurotoxin and several nontoxic components, designated nontoxic nonhemagglutinin (HA), HA1 (HA-33), HA2 (HA-17), HA3a (HA-22-23), and HA3b (HA-53). The HA3b subcomponent seems to play an important role cooperatively with HA1 in the internalization of the toxin by gastrointestinal epithelial cells via binding of these subcomponents to specific oligosaccharides. In this study, we investigated the sugar-binding specificity of the HA3b subcomponent using recombinant protein fused to glutathione S-transferase and determined the three-dimensional structure of the HA3a-HA3b complex based on X-ray crystallography. The crystal structure was determined at a resolution of 2.6 Å. HA3b contains three domains, domains I to III, and the structure of domain I resembles HA3a. In crystal packing, three HA3a-HA3b molecules are assembled to form a three-leaved propeller-like structure. The three HA3b domain I and three HA3a alternate, forming a trimer of dimers. In a database search, no proteins with high structural homology to any of the domains (Z score > 10) were found. Especially, HA3a and HA3b domain I, mainly composed of β-sheets, reveal a unique fold. In binding assays, HA3b bound sialic acid with high affinity, but did not bind galactose, N-acetylgalactosamine, or N-acetylglucosamine. The electron density of liganded N-acetylneuraminic acid was determined by crystal soaking. In the sugar-complex structure, the N-acetylneuraminic acid-binding site was located in the cleft formed between domains II and III of HA3b. This report provides the first determination of the three-dimensional structure of the HA3a-HA3b complex and its sialic acid binding site. Our results will provide useful information for elucidating the mechanism of assembly of the C16S toxin and for understanding the interactions with oligosaccharides on epithelial cells and internalization of the botulinum toxin complex.     

Crystal Structure of the Human Rad9-Hus1-Rad1 Clamp

Three evolutionarily conserved proteins, Rad9, Hus1, and Rad1, form a heterotrimeric 9-1-1 complex that plays critical roles in cellular responses to DNA damage by activating checkpoints and by recruiting DNA repair enzymes to DNA lesions. We have determined the crystal structure of the human Rad9 (residues 1-272)-Hus1-Rad1 complex at 2.5 Å resolution. The 9^1-272-1-1 complex forms a closed ring, with each subunit having a similar structure. Despite its high level of similarity to proliferating cell nucleus antigen in terms of overall structure, the 9^1-272-1-1 complex exhibits notable differences in local structures, including interdomain connecting loops, H2 and H3 helices, and loops in the vicinity of the helices of each subunit. These local structural variations provide several unique features to the 9-1-1 heterotrimeric complex—including structures of intermolecular interfaces and the inner surface around the central hole, and different electrostatic potentials at and near the interdomain connecting loops of each 9-1-1 subunit—compared to the proliferating cell nucleus antigen trimer. We propose that these structural features allow the 9-1-1 complex to bind to a damaged DNA during checkpoint control and to serve as a platform for base excision repair. We also show that the 9^1-272-1-1 complex, but not the full-length 9-1-1 complex, forms a stable complex with the 5′ recessed DNA, suggesting that the C-terminal tail of Rad9 is involved in the regulation of the 9-1-1 complex in DNA binding.     

How to Switch Off a Histidine Kinase: Crystal Structure of Geobacillus stearothermophilus KinB with the inhibitor Sda

Entry to sporulation in bacilli is governed by a histidine kinase phosphorelay, a variation of the predominant signal transduction mechanism in prokaryotes. Sda directly inhibits sporulation histidine kinases in response to DNA damage and replication defects. We determined a 2.0-Å-resolution X-ray crystal structure of the intact cytoplasmic catalytic core [comprising the dimerization and histidine phosphotransfer domain (DHp domain), connected to the ATP binding catalytic domain] of the Geobacillus stearothermophilus sporulation kinase KinB complexed with Sda. Structural and biochemical analyses reveal that Sda binds to the base of the DHp domain and prevents molecular transactions with the DHp domain to which it is bound by acting as a simple molecular barricade. Sda acts to sterically block communication between the catalytic domain and the DHp domain, which is required for autophosphorylation, as well as to sterically block communication between the response regulator Spo0F and the DHp domain, which is required for phosphotransfer and phosphatase activities.     

Structure of a Prokaryotic Sodium Channel Pore Reveals Essential Gating Elements and an Outer Ion Binding Site Common to Eukaryotic Channels

Voltage-gated sodium channels (Na_Vs) are central elements of cellular excitation. Notwithstanding advances from recent bacterial Na_V (BacNa_V) structures, key questions about gating and ion selectivity remain. Here, we present a closed conformation of Na_VAe1p, a pore-only BacNa_V derived from Na_VAe1, a BacNa_V from the arsenite oxidizer Alkalilimnicola ehrlichei found in Mono Lake, California, that provides insight into both fundamental properties. The structure reveals a pore domain in which the pore-lining S6 helix connects to a helical cytoplasmic tail. Electrophysiological studies of full-length BacNa_Vs show that two elements defined by the Na_VAe1p structure, an S6 activation gate position and the cytoplasmic tail “neck”, are central to BacNa_V gating. The structure also reveals the selectivity filter ion entry site, termed the “outer ion” site. Comparison with mammalian voltage-gated calcium channel (Ca_V) selectivity filters, together with functional studies, shows that this site forms a previously unknown determinant of Ca_V high-affinity calcium binding. Our findings underscore commonalities between BacNa_Vs and eukaryotic voltage-gated channels and provide a framework for understanding gating and ion permeation in this superfamily.     

Crystal Structure of a Thermophilic GrpE Protein: Insight into Thermosensing Function for the DnaK Chaperone System

A homodimeric GrpE protein functions as a nucleotide exchange factor of the eubacterium DnaK molecular chaperone system. The co-chaperone GrpE accelerates ADP dissociation from, and promotes ATP binding to, DnaK, which cooperatively facilitates the DnaK chaperone cycle with another co-chaperone, DnaJ. GrpE characteristically undergoes two-step conformational changes in response to elevation of the environmental temperature. In the first transition at heat-shock temperatures, a fully reversible and functionally deficient structural alteration takes place in GrpE, and then the higher temperatures lead to the irreversible dissociation of the GrpE dimer into monomers as the second transition. GrpE is also thought to be a thermosensor of the DnaK system, since it is the only member of the DnaK system that changes its structure reversibly and loses its function at heat-shock temperatures of various organisms. We here report the crystal structure of GrpE from Thermus thermophilus HB8 (GrpE_Tth) at 3.23 Å resolution. The resolved structure is compared with that of GrpE from mesophilic Escherichia coli (GrpE_Eco), revealing structural similarities, particularly in the DnaK interaction regions, and structural characteristics for the thermal stability of GrpE_Tth. In addition, the structure analysis raised the possibility that the polypeptide chain in the reported GrpE_Eco structure was misinterpreted. Comparison of these two GrpE structures combined with the results of limited proteolysis experiments provides insight into the protein dynamics of GrpE_Tth correlated with the shift of temperature, and also suggests that the localized and partial unfolding at the plausible DnaK interaction sites of GrpE_Tth causes functional deficiency of nucleotide exchange factor in response to the heat shock.     

Oligomeric State in the Crystal Structure of Modular FAD Synthetase Provides Insights into Its Sequential Catalysis in Prokaryotes

The crystal structure of the modular flavin adenine dinucleotide (FAD) synthetase from Corynebacterium ammoniagenes has been solved at 1.95 Å resolution. The structure of C. ammoniagenes FAD synthetase presents two catalytic modules—a C-terminus with ATP-riboflavin kinase activity and an N-terminus with ATP-flavin mononucleotide (FMN) adenylyltransferase activity—that are responsible for the synthesis of FAD from riboflavin in two sequential steps. In the monomeric structure, the active sites from both modules are placed 40 Å away, preventing the direct transfer of the product from the first reaction (FMN) to the second catalytic site, where it acts as substrate. Crystallographic and biophysical studies revealed a hexameric assembly formed by the interaction of two trimers. Each trimer presents a head-tail configuration, with FMN adenylyltransferase and riboflavin kinase modules from different protomers approaching the active sites and allowing the direct transfer of FMN. Experimental results provide molecular-level evidences of the mechanism of the synthesis of FMN and FAD in prokaryotes in which the oligomeric state could be involved in the regulation of the catalytic efficiency of the modular enzyme.     

Crystal structure of a core domain of stomatin from Pyrococcus horikoshii Illustrates a novel trimeric and coiled-coil fold

Stomatin is a major integral membrane protein of human erythrocytes, the absence of which is associated with a form of hemolytic anemia known as hereditary stomatocytosis. However, the function of stomatin is not fully understood. An open reading frame, PH1511, from the hyperthermophilic archaeon Pyrococcus horikoshii encodes p-stomatin, a prokaryotic stomatin. Here, we report the first crystal structure of a stomatin ortholog, the core domain of the p-stomatin PH1511p (residues 56-234 of PH1511p, designated as PhSto^CD). PhSto^CD forms a novel homotrimeric structure. Three α/β domains form a triangle of about 50 Å on each side, and three α-helical segments of about 60 Å in length extend from the apexes of the triangle. The α/β domain of PhSto^CD is partly similar in structure to the band-7 domain of mouse flotillin-2. While the α/β domain is relatively rigid, the α-helical segment shows conformational flexibility, adapting to the neighboring environment. One α-helical segment forms an anti-parallel coiled coil with another α-helical segment from a symmetry-related molecule. The α-helical segment shows a heptad repeat pattern, and mainly hydrophobic residues form a coiled-coil interface. According to chemical cross-linking experiments, PhSto^CD would be able to assemble into an oligomeric form. The coiled-coil fold observed in the crystal probably contributes to self-association.