Interaction of the Hsp70 molecular chaperone, DnaK, with its cochaperone DnaJ

Chaperones of the Hsp70 family bind to unfolded or partially folded polypeptides to facilitate many cellular processes. ATP hydrolysis and substrate binding, the two key molecular activities of this chaperone, are modulated by the cochaperone DnaJ. By using both genetic and biochemical approaches, we provide evidence that DnaJ binds to at least two sites on the Escherichia coli Hsp70 family member DnaK: under the ATPase domain in a cleft between its two subdomains and at or near the pocket of substrate binding. The lower cleft of the ATPase domain is defined as a binding pocket for the J-domain because (i) a DnaK mutation located in this cleft (R167H) is an allele-specific suppressor of the binding defect of the DnaJ mutation, D35N and (ii) alanine substitution of two residues close to R167 in the crystal structure, N170A and T173A, significantly decrease DnaJ binding. A second binding determinant is likely to be in the substrate-binding domain because some DnaK mutations in the vicinity of the substrate-binding pocket are defective in either the affinity (G400D, G539D) or rate (D526N) of both peptide and DnaJ binding to DnaK. Binding of DnaJ may propagate conformational changes to the nearby ATPase catalytic center and substrate-binding sites as well as facilitate communication between these two domains to alter the molecular properties of Hsp70.

Force-mediated kinetics of single P-selectin/ligand complexes observed by atomic force microscopy

Leukocytes roll along the endothelium of postcapillary venules in response to inflammatory signals. Rolling under the hydrodynamic drag forces of blood flow is mediated by the interaction between selectins and their ligands across the leukocyte and endothelial cell surfaces. Here we present force-spectroscopy experiments on single complexes of P-selectin and P-selectin glycoprotein ligand-1 by atomic force microscopy to determine the intrinsic molecular properties of this dynamic adhesion process. By modeling intermolecular and intramolecular forces as well as the adhesion probability in atomic force microscopy experiments we gain information on rupture forces, elasticity, and kinetics of the P-selectin/P-selectin glycoprotein ligand-1 interaction. The complexes are able to withstand forces up to 165 pN and show a chain-like elasticity with a molecular spring constant of 5.3 pN nm−1 and a persistence length of 0.35 nm. The dissociation constant (off-rate) varies over three orders of magnitude from 0.02 s−1 under zero force up to 15 s−1 under external applied forces. Rupture force and lifetime of the complexes are not constant, but directly depend on the applied force per unit time, which is a product of the intrinsic molecular elasticity and the external pulling velocity. The high strength of binding combined with force-dependent rate constants and high molecular elasticity are tailored to support physiological leukocyte rolling.

Purification and characterization of acetone carboxylase from Xanthobacter strain Py2

Acetone metabolism in the aerobic bacterium Xanthobacter strain Py2 proceeds by a carboxylation reaction forming acetoacetate as the first detectable product. In this study, acetone carboxylase, the enzyme catalyzing this reaction, has been purified to homogeneity and characterized. Acetone carboxylase was comprised of three polypeptides with molecular weights of 85,300, 78,300, and 19,600 arranged in an α2β2γ2 quaternary structure. The carboxylation of acetone was coupled to the hydrolysis of ATP and formation of 1 mol AMP and 2 mol inorganic phosphate per mol acetoacetate formed. ADP was also formed during the course of acetone consumption, but only accumulated at low, substoichiometric levels (≈10% yield) relative to acetoacetate. Inorganic pyrophosphate could not be detected as an intermediate or product of acetone carboxylation. In the absence of CO2, acetone carboxylase catalyzed the acetone-dependent hydrolysis of ATP to form both ADP and AMP, with ADP accumulating to higher levels than AMP during the course of the assays. Acetone carboxylase did not have inorganic pyrophosphatase activity. Acetone carboxylase exhibited a Vmax for acetone carboxylation of 0.225 μmol acetoacetate formed min−1⋅mg−1 at 30°C and pH 7.6 and apparent Km values of 7.80 μM (acetone), 122 μM (ATP), and 4.17 mM (CO2 plus bicarbonate). These studies reveal molecular properties of the first bacterial acetone-metabolizing enzyme to be isolated and suggest a novel mechanism of acetone carboxylation coupled to ATP hydrolysis and AMP and inorganic phosphate formation.

The vibrational energy flow transition in organic molecules: Theory meets experiment

Most large dynamical systems are thought to have ergodic dynamics, whereas small systems may not have free interchange of energy between degrees of freedom. This assumption is made in many areas of chemistry and physics, ranging from nuclei to reacting molecules and on to quantum dots. We examine the transition to facile vibrational energy flow in a large set of organic molecules as molecular size is increased. Both analytical and computational results based on local random matrix models describe the transition to unrestricted vibrational energy flow in these molecules. In particular, the models connect the number of states participating in intramolecular energy flow to simple molecular properties such as the molecular size and the distribution of vibrational frequencies. The transition itself is governed by a local anharmonic coupling strength and a local state density. The theoretical results for the transition characteristics compare well with those implied by experimental measurements using IR fluorescence spectroscopy of dilution factors reported by Stewart and McDonald [Stewart, G. M. & McDonald, J. D. (1983) J. Chem. Phys. 78, 3907–3915].

Mobilization of MHC class I molecules from late endosomes to the cell surface following activation of CD34-derived human Langerhans cells

Langerhans cells are a subset of dendritic cells (DCs) found in the human epidermis with unique morphological and molecular properties that enable their function as “sentinels” of the immune system. DCs are pivotal in the initiation and regulation of primary MHC class I restricted T lymphocyte immune responses and are able to present both endogenous and exogenous antigen onto class I molecules. Here, we study the MHC class I presentation pathway following activation of immature, CD34-derived human Langerhans cells by lipopolysaccharide (LPS). LPS induces an increase in all components of the MHC class I pathway including the transporter for antigen presentation (TAP), tapasin and ERp57, and the immunoproteasome subunits LMP2 and LMP7. Moreover, in CD34-derived Langerhans cells, the rapid increase in expression of MHC class I molecules seen at the cell surface following LPS activation is because of mobilization of MHC class I molecules from HLA-DM positive endosomal compartments, a pathway not seen in monocyte-derived DCs. Mobilization of class I from this compartment is primaquine sensitive and brefeldin A insensitive. These data demonstrate the regulation of the class I pathway in concert with the maturation of the CD34-derived Langerhans cells and suggest potential sites for antigen loading of class I proteins.

Direct molecular level measurements of the electrostatic properties of a protein surface

In this work, we used direct measurements with the surface force apparatus to determine the pH-dependent electrostatic charge density of a single binding face of streptavidin. Mean field calculations have been used with considerable success to model electrostatic potential fields near protein surfaces, but these models and their inherent assumptions have not been tested directly at the molecular level. Using the force apparatus and immobilized, oriented monolayers of streptavidin, we measured a pI of 5–5.5 for the biotin-binding face of the protein. This differs from the pI of 6.3 for the soluble protein and confirms that we probed the local electrostatic features of the macromolecule. With finite difference solutions of the linearized Poisson–Boltzmann equation, we then calculated the pH-dependent charge densities adjacent to the same face of the protein. These calculated values agreed quantitatively with those obtained by direct force measurements. Although our study focuses on the pH-dependence of surface electrostatics, this direct approach to probing the electrostatic features of proteins is applicable to investigations of any perturbations that alter the charge distribution of the surfaces of immobilized molecules.

A new high molecular weight immunoglobulin class from the carcharhine shark: implications for the properties of the primordial immunoglobulin.

All immunoglobulins and T-cell receptors throughout phylogeny share regions of highly conserved amino acid sequence. To identify possible primitive immunoglobulins and immunoglobulin-like molecules, we utilized 3' RACE (rapid amplification of cDNA ends) and a highly conserved constant region consensus amino acid sequence to isolate a new immunoglobulin class from the sandbar shark Carcharhinus plumbeus. The immunoglobulin, termed IgW, in its secreted form consists of 782 amino acids and is expressed in both the thymus and the spleen. The molecule overall most closely resembles mu chains of the skate and human and a new putative antigen binding molecule isolated from the nurse shark (NAR). The full-length IgW chain has a variable region resembling human and shark heavy-chain (VH) sequences and a novel joining segment containing the WGXGT motif characteristic of H chains. However, unlike any other H-chain-type molecule, it contains six constant (C) domains. The first C domain contains the cysteine residue characteristic of C mu1 that would allow dimerization with a light (L) chain. The fourth and sixth domains also contain comparable cysteines that would enable dimerization with other H chains or homodimerization. Comparison of the sequences of IgW V and C domains shows homology greater than that found in comparisons among VH and C mu or VL, or CL thereby suggesting that IgW may retain features of the primordial immunoglobulin in evolution.

Human fatty acid synthase: properties and molecular cloning.

Fatty acid synthase (FAS; EC 2.3.1.85) was purified to near homogeneity from a human hepatoma cell line, HepG2. The HepG2 FAS has a specific activity of 600 nmol of NADPH oxidized per min per mg, which is about half that of chicken liver FAS. All the partial activities of human FAS are comparable to those of other animal FASs, except for the beta-ketoacyl synthase, whose significantly lower activity is attributable to the low 4'-phosphopantetheine content of HepG2 FAS. We cloned the human brain FAS cDNA. The cDNA sequence has an open reading frame of 7512 bp that encodes 2504 amino acids (M(r), 272,516). The amino acid sequence of the human FAS has 79% and 63% identity, respectively, with the sequences of the rat and chicken enzymes. Northern analysis revealed that human FAS mRNA was about 9.3 kb in size and that its level varied among human tissues, with brain, lung, and liver tissues showing prominent expression. The nucleotide sequence of a segment of the HepG2 FAS cDNA (bases 2327-3964) was identical to that of the cDNA from normal human liver and brain tissues, except for a 53-bp sequence (bases 3892-3944) that does not alter the reading frame. This altered sequence is also present in HepG2 genomic DNA. The origin and significance of this sequence variance in the HepG2 FAS gene are unclear, but the variance apparently does not contribute to the lower activity of HepG2 FAS.