1.2 Angstroms crystal structure of the S. pneumoniae PhtA histidine triad domain a novel zinc binding fold.

The recently described pneumococcal histidine triad protein family has been shown to be highly conserved within the pneumococcus. As part of our structural genomics effort on proteins from Streptococcus pneumoniae, we have expressed, crystallised and solved the structure of PhtA-166-220 at 1.2 Angstroms using remote SAD with zinc. The structure of PhtA-166-220 shows no similarity to any protein structure. The overall fold contains 3beta-strands and a single short alpha-helix. The structure appears to contain a novel zinc binding motif. The remaining 4 histidine triad repeats from PhtA have been modelled based on the crystal structure of the PhtA histidine triad repeat 2. From this modelling work, we speculate that only three of the five histidine triad repeats contain the residues in the correct geometry to allow the binding of a zinc ion.

Detergent structure in crystals of the integral membrane light-harvesting complex LH2 from Rhodopseudomonas acidophila strain 10050.

Integral membrane proteins are solubilized by their incorporation into a detergent micelle. The detergent micelle has a critical influence on the formation of a three-dimensional crystal lattice. The bulk detergent phase is not seen in X-ray crystal structures of integral membrane proteins, due to its disordered character. Here, we describe the detergent structure present in crystals of the peripheral light-harvesting complex of the purple bacteria Rhodopseudomonas acidophila strain 10050 at a maximal resolution of 12A as determined by neutron crystallography. The LH2 molecule has a toroidal shape and spans the membrane completely in vivo. A volume of 16% of the unit cell could be ascribed to detergent tails, localized on both the inner and outer hydrophobic surfaces of the molecule. The detergent tail volumes were found to be associated with individual LH2 molecules and had no direct role in the formation of the crystalline lattice.

High-throughput screens for postgenomics: studies of protein crystallization using microsystems technology.

This paper describes the fabrication of a micromachined miniaturized array of chambers in a 2-mm-thick single crystal (100) silicon substrate for the combinatorial screening of the conditions required for protein crystallization screening (including both temperature and the concentration of crystallization agent). The device was fabricated using standard photolithography techniques, reactive ion etching (RIE) and anisotropic silicon wet etching to produce an array of 10 x 10 microchambers, with each element having a volume of 5 microL. A custom-built temperature controller was used to drive two peltier elements in order to maintain a temperature gradient (between 12 and 40 degrees C) across the device. The performance of the microsystem was illustrated by studying the crystallization of a model protein, hen egg white lysozyme. The crystals obtained were studied using X-ray diffraction at room temperature and exhibited 1.78 A resolution. The problems of delivering a robust crystallization protocol, including issues of device fabrication, delivery of a reproducible temperature gradient, and overcoming evaporation are described.

Structural factors which control the position of the Q(y) absorption band of bacteriochlorophyll a in purple bacterial antenna complexes.

This paper presents a concise review of the structural factors which control the energy of the Q(y) absorption band of bacteriochlorophyll a in purple bacterial antenna complexes. The energy of these Q(y) absorption bands is important for excitation energy transfer within the bacterial photosynthetic unit.

Identification of a surface on the beta-propeller protein RACK1 that interacts with the cAMP-specific phosphodiesterase PDE4D5.

A strategy of mutagenesis followed by yeast two-hybrid assay was used to determine the sites on the WD-repeat protein Receptor for Activated C Kinase 1 (RACK1) necessary for it to interact with the cAMP-specific phosphodiesterase isoform PDE4D5. Analysis of deletion mutations demonstrated that WD-repeats 5-7, inclusively, of RACK1 contained the major site for interaction with PDE4D5. A reverse two-hybrid screen focusing on WD-repeats 5-7 of RACK1 isolated 11 single amino acid mutations from within this region that blocked the interaction. The ability of these mutations to block the interaction was confirmed by "pull-down" assays using bacterially expressed glutathione-S-transferase (GST)-RACK1 and mammalian cell-expressed PDE4D5. A model of RACK1 structure, based on the structural similarity of RACK1 to other beta-propeller WD-repeat proteins, indicated that the majority of the amino acids identified by mutagenesis are clustered in a discrete surface of RACK1. We propose that this surface of RACK1 is the major site for its interaction with the unique amino-terminal region of PDE4D5.

The crystallographic structure of the B800-820 LH3 light-harvesting complex from the purple bacteria Rhodopseudomonas acidophila strain 7050.

The B800-820, or LH3, complex is a spectroscopic variant of the B800-850 LH2 peripheral light-harvesting complex. LH3 is synthesized by some species and strains of purple bacteria when growing under what are generally classed as "stressed" conditions, such as low intensity illumination and/or low temperature (<30 degrees C). The apoproteins in these complexes modify the absorption properties of the chromophores to ensure that the photosynthetic process is highly efficient. The crystal structure of the B800-820 light-harvesting complex, an integral membrane pigment-protein complex, from the purple bacteria Rhodopseudomonas (Rps.) acidophila strain 7050 has been determined to a resolution of 3.0 A by molecular replacement. The overall structure of the LH3 complex is analogous to that of the LH2 complex from Rps. acidophila strain 10050. LH3 has a nonameric quaternary structure where two concentric cylinders of alpha-helices enclose the pigment molecules bacteriochlorophyll a and carotenoid. The observed spectroscopic differences between LH2 and LH3 can be attributed to differences in the primary structure of the apoproteins. There are changes in hydrogen bonding patterns between the coupled Bchla molecules and the protein that have an effect on the conformation of the C3-acetyl groups of the B820 molecules. The structure of LH3 shows the important role that the protein plays in modulating the characteristics of the light-harvesting system and indicates the mechanisms by which the absorption properties of the complex are altered to produce a more efficient light-harvesting component.

The crystal structure of tetanus toxin Hc fragment complexed with a synthetic GT1b analogue suggests cross-linking between ganglioside receptors and the toxin.

Tetanus toxin, a member of the family of Clostridial neurotoxins, is one of the most potent toxins known. The crystal structure of the complex of the COOH-terminal fragment of the heavy chain with an analogue of its ganglioside receptor, GT1b, provides the first direct identification and characterization of the ganglioside-binding sites. The ganglioside induces cross-linking by binding to two distinct sites on the Hc molecule. The structure sheds new light on the binding of Clostridial neurotoxins to receptors on neuronal cells and provides important information relevant to the design of anti-tetanus and anti-botulism therapeutic agents.

Probing the interface between membrane proteins and membrane lipids by X-ray crystallography.

Biological membranes are composed of a complex mixture of lipids and proteins, and the membrane lipids support several key biophysical functions, in addition to their obvious structural role. Recent results from X-ray crystallography are shedding new light on the precise molecular details of the protein-lipid interface.

Hormone interactions to Leu-rich repeats in the gonadotropin receptors. I. Analysis of Leu-rich repeats of human luteinizing hormone/chorionic gonadotropin receptor and follicle-stimulating hormone receptor.

The luteinizing hormone receptor (LHR) and follicle-stimulating hormone receptor (FSHR) have an approximately 350-amino acid-long, N-terminal extracellular exodomain. This exodomain binds hormone with high affinity and specificity and contains eight to nine putative Leu-rich repeat (LRR) sequences. LRRs are known to assume the horseshoe structure in ribonuclease inhibitors, and the inner lining of the horseshoe consists of the beta-stranded Leu/Ile-X-Leu/Ile motif. In the case of ribonuclease inhibitors, these beta strands interact with ribonuclease. However, it is unclear whether the putative LRRs of LHR and FSHR play any role in the structure and function. In this work, the beta-stranded Leu/Ile residues in all LRRs of the human LHR and FSHR were Ala-scanned and characterized. In addition, the 23 residues around LRR2 of LHR were Ala-scanned. The results show that beta-stranded Leu and Ile residues in all LRRs are important but not equally. These Leu/Ile-X-Leu/Ile motifs appear to form the hydrophobic core of the LRR loop, crucial for the LRR structure. Interestingly, the hot spots are primarily in the upstream and downstream LRRs of the LHR exodomain, whereas important LRRs spread throughout the FSHR exodomain. This may explain the distinct hormone specificity despite the structural similarity of the two receptors.

Hormone interactions to Leu-rich repeats in the gonadotropin receptors. II. Analysis of Leu-rich repeat 4 of human luteinizing hormone/chorionic gonadotropin receptor.

The luteinizing hormone receptor (LHR) consists of an approximately 350-amino acid-long N-terminal extracellular exodomain and a membrane-associated endodomain of similar size. Human chorionic gonadotropin (hCG) binds to the exodomain, and then hCG/exodomain complex is thought to make a secondary contact with the endodomain and generate hormone signals. The sequence alignment of the exodomain shows imperfectly matching eight to nine Leu-rich repeats (LRRs). In the preceding article (Song, Y., Ji, I., Beauchamp, J., Isaacs, N., and Ji, T. (2001) J. Biol. Chem. 276, 3426-3435), we have shown that LRR2 and LRR4 are crucial for hormone binding. In this work, we have examined the residues of LRR4, in particular Leu(103) and Ile(105) in the putative beta strand. Our data show that Leu(103) and Ile(105) are involved in the specific, hydrophobic interaction of the LRR4 loop, likely to form the hydrophobic core. This loop is crucial for the structural integrity of all of the LRRs. In contrast, the downstream sequence consisting of Asn(107), Thr(108), Gly(109), and Ile(110) of LRR4 is crucial for cAMP induction but not for hormone binding, folding, and surface expression. This implicates, for the first time, its involvement in the interaction with the endodomain and signal generation. The evidence for the interaction is presented in the following article.

Ubiquinone binding, ubiquinone exclusion, and detailed cofactor conformation in a mutant bacterial reaction center.

The X-ray crystal structure of a Rhodobacter sphaeroides reaction center with the mutation Ala M260 to Trp (AM260W) has been determined. Diffraction data were collected that were 97.6% complete between 30.0 and 2.1 A resolution. The electron density maps confirm the conclusions of a previous spectroscopic study, that the Q(A) ubiquinone is absent from the AM260W reaction center (Ridge, J. P., van Brederode, M. E., Goodwin, M. G., van Grondelle, R., and Jones, M. R. (1999) Photosynthesis Res. 59, 9-26). Exclusion of the Q(A) ubiquinone caused by the AM260W mutation is accompanied by a change in the packing of amino acids in the vicinity of the Q(A) site that form part of a loop that connects the DE and E helices of the M subunit. This repacking minimizes the volume of the cavity that results from the exclusion of the Q(A) ubiquinone, and further space is taken up by a feature in the electron density maps that has been modeled as a chloride ion. An unexpected finding is that the occupancy of the Q(B) site by ubiquinone appears to be high in the AM260W crystals, and as a result the position of the Q(B) ubiquinone is well-defined. The high quality of the electron density maps also reveals more precise information on the detailed conformation of the reaction center carotenoid, and we discuss the possibility of a bonding interaction between the methoxy group of the carotenoid and residue Trp M75. The conformation of the 2-acetyl carbonyl group in each of the reaction center bacteriochlorins is also discussed.

An examination of how structural changes can affect the rate of electron transfer in a mutated bacterial photoreaction centre.

A series of reaction centres bearing mutations at the (Phe) M197 position were constructed in the photosynthetic bacterium Rhodobacter sphaeroides. This residue is adjacent to the pair of bacteriochlorophyll molecules (P(L) and P(M)) that is the primary donor of electrons (P) in photosynthetic light-energy transduction. All of the mutations affected the optical and electrochemical properties of the P bacteriochlorophylls. A mutant reaction centre with the change Phe M197 to Arg (FM197R) was crystallized, and a structural model constructed at 2.3 A (1 A=0.1 nm) resolution. The mutation resulted in a change in the structure of the protein at the interface region between the P bacteriochlorophylls and the monomeric bacteriochlorophyll that is the first electron acceptor (B(L)). The new Arg residue at the M197 position undergoes a significant reorientation, creating a cavity at the interface region between P and B(L). The acetyl carbonyl substituent group of the P(M) bacteriochlorophyll undergoes an out-of-plane rotation, which decreases the edge-to-edge distance between the macrocycles of P(M) and B(L). In addition, two new buried water molecules partially filled the cavity that is created by the reorientation of the Arg residue. These waters are in a suitable position to connect the macrocycles of P and B(L) via three hydrogen bonds. Transient absorption measurements show that, despite an inferred decrease in the driving force for primary electron transfer in the FM197R reaction centre, there is little effect on the overall rate of the primary reaction in the bulk of the reaction-centre population. Examination of the X-ray crystal structure reveals a number of small changes in the structure of the reaction centre in the interface region between the P and B(L) bacteriochlorophylls that could account for this faster-than-predicted rate of primary electron transfer.

Structural consequences of the replacement of glycine M203 with aspartic acid in the reaction center from Rhodobacter sphaeroides.

Reaction centers with the double mutation Phe M197 to Arg and Gly M203 to Asp (FM197R/GM203D) have been crystallized from an antenna-deficient strain of Rhodobacter sphaeroides, and the structure has been determined at 2.7 A resolution. Unlike in reaction centers with a single FM197R mutation, the Arg M197 residue in the FM197R/GM203D reaction center adopts a position similar to that of the native Phe residue in the wild-type reaction center. Asp M203 is packed in such a way that the gamma-carboxy group interacts with the backbone carbonyl of Arg M197. The Asp M203 residue takes up part of the volume that is occupied in the wild-type reaction center by a water molecule. This water has been proposed to form a hydrogen bond interaction with the 9-keto carbonyl group of the active branch accessory bacteriochlorophyll, particularly when the primary donor bacteriochlorophylls are oxidized. The GM203D mutation therefore appears to remove the possibility of this hydrogen bond interaction by exclusion of this water molecule, as well as altering the local dielectric environment of the 9-keto carbonyl group. We examine whether the observed structural changes can provide new or alternative explanations for the absorbance and electron-transfer properties of reaction centers with the FM197R and GM203D mutations.

The structures of the H(C) fragment of tetanus toxin with carbohydrate subunit complexes provide insight into ganglioside binding.

The entry of tetanus neurotoxin into neuronal cells proceeds through the initial binding of the toxin to gangliosides on the cell surface. The carboxyl-terminal fragment of the heavy chain of tetanus neurotoxin contains the ganglioside-binding site, which has not yet been fully characterized. The crystal structures of native H(C) and of H(C) soaked with carbohydrates reveal a number of binding sites and provide insight into the possible mode of ganglioside binding.

X-ray crystal structure of the YM210W mutant reaction centre from Rhodobacter sphaeroides.

The X-ray crystal structure of a reaction centre from Rhodobacter sphaeroides with a mutation of tyrosine M210 to tryptophan (YM210W) has been determined to a resolution of 2.5 A. Structural conservation is very good throughout the body of the protein, with the tryptophan side chain adopting a position in the mutant complex closely resembling that of the tyrosine in the wild-type complex. The spectroscopic properties of the YM210W reaction centre are discussed with reference to the structural data, with particular focus on evidence that the introduction of the bulkier tryptophan in place of the native tyrosine may cause a small tilt of the macrocycle of the B(L) monomeric bacteriochlorophyll.

Structural details of an interaction between cardiolipin and an integral membrane protein.

Anionic lipids play a variety of key roles in biomembrane function, including providing the immediate environment for the integral membrane proteins that catalyze photosynthetic and respiratory energy transduction. Little is known about the molecular basis of these lipid-protein interactions. In this study, x-ray crystallography has been used to examine the structural details of an interaction between cardiolipin and the photoreaction center, a key light-driven electron transfer protein complex found in the cytoplasmic membrane of photosynthetic bacteria. X-ray diffraction data collected over the resolution range 30.0-2.1 A show that binding of the lipid to the protein involves a combination of ionic interactions between the protein and the lipid headgroup and van der Waals interactions between the lipid tails and the electroneutral intramembrane surface of the protein. In the headgroup region, ionic interactions involve polar groups of a number of residues, the protein backbone, and bound water molecules. The lipid tails sit along largely hydrophobic grooves in the irregular surface of the protein. In addition to providing new information on the immediate lipid environment of a key integral membrane protein, this study provides the first, to our knowledge, high-resolution x-ray crystal structure for cardiolipin. The possible significance of this interaction between an integral membrane protein and cardiolipin is considered.

Methods for X-ray diffraction analysis of macromolecular structures.

A modern approach to protein crystallography relies as much on molecular biology as on the 'core' crystallographic disciplines. Some recent, biologically significant structure determinations have demonstrated this and show the importance of new third generation synchrotron sources. Novel uses of well known phasing techniques have also been valuable in these structure determinations. For the majority of structures, advances in phasing techniques, data collection and processing and the associated computer programs have led to more effective structure determinations.

Derivative manipulation in the structure solution of the integral membrane LH2 complex.

The structure of the peripheral light-harvesting complex from Rhodopseudomonas acidophila strain 10050 was determined by multiple isomorphous replacement methods. The derivatization of the crystals was augmented by the addition of a backsoaking stage. The soak/backsoaked data comparison had greater isomorphism and showed simpler Patterson maps than the standard native/soak comparison. Amplitudes from the derivatized then backsoaked crystals and from the derivatized crystals were compared in order to extract a subset of heavy-atom sites. Using this information, the full array of sites were found from a derivative/native comparison, eventually leading to excellent electron-density maps.

The two types of 3-dehydroquinase have distinct structures but catalyze the same overall reaction.

The structures of enzymes catalyzing the reactions in central metabolic pathways are generally well conserved as are their catalytic mechanisms. The two types of 3-dehydroquinate dehydratase (DHQase) are therefore most unusual since they are unrelated at the sequence level and they utilize completely different mechanisms to catalyze the same overall reaction. The type I enzymes catalyze a cis-dehydration of 3-dehydroquinate via a covalent imine intermediate, while the type II enzymes catalyze a trans-dehydration via an enolate intermediate. Here we report the three-dimensional structures of a representative member of each type of biosynthetic DHQase. Both enzymes function as part of the shikimate pathway, which is essential in microorganisms and plants for the biosynthesis of aromatic compounds including folate, ubiquinone and the aromatic amino acids. An explanation for the presence of two different enzymes catalyzing the same reaction is presented. The absence of the shikimate pathway in animals makes it an attractive target for antimicrobial agents. The availability of these two structures opens the way for the design of highly specific enzyme inhibitors with potential importance as selective therapeutic agents.