Extracellular matrix fibronectin initiates endothelium-dependent arteriolar dilatation via the heparin-binding, matricryptic RWRPK sequence of the first type III repeat of fibrillar fibronectin.

The local arteriolar dilatation produced by contraction of skeletal muscle is dependent upon multiple signalling mechanisms. In addition to the many metabolic signals that mediate this vasodilatation, we show here that the extracellular matrix protein fibronectin also contributes to the response. This vasodilatory signal requires the heparin-binding matricryptic RWRPK sequence in the first type III repeat of fibrillar fibronectin.

Chimeric fibronectin matrix mimetic as a functional growth- and migration-promoting adhesive substrate.

Therapeutic protein engineering combines genetic, biochemical, and functional information to improve existing proteins or invent new protein technologies. Using these principles, we developed an approach to deliver extracellular matrix (ECM) fibronectin-specific signals to cells. Fibronectin matrix assembly is a cell-dependent process that converts the inactive, soluble form of fibronectin into biologically-active ECM fibrils.

Assembly of the stator in Escherichia coli ATP synthase. Complexation of alpha subunit with other F1 subunits is prerequisite for delta subunit binding to the N-terminal region of alpha.

Alpha subunit of Escherichia coli ATP synthase was expressed with a C-terminal 6-His tag and purified. Pure alpha was monomeric, was competent in nucleotide binding, and had normal N-terminal sequence. In F1 subunit dissociation/reassociation experiments it supported full reconstitution of ATPase, and reassociated complexes were able to bind to F1-depleted membranes with restoration of ATP-driven proton pumping.

Analysis of sequence determinants of F1Fo-ATP synthase in the N-terminal region of alpha subunit for binding of delta subunit.

The stator in F(1)F(o)-ATP synthase resists strain generated by rotor torque. In Escherichia coli, the b(2)delta subunit complex comprises the stator, bound to subunit a in F(o) and to the alpha(3)beta(3) hexagon of F(1). Previous work has shown that N-terminal residues of alpha subunit are involved in binding delta.

Quantitative determination of direct binding of b subunit to F1 in Escherichia coli F1F0-ATP synthase.

The stator in F(1)F(0)-ATP synthase resists strain generated by rotor torque. In Escherichia coli, the b(2)delta subunit complex comprises the stator, bound to subunit a in F(0) and to the alpha(3)beta(3) hexagon of F(1). To quantitatively characterize binding of b subunit to the F(1) alpha(3)beta(3) hexagon, we developed fluorimetric assays in which wild-type F(1), or F(1) enzymes containing introduced Trp residues, were titrated with a soluble portion of the b subunit (b(ST34-156)).

F1F0-ATP synthase. Binding of delta subunit to a 22-residue peptide mimicking the N-terminal region of alpha subunit.

The stator in F(1)F(0)-ATP synthase resists strain generated by rotor torque. In Escherichia coli the b(2)delta subunit complex comprises the stator, bound to subunit a in F(0) and to alpha(3)beta(3) hexagon of F(1). Proteolysis and cross-linking had suggested that N-terminal residues of alpha subunit are involved in binding delta.

Identification of the F1-binding surface on the delta-subunit of ATP synthase.

The stator function in ATP synthase was studied by a combined mutagenesis and fluorescence approach. Specifically, binding of delta-subunit to delta-depleted F(1) was studied. A plausible binding surface on delta-subunit was identified from conservation of amino acid sequence and the high resolution NMR structure.

Quantitative determination of binding affinity of delta-subunit in Escherichia coli F1-ATPase: effects of mutation, Mg2+, and pH on Kd.

To study the stator function in ATP synthase, a fluorimetric assay has been devised for quantitative determination of binding affinity of delta-subunit to Escherichia coli F(1)-ATPase. The signal used is that of the natural tryptophan at residue delta28, which is enhanced by 50% upon binding of delta-subunit to alpha(3)beta(3)gammaepsilon complex. K(d) for delta binding is 1.

Cysteine-reactive fluorescence probes of catalytic sites of ATP synthase.

We searched for new fluorescent probes of catalytic-site nucleotide binding in F(1)F(0)-ATP synthase by introducing Cys mutations at positions in or close to catalytic sites and then reacting Cys-mutant F(1) with thiol-reactive fluorescent probes. Four suitable mutant/probe combinations were identified. beta F410C labeled by 7-fluorobenz-2-oxa-1,3-diazole-4-sulfonamide (ABD-F) gave very large signal changes in response to nucleotide, allowing facile measurement of fluorescence and nucleotide-binding parameters, not only in F(1) but also in F(1)F(0).

Purification and characterization of N-glycosylation mutant mouse and human P-glycoproteins expressed in Pichia pastoris cells.

P-glycoprotein confers multidrug resistance in mammalian cells and basic structure-function studies of it are germane to anti-cancer and anti-AIDS therapy. Pure, detergent-soluble mouse MDR3 and human MDR1 P-glycoproteins have recently been obtained in sufficient quantity for high-resolution structure analysis after expression in Pichia pastoris (N. Lerner-Marmarosh et al.

Cysteines 431 and 1074 are responsible for inhibitory disulfide cross-linking between the two nucleotide-binding sites in human P-glycoprotein.

Human wild-type and Cys-less P-glycoproteins were expressed in Pichia pastoris and purified in high yield in detergent-soluble form. Both ran on SDS gels as a single 140-kDa band in the presence of reducing agent and showed strong verapamil-stimulated ATPase activity in the presence of added lipid. The wild type showed spontaneous formation of higher molecular mass species in the absence of reducing agent, and its ATPase was activated by dithiothreitol.

Investigation of the role of glutamine-471 and glutamine-1114 in the two catalytic sites of P-glycoprotein.

P-glycoprotein, also known as multidrug resistance protein, pumps drugs out of cells using ATP hydrolysis as the energy source. Glutamine-471 and the corresponding glutamine-1114 in the two catalytic sites of P-glycoprotein are conserved in ABC transporters. X-ray structures show that they lie close to the bound nucleotide.

Conserved walker A Ser residues in the catalytic sites of P-glycoprotein are critical for catalysis and involved primarily at the transition state step.

P-glycoprotein mutants S430A/T and S1073A/T, affecting conserved Walker A Ser residues, were characterized to elucidate molecular roles of the Ser and functioning of the two P-glycoprotein catalytic sites. Results showed the Ser-OH is critical for MgATPase activity and formation of the normal transition state, although not for initial MgATP binding. Mutation to Ala in either catalytic site abolished MgATPase and transition state formation in both sites, whereas Thr mutants had similar MgATPase to wild-type.

Importance of F1-ATPase residue alpha-Arg-376 for catalytic transition state stabilization.

The functional role of essential residue alpha-Arg-376 in the catalytic site of F1-ATPase was studied. The mutants alpha R376C, alpha R376Q, and alpha R376K were constructed, and combined with the mutation beta Y331W, to investigate catalytic site nucleotide-binding parameters, and to assess catalytic transition state formation by measurement of MgADP-fluoroaluminate binding. Each mutation caused large impairment of ATP synthesis and hydrolysis.

Tryptophan substitutions surrounding the nucleotide in catalytic sites of F1-ATPase.

Novel tryptophan substitutions, surrounding the nucleotide bound in catalytic sites, were introduced into Escherichia coli F1-ATPase. The mutant enzymes were purified and studied by fluorescence spectroscopy. One cluster of Trp substitutions, consisting of beta-Trp-404, beta-Trp-410, beta-Asp-158 (lining the adenine-binding pocket), and beta-Trp-153 (close to the alpha/beta-phosphates), showed the same fluorescence responses to MgADP, MgAMPPNP, and MgATP and the same nucleotide binding pattern with MgADP and MgAMPPNP, with one site of higher and two sites of lower affinity.

Mg2+ coordination in catalytic sites of F1-ATPase.

Coordination of the Mg2+ ion in Mg-nucleotide substrates by amino acid residue side chains in the catalytic site of Escherichia coli F1-ATPase was investigated. From the X-ray structure of the mitochondrial enzyme [Abrahams, J. P.

F1-ATPase, roles of three catalytic site residues.

Three critical residues, beta-Lys-155, beta-Asp-242, and beta-Glu-181, situated close to the gamma-phosphate of MgATP in F1-ATPase catalytic sites, were investigated. The mutations betaK155Q, betaD242N, and betaE181Q were each combined with the betaY331W mutation; the fluorescence signal of beta-Trp-331 was used to determine MgATP, MgADP, ATP, and ADP binding parameters for the three catalytic sites of the enzyme. The quantitative contribution of side chains to binding energy at all three catalytic sites was calculated.

Mutagenesis and reversion analysis of residue Met-209 of the beta-subunit of Escherichia coli ATP synthase.

Residue beta-Met-209 is conserved in all known F1-ATPase sequences, and the mutation beta M209I in Escherichia coli causes profound inhibition of ATP synthesis and hydrolysis. Based on the properties of this mutant it had previously been proposed that residue beta-209 lies close to the site of catalysis. Two approaches were used to study this residue further.

alpha-Aspartate 261 is a key residue in noncatalytic sites of Escherichia coli F1-ATPase.

X-ray structure analysis of the noncatalytic sites of F1-ATPase revealed that residue alpha-Asp261 lies close to the Mg of bound Mg-5'-adenylyl-beta,gamma-imidodiphosphate. Here, the mutation alpha D261N was generated in Escherichia coli and combined with the alpha R365W mutation, allowing nucleotide binding at F1 noncatalytic sites to be specifically monitored by tryptophan fluorescence spectroscopy. Purified alpha D261N/alpha R365W F1-ATPase showed catalytic activity similar to wild-type.

Cooperativity and stoichiometry of substrate binding to the catalytic sites of Escherichia coli F1-ATPase. Effects of magnesium, inhibitors, and mutation.

The fluorescence of residue Trp beta 331 in beta Y331W mutant Escherichia coli F1-ATPase was used as reporter probe to investigate the effects of magnesium ions, inhibitors, and mutation on substrate (ATP) binding stoichiometry and cooperativity. It was found that Mg2+ is required for catalytic site binding cooperativity. In the absence of magnesium, ATP bound to three independent catalytic sites, each with Kd = 76 microM.

Tryptophan fluorescence provides a direct probe of nucleotide binding in the noncatalytic sites of Escherichia coli F1-ATPase.

Tryptophan fluorescence was investigated as a tool to study the noncatalytic nucleotide-binding sites of Escherichia coli F1-ATPase. Site-directed mutagenesis, affinity labeling, and lin-benzo-ATP binding studies had shown that residues alpha R365 and beta Y354 are located close to the base moiety of bound nucleotide; here, we mutagenized each to tryptophan. The new tryptophans gave a fluorescence signal indicating an environment of high (alpha W365) or intermediate (beta W354) polarity in unoccupied sites.

Tryptophan-free Escherichia coli F1-ATPase.

We have engineered a mutant form of Escherichia coli F1-ATPase which is tryptophan-free and contains five mutations, namely delta W28L/alpha W513F/gamma W108Y/gamma W206Y/beta W107F. A strain carrying all five mutations grew normally by oxidative phosphorylation. Purified mutant F1-ATPase showed Vmax and Km both 65% higher than wild-type, resulting in kcat/Km the same as wild-type.

Specific placement of tryptophan in the catalytic sites of Escherichia coli F1-ATPase provides a direct probe of nucleotide binding: maximal ATP hydrolysis occurs with three sites occupied.

Residue beta Y331 of Escherichia coli F1-ATPase is known from previous affinity labeling, mutagenesis, and lin-benzo-ADP binding experiments to interact directly with the adenine moiety of substrates bound in catalytic sites. Here we mutagenized beta Y331 to tryptophan. Mutant cells grew well on succinate or limiting glucose; purified mutant F1 had kappa cat/Km and lin-benzo-ADP binding characteristics similar to wild type.

Lysine 155 in beta-subunit is a catalytic residue of Escherichia coli F1 ATPase.

The "homology A" ("glycine-rich" or "P-loop") consensus sequence occurs in the catalytic sites of F1F0 ATP synthase enzymes. The conserved lysine of this motif is beta-subunit Lys-155 in Escherichia coli F1. The role of this lysine in binding and catalysis at the high affinity ATP binding site was studied with the mutants beta K155Q and beta K155E by measuring the rates of ATP binding/release, ATP hydrolysis/synthesis, and Pi release as a function of pH varied from 5.

Combined application of site-directed mutagenesis, 2-azido-ATP labeling, and lin-benzo-ATP binding to study the noncatalytic sites of Escherichia coli F1-ATPase.

Noncatalytic nucleotide sites of Escherichia coli F1-ATPase were studied by site-directed mutagenesis, covalent photolabeling with 2-azido-ATP, and lin-benzo-ATP binding. In wild-type, 89% of 2-azido-ATP label was bound to beta-subunit, whereas in the beta Y354F mutant, 95% of the label was bound to alpha-subunit. In the alpha R365Y mutant, label was seen on both alpha (38%) and beta (62%); whereas in the alpha R365F mutant, 93% was on beta.