The structures of Thermoplasma volcanium phosphoribosyl pyrophosphate synthetase bound to ribose-5-phosphate and ATP analogs

Phosphoribosyl pyrophosphate (PRPP) synthetase catalyzes the transfer of the pyrophosphate group from ATP to ribose-5-phosphate (R5P) yielding PRPP and AMP. PRPP is an essential metabolite that plays a central role in cellular metabolism. The enzyme from a thermophilic archaeon Thermoplasma volcanium (Tv) was expressed in Escherichia coli, crystallized, and its X-ray molecular structure was determined in a complex with its substrate R5P and with substrate analogs β,γ-methylene ATP and ADP in two monoclinic crystal forms, P2₁. The β,γ-methylene ATP- and the ADP-bound binary structures were determined from crystals grown from ammonium sulfate solutions; these crystals diffracted to 1.8 Å and 1.5 Å resolutions, respectively. Crystals of the ternary complex with ADP-Mg²⁺ and R5P were grown from a polyethylene glycol solution in the absence of sulfate ions, and they diffracted to 1.8 Å resolution; the unit cell is approximately double the size of the unit cell of the crystals grown in the presence of sulfate. The Tv PRPP synthetase adopts two conformations, open and closed, at different stages in the catalytic cycle. The binding of substrates, R5P and ATP, occurs with PRPP synthetase in the open conformation, whereas catalysis presumably takes place with PRPP synthetase in the closed conformation. The Tv PRPP synthetase forms a biological dimer in contrast to the tetrameric or hexameric quaternary structures of the Methanocaldococcus jannaschii and Bacillus subtilis PRPP synthetases, respectively.     

Structural basis of the LOV1 dimerization of Arabidopsis phototropins 1 and 2

Phototropin (phot) is a blue-light receptor protein that triggers phototropic responses, chloroplast relocation, and stomata opening to maximize the efficiency of photosynthesis in higher plants. Phot is composed of three functional domains. The N-terminal half folds into two light-oxygen-voltage-sensing domains called LOV1 and LOV2, each binding a flavin mononucleotide to absorb blue light. The C-terminal half is a serine/threonine kinase domain that causes light-dependent autophosphorylation leading to cellular signaling cascades. LOV2 domain is primarily responsible for activation of the kinase, and LOV1 domain is thought to act as a dimerization site and to regulate sensitivity to activation by blue light. Here we show the crystal structures of LOV1 domains of Arabidopsis phot1 and phot2 in the dark at resolutions of 2.1 Å and 2.0 Å, respectively. Either LOV1 domain forms a dimer through face-to-face association of β-scaffolds in the crystallographic asymmetric unit. Three types of interactions stabilizing the dimer structures found are as follows: contacts of side chains in their β-scaffolds, hydrophobic interactions of a short helix found in the N-terminus of a subunit with the β-scaffolds of both subunits, and hydrogen bonds mediated by hydration water molecules filling the dimer interface. The critical residues for dimerization are Cys261, forming a disulfide bridge between subunits in phot1-LOV1 domain, and Thr217 and Met232 in phot2-LOV1. The topology in homodimeric associations of the LOV1 domains is discussed when referring to those of homodimers or heterodimers of light-oxygen-voltage-sensing or Per-ARNT-Sim domains. The present results also provide clues to understanding structural basis in dimeric interactions of Per-ARNT-Sim protein modules in cellular signaling.     

The Three-dimensional Structure of a Mycobacterial DapD Provides Insights into DapD Diversity and Reveals Unexpected Particulars about the Enzymatic Mechanism

The enzyme tetrahydrodipicolinate N-succinyltransferase (DapD) is part of the L-lysine biosynthetic pathway. This pathway is crucial for the survival of the pathogen Mycobacterium tuberculosis (Mtb) and, consequently, the enzymes of the pathway are potential drug targets. We report here the crystal structures of Mtb-DapD and of Mtb-DapD in complex with the co-factor succinyl-CoA (SCoA) at 2.15 Å and 1.97 Å resolution, respectively. Each subunit of the trimeric enzyme consists of three domains, of which the second, a left-handed, parallel β-helix (LβH domain), is the common structural motif of enzymes belonging to the hexapeptide repeat superfamily. The trimeric quaternary structure is stabilized by Mg²⁺ and Na⁺ located on the 3-fold axis. The binary complex of Mtb-DapD and SCoA reveals the binding mode(s) of the co-factor and a possible covalent reaction intermediate. The N-terminal domain of Mtb-DapD exhibits a unique architecture, including an interior water-filled channel, which allows access to a magnesium ion located at the 3-fold symmetry axis.     

Active-site structure of class IV adenylyl cyclase and transphyletic mechanism

Adenylyl cyclases (ACs) belonging to three nonhomologous classes (II, III, and IV) have been structurally characterized, enabling a comparison of the mechanisms of cyclic adenosine 3′,5′-monophosphate biosynthesis. We report the crystal structures of three active-site complexes for Yersinia pestis class IV AC (AC-IV)—two with substrate analogs and one with product. Mn²⁺ binds to all three phosphates, and to Glu12 and Glu136. Electropositive residues Lys14, Arg63, Lys76, Lys111, and Arg113 also form hydrogen bonds to phosphates. The conformation of the analogs is suitable for in-line nucleophilic attack by the ribose O3′ on α-phosphate (distance ∼ 4 Å). In the product complex, a second Mn ion is observed to be coordinated to both ribose 2′ oxygen and ribose 3′ oxygen. Observation of both metal sites, together with kinetic measurements, provides strong support for a two-cation mechanism. Eleven active-site mutants were also made and kinetically characterized. These findings and comparisons with class II and class III enzymes enable a detailed transphyletic analysis of the AC mechanism. Consistent with its lack of coordination to purine, Y. pestis AC-IV cyclizes both ATP and GTP. As in other classes of AC, the ribose is loosely bound, and as in class III, no base appears to ionize the O3′ nucleophile. Different syn/anti conformations suggest that the mechanism involves a conformational transition, and further evidence suggests a role for ribosyl pseudorotation. With resolutions of 1.6-1.7 Å, these are the most detailed active-site ligand complexes for any class of this ubiquitous signaling enzyme.     

Nautilus pompilius hemocyanin: 9 A cryo-EM structure and molecular model reveal the subunit pathway and the interfaces between the 70 functional units

Hemocyanins are giant extracellular oxygen carriers in the hemolymph of many molluscs. Nautilus pompilius (Cephalopoda) hemocyanin is a cylindrical decamer of a 350 kDa polypeptide subunit that in turn is a “pearl-chain” of seven different functional units (FU-a to FU-g). Each globular FU has a binuclear copper centre that reversibly binds one O₂ molecule, and the 70-FU decamer is a highly allosteric protein. Its primary structure and an 11 Å cryo-electron microscopy (cryo-EM) structure have recently been determined, and the crystal structures of two related FU types are available in the databanks. However, in molluscan hemocyanin, the precise subunit pathway within the decamer, the inter-FU interfaces, and the allosteric unit are still obscure, but this knowledge is crucial to understand assembly and allosterism of these proteins. Here we present the cryo-EM structure of Nautilus hemocyanin at 9.1 Å resolution (FSC_1/2-bit criterion), and its molecular model obtained by rigid-body fitting of the individual FUs. In this model we identified the subunit dimer, the subunit pathway, and 15 types of inter-FU interface. Four interface types correspond to the association mode of the two protomers in the published Octopus FU-g crystal. Other interfaces explain previously described morphological structures such as the fenestrated wall (which shows D5 symmetry), the three horizontal wall tiers, the major and minor grooves, the anchor structure and the internal collar (which unexpectedly has C5 symmetry). Moreover, the potential calcium/magnesium and N-glycan binding sites have emerged. Many interfaces have amino acid constellations that might transfer allosteric interaction between FUs. From their topologies we propose that the prime allosteric unit is the oblique segment between major and minor groove, consisting of seven FUs from two different subunits. Thus, the 9 Å structure of Nautilus hemocyanin provides fundamentally new insight into the architecture and function of molluscan hemocyanins.     

Crystal Structure of an Essential Enzyme in Seed Starch Degradation: Barley Limit Dextrinase in Complex with Cyclodextrins

Barley limit dextrinase [Hordeum vulgare limit dextrinase (HvLD)] catalyzes the hydrolysis of α-1,6 glucosidic linkages in limit dextrins. This activity plays a role in starch degradation during germination and presumably in starch biosynthesis during grain filling. The crystal structures of HvLD in complex with the competitive inhibitors α-cyclodextrin (CD) and β-CD are solved and refined to 2.5 Å and 2.1 Å, respectively, and are the first structures of a limit dextrinase. HvLD belongs to glycoside hydrolase 13 family and is composed of four domains: an immunoglobulin-like N-terminal eight-stranded β-sandwich domain, a six-stranded β-sandwich domain belonging to the carbohydrate binding module 48 family, a catalytic (β/α)₈-like barrel domain that lacks α-helix 5, and a C-terminal eight-stranded β-sandwich domain of unknown function. The CDs are bound at the active site occupying carbohydrate binding subsites + 1 and + 2. A glycerol and three water molecules mimic a glucose residue at subsite − 1, thereby identifying residues involved in catalysis. The bulky Met440, a unique residue at its position among α-1,6 acting enzymes, obstructs subsite − 4. The steric hindrance observed is proposed to affect substrate specificity and to cause a low activity of HvLD towards amylopectin. An extended loop (Asp513-Asn520) between β5 and β6 of the catalytic domain also seems to influence substrate specificity and to give HvLD a higher affinity for α-CD than pullulanases. The crystal structures additionally provide new insight into cation sites and the concerted action of the battery of hydrolytic enzymes in starch degradation.     

The asymmetric trimeric architecture of [2Fe-2S] IscU: implications for its scaffolding during iron-sulfur cluster biosynthesis

IscU is a key component of the ISC machinery and is involved in the biogenesis of iron-sulfur (Fe-S) proteins. IscU serves as a scaffold for assembly of a nascent Fe-S cluster prior to its delivery to an apo protein. Here, we report the first crystal structure of IscU with a bound [2Fe-2S] cluster from the hyperthermophilic bacterium Aquifex aeolicus, determined at a resolution of 2.3 Å, using multiwavelength anomalous diffraction of the cluster. The holo IscU formed a novel asymmetric trimer that harbored only one [2Fe-2S] cluster. One iron atom of the cluster was coordinated by the S^γ atom of Cys36 and the N^ε atom of His106, and the other was coordinated by the S^γ atoms of Cys63 and Cys107 on the surface of just one of the protomers. However, the cluster was buried inside the trimer between the neighboring protomers. The three protomers were conformationally distinct from one another and associated around a noncrystallographic pseudo-3-fold axis. The three flexible loop regions carrying the ligand-binding residues (Cys36, Cys63, His106 and Cys107) and the N-terminal α1 helices were positioned at the interfaces and underwent substantial conformational rearrangement, which stabilized the association of the asymmetric trimer. This unique trimeric A. aeolicus holo-IscU architecture was clearly distinct from other known monomeric apo-IscU/SufU structures, indicating that asymmetric trimer organization, as well as its association/dissociation, would be involved in the scaffolding function of IscU.     

Nucleotide Binding States of Subunit A of the A-ATP Synthase and the Implication of P-Loop Switch in Evolution

The crystal structures of the nucleotide-empty (A_E), 5′-adenylyl-β,γ-imidodiphosphate (A_PNP)-bound, and ADP (A_DP)-bound forms of the catalytic A subunit of the energy producer A₁A_O ATP synthase from Pyrococcus horikoshii OT3 have been solved at 2.47 Å and 2.4 Å resolutions. The structures provide novel features of nucleotide binding and depict the residues involved in the catalysis of the A subunit. In the A_E form, the phosphate analog SO₄²⁻ binds, via a water molecule, to the phosphate binding loop (P-loop) residue Ser238, which is also involved in the phosphate binding of ADP and 5′-adenylyl-β,γ-imidodiphosphate. Together with amino acids Gly234 and Phe236, the serine residue stabilizes the arched P-loop conformation of subunit A, as shown by the 2.4-Å structure of the mutant protein S238A in which the P-loop flips into a relaxed state, comparable to the one in catalytic β subunits of F₁F_O ATP synthases. Superposition of the existing P-loop structures of ATPases emphasizes the unique P-loop in subunit A, which is also discussed in the light of an evolutionary P-loop switch in related A₁A_O ATP synthases, F₁F_O ATP synthases, and vacuolar ATPases and implicates diverse catalytic mechanisms inside these biological motors.     

Crystal structure of a glutamate/aspartate binding protein complexed with a glutamate molecule: structural basis of ligand specificity at atomic resolution

The crystal structure of a periplasmic l-aspartate/l-glutamate binding protein (DEBP) from Shigella flexneri complexed with an l-glutamate molecule has been determined and refined to an atomic resolution of 1.0 Å. There are two DEBP molecules in the asymmetric unit. The refined model contains 4462 non-hydrogen protein atoms, 730 water molecules, 2 bound glutamate molecules, and 2 Tris molecules from the buffer used in crystallization. The final R_cryst and R_free factors are 13.61% and 16.89%, respectively. The structure has root-mean-square deviations of 0.016 Å from standard bond lengths and 2.35° from standard bond angles.The DEBP molecule is composed of two similarly folded domains separated by the ligand binding region. Both domains contain a central five-stranded β-sheet that is surrounded by several α-helices. The two domains are linked by two antiparallel β-strands. The overall shape of DEBP is that of an ellipsoid approximately 55 Å × 45 Å × 40 Å in size.The binding of ligand to DEBP is achieved mostly through hydrogen bonds between the glutamate and side-chain and main-chain groups of DEBP. Side chains of residues Arg24, Ser72, Arg75, Ser90, and His164 anchor the deprotonated γ-carboxylate group of the glutamate with six hydrogen bonds. Side chains of Arg75 and Arg90 form salt bridges with the deprotonated α-carboxylate group, while the main-chain amide groups of Thr92 and Thr140 form hydrogen bonds with the same group. The positively charged α-amino group of the l-glutamate forms salt bridge interaction with the side-chain carboxylate group of Asp182 and hydrogen bond interaction with main-chain carbonyl oxygen of Ser90. In addition to these hydrogen bond and electrostatic interactions, other interactions may also play important roles. For example, the two methylene groups from the glutamate form van der Waals interactions with hydrophobic side chains of DEBP.Comparisons with several other periplasmic amino acid binding proteins indicate that DEBP residues involved in the binding of α-amino and α-carboxylate groups of the ligand and the pattern of hydrogen bond formation between these groups are very well conserved, but the binding pocket around the ligand side chain is not, leading to the specificity of DEBP. We have identified structural features of DEBP that determine its ability of binding glutamate and aspartate, two molecules with different sizes, but discriminating against very similar glutamine and asparagine molecules.     

Conformational Changes in a Plant Ketol-Acid Reductoisomerase upon Mg and NADPH Binding as Revealed by Two Crystal Structures

Ketol-acid reductoisomerase (KARI; EC 1.1.1.86) is an enzyme in the branched-chain amino acid biosynthesis pathway where it catalyzes the conversion of 2-acetolactate into (2R)-2,3-dihydroxy-3-isovalerate or the conversion of 2-aceto-2-hydroxybutyrate into (2R,3R)-2,3-dihydroxy-3-methylvalerate. KARI catalyzes two reactions—alkyl migration and reduction—and requires Mg²⁺ and NADPH for activity. To date, the only reported structures for a plant KARI are those of the spinach enzyme-Mn²⁺-(phospho)ADP ribose-(2R,3R)-2,3-dihydroxy-3-methylvalerate complex and the spinach KARI-Mg²⁺-NADPH-N-hydroxy-N-isopropyloxamate complex, where N-hydroxy-N-isopropyloxamate is a predicted transition-state analog. These studies demonstrated that the enzyme consists of two domains, N-domain and C-domain, with the active site at the interface of these domains. Here, we have determined the structures of the rice KARI-Mg²⁺ and rice KARI-Mg²⁺-NADPH complexes to 1.55 Å and 2.80 Å resolutions, respectively. In comparing the structures of all the complexes, several differences are observed. Firstly, the N-domain is rotated up to 15° relative to the C-domain, expanding the active site by up to 4 Å. Secondly, an α-helix in the C-domain that includes residues V510-T519 and forms part of the active site moves by ∼ 3.9 Å upon binding of NADPH. Thirdly, the 15 C-terminal amino acid residues in the rice KARI-Mg²⁺ complex are disordered. In the rice KARI-Mg²⁺-NADPH complex and the spinach KARI structures, many of the 15 residues bind to NADPH and the N-domain and cover the active site. Fourthly, the location of the metal ions within the active site can vary by up to 2.7 Å. The new structures allow us to propose that an induced-fit mechanism operates to (i) allow substrate to enter the active site, (ii) close over the active site during catalysis, and (iii) open the active site to facilitate product release.     

Analysis of the Open and Closed Conformations of the β Subunits in Thermophilic F1-ATPase by Solution NMR

F₁-ATPase, composed of α, β, γ, δ, and ? subunits, is a unique enzyme in terms of its rotational catalytic activity. The smallest unit showing this function is the α₃β₃γ complex. We have investigated the α₃β₃γ?^ΔC (?^ΔC, truncated ?) complex from thermophilic Bacillus PS3 (TF₁′, 360 kDa) in the solution state by using the combination of extensive deuteration, segmental-labeling, and CRINEPT (cross-correlated relaxation-enhanced polarization transfer) NMR. Well-resolved CRINEPT-HMQC (heteronuclear multiple-quantum correlation) spectra of partially ¹⁵N-labeled TF₁′ were obtained for this huge and asymmetric protein complex. The spectrum of the C-terminal domain of the β subunit revealed that the open form of the β subunit in the TF₁′ complex is similar to that of the free β monomer. The open β subunit in the TF₁′ complex does not exhibit high affinity for nucleotides unlike the monomer, but this is in agreement with the results of single-molecule analysis of TF₁α₃β₃γ. On the other hand, the closed form of the β subunit in the TF₁′ complex was shown to be distinct from that of the nucleotide-bound β monomer. This is consistent with a previous report that the closed form of the TF₁β monomer could be a catalytically activated state. The loop between the N-terminal β-barrel and the central domain is highly flexible in the TF₁′ complex, in contrast to that in the α₃β₃ hexamer, suggesting that it is affected by the presence of the γ subunit in this area.     

Atomic structure of mutant PPARγ LBD complexed with 15d-PGJ2: Novel modulation mechanism of PPARγ/RXRα function by covalently bound ligands

15-deoxy-Δ^12,14-prostaglandin J₂ (15d-PGJ₂) activates a nuclear receptor heterodimer, peroxisome proliferators-activated receptor γ (PPARγ)/ retinoid X receptor (RXRα) through covalent binding to Cys285 in PPARγ ligand-binding domain (LBD). Here, we present the 1.9 Å crystal structure of C285S mutant LBD complexed with 15d-PGJ₂, corresponding to the non-covalently bound state. The ligand lies adjacent to a hydrogen-bond network around the helix H2 and the nearby β-sheet. Comparisons with previous structures clarified the relationships between PPARγ function and conformational alterations of LBD during the process of covalently binding ligands, such as 15d-PGJ₂, and thus suggested a mechanism, by which these ligands modulate PPARγ/RXRα function through conformational changes of the loop following helix H2′ and the β-sheet.     

Crystal Structure of the GalNAc/Gal-Specific Agglutinin from the Phytopathogenic Ascomycete Sclerotinia sclerotiorum Reveals Novel Adaptation of a β-Trefoil Domain

A lectin from the phytopathogenic ascomycete Sclerotinia sclerotiorum that shares only weak sequence similarity with characterized fungal lectins has recently been identified. S. sclerotiorum agglutinin (SSA) is a homodimeric protein consisting of two identical subunits of ∼ 17 kDa and displays specificity primarily towards Gal/GalNAc. Glycan array screening indicates that SSA readily interacts with Gal/GalNAc-bearing glycan chains. The crystal structures of SSA in the ligand-free form and in complex with the Gal-β1,3-GalNAc (T-antigen) disaccharide have been determined at 1.6 and 1.97 Å resolution, respectively. SSA adopts a β-trefoil domain as previously identified for other carbohydrate-binding proteins of the ricin B-like lectin superfamily and accommodates terminal non-reducing galactosyl and N-acetylgalactosaminyl glycans. Unlike other structurally related lectins, SSA contains a single carbohydrate-binding site at site α. SSA reveals a novel dimeric assembly markedly dissimilar to those described earlier for ricin-type lectins. The present structure exemplifies the adaptability of the β-trefoil domain in the evolution of fungal lectins.     

Structure of the type III secretion recognition protein YscU from Yersinia enterocolitica

The inner-membrane protein YscU has an important role during the assembly of the Yersinia enterocolitica type III secretion injectisome. Its cytoplasmic domain (YscU^C) recognizes translocators as individual substrates in the export hierarchy. Activation of YscU entails autocleavage at a conserved NPTH motif. Modification of this motif markedly changes the properties of YscU, including translocator export cessation and production of longer injectisome needles. We determined the crystal structures of the uncleaved variants N263A and N263D of YscU^C at 2.05 Å and 1.55 Å resolution, respectively. The globular domain is found to consist of a central, mixed β-sheet surrounded by α-helices. The NPTH motif forms a type II β-turn connecting two β-strands. NMR analysis of cleaved and uncleaved YscU^C indicates that the global structure of the protein is retained in cleaved YscU^C. The structure of YscU^C variant N263D reveals that wild type YscU^C is poised for cleavage due to an optimal reaction geometry for nucleophilic attack of the scissile bond by the side chain of Asn263. In vivo analysis of N263Q and H266A/R314A YscU variants showed a phenotype that combines the absence of translocator secretion with normal needle-length control. Comparing the structure of YscU to those of related proteins reveals that the linker domain between the N-terminal transmembrane domain and the autocleavage domain can switch from an extended to a largely α-helical conformation, allowing for optimal positioning of the autocleavage domain during injectisome assembly.     

Structural and molecular characterization of the prefoldin beta subunit from Thermococcus strain KS-1

Prefoldin (PFD) is a heterohexameric molecular chaperone that is found in eukaryotic cytosol and archaea. PFD is composed of α and β subunits and forms a “jellyfish-like” structure. PFD binds and stabilizes nascent polypeptide chains and transfers them to group II chaperonins for completion of their folding. Recently, the whole genome of Thermococcus kodakaraensis KOD1 was reported and shown to contain the genes of two α and two β subunits of PFD. The genome of Thermococcus strain KS-1 also possesses two sets of α (α1 and α2) and β subunits (β1 and β2) of PFD (TsPFD). However, the functions and roles of each of these PFD subunits have not been investigated in detail. Here, we report the crystal structure of the TsPFD β1 subunit at 1.9 Å resolution and its functional analysis. TsPFD β1 subunits form a tetramer with four coiled-coil tentacles resembling the jellyfish-like structure of heterohexameric PFD. The β hairpin linkers of β1 subunits assemble to form a β barrel “body” around a central fourfold axis. Size-exclusion chromatography and multi-angle light-scattering analyses show that the β1 subunits form a tetramer at pH 8.0 and a dimer of tetramers at pH 6.8. The tetrameric β1 subunits can protect against aggregation of relatively small proteins, insulin or lysozyme. The structural and biochemical analyses imply that PFD β1 subunits act as molecular chaperones in living cells of some archaea.     

Monolignol acylation and lignin structure in some nonwoody plants: a 2D NMR study

Lignins from three nonwoody angiosperms were analyzed by 2D NMR revealing important differences in their molecular structures. The Musatextilis milled-wood-lignin (MWL), with a syringyl-to-guaiacyl (S/G) ratio of 9, was strongly acylated (near 85% of side-chains) at the γ-carbon by both acetates and p-coumarates, as estimated from ¹H-¹³C correlations in C_γ-esterified and C_γ-OH units. The p-coumarate H_3,5-C_3,5 correlation signal was completely displaced by acetylation, and disappeared after alkali treatment, indicating that p-coumaric acid was esterified maintaining its free phenolic group. By contrast, the Cannabissativa MWL (S/G ∼0.8) was free of acylating groups, and the Agavesisalana MWL (S/G ∼4) showed high acylation degree (near 80%) but exclusively with acetates. Extensive C_γ-acylation results in the absence (in M.textilis lignin) or low abundance (4% in A.sisalana lignin) of β-β′ resinol linkages, which require free C_γ-OH to form the double tetrahydrofuran ring. However, minor signals revealed unusual acylated β-β′ structures confirming that acylation is produced at the monolignol level, in agreement with chromatographic identification of γ-acetylated sinapyl alcohol among the plant extractives. In contrast, resinol substructures involved 22% side-chains in the C.sativa MWL. The ratio between β-β′ and β-O-4′ side-chains in these and other MWL varied from 0.32 in C.sativa MWL to 0.02 in M.textilis MWL, and was inversely correlated with the degree of acylation. The opposite was observed for the S/G ratio that was directly correlated with the acylation degree. Monolignol acylation is discussed as a mechanism potentially involved in the control of lignin structure.     

Structure of the Alkalohyperthermophilic Archaeoglobus fulgidus Lipase Contains a Unique C-Terminal Domain Essential for Long-Chain Substrate Binding

Several crystal structures of AFL, a novel lipase from the archaeon Archaeoglobus fulgidus, complexed with various ligands, have been determined at about 1.8 Å resolution. This enzyme has optimal activity in the temperature range of 70-90 °C and pH 10-11. AFL consists of an N-terminal α/β-hydrolase fold domain, a small lid domain, and a C-terminal β-barrel domain. The N-terminal catalytic domain consists of a 6-stranded β-sheet flanked by seven α-helices, four on one side and three on the other side. The C-terminal lipid binding domain consists of a β-sheet of 14 strands and a substrate covering motif on top of the highly hydrophobic substrate binding site. The catalytic triad residues (Ser136, Asp163, and His210) and the residues forming the oxyanion hole (Leu31 and Met137) are in positions similar to those of other lipases. Long-chain lipid is located across the two domains in the AFL-substrate complex. Structural comparison of the catalytic domain of AFL with a homologous lipase from Bacillus subtilis reveals an opposite substrate binding orientation in the two enzymes. AFL has a higher preference toward long-chain substrates whose binding site is provided by a hydrophobic tunnel in the C-terminal domain. The unusually large interacting surface area between the two domains may contribute to thermostability of the enzyme. Two amino acids, Asp61 and Lys101, are identified as hinge residues regulating movement of the lid domain. The hydrogen-bonding pattern associated with these two residues is pH dependent, which may account for the optimal enzyme activity at high pH. Further engineering of this novel lipase with high temperature and alkaline stability will find its use in industrial applications.     

Crystal Structure of the Light-Driven Chloride Pump Halorhodopsin from Natronomonas pharaonis

The light-driven chloride pump halorhodopsin from Natronomonas pharaonis (phR) crystallised into the monoclinic space group C2, with a phR trimer per the asymmetric unit. Diffraction data at 2.0-Å resolution showed that the carotenoid bacterioruberin binds to crevices between adjacent protein subunits in the trimeric assembly. Besides seven transmembrane helices (A to G) that characterise archaeal rhodopsins, the phR protomer possesses an amphipathic α-helix (A′) at the N-terminus. This helix, together with a long loop between helices B and C, forms a hydrophobic cap that covers the extracellular surface and prevents a rapid ion exchange between the active centre and the extracellular medium. The retinal bound to Lys256 in helix G takes on an all-trans configuration with the Schiff base being hydrogen-bonded to a water molecule. The Schiff base also interacts with Asp252 and a chloride ion, the latter being fixed by two polar groups (Thr126 and Ser130) in helix C. In the anion uptake pathway, four ionisable residues (Arg123, Glu234, Arg176 and His100) and seven water molecules are aligned to form a long hydrogen-bonding network. Conversely, the cytoplasmic half is filled mostly by hydrophobic residues, forming a large energetic barrier against the transport of anion. The height of this barrier would be lowered substantially if the cytoplasmic half functions as a proton/HCl antiporter. Interestingly, there is a long cavity extending from the main-chain carbonyl of Lys256 to Thr71 in helix B. This cavity, which is commonly seen in halobacterial light-driven proton pumps, is one possible pathway that is utilised for a water-mediated proton transfer from the cytoplasmic medium to the anion, which is relocated to the cytoplasmic channel during the photocycle.     

Vancomycin forms ligand-mediated supramolecular complexes

The emergence of resistance to vancomycin and related glycopeptide antibiotics is spurring efforts to develop new antimicrobial therapeutics. High-resolution structural information about antibiotic-ligand recognition should prove valuable in the rational design of improved drugs. We have determined the X-ray crystal structure of the complex of vancomycin with N-acetyl-d-Ala-d-Ala, a mimic of the natural muramyl peptide target, and refined this structure at a resolution of 1.3 Å to R and R_free values of 0.172 and 0.195, respectively. The crystal asymmetric unit contains three back-back vancomycin dimers; two of these dimers participate in ligand-mediated face-face interactions that produce an infinite chain of molecules running throughout the crystal. The third dimer packs against the side of a face-face interface in a tight “side-side” interaction that involves both polar contacts and burial of hydrophobic surface. The trimer of dimers found in the asymmetric unit is essentially identical to complexes seen in three other crystal structures of glycopeptide antibiotics complexed with peptide ligands. These four structures are derived from crystals belonging to different space groups, suggesting that the trimer of dimers may not be simply a crystal packing artifact and prompting us to ask if ligand-mediated oligomerization could be observed in solution. Using size-exclusion chromatography, dynamic light scattering, and small-angle X-ray scattering, we demonstrate that vancomycin forms discrete supramolecular complexes in the presence of tripeptide ligands. Size estimates for these complexes are consistent with assemblies containing four to six vancomycin monomers.