Preparation of single crystals of a yolk lipoprotein

Single crystals of a yolk lipoprotein from two species of lamprey oocytes have been prepared and shown to belong to the monoclinic space group C2. The unit cell dimensions combined with molecular weight determinations support the idea that the lipoprotein is a symmetrical dimer centered on a crystallographic 2-fold symmetry axis. The lipid content is estimated to be about 15% (w/w) and the crystals are suitable for structural studies by single crystal X-ray analysis. The X-ray results are correlated with those obtained by electron diffraction.     

Crystal structure of the cytochrome P-450CAM active site mutant Thr252Ala.

The crystal structure of a cytochrome P-450CAM site-directed mutant in which the active site Thr252 has been replaced with an Ala (Thr252Ala) has been refined to an R factor of 0.18 at 2.2 A. According to sequence alignments (Nelson & Strobel, 1989), Thr252 is highly conserved among P-450 enzymes. The crystallographic structure of ferrous camphor- and carbon monoxide-bound P-450CAM (Raag & Poulos, 1989b) suggests that Thr252 is a key active site residue, forming part of the dioxygen-binding site. Mutation of the active site threonine to alanine produces an enzyme in which substrate hydroxylation is uncoupled from electron transfer. Specifically, hydrogen peroxide and "excess" water are produced instead of the product, 5-exo-hydroxycamphor. The X-ray structure has revealed that a local distortion in the distal helix between Gly248 and Thr252 becomes even more severe in the Thr252Ala mutant. Furthermore, a solvent molecule not present in the native enzyme is positioned in the dioxygen-binding region of the mutant enzyme active site. In this location, the solvent molecule could sterically interfere with and destabilize dioxygen binding. In addition, the active site solvent molecule is connected, via a network of hydrogen bonds, with an internal solvent channel which links distal helix residues to a buried Glu side chain. Thus, solvent protons appear to be much more accessible to dioxygen in the mutant than in the wild-type enzyme, a factor which may promote hydrogen peroxide and/or water production instead of substrate hydroxylation. On the basis of crystallographic and mutagenesis data, a proton delivery pathway involving residues Lys178/Arg186, Asp251, and Thr252 is proposed for wild-type P-450CAM. Coordinates of structures discussed in this paper have been submitted to the Brookhaven Protein Data Bank (Bernstein et al., 1977).     

Formation, crystal structure, and rearrangement of a cytochrome P-450cam iron-phenyl complex

Cytochrome P-450cam reacts with phenyldiazene (PhN = NH), or less efficiently with phenylhydrazine, to give a catalytically inactive complex with an absorption maximum at 474 nm. The prosthetic group extracted anaerobically from the inactivated protein has the spectroscopic properties of a sigma phenyl-iron complex and rearranges, on exposure to air and acid, to an approximately equal mixture of the four N-phenylprotoporphyrin IX regioisomers. The crystal structure of the intact protein complex, refined at 1.9-A resolution to an R factor of 20%, confirms that the phenyl group is directly bonded through one of its carbons to the iron atom. The phenyl ring is tilted from the heme normal by about 10 degrees in the opposite direction from that in which carbon monoxide tilts when bound to P-450cam. Camphor, the natural substrate for P-450cam, is larger than a phenyl group and hydrogen bonds to Tyr 96, the only hydrophilic residue near the active site. Electron density in the active site in addition to that contributed by the phenyl group suggests that two water molecules occupy part of the camphor binding site but are not within hydrogen-bonding distance of Tyr 96. As observed in a previous crystallographic study of inhibitor-P-450cam complexes [Poulos, T.L., & Howard, A.J. (1987) Biochemistry 26, 8165-8174], there are large changes in both the atomic positions and mobilities of the residues in the proposed substrate access channel region of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure of the lamprey yolk lipid-protein complex lipovitellin-phosvitin at 2.8 A resolution

The X-ray crystallographic structure of the lipid-protein complex lipovitellin-phosvitin has been determined with the multiple isomorphous replacement method using four heavy-atom derivatives. Lamprey yolk lipovitellin-phosvitin is a dimeric molecule of molecular weight 352,000. The monomer consists of three polypeptide chains. The smallest is known as phosvitin and has an extremely high phosphoserine content. The monomeric unit also contains about 16% (w/w) of non-covalently bound lipid, probably in a monolayer or bilayer-like configuration. Within each monomer is a "cavity" or region of low electron density. The cavity has a volume of about 68,000 A3 and is believed to contain the lipid in a presumably disordered state. The cavity is roughly conical in shape and is lined on two sides by seven and eight-stranded antiparallel beta-sheets. The base of the cavity opens away from the intersubunit interface, but appears partially closed off from solvent regions by additional antiparallel beta-sheet structure. The beta-sheets lining the sides of the cavity are surrounded by a shell of two curved layers of 16 interconnected helices. The helices in either layer of the shell are all roughly parallel to each other and antiparallel to all of the helices of the other layer. The connectivity of the helices resembles a "superhelix" and is different from the connectivities seen in proteins containing four-helix bundles. There are an estimated 1300 amino acids in lamprey lipovitellin-phosvitin and almost 1000 alanine residues have been modeled into electron density. The remaining residues are assumed to be disordered.     

The structural basis for substrate-induced changes in redox potential and spin equilibrium in cytochrome P-450CAM

The crystal structures of cytochrome P-450CAM complexed with the alternative substrates norcamphor and adamantanone have been refined at 2.0-A resolution and compared with the native, camphor-bound form of the enzyme. Norcamphor lacks the 8-, 9-, and 10-methyl groups of camphor. Thus, specific interactions between these groups and phenylalanine 87 and valines 247 and 295 are missing in the norcamphor complex. As a result, norcamphor binds about 0.9 A further from the oxygen-binding site than does camphor, which allows sufficient room for a water molecule or hydroxide ion to remain coordinated with the heme iron atom. The larger adamantanone occupies a position closer to that of camphor and, as in the camphor-bound enzyme, the heme iron remains pentacoordinate with no solvent molecule coordinated as a sixth ligand. A comparison of crystallographic temperature factors indicates that norcamphor is more "loosely" bound than are either camphor or adamantanone, as might be expected from the relative sizes of the different substrates. The looser fit of norcamphor in the active-site pocket results in a less specific pattern of hydroxylation. The presence of an aqua ligand is the likely structural basis for the norcamphor-P-450CAM complex having both a lower redox potential and higher percentage of low-spin heme than do either the camphor-P-450CAM or adamantanone-P-450CAM complexes.     

Functional implications of interleukin-1 beta based on the three-dimensional structure.

The molecular structure of interleukin-1 beta, a hormone-like cytokine with roles in several disease processes, has been determined at 2.0 A resolution and refined to a crystallographic R-factor of 0.19. The framework of this molecule consists of 12 antiparallel beta-strands exhibiting pseudo-3-fold symmetry. Six of the strands make up a beta-barrel with polar residues concentrated at either end. Analysis of the three-dimensional structure, together with results from site-directed mutagenesis and biochemical and immunological studies, suggest that the core of the beta-barrel plays an important functional role. A large patch of charged residues on one end of the barrel is proposed as the binding surface with which IL-1 interacts with its receptor.     

Cytochrome P450cam: crystallography, oxygen activation, and electron transfer.

Several crystal structures of various substrate and inhibited complexes of the camphor monoxygenase, cytochrome P450cam from Pseudomonas putida, are now available. These structures, together with mutagenesis, biochemical, and biophysical studies, have allowed for a detailed penetration into the problem of how P450s activate molecular oxygen, control stereoselectivity, and transfer electrons. This review will provide a summary of the crystallographic work in light of what these structures have taught us about P450 function.     

Crystal structures of cytochrome P-450CAM complexed with camphane, thiocamphor, and adamantane: factors controlling P-450 substrate hydroxylation

X-ray crystal structures have been determined for complexes of cytochrome P-450CAM with the substrates camphane, adamantane, and thiocamphor. Unlike the natural substrate camphor, which hydrogen bonds to Tyr96 and is metabolized to a single product, camphane, adamantane and thiocamphor do not hydrogen bond to the enzyme and all are hydroxylated at multiple positions. Evidently the lack of a substrate-enzyme hydrogen bond allows substrates greater mobility in the active site, explaining this lower regiospecificity of metabolism as well as the inability of these substrates to displace the distal ligand to the heme iron. Tyr96 is a ligand, via its carbonyl oxygen atom, to a cation that is thought to stabilize the camphor-P-450CAM complex [Poulos, T. L., Finzel, B. C., & Howard, A. J. (1987) J. Mol. Biol. 195, 687-700]. The occupancy and temperature factor of the cationic site are lower and higher, respectively, in the presence of the non-hydrogen-bonding substrates investigated here than in the presence of camphor, underscoring the relationship between cation and substrate binding. Thiocamphor gave the most unexpected orientation in the active site of any of the substrates we have investigated to date. The orientation of thiocamphor is quite different from that of camphor. That is, carbons 5 and 6, at which thiocamphor is primarily hydroxylated [Atkins, W. M., & Sligar, S. G. (1988) J. Biol. Chem. 263, 18842-18849], are positioned near Tyr96 rather than near the heme iron. Therefore, the crystallographically observed thiocamphor-P-450CAM structure may correspond to a nonproductive complex. Disordered solvent has been identified in the active site in the presence of uncoupling substrates that channel reducing equivalents away from substrate hydroxylation toward hydrogen peroxide and/or "excess" water production. A buried solvent molecule has also been identified, which may promote uncoupling by moving from an internal location to the active site in the presence of highly mobile substrates.     

Integration of light-controlled neuronal firing and fast circuit imaging

For understanding normal and pathological circuit function, capitalizing on the full potential of recent advances in fast optical neural circuit control will depend crucially on fast, intact-circuit readout technology. First, millisecond-scale optical control will be best leveraged with simultaneous millisecond-scale optical imaging. Second, both fast circuit control and imaging should be adaptable to intact-circuit preparations from normal and diseased subjects. Here we illustrate integration of fast optical circuit control and fast circuit imaging, review recent work demonstrating utility of applying fast imaging to quantifying activity flow in disease models, and discuss integration of diverse optogenetic and chemical genetic tools that have been developed to precisely control the activity of genetically specified neural populations. Together these neuroengineering advances raise the exciting prospect of determining the role-specific cell types play in modulating neural activity flow in neuropsychiatric disease.     

Site-directed mutageneses of rat liver cytochrome P-450d: catalytic activities toward benzphetamine and 7-ethoxycoumarin

Catalytic activities toward benzphetamine and 7-ethoxycoumarin of 11 distal mutants, 9 proximal mutants, and 3 aromatic mutants of rat liver cytochrome P-450d were studied. A distal mutant Thr319Ala was not catalytically active toward benzphetamine, while this mutant retained activity toward 7-ethoxycoumarin. Distal mutants Gly316Glu, Thr319Ala, and Thr322Ala displayed higher activities (kcat/Km) toward 7-ethoxycoumarin that were 2.4-4.7-fold higher than that of the wild-type enzyme. Although kcat/Km values of four multiple distal mutants toward benzphetamine were less than half that of the wild type, activities of these mutants toward 7-ethoxycoumarin were almost the same as or higher than the wild-type activity toward this substrate. The distal double mutant Glu318Asp, Phe325Tyr showed 6-fold higher activity than the wild-type P-450d toward 7-ethoxycoumarin. Activities of the proximal mutants Lys453Glu and Arg455Gly toward both substrates were much lower (less than one-seventh) than the corresponding wild-type activities. Catalytic activities of three aromatic mutants, Phe425Leu, Pro427Leu, and Phe430Leu, toward benzphetamine were less than 7% of that of the wild type, while the activities of these aromatic mutants toward 7-ethoxycoumarin were more than 2.5 times higher than the wild-type activity toward this substrate. From these findings, in conjunction with a molecular model for P-450d, we suggest that (1) the relative importance to catalysis of various distal helix amino acids differs depending on the substrate and that these differences are associated with the size, shape, and flexibility of the substrate and (2) the proximal residue Lys453 appears to play a critical role in the catalytic activity of P-450d, perhaps by participating in forming an intermolecular electron-transfer complex.