Distinct functions of Cdk5(Y15) phosphorylation and Cdk5 activity in stress fiber formation and organization

Previous studies have shown that Cdk5 promotes lens epithelial cell adhesion. Here we use a cell spreading assay to investigate the mechanism of this effect. As cells spread, forming matrix adhesions and stress fibers, Cdk5(Y15) phosphorylation and Cdk5 kinase activity increased. Cdk5(Y15) phosphorylation was inhibited by PP1, a Src family kinase inhibitor. To identify the PP1-sensitive kinase, we transfected cells with siRNA oligonucleotides for cSrc and related kinases. Only cSrc siRNA oligonucleotides inhibited Cdk5(Y15) phosphorylation. Cdk5(pY15) and its activator, p35, colocalized with actin in stress fibers. To examine Cdk5 function, we inhibited Cdk5 activity under conditions that also prevent phosphorylation at Y15: expression of kinase inactive mutations Cdk5(Y15F) and Cdk5(K33T), and siRNA suppression of Cdk5. Stress fiber formation was severely inhibited. To distinguish between a requirement for Cdk5 kinase activity and a possible adaptor role for Cdk5(pY15), we used two methods that inhibit kinase activity without inhibiting phosphorylation at Y15: pharmacological inhibition with olomoucine and expression of the kinase inactive mutation, Cdk5(D144N). Stress fiber organization was altered, but stress fiber formation was not blocked. These findings indicate that Cdk5(Y15) phosphorylation and Cdk5 activity have distinct functions required for stress fiber formation and organization, respectively.     

The dependence of the conductance of the hemocyanin channel on applied potential and ionic concentration with mono- and divalent cations

Incorporation of Megatura crenulata hemocyanin into phosphatidylcholine black lipid membranes results in the formation of ion channels. Channel properties depend on many factors, three of which are examined in this work: type and concentration of electrolyte and applied voltage. Eight cations at different concentrations have been used. Instantaneous conductance of the channel is a saturating function of both applied voltage and ionic strength of the bathing solution with monovalent cations, but only of ionic strength with divalent cations. Steady-state voltage-conductance relations are nonlinear for both signs but show slight saturation with ionic strength. Relaxation towards the steady state can be fitted by two exponentials with different time constants. All experimental data are fitted postulating the existence of a mechanism of voltage gating of the channel, and of discrete negative charge near its mouth. Specific and nonspecific binding of cations is required.     

Structural Characterization of the Predominant Family of Histidine Kinase Sensor Domains

Histidine kinase (HK) receptors are used ubiquitously by bacteria to monitor environmental changes, and they are also prevalent in plants, fungi, and other protists. Typical HK receptors have an extracellular sensor portion that detects a signal, usually a chemical ligand, and an intracellular transmitter portion that includes both the kinase domain itself and the site for histidine phosphorylation. While kinase domains are highly conserved, sensor domains are diverse. HK receptors function as dimers, but the molecular mechanism for signal transduction across cell membranes remains obscure. In this study, eight crystal structures were determined from five sensor domains representative of the most populated family, family HK1, found in a bioinformatic analysis of predicted sensor domains from transmembrane HKs. Each structure contains an inserted repeat of PhoQ/DcuS/CitA (PDC) domains, and similarity between sequence and structure is correlated across these and other double-PDC sensor proteins. Three of the five sensors crystallize as dimers that appear to be physiologically relevant, and comparisons between ligated structures and apo-state structures provide insights into signal transmission. Some HK1 family proteins prove to be sensors for chemotaxis proteins or diguanylate cyclase receptors, implying a combinatorial molecular evolution.     

Mutations in the gene encoding C8orf38 block complex I assembly by inhibiting production of the mitochondria-encoded subunit ND1

The assembly of complex I (NADH-ubiquinone oxidoreductase) is a complicated process, requiring the integration of 45 subunits encoded by both nuclear and mitochondrial DNAs into a structure of approximately 1 MDa. A number of “assembly factors” that aid complex I biogenesis have recently been described, including C8orf38. This protein was identified as an assembly factor by its evolutionary conservation in organisms containing complex I and by a C8orf38 mutation in a patient presenting with Leigh syndrome and isolated complex I deficiency. In this report, we have undertaken the characterization of C8orf38 and its role in complex I assembly. Analysis of mitochondria from fibroblasts of a patient harboring a C8orf38 mutation showed almost undetectable levels of steady-state complex I and defective biogenesis of the mtDNA-encoded subunit ND1. Complementation with wild-type C8orf38 restored the levels of both ND1 and complex I, confirming the C8orf38 mutation as the cause of the complex I defect in the patient. In the absence of ND1 in patient cells, early- and mid-stage intermediate complexes were still formed; however, assembly of late-stage intermediates was impaired, indicating a convergence point in the assembly process. While C8orf38 appears to behave at a step in complex I biogenesis similar to that of the assembly factor C20orf7, complementation studies showed that both proteins are required for ND1 synthesis/stabilization. We conclude that C8orf38 is a crucial factor required for the translation and/or integration of ND1 into an early-stage assembly intermediate and that mutation of C8orf38 disrupts the initial stages of complex I biogenesis.     

1.25 A resolution crystal structures of human haemoglobin in the oxy, deoxy and carbonmonoxy forms

The most recent refinement of the crystallographic structure of oxyhaemoglobin (oxyHb) was completed in 1983, and differences between this real-space refined model and later R state models have been interpreted as evidence of crystallisation artefacts, or numerous sub-states. We have refined models of deoxy, oxy and carbonmonoxy Hb to 1.25 A resolution each, and compare them with other Hb structures. It is shown that the older structures reflect the software used in refinement, and many differences with newer structures are unlikely to be physiologically relevant. The improved accuracy of our models clarifies the disagreement between NMR and X-ray studies of oxyHb, the NMR experiments suggesting a hydrogen bond to exist between the distal histidine and oxygen ligand of both the alpha and beta-subunits. The high-resolution crystal structure also reveals a hydrogen bond in both subunit types, but with subtly different geometry which may explain the very different behaviour when this residue is mutated to glycine in alpha or beta globin. We also propose a new set of relatively fixed residues to act as a frame of reference; this set contains a similar number of atoms to the well-known "BGH" frame yet shows a much smaller rmsd value between R and T state models of HbA.     

Identification of four distinct serine proteinase inhibitors in rat skeletal muscle

The serine proteinase inhibitory capacity in the cytosolic fraction of rat skeletal muscle tissue is accounted for by several discrete inhibitory activities. Three of these activities are identical with the proteinase inhibitors α₁-proteinase inhibitor, rat proteinase inhibitor I and rat proteinase inhibitor I I respectively, which have been recently characterized as major serine proteinase inhibitors in rat serum (Kuehn, L., Rutschmann, M., Dahlmann, B. and Reinauer, H. (1984) Biochem. J. $\text{218}$, in the press). The other inhibitor molecule, having an M_r of about 15 000, appears to be an endogeneous inhibitor.     

The Structural Domains of Pseudomonas aeruginosa Phosphorylcholine Phosphatase Cooperate in Substrate Hydrolysis: 3D Structure and Enzymatic Mechanism

Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen. It colonizes different tissues by the utilization of diverse mechanisms. One of these may involve the breakdown of the host cell membrane through the sequential action of hemolytic phospholipase C and phosphorylcholine phosphatase (PchP). The action of hemolytic phospholipase C on phosphatidylcholine produces phosphorylcholine, which is hydrolyzed to choline (Cho) and inorganic phosphate by PchP. The available biochemical data on this enzyme demonstrate the involvement of two Cho-binding sites in the catalytic cycle and in enzyme regulation. The crystal structure of P. aeruginosa PchP has been determined. It folds into three structural domains. The first domain harbors all the residues involved in catalysis and is well conserved among the haloacid dehalogenase superfamily of proteins. The second domain is characteristic of PchP and is involved in the recognition of the Cho moiety of the substrate. The third domain stabilizes the relative position of the other two. Fortuitously, the crystal structure of PchP captures molecules of Bistris (2‐[bis(2‐hydroxyethyl)amino]‐2‐(hydroxymethyl)propane‐1,3‐diol) at the active site and at an additional site. This represents two catalytically relevant complexes with just one or two inhibitory Bistris molecules and provides the basis of the PchP function and regulation. Site‐directed mutagenesis along with biochemical experiments corroborates the structural observations and demonstrates the interplay between different sites for Cho recognition and inhibition. The structural comparison of PchP with other phosphatases of the haloacid dehalogenase family provides a three‐dimensional picture of the conserved catalytic cycle and the structural basis for the recognition of the diverse substrate molecules.     

Insights into the carboxyltransferase reaction of pyruvate carboxylase from the structures of bound product and intermediate analogs

Pyruvate carboxylase (PC) is a biotin-dependent enzyme that catalyzes the MgATP- and bicarbonate-dependent carboxylation of pyruvate to oxaloacetate, an important anaplerotic reaction in central metabolism. The carboxyltransferase (CT) domain of PC catalyzes the transfer of a carboxyl group from carboxybiotin to the accepting substrate, pyruvate. It has been hypothesized that the reactive enolpyruvate intermediate is stabilized through a bidentate interaction with the metal ion in the CT domain active site. Whereas bidentate ligands are commonly observed in enzymes catalyzing reactions proceeding through an enolpyruvate intermediate, no bidentate interaction has yet been observed in the CT domain of PC. Here, we report three X-ray crystal structures of the Rhizobium etli PC CT domain with the bound inhibitors oxalate, 3-hydroxypyruvate, and 3-bromopyruvate. Oxalate, a stereoelectronic mimic of the enolpyruvate intermediate, does not interact directly with the metal ion. Instead, oxalate is buried in a pocket formed by several positively charged amino acid residues and the metal ion. Furthermore, both 3-hydroxypyruvate and 3-bromopyruvate, analogs of the reaction product oxaloacetate, bind in an identical manner to oxalate suggesting that the substrate maintains its orientation in the active site throughout catalysis. Together, these structures indicate that the substrates, products and intermediates in the PC-catalyzed reaction are not oriented in the active site as previously assumed. The absence of a bidentate interaction with the active site metal appears to be a unique mechanistic feature among the small group of biotin-dependent enzymes that act on α-keto acid substrates.     

Complete predicted three-dimensional structure of the facilitator transmembrane protein and hepatitis C virus receptor CD81: conserved and variable structural domains in the tetraspanin superfamily

Tetraspanins are a superfamily of transmembrane proteins implicated in cellular development, motility, and activation through their interactions with a large range of proteins and with specific membrane microdomains. The complete three-dimensional structure of the tetraspanin CD81 has been predicted by molecular modeling and from the crystallographic structure of the EC2 large extracellular domain. Periodicity of sequence conservation, homology modeling, secondary structure prediction, and protein docking were used. The transmembrane domain appears organized as a four-stranded left-handed coiled coil directly connecting to two helices of the EC2. A smaller extracellular loop EC1 contains a small largely hydrophobic beta-strand that packs in a conserved hydrophobic groove of the EC2. The palmitoylable intracellular N-terminal segment forms an amphipathic membrane-parallel helix. Structural variability occurs mainly in an hypervariable subdomain of the EC2 and in intracellular regions. Therefore, the variable interaction selectivity of tetraspanins originates both from sequence variability within structurally conserved domains and from the occurrence of small structurally variable domains. In CD81 and other tetraspanins, the numerous membrane-exposed aromatic residues are asymmetrically clustered and protrude on one side of the transmembrane domain. This may represent a functional specialization of these two sides for interactions with cholesterol, proteins, or membrane microdomains.     

Sensitivity of Na+-coupled d-glucose uptake,Mg2+-ATPase and sucrase to perturbations of the fluidity of brush-border membrane vesicles induced by n-aliphatic alcohols

The aim of our work is to show the importance of the role of hydrophobic bonds in maintaining Mg²⁺-ATPase or sucrase activity and Na⁺-coupled d-glucose uptake normal for the brush border of rat enterocytes. The activity of the two enzymes and the d-glucose uptake were therefore measured under the action of $\text{n-}\text{aliphatic alcohols}$ and related to the fluidity determined by ESR. Three concentrations were used for the first eight alcohols, those of octanol being about 1500-times lower than those of methanol. For each alcohol the d-glucose uptake and the fluidity were linear functions of the logarithm of the concentration, the linear regressions being practically parallel and equidistant. The concentrations ($\text{C}$) of the eight alcohols inhibiting the d-glucose uptake by 80% were similar to those increasing the membrane fluidity by 3%. The linear relationship which existed in both cases between log 1 / $\text{C}$ and log $\text{P, P}$ being octanol / water partition coefficients of the alcohols, was evidence of great sensitivity to the hydrophobic effect of the alcohols. Only the first alcohols, however, produced any notable inhibition of Mg²⁺-ATPase and sucrase. Hydrophobic bonds are thus shown to have little influence in maintaining the activity of Mg²⁺-ATPase and sucrase, but they modulate the Na⁺-coupled d-glucose uptake.     

Crystal structures of native and inactivated cis-3-chloroacrylic acid dehalogenase: Implications for the catalytic and inactivation mechanisms

The isomeric mixture of cis- and trans-1,3-dichloropropene constitutes the active component of a widely used nematocide known as Telone II®. The mixture is processed by various soil bacteria to acetaldehyde through the 1,3-dichloropropene catabolic pathway. The pathway relies on an isomer-specific hydrolytic dehalogenation reaction catalyzed by cis- or trans-3-chloroacrylic acid dehalogenase, known respectively as cis-CaaD and CaaD. Previous sequence analysis and crystallographic studies of the native and covalently modified enzymes identified Pro-1, His-28, Arg-70, Arg-73, Tyr-103, and Glu-114 as key binding and catalytic residues in cis-CaaD. Mutagenesis of these residues confirmed their importance to the dehalogenation reaction. Crystal structures of the native enzyme (2.01 Å resolution) and the enzyme covalently modified at the Pro-1 nitrogen by 2-hydroxypropanoate (1.65 Å resolution) are reported here. Both structures are at a resolution higher than previously reported (2.75 Å and 2.1 Å resolution, respectively). The conformation of the covalent adduct is strikingly different from that previously reported due to its interaction with a 7-residue loop (Thr-32 to Leu-38). The participation of another active site residue, Arg-117, in catalysis and inactivation was also examined. The implications of the combined findings for the mechanisms of catalysis and inactivation are discussed.     

Effects of ethanol and 3,4,-dihydron-2,2-dimethyl-2h-1-benzopyran-6-butyric acid on the solubility of sickle hemoglobin.

The effect of 3,4-dihydro-2,2-dimethyl-2H-1 benzopyran-6-butyric acid (DBA) on the solubility of deoxy-Hb S was evaluated by measuring saturation concentration, c_sat. Plots of c_sat versus DBA concentration in the presence or absence of ethanol gave two parallel lines, indicative of the additive fashion in which ethanol and DBA increase the solubility of deoxy-Hb S. At a $\text{DBA}\text{Hb}$ molar ratio of 10:1, c_sat was increased 16%. Ethanol alone increased c_sat comparably, but at a much higher molar excess (200:1). DBA had no effect on the oxygenation parameters of Hb S. Complementary solubility studies using the salting-out method showed that DBA had no effect on deoxy-Hb S, but decreased the solubility of deoxy-Hb A, oxy-Hb A and oxy-Hb S. Hence, no correlation exists between the effect of DBA on the solubility of deoxy-Hb S measured as c_sat and that measured by the salting-out technique.     

Properties and structures of the influenza and HIV fusion peptides on lipid membranes: implications for a role in fusion

The fusion peptides of HIV and influenza virus are crucial for viral entry into a host cell. We report the membrane-perturbing and structural properties of fusion peptides from the HA fusion protein of influenza virus and the gp41 fusion protein of HIV. Our goals were to determine: 1), how fusion peptides alter structure within the bilayers of fusogenic and nonfusogenic lipid vesicles and 2), how fusion peptide structure is related to the ability to promote fusion. Fluorescent probes revealed that neither peptide had a significant effect on bilayer packing at the water-membrane interface, but both increased acyl chain order in both fusogenic and nonfusogenic vesicles. Both also reduced free volume within the bilayer as indicated by partitioning of a lipophilic fluorophore into membranes. These membrane ordering effects were smaller for the gp41 peptide than for the HA peptide at low peptide/lipid ratio, suggesting that the two peptides assume different structures on membranes. The influenza peptide was predominantly helical, and the gp41 peptide was predominantly antiparallel beta-sheet when membrane bound, however, the depths of penetration of Trps of both peptides into neutral membranes were similar and independent of membrane composition. We previously demonstrated: 1), the abilities of both peptides to promote fusion but not initial intermediate formation during PEG-mediated fusion and 2), the ability of hexadecane to compete with this effect of the fusion peptides. Taken together, our current and past results suggest a hypothesis for a common mechanism by which these two viral fusion peptides promote fusion.     

Structural and Functional Analyses of β-Glucosidase 3B from Thermotoga neapolitana: A Thermostable Three-Domain Representative of Glycoside Hydrolase 3

Based on sequence and phylogenetic analyses, glycoside hydrolase (GH) family 3 can be divided into several clusters that differ in the length of their primary sequences. However, structural data on representatives of GH3 are still scarce, since only three of their structures are known and only one of them has been thoroughly characterized—that of an exohydrolase from barley. To allow a deeper structural understanding of the GH3 family, we have determined the crystal structure of the thermostable β-glucosidase from Thermotoga neapolitana, which has potentially important applications in environmentally friendly industrial biosynthesis at a resolution of 2.05 Å. Selected active-site mutants have been characterized kinetically, and the structure of the mutant D242A is presented at 2.1 Å resolution. Bgl3B from Th. neapolitana is the first example of a GH3 glucosidase with a three-domain structure. It is composed of an (α/β)₈ domain similar to a triose phosphate isomerase barrel, a five-stranded α/β sandwich domain (both of which are important for active-site organization), and a C-terminal fibronectin type III domain of unknown function. Remarkably, the direction of the second β-strand of the triose phosphate isomerase barrel domain is reversed, which has implications for the active-site shape. The active site, at the interface of domains 1 and 2, is much more open to solvent than the corresponding site in the structurally homologous enzyme from barley, and only the − 1 site is well defined. The structures, in combination with kinetic studies of active-site variants, allow the identification of essential catalytic residues (the nucleophile D242 and the acid/base E458), as well as other residues at the − 1 subsite, including D58 and W243, which, by mutagenesis, are shown to be important for substrate accommodation/interaction. The position of the fibronectin type III domain excludes a direct participation of this domain in the recognition of small substrates, although it may be involved in the anchoring of the enzyme on large polymeric substrates and in thermostability.     

Removal of both Ycf48 and Psb27 in Synechocystis sp. PCC 6803 disrupts Photosystem II assembly and alters QA oxidation in the mature complex

The Photosystem II (PS II) assembly factors Psb27 and Ycf48 are transiently associated with PS II during its biogenesis and repair pathways. We investigated the function of these proteins by constructing knockout mutants in Synechocystis sp. PCC 6803. In ΔYcf48 cells, PS II electron transfer and stable oxygen evolution were perturbed. Additionally, Psb27 was required for photoautotrophic growth of cells lacking Ycf48 and assembly beyond the RC47 assembly complex in ΔYcf48:ΔPsb27 cells was impeded. Our results suggest the RC47 complex formed in ΔYcf48 cells is defective and that this deficiency is exacerbated if CP43 binds in the absence of Psb27.     

The Interaction between Thermodynamic Stability and Buried Free Cysteines in Regulating the Functional Half-Life of Fibroblast Growth Factor-1

Protein biopharmaceuticals are an important and growing area of human therapeutics; however, the intrinsic property of proteins to adopt alternative conformations (such as during protein unfolding and aggregation) presents numerous challenges, limiting their effective application as biopharmaceuticals. Using fibroblast growth factor-1 as model system, we describe a cooperative interaction between the intrinsic property of thermostability and the reactivity of buried free-cysteine residues that can substantially modulate protein functional half-life. A mutational strategy that combines elimination of buried free cysteines and secondary mutations that enhance thermostability to achieve a substantial gain in functional half-life is described. Furthermore, the implementation of this design strategy utilizing stabilizing mutations within the core region resulted in a mutant protein that is essentially indistinguishable from wild type as regard protein surface and solvent structure, thus minimizing the immunogenic potential of the mutations. This design strategy should be generally applicable to soluble globular proteins containing buried free-cysteine residues.