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.

Cystine knots.

Recent reports of the crystal structure of the glycoprotein hormone human chorionic gonadotrophin show that cystine knots are proving to be versatile structural motifs that enable the construction of a variety of proteins with different functional properties. Because of their shape, there appears to be an intrinsic requirement for the cystine-knot growth factors to form dimers. This extra level of organization increases the variety of structures built around this simple structural motif.

Light-harvesting mechanisms in purple photosynthetic bacteria.

The processes by which photosynthetic bacteria capture light and transfer the energy to the reaction centre continue to be studied using an array of methodologies, both physical and biological. With the publication this year of the crystal structure of the LH2 complex from Rhodopseudomonas acidophila and the projection structure of the LH1 complex from Rhodospirillum rubrum, structural models now exist for all the components in the bacterial photosynthetic apparatus.

Pigment-pigment interactions and energy transfer in the antenna complex of the photosynthetic bacterium Rhodopseudomonas acidophila.

BACKGROUND: Photosynthesis starts with the absorption of solar radiation by antenna pigment molecules. In purple bacteria these chromophores, (bacteriochlorophyll a and carotenoid) are embedded in the membrane; they are non-covalently bound to apoproteins which have the ability to modulate the chromophores' absorbing characteristics. The first structure of the bacterial antenna complex from Rhodopseudomonas acidophila, strain 10050, shows a ring of nonameric symmetry. Two concentric cylinders of apoproteins enclose the pigment molecules. The current resolution of the structure, to 2.5 A, allows us to begin to explore the mechanism of energy transfer among these pigments. RESULTS: The mechanism of energy transfer, from the short- to long-wavelength-absorbing pigments, is largely determined by the relative distances and orientations of the chromophores. In this paper we provide evidence that energy transfer between the B800 and B850 bacteriochlorophylls is largely via Förster induced dipole-dipole resonance. Strong Coulombic (exciton) coupling among the 18 short distanced chromophores in the B850 macrocycle is promoted by good alignment of the Qy dipoles. Singlet-singlet energy transfer from carotenoid to the B800 macrocycle appears to be minimal, with most of the energy transfer going to B850. The higher energy state of both chromophores dominates in more complex situations. CONCLUSIONS: The structure of the antenna complex not only shows Nature at its most aesthetic but also illustrates how clever and efficient the energy transfer mechanism has become, with singlet-singlet excitation being passed smoothly down the spectral gradient to the reaction centre.

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.

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.

Crystallographic characterization of tetanus toxin fragment C.

The C-terminal fragment from tetanus toxin has been crystallized. The 50 kDa protein forms prismatic crystals with an orthorhombic unit cell of dimensions a = 64.03 A, b = 76.31 A and c = 135.3 A. The space group is P2(1)2(1)2(1). Assuming one molecule per asymmetric unit, the solvent occupies 63% of the unit cell.

Crystallographic characterization of pertactin, a membrane-associated protein from Bordetella pertussis.

Pertactin, a membrane-associated protein of Bordetella pertussis, has been crystallized in the presence of 28% ammonium sulphate. The space group is P6(3)22 with cell dimensions a = b = 178.2 A and c = 106.8 A. The crystals diffract to 3.3 A using a rotating anode source and are suitable for an X-ray structure determination. Assuming one molecule in the asymmetric unit, 70% of the cell is occupied by solvent.

Crystallization and preliminary crystallographic data of the Fab fragment of an anti-phenylalanine hydroxylase monoclonal antibody.

Two crystal habits, one rod shaped and the other square prismatic, of the Fab fragment of a monoclonal anti-phenylalanine hydroxylase antibody have been grown using the method of vapour phase diffusion against polyethylene glycol 6000. The square prisms diffract to better than 2.8 A, belong to the space group P1 and have unit cell parameters a = 41.8 A, b = 50.3 A, c = 114.7 A, alpha = 97.6 degrees, beta = 91.7 degrees, gamma = 91.0 degrees, while the rod-shaped crystals belong to the space group P212121, have unit cell parameters a = 105.6 A, b = 119.8 A, c = 82.2 A and diffract to 3.5 A resolution.

Crystallization of the B800-820 light-harvesting complex from Rhodopseudomonas acidophila strain 7750.

The B800-820 light-harvesting complex, an integral membrane protein, from Rhodopseudomonas acidophila strain 7750 has been crystallized. The tabular plates have a hexagonal unit cell of a = b = 121.8 A and c = 283.1 A and belong to the space group R32. X-ray diffraction data have been collected to 6 A resolution, using an area detector on a rotating anode source. The B800-820 light-harvesting complex is comprised of four low molecular weight apoproteins (B800-820 alpha 1, B800-820 alpha 2, B800-820 beta 1 and B800-820 beta 2). Polyacrylamide gel electrophoresis shows that the complex exists as an oligomeric assembly, with an apparent molecular weight of 92,000.

Apoprotein structure in the LH2 complex from Rhodopseudomonas acidophila strain 10050: modular assembly and protein pigment interactions.

The refined structure of the peripheral light-harvesting complex from Rhodopseudomonas acidophila strain 10050 reveals a membrane protein with protein-protein interactions in the trans-membrane region exclusively of a van der Waals nature. The dominant factors in the formation of the complex appear to be extramembranous hydrogen bonds (suggesting that each apoprotein must achieve a fold close to its final structure in order to oligomerize), protein-pigment and pigment-pigment interactions within the membrane-spanning region. The pigment molecules are known to play an important role in the formation of bacterial light-harvesters, and their extensive mediation of structural contacts within the membrane bears this out. Amino acid residues determining the secondary structure of the apoproteins influence the oligomeric state of the complex. The assembly of the pigment array is governed by the apoproteins of LH2. The particular environment of each of the pigment molecules is, however, influenced directly by few protein contacts. These contacts produce functional effects that are not attributable to a single cause, e.g. the arrangement of an overlapping cycle of chromophores not only provides energy delocalisation and storage properties, but also has consequences for oligomer size, pigment distortion modes and pigment chemical environment, all of which modify the precise function of the complex. The evaluation of site energies for the pigment array requires the consideration of a number of effects, including heterogeneous pigment distortions, charge distributions in the local environment and mechanical interactions.

Crystallization of a type II dehydroquinase from Mycobacterium tuberculosis.

A type II 3-dehydroquinase from Mycobacterium tuberculosis has been crystallized in the presence of 6% polyethyleneglycol 6000. Data from these crystals have been collected to a resolution of 2.2 A on a rotating anode X-ray source. The space group has been determined as F23 with unit cell dimensions of a = b = c = 127.8 A. There is one molecule in the asymmetric unit.

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.

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.

Determination of residues important in hormone binding to the extracellular domain of the luteinizing hormone/chorionic gonadotropin receptor by site-directed mutagenesis and modeling.

The LH/CG receptor (LH/CG-R) belongs to the family of glycoprotein hormone G protein-coupled receptors. The extracellular domain of LH/CG-R is associated with high ligand-binding affinity and contains leucine-rich repeats (LRRs). With the goal of identifying essential amino acid residues involved in ligand binding, we replaced several conserved ionizable residues in the rat LH/CG-R with ones of opposite charge. The expression of these mutants was assessed by binding studies and Western blots after COS-7 cells were transiently transfected with wild type and mutant receptor cDNAs. The charge inversion of each of Lys40, Lys104, Asp118, Glu132, and Asp135 with Asp or Lys resulted in no detectable human CG binding in intact or solubilized cells; as control, a Lys40-->Arg replacement yielded a mutant with characteristics of the wild type receptor. Western analysis showed that the Lys40-->Arg mutant expressed at a level comparable to that of wild type receptor and, like wild type, exhibited a predominant immunoreactive mature form of LH/CG-R. Each of the five charge inversion mutants expressed at a lower level than wild type as assessed by immunoreactivity, and the levels of the Lys40-->Asp and Glu132-->Lys mutants were particularly low. The ratio of the mature to immature form of the receptor was high, i.e. like that of wild type, for the Glu132-->Lys and Asp135-->Lys replacements; the three other charge inversion mutants exhibited less mature than immature forms of the receptor. To aid in interpreting these results, we developed a model incorporating residues 27-235 of the extracellular domain of the rat LH/CG-R based on the crystal structure of the porcine ribonuclease inhibitor. Sequence homology and alignment revealed nine LRRs, with flanking cysteine clusters as found in a number of LRR proteins. Our model suggested that the Lys replacements of Glu132 and Asp135, i.e. those mutants that formed mature receptors, would disrupt the regional negative charge of the receptor. We propose that these residues are either directly involved in hormone binding or indirectly by disruption of the charge of an important binding surface.

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 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 and selective destruction of shared epitopes in human chorionic gonadotropin beta subunit.

The feasibility of producing epitope-specific antigens by mutation of the gene is demonstrated, the aim being to eliminate unwanted surface epitopes yet allowing the natural folding of the protein to maintain the desired epitope(s). The model protein is the beta subunit of human chorionic gonadotropin (hCG beta) which previously has been used as an immunological contraceptive vaccine but has extensive cross-reaction with human luteinizing hormone. Of a series of mutants made, the mutant with substitutions of Glu for Arg 68, Ser for Arg 74, His for Gly 75 and His for Val 79, lost the ability to react with a panel of cross-reacting monoclonal antibodies while retaining the discontinuous and linear epitopes specific to the holo-hormone. In addition, allocation of amino acid residues to established epitope clusters could be made: residues 24, 25, 68 and 71 probably contribute to the cluster termed beta 3, residues 20, 21, 22, 75 and 77 to cluster beta 6 and residue 68 to clusters beta 2, beta 4 and beta 5.