Subcellular targeting by membrane lipids.

The reversible localization of signaling proteins to both the plasma and the internal membranes of cells is critical for the selective activation of downstream functions and depends on interactions with both proteins and membrane lipids. New structural and biochemical analyses of C1, C2, PH, FYVE, FERM and other domains have led to an unprecedented amount of information on the molecular interactions of these signaling proteins with regulatory lipids. A wave of studies using GFP-tagged membrane binding domains as reporters has led to new quantitative insights into the kinetics of these signaling mechanisms.

Structure of the regulatory complex of Escherichia coli IIIGlc with glycerol kinase.

The phosphocarrier protein IIIGlc is an integral component of the bacterial phosphotransferase (PTS) system. Unphosphorylated IIIGlc inhibits non-PTS carbohydrate transport systems by binding to diverse target proteins. The crystal structure at 2.6 A resolution of one of the targets, glycerol kinase (GK), in complex with unphosphorylated IIIGlc, glycerol, and adenosine diphosphate was determined. GK contains a region that is topologically identical to the adenosine triphosphate binding domains of hexokinase, the 70-kD heat shock cognate, and actin. IIIGlc binds far from the catalytic site of GK, indicating that long-range conformational changes mediate the inhibition of GK by IIIGlc. GK and IIIGlc are bound by hydrophobic and electrostatic interactions, with only one hydrogen bond involving an uncharged group. The phosphorylation site of IIIGlc, His90, is buried in a hydrophobic environment formed by the active site region of IIIGlc and a 3(10) helix of GK, suggesting that phosphorylation prevents IIIGlc binding to GK by directly disrupting protein-protein interactions.

Regulation of an enzyme by phosphorylation at the active site.

The isocitrate dehydrogenase of Escherichia coli is an example of a ubiquitous class of enzymes that are regulated by covalent modification. In the three-dimensional structure of the enzyme-substrate complex, isocitrate forms a hydrogen bond with Ser113, the site of regulatory phosphorylation. The structures of Asp113 and Glu113 mutants, which mimic the inactivation of the enzyme by phosphorylation, show minimal conformational changes from wild type, as in the phosphorylated enzyme. Calculations based on observed structures suggest that the change in electrostatic potential when a negative charge is introduced either by phosporylation or site-directed mutagenesis is sufficient to inactivate the enzyme. Thus, direct interaction at a ligand binding site is an alternative mechanism to induced conformational changes from an allosteric site in the regulation of protein activity by phosphorylation.

The adenylyl and guanylyl cyclase superfamily.

New structures solved in 1997 revealed that the adenylyl cyclase core consists of a pair of catalytic domains arranged in a wreath. Homologous catalytic domains are arranged in diverse adenylyl and guanylyl cyclases as symmetric homodimers or pseudosymmetric heterodimers. The kinship of the adenylyl and guanylyl cyclases has been confirmed by the structure-based interconversion of their nucleotide specificities. Catalysis is activated when two metal-binding aspartate residues on one domain are juxtaposed with a key aspargine-arginine pair on the other. Allosteric activators of mammalian adenylyl cyclase, forskolin and the stimulatory G protein alpha subunit, promote the catalytically optimal juxtaposition of the two domains.

Protein kinase C and phospholipase C: bilayer interactions and regulation.

Protein kinase C and phospholipase C are interfacially active modular enzymes that contain multiple membrane-binding domains. During the past two years, 3D structures and functional data have been reported for the key domains: pleckstrin homology, protein kinase C homology-1 and -2, and the phospholipase C catalytic domain. Roles for membrane bilayer structure and lipid microdomains have become clearly domains has shown how the domains work together to coordinate regulation.

Floundering about at cell membranes: a structural view of phospholipid signaling.

Structures are now available for the majority of the enzyme families involved in the phosphorylation, dephosphorylation and hydrolysis of signaling phospholipids. Lipid kinase and phosphatase structures recapitulate catalytic motifs involved in protein phosphorylation and dephosphorylation, whereas cytosolic phospholipase A(2) manifests novel catalytic geometry. Structures have been determined for most known intracellular phospholipid 'receptor' domains, both those that bind membrane-embedded phospholipids and those that bind lipid monomers.

Structure of 3-isopropylmalate dehydrogenase in complex with NAD+: ligand-induced loop closing and mechanism for cofactor specificity.

BACKGROUND: The leucine biosynthetic enzyme 3-isopropylmalate dehydrogenase (IMDH) belongs to a unique class of bifunctional decarboxylating dehydrogenases. The two best-known members of this family, IMDH and isocitrate dehydrogenase (IDH), share a common structural framework and catalytic mechanism but have different substrate and cofactor specificities. IMDH is NAD(+)-dependent, while IDHs occur in both NAD(+)-dependent and NADP(+)-dependent forms. RESULTS: We have co-crystallized Thermus thermophilus IMDH with NAD+ and have determined the structure at 2.5 A resolution. NAD+ binds in an extended conformation. Comparisons with the structure in the absence of cofactor show that binding induces structural changes of up to 2.5 A in the five loops which form the dinucleotide-binding site. The adenine and diphosphate moieties of NAD+ are bound via interactions which are also present in the NADP(+)-IDH complex. Amino acids which interact with the NADP+ 2'-phosphate in IDH are substituted or absent in IMDH. The adenosine ribose forms two hydrogen bonds with Asp278, and the nicotinamide and nicotinamide ribose interact with Glu87 and Asp78, all unique to IMDH. CONCLUSIONS: NAD+ binding induces a conformational transition in IMDH, resulting in a structure that is intermediate between the most 'open' and 'closed' decarboxylating dehydrogenase conformations. Physiological specificity of IMDH for NAD+ versus NADP+ can be explained by the unique interaction between Asp278 and the free 2'-hydroxyl of the NAD+ adenosine, discrimination against the presence of the 2'-phosphate by the negative charge on Asp278, and the absence of potential favorable interactions with the 2'-phosphate of NADP+.

Coexpression of cloned alpha(1B), beta(2a), and alpha(2)/delta subunits produces non-inactivating calcium currents similar to those found in bovine chromaffin cells.

Chromaffin cells express N-type calcium channels identified on the basis of their high sensitivity to block by omega-conotoxin GVIA (omega-CgTx GVIA). In contrast to neuronal N-type calcium currents that inactivate during long depolarizations and that require negative holding potentials to remove inactivation, many chromaffin cells exhibit N-type calcium channel currents that show little inactivation during maintained depolarizations and that exhibit no decrease in channel availability at depolarized holding potentials. N-type calcium channels are thought to be produced by combination of the pore-forming alpha(1B) subunit and accessory beta and alpha(2)/delta subunits. To examine the molecular composition of the non-inactivating N-type calcium channel, we cloned the alpha(1B) and accessory beta (beta(1b), beta(1c,) beta(2a), beta(2b), and beta(3a)) subunits found in bovine chromaffin cells. Expression of the subunits in either Xenopus oocytes or human embryonic kidney 293 cells produced high-threshold calcium currents that were blocked by omega-CgTx GVIA. Coexpression of bovine alpha(1B) with beta(1b), beta(1c), beta(2b), or beta(3a) produced currents that were holding potential dependent. In contrast, coexpression of bovine alpha(1B) with beta(2a) produced holding potential-independent calcium currents that closely mimicked native non-inactivating currents, suggesting that non-inactivating N-type channels consist of bovine alpha(1B), alpha(2)/delta, and beta(2a).

Design and structural analysis of alternative hydrophobic core packing arrangements in bacteriophage T4 lysozyme.

An attempt has been made to design modified core-packing arrangements in bacteriophage T4 lysozyme. Alternative replacements of the buried residues Leu99, Met102, Val111 and Phe153 were selected using packing calculations and energy minimization. To test the design procedure, a series of multiple mutants was constructed culminating in the replacement L99F/M102L/V111I/F153L. These variants decrease the stability of T4 lysozyme by approximately 0 to 2 kcal/mol. The crystal structures of a number of the variants were determined. In the variant in which Val111 was replaced by Ile, alpha-helix 107-114 moved by approximately 1.5 A, breaking the hydrogen bond between the backbone carbonyl group of Thr109 and the backbone amide group of Gly113. This conformational change was not anticipated by the design procedure. Compensating interactions of magnitude up to 1.1 kcal/mol occur for some sets of mutations, while other sets display nearly additive stability changes. Within experimental error, the stability of the double mutant V111F/F153L is additive, with delta delta G different by only 0.1 kcal/mol from the sum of the two single mutants. The quadruple mutant L99F/M102L/V111I/F153L is destabilized by 0.5 kcal/mol, compared to delta delta G = -1.6 kcal/mol for the sum of the four single mutants. Multiple mutants show smaller overall structural changes from wild-type than M102L or V111I alone. Co-operative changes in structure and stability can be rationalized in terms of specific structural differences between single and multiple mutants. Genuine repacking of the hydrophobic core of T4 lysozyme with minimal effects on structure, stability and activity thus appears to have been achieved.

Expression, characterization, and crystallization of the catalytic core of rat phosphatidylinositide-specific phospholipase C delta 1.

Phosphatidylinositide-specific phospholipase Cs (PI-PLCs) catalyze the calcium-dependent hydrolysis of phosphatidylinositides in response to diverse stimuli in higher eukaryotes. Mammalian PI-PLCs contain divergent regulatory regions, but all share three conserved regions: an N-terminal pleckstrin homology (PH) domain, X, and Y. We report the high-level expression and characterization of a recombinant "catalytic core" of rat PI-PLC delta 1 that contains the catalytically essential X and Y regions, but not the PH domain. The expressed protein, PI-PLC delta delta 1-134, is catalytically active versus phosphatidylinositol 4,5-bisphosphate in deoxycholate micelles with a K(m) of 182 microM and a Vmax of 27 mumol/min/mg. PI-PLC delta delta 1-134 is monomeric and monodisperse as judged by dynamic light scattering. Far-UV CD indicates a structure with approximately 35% alpha-helix. A reversible change in the near-UV CD spectrum is observed on addition of calcium, suggesting that calcium can bind PI-PLC delta delta 1-134 in the absence of phospholipid. Triclinic crystals of PI-PLC delta delta 1-134 have been obtained that diffract beyond 2.4 A resolution under cryogenic conditions. Based on Vm = 2.72 Da/A3 and on the self-rotation function, there are two PI-PLC delta delta 1-134 molecules per asymmetric unit that are related to each other by a noncrystallographic axis of approximate twofold symmetry parallel to a.

Second-site suppression of regulatory phosphorylation in Escherichia coli isocitrate dehydrogenase.

Inactivation of Escherichia coli isocitrate dehydrogenase upon phosphorylation at S113 depends upon the direct electrostatic repulsion of the negatively charged gamma-carboxylate of isocitrate by the negatively charged phosphoserine. The effect is mimicked by replacing S113 with aspartate or glutamate, which reduce performance (kcat/K(i).isocitrat/ Km.NADP) by a factor of 10(7). Here, we demonstrate that the inactivating effects of the electrostatic repulsion are completely eliminated by a second-site mutation, and provide the structural basis for this striking example of intragenic suppression. N115 is adjacent to S113 on one face of the D-helix, interacts with isocitrate and NADP+, and has been postulated to serve in both substrate binding and in catalysis. The single N115L substitution reduces affinity for isocitrate by a factor of 50 and performance by a factor of 500. However, the N115L substitution completely suppresses the inactivating electrostatic effects of S113D or S113E: the performance of the double mutants is 10(5) higher than the S113D and S113E single mutants. These mutations have little effect on the kinetics of alternative substrates, which lack the charged gamma-carboxylate of isocitrate. Both glutamate and aspartate at site 113 remain fully ionized in the presence of leucine. In the crystal structure of the N115L mutant, the leucine adopts a different conformer from the wild-type asparagine. Repacking around the leucine forces the amino-terminus of the D-helix away from the rest of the active site. The hydrogen bond between E113 and N115 in the S113E single mutant is broken in the S113E/N115L mutant, allowing the glutamate side chain to move away from the gamma-carboxylate of isocitrate. These movements increase the distance between the carboxylates, diminish the electrostatic repulsion, and lead to the remarkably high activity of the S113E/N115L mutant.

Taxonomy and function of C1 protein kinase C homology domains.

C1 domains are compact alpha/beta structural units of about 50 amino acids which tightly bind two zinc ions. These domains were first discovered as the loci of phorbol ester and diacylglycerol binding to conventional protein kinase C isozymes, which contain 2 C1 domains (C1A and C1B) in their N-terminal regulatory regions. We present a comprehensive list of 54 C1 domains occurring singly or doubly in 34 different proteins. Many C1 domains and C1 domain-containing proteins bind phorbol esters, but many others do not. By combining analysis of 54 C1 domain sequences with information from previously reported solution and crystal structure determinations and site-directed mutagenesis, profiles are derived and used to classify C1 domains. Twenty-six C1 domains fit the profile for phorbol-ester binding and are termed "typical." Twenty-eight other domains fit the profile for the overall C1 domain fold but do not fit the profile for phorbol ester binding, and are termed "atypical." Proteins containing typical C1 domains are predicted to be regulated by diacylglycerol, whereas those containing only atypical domains are not.

Hydrophobic core repacking and aromatic-aromatic interaction in the thermostable mutant of T4 lysozyme Ser 117-->Phe.

The T4 lysozyme mutant Ser 117-->Phe was isolated fortuitously and found to be more thermostable than wild-type by 1.1-1.4 kcal/mol. In the wild-type structure, the side chain of Ser 117 is in a sterically restricted region near the protein surface and forms a short hydrogen bond with Asn 132. The crystal structure of the S117F mutant shows that the introduced Phe side chain rotates by about 150 degrees about the C alpha-C beta bond relative to wild type and is buried in the hydrophobic core of the protein. Burial of Phe 117 is accommodated by rearrangements of the surrounding side chains of Leu 121, Leu 133, and Phe 153 and by main-chain shifts, which result in a minimal increase in packing density. The benzyl rings of Phe 117 and Phe 153 form a near-optimal edge-face interaction in the mutant structure. This aromatic-aromatic interaction, as well as increased hydrophobic stabilization and elimination of a close contact in the wild-type protein, apparently compensate for the loss of a hydrogen bond and the possible cost of structural rearrangements in the mutant. The structure illustrates the ability of a protein to accommodate a surprisingly large structural change in a manner that actually increases thermal stability. The mutant has activity about 10% that of wild-type, supportive of the prior hypothesis (Grütter, M.G. & Matthews, B.W., 1982, J. Mol. Biol. 154, 525-535) that the peptidoglycan substrate of T4 lysozyme makes extended contacts with the C-terminal domain in the vicinity of Ser 117.

Characterization and crystallization of a minimal catalytic core domain from mammalian type II adenylyl cyclase.

Adenylyl cyclases play a pivotal role in signal transduction by carrying out the regulated synthesis of cyclic AMP. The nine cloned mammalian adenylyl cyclases all share two conserved regions of sequence, C1 and C2, which are homologous to each other and are together responsible for catalytic activity. Recombinant C1 and C2 domains catalyze the synthesis of cyclic AMP when they are mixed and activated by forskolin, and C2 domains alone also manifest reduced levels of forskolin-stimulated enzyme activity. Using limited proteolysis and mass spectrometry, we have mapped the boundaries of a minimal stable and active C2 catalytic domain to residues 871-1090 of type II adenylyl cyclase. We report the properties and crystallization of this trimmed domain, termed IIC2-delta 4. Crystals belong to space group P4n2(1)2, where n = 1 or 3; a = b = 81.3, and c = 180.5 A; and there are two molecules per asymmetric unit related by an approximate body centering operation. Flash-frozen crystals diffract anisotropically to 2.2 A along the c* direction and to 2.8 A along the a* and b* directions using synchrotron radiation.

The variability of response of scoliotic spines to segmental spinal instrumentation.

Ninety-two patients (71 idiopathic, 15 neurogenic, 2 Marfan's syndrome, 1 Down's syndrome, 1 osteogenesis imperfecta, 1 Prader-Willi syndrome, 1 Klippel-Trenaunay-Weber syndrome), ages 9-49 years, had posterior spine fusion and stabilization with Luque L-rods and sublaminar segmental wires (SSI) for progressive scoliosis. There were no neurologic complications. The average preoperative major curve of 52 degrees (37-113 degrees) initially corrected to 30 degrees (6-94 degrees), and at last follow-up (range, 2-7 years), was 33 degrees (8-90 degrees). There was marked variability in curvature correction and maintenance of correction, with 14 patients progressively decreasing their curvatures postoperatively. Average preoperative Pedriolle vertebral rotation angle for all 92 patients was 16 degrees (4-26 degrees), which initially corrected to 14 degrees (0-24 degrees), and at last follow-up remained 14 degrees (0-23 degrees). Although SSI had little influence on rotation, 12 patients progressively decreased their rotational deformity after operation. Aside from the positive influence of curve flexibility on the degree of postoperative curve correction, the authors could not identify factors explaining how curvature and vertebral rotation responded so variably to SSI. The authors recommend continued use of SSI to stabilize curvatures in osteopenic patients (particularly those with neuromuscular disease), but rotational deformity will probably persist.

Postoperative Clostridium difficile pseudomembranous colitis in idiopathic scoliosis. A brief clinical report.

The authors report two healthy young patients with progressive idiopathic scoliosis, both without allergies or histories of gastrointestinal disorders, who received perioperative preventive cephalosporin antibiotics, and developed explosive diarrhea postoperatively, confirmed as Clostridium difficile pseudomembranous colitis by stool toxin assay. Both patients had initially recovered uneventfully after posterior fusion and Cotrel-Dubousset instrumentation. Their youth, health, diagnosis, and lack of nosocomial factors made colitis unexpected. The two cases were sporadic, occurring 2 years apart over a 12-year observation period. Symptoms and signs of colitis for the two patients were markedly varied as to time of onset, order of appearance, and severity. Unexplained fever before onset of diarrhea led to renewed cephalosporin administration, potentially exacerbating the colitis. Initial symptoms and signs were nonspecific; appropriate treatment had to begin before diagnosis could be confirmed by stool toxin asay, which requires 2 days.

Intraoperative use of the mobile gamma camera in localizing and excising osteoid osteomas of the spine.

Three patients (ages 12, 13, and 15 years) had histologically verified osteoid osteomas of the spine (specifically in the facet processes of L3, L2, and C5) which were localized preoperatively with nuclear scintigraphy utilizing technetium 99m diphosphonate. Intraoperative nuclear scintigraphy utilizing the mobile gamma camera was essential in both localizing the tumor during surgical excision and ensuring adequacy of excision. The authors strongly recommend intraoperative scintigraphy, and en bloc resection rather than shelling out and curettage. All three patients required facet process excision for successful tumor removal, and therefore required local fusion with internal fixation.

N- and P/Q-type Ca2+ channels in adrenal chromaffin cells.

Ca2+ is the most ubiquitous second messenger found in all cells. Alterations in [Ca2+]i contribute to a wide variety of cellular responses including neurotransmitter release, muscle contraction, synaptogenesis and gene expression. Voltage-dependent Ca2+ channels, found in all excitable cells (Hille 1992), mediate the entry of Ca2+ into cells following depolarization. Ca2+ channels are composed of a large pore-forming subunit, called the alpha1 subunit, and several accessory subunits. Ten different alpha1 subunit genes have been identified and classified into three families, Ca(v1-3) (Dunlap et al. 1995, Catterall 2000). Each alpha1 gene produces a unique Ca2+ channel. Although chromaffin cells express several different types of Ca2+ channels, this review will focus on the Cav(2.1) and Cav(2.2) channels, also known as P/Q- and N-type respectively (Nowycky et al. 1985, Llinas et al. 1989b, Wheeler et al. 1994). These channels exhibit physiological and pharmacological properties similar to their neuronal counterparts. N-, P/Q and to a lesser extent R-type Ca2+ channels are known to regulate neurotransmitter release (Hirning et al. 1988, Horne & Kemp 1991, Uchitel et al. 1992, Luebke et al. 1993, Takahashi & Momiyama 1993, Turner et al. 1993, Regehr & Mintz 1994, Wheeler et al. 1994, Wu & Saggau 1994, Waterman 1996, Wright & Angus 1996, Reid et al. 1997). N- and P/Q-type Ca2+ channels are abundant in nerve terminals where they colocalize with synaptic vesicles. Similarly, these channels play a role in neurotransmitter release in chromaffin cells (Garcia et al. 2006). N- and P/Q-type channels are subject to many forms of regulation (Ikeda & Dunlap 1999). This review pays particular attention to the regulation of N- and P/Q-type channels by heterotrimeric G-proteins, interaction with SNARE proteins, and channel inactivation in the context of stimulus-secretion coupling in adrenal chromaffin cells.

The sugar kinase/heat shock protein 70/actin superfamily: implications of conserved structure for mechanism.

Sugar kinases, stress-70 proteins, and actin belong to a superfamily defined by a fold consisting of two domains with the topology beta beta beta alpha beta alpha beta alpha. These enzymes catalyze ATP phosphoryl transfer or hydrolysis coupled to a large conformational change in which the two domains close around the nucleotide. The beta 1-beta 2 turns of each domain form hydrogen bonds with ATP phosphates, and conserved Asp, Glu or Gln residues coordinate Mg2+ or Ca2+ through bound waters. The activity of superfamily members is regulated by various effectors, some of which act by promoting or inhibiting the conformational change. Nucleotide hydrolysis eliminates interdomain bridging interactions between the second beta 1-beta 2 turn and the ATP gamma-phosphate. This is proposed to destabilize the closed conformation and affect the orientation of the two domains, which might in turn regulate the activity of kinase oligomers, stress-70 protein-protein complexes, and actin filaments.

Signaling and subcellular targeting by membrane-binding domains.

Protein kinase C homology-1 and -2, FYVE, and pleckstrin homology domains are ubiquitous in eukaryotic signal transduction and membrane-trafficking proteins. These domains regulate subcellular localization and protein function by binding to lipid ligands embedded in cell membranes. Structural and biochemical analysis of these domains has shown that their molecular mechanisms of membrane binding depend on a combination of specific and nonspecific interactions with membrane lipids. In vivo studies of green fluorescent protein fusions have highlighted the key roles of these domains in regulating protein localization to plasma and internal membranes in cells.