Novobiocin is an antibiotic which binds to a 24 kDa fragment from the B subunit of DNA gyrase. Naturally occurring resistance arises from mutation of Arg-136 which hydrogen bonds to the coumarin ring of novobiocin. We have applied calorimetry to characterize the binding of novobiocin to wild-type and R136H mutant 24 kDa fragments. Upon mutation, the Kd increases from 32 to 1200 nM at 300 K. The enthalpy of binding is more favorable for the mutant (DeltaH degrees shifts from -12.1 to -17.5 kcal/mol), and the entropy of binding is much less favorable (TDeltaS degrees changes from -1.8 to -9.4 kcal/mol). Both of these changes are in the direction opposite to that expected if the loss of the Arg residue reduces hydrogen bonding. The change in heat capacity at constant pressure upon binding (DeltaCp) shifts from -295 to -454 cal mol-1 K-1. We also report the crystal structure, at 2.3 A resolution, of a complex between the R136H 24 kDa fragment and novobiocin. Although the change in DeltaCp often would be interpreted as reflecting increased burial of hydrophobic surface on binding, this structure reveals a small decrease. Furthermore, an ordered water molecule is sequestered into the volume vacated by removal of the guanidinium group. There are large discrepancies when the measured thermodynamic parameters are compared to those estimated from the structural data using empirical relationships. These differences seem to arise from the effects of sequestering ordered water molecules upon complexation. The water-mediated hydrogen bonds linking novobiocin to the mutant protein make a favorable enthalpic contribution, whereas the immobilization of the water leads to an entropic cost and a reduction in the heat capacity of the system. Such a negative contribution to DeltaCp, DeltaH degrees , and TDeltaS degrees appears to be a general property of water molecules that are sequestered when ligands bind to proteins.

Triclosan is used widely as an antibacterial agent in dermatological products, mouthwashes, and toothpastes. Recent studies imply that antibacterial activity results from binding to enoyl (acyl carrier protein) reductase (EACPR, EC 1.3.1.9). We first recognized the ability of triclosan to inhibit EACPR from Escherichia coli in a high throughput screen where the enzyme and test compound were preincubated with NAD(+), which is a product of the reaction. The concentration of triclosan required for 50% inhibition approximates to 50% of the enzyme concentration, indicating that the free compound is depleted by binding to EACPR. With no preincubation or added NAD(+), the degree of inhibition by 150 nM triclosan increases gradually over several minutes. The onset of inhibition is more rapid when NAD(+) is added. Gel filtration and mass spectrometry show that inhibition by triclosan is reversible. Steady-state assays were designed to avoid depletion of free inhibitor and changes in the degree of inhibition. The results suggest that triclosan binds to E-NAD(+) complex, with a dissociation constant around 20-40 pM. Triclosan follows competitive kinetics with respect to NADH, giving an inhibition constant of 38 pM at zero NADH and saturating NAD(+). Uncompetitive kinetics are observed when NAD(+) is varied, giving an inhibition constant of 22 pM at saturating NAD(+). By following regain of catalytic activity after dilution of EACPR that had been preincubated with triclosan and NAD(+), the rate constant for dissociation of the inhibitor (k(off)) is measured as 1.9 x 10(-4) s(-1). The association rate constant (k(on)) is estimated as 2.6 x 10(7) s(-1) M(-1) by monitoring the onset of inhibition during assays started by addition of EACPR. As expected, the ratio k(off)/k(on) = 7.1 pM is similar to the inhibition constants from the steady-state studies. The crystal structure of E. coli EACPR in a complex with coenzyme and triclosan has been determined at 1.9 A resolution, showing that this compound binds in a similar site to the diazaborine inhibitors. The high affinity of triclosan appears to be due to structural similarity to a tightly bound intermediate in catalysis.

No abstract

No abstract

Inhibition of p38alpha MAP kinase is a potential approach for the treatment of inflammatory disorders. MKK6-dependent phosphorylation on the activation loop of p38alpha increases its catalytic activity and affinity for ATP. An inhibitor, BIRB796, binds at a site used by the purine moiety of ATP and extends into a "selectivity pocket", which is not used by ATP. It displaces the Asp168-Phe169-Gly170 motif at the start of the activation loop, promoting a "DFG-out" conformation. Some other inhibitors bind only in the purine site, with p38alpha remaining in a "DFG-in" conformation. We now demonstrate that selectivity pocket compounds prevent MKK6-dependent activation of p38alpha in addition to inhibiting catalysis by activated p38alpha. Inhibitors using only the purine site do not prevent MKK6-dependent activation. We present kinetic analyses of seven inhibitors, whose crystal structures as complexes with p38alpha have been determined. This work includes four new crystal structures and a novel assay to measure K(d) for nonactivated p38alpha. Selectivity pocket compounds associate with p38alpha over 30-fold more slowly than purine site compounds, apparently due to low abundance of the DFG-out conformation. At concentrations that inhibit cellular production of an inflammatory cytokine, TNFalpha, selectivity pocket compounds decrease levels of phosphorylated p38alpha and beta. Stabilization of a DFG-out conformation appears to interfere with recognition of p38alpha as a substrate by MKK6. ATP competes less effectively for prevention of activation than for inhibition of catalysis. By binding to a different conformation of the enzyme, compounds that prevent activation offer an alternative approach to modulation of p38alpha.