Crystal structures of substrate binding to Bacillus subtilis holo-(acyl carrier protein) synthase reveal a novel trimeric arrangement of molecules resulting in three active sites.

BACKGROUND: Holo-(acyl carrier protein) synthase (AcpS), a member of the phosphopantetheinyl transferase superfamily, plays a crucial role in the functional activation of acyl carrier protein (ACP) in the fatty acid biosynthesis pathway. AcpS catalyzes the attachment of the 4'-phosphopantetheinyl moiety of coenzyme A (CoA) to the sidechain of a conserved serine residue on apo-ACP. RESULTS: We describe here the first crystal structure of a type II ACP from Bacillus subtilis in complex with its activator AcpS at 2.3 A. We also have determined the structures of AcpS alone (at 1.8 A) and AcpS in complex with CoA (at 1.5 A). These structures reveal that AcpS exists as a trimer. A catalytic center is located at each of the solvent-exposed interfaces between AcpS molecules. Site-directed mutagenesis studies confirm the importance of trimer formation in AcpS activity. CONCLUSIONS: The active site in AcpS is only formed when two AcpS molecules dimerize. The addition of a third molecule allows for the formation of two additional active sites and also permits a large hydrophobic surface from each molecule of AcpS to be buried in the trimer. The mutations Ile5-->Arg, Gln113-->Glu and Gln113-->Arg show that AcpS is inactive when unable to form a trimer. The co-crystal structures of AcpS-CoA and AcpS-ACP allow us to propose a catalytic mechanism for this class of 4'-phosphopantetheinyl transferases.

Crystal structures of a mutant (betaK87T) tryptophan synthase alpha2beta2 complex with ligands bound to the active sites of the alpha- and beta-subunits reveal ligand-induced conformational changes.

Three-dimensional structures are reported for a mutant (betaK87T) tryptophan synthase alpha2beta2 complex with either the substrate L-serine (betaK87T-Ser) or product L-tryptophan (betaK87T-Trp) at the active site of the beta-subunit, in which both amino acids form external aldimines with the coenzyme, pyridoxal phosphate. We also present structures with L-serine bound to the beta site and either alpha-glycerol 3-phosphate (betaK87T-Ser-GP) or indole-3-propanol phosphate (betaK87T-Ser-IPP) bound to the active site of the alpha-subunit. The results further identify the substrate and product binding sites in each subunit and provide insight into conformational changes that occur upon formation of these complexes. The two structures having ligands at the active sites of both alpha- and beta-subunits reveal an important new feature, the ordering of alpha-subunit loop 6 (residues 179-187). Closure of loop 6 isolates the active site of the alpha-subunit from solvent and results in interaction between alphaThr183 and the catalytic residue alphaAsp60. Other conformational differences between the wild type and these two mutant structures include a rigid-body rotation of the alpha-subunit of approximately 5 degrees relative to the beta-subunit and large movements of part of the beta-subunit (residues 93-189) toward the rest of the beta-subunit. Much smaller differences are observed in the betaK87T-Ser structure. Remarkably, binding of tryptophan to the beta active site results in conformational changes very similar to those observed in the betaK87T-Ser-GP and betaK87T-Ser-IPP structures, with exception of the disordered alpha-subunit loop 6. These large-scale changes, the closure of loop 6, and the movements of a small number of side chains in the alpha-beta interaction site provide a structural base for interpreting the allosteric properties of tryptophan synthase.

Exchange of K+ or Cs+ for Na+ induces local and long-range changes in the three-dimensional structure of the tryptophan synthase alpha2beta2 complex.

Monovalent cations activate the pyridoxal phosphate-dependent reactions of tryptophan synthase and affect intersubunit communication in the alpha2beta2 complex. We report refined crystal structures of the tryptophan synthase alpha2beta2 complex from Salmonella typhimurium in the presence of K+ at 2.0 angstrom and of Cs+ at 2.3 angstrom. Comparison of these structures with the recently refined structure in the presence of Na+ shows that each monovalent cation binds at approximately the same position about 8 angstrom from the phosphate of pyridoxal phosphate. Na+ and K+ are coordinated to the carbonyl oxygens of beta Phe-306, beta Ser-308, and beta Gly-232 and to two or one water molecule, respectively. Cs+ is coordinated to the carbonyl oxygens of beta Phe-306, beta Ser-308, beta Gly-232, beta Val-231, beta Gly-268 and beta Leu-304. A second binding site for Cs+ is located in the beta/beta interface on the 2-fold axis with four carbonyl oxygens in the coordination sphere. In addition to local changes in structure close to the cation binding site, a number of long-range changes are observed. The K+ and Cs+ structures differ from the Na+ structure with respect to the positions of beta Asp-305, beta Lys-167, and alpha Asp-56. One unexpected result of this investigation is the movement of the side chains of beta Phe-280 and beta Tyr-279 from a position partially blocking the tunnel in the Na+ structure to a position lining the surface of the tunnel in the K+ and Cs+ structures. The results provide a structural basis for understanding the effects of cations on activity and intersubunit communication.

Ligand-mediated changes in the tryptophan synthase indole tunnel probed by nile red fluorescence with wild type, mutant, and chemically modified enzymes.

The bacterial tryptophan synthase alpha 2 beta 2 complex contains an unusual structural feature: an intramolecular tunnel that channels indole from the active site of the alpha subunit to the active site of the beta subunit 25 A away. Here we investigate the role of the tunnel in communication between the alpha and beta subunits using the polarity-sensitive fluorescent probe, Nile Red. Interaction of Nile Red in the nonpolar tunnel near beta subunit residues Cys-170 and Phe-280 is supported by studies with enzymes altered at these positions. Restricting the tunnel by enlarging Cys-170 by chemical modification or mutagenesis decreases the fluorescence of Nile Red by 30-70%. Removal of a partial restriction in the tunnel by replacing Phe-280 by Cys or Ser increases the fluorescence of Nile Red more than 2-fold. A binding site for Nile Red in this region near the pyridoxal phosphate coenzyme of the beta subunit is further supported by iodide quenching and fluorescence energy transfer experiments and by molecular modeling based on the three-dimensional structure of the alpha 2 beta 2 complex. Finally, studies using Nile Red as a sensitive probe of conformational changes in the tunnel reveal that allosteric ligands (alpha subunit) or active site ligands (beta subunit) decrease the fluorescence of Nile Red. We speculate that allosteric and active site ligands induce a tunnel restriction near Phe-280 that serves as a gate to control passage of indole through the tunnel.

Kinetics and mechanism of autoprocessing of human immunodeficiency virus type 1 protease from an analog of the Gag-Pol polyprotein.

Upon renaturation, the polyprotein MBP-delta TF-Protease-delta Pol, consisting of HIV-1 protease and short native sequences from the trans-frame protein (delta TF) and the polymerase (delta Pol) fused to the maltose-binding protein (MBP) of Escherichia coli, undergoes autoprocessing to produce the mature protease in two steps. The initial step corresponds to cleavage of the N-terminal sequence to release the protein intermediate Protease-delta Pol, which has enzymatic activity comparable to that of the mature enzyme. Subsequently, the mature enzyme is formed by a slower cleavage at the C terminus. The rate of increase in enzymatic activity is identical to that of the appearance of MBP-delta TF and the disappearance of the MBP-delta TF-Protease-delta Pol. Initial rates are linearly dependent on the protein concentration, indicating that the N-terminal cleavage is first-order in protein concentration. The reaction is competitively inhibited by pepstatin A and has a pH rate profile similar to that of the mature enzyme. These results and molecular modeling studies are discussed in terms of a mechanism in which a dimeric full-length fusion protein must form prior to rate-limiting intramolecular cleavage of the N-terminal sequence that leads to an increase in enzymatic activity.

Synthesis and crystallographic analysis of two rhizopuspepsin inhibitor complexes.

The crystal structures of rhizopuspepsin complexed with two oligopeptide inhibitors have been determined. CP-69,799, an azahomostatine dipeptide isostere, had previously been associated with a displacement of the C-terminal subdomain of endothiapepsin [Sali, A., Veerapandian, B., Cooper, J. B., Foundling, S. I., Hoover, D. J., & Blundell, T. L. (1989) EMBO J. 8, 2179-2188]. Here, we report the measurement of two data sets, one from crystals soaked in the inhibitor and the other from protein crystallized in the presence of excess inhibitor. In neither case is there any significant movement of the C-terminal subdomain of the rhizopuspepsin. The data suggest that the energy associated with any conformational change is small and is overcome by the crystal packing forces. The second inhibitor, a hydrated difluorostatone, was examined in a search for transition-state analogs that could cast further light on the mechanism of action [Suguna, K., Padlan, E. A., Smith, C. W., Carlson, W. D., & Davies, D. R. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 7009-7013]. The gem-diol provides a set of contact distances with the enzyme that mimic the interactions with the tetrahedral intermediate of the substrate during catalysis. These data provide support for the suggestion that the polarization of the keto group of the peptide substrate is enhanced by a hydrogen bond from the OD1 of Asp 35 (Suguna et al., 1987).

Structures of complexes of rhizopuspepsin with pepstatin and other statine-containing inhibitors.

The three-dimensional structures of the complexes of the aspartic proteinase from Rhizopus chinensis (Rhizopuspepsin, EC 3.4.23.6) with pepstatin and two pepstatin-like peptide inhibitors of renin have been determined by X-ray diffraction methods and refined by restrained least-squares procedures. The inhibitors adopt an extended conformation and lie in the deep groove located between the two domains of the enzyme. Inhibitor binding is accompanied by a conformational change at the "flap," a beta-hairpin loop region, that projects over the binding cleft and closes down over the inhibitor, excluding water molecules from the vicinity of the scissile bond. The hydroxyl group of the central statyl residue of the inhibitors replaces the water molecule found between the two active aspartates, Asp-35 and Asp-218, in the native structure. The refined structures provide additional data to define the specific subsites of the enzyme and also show a system of hydrogen bonding to the inhibitor backbone similar to that observed for a reduced inhibitor.