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.

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.

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.

Auristatins, synthetic analogues of the antineoplastic natural product Dolastatin 10, are ultrapotent cytotoxic microtubule inhibitors that are clinically used as payloads in antibody-drug conjugates (ADCs). The design and synthesis of several new auristatin analogues with N-terminal modifications that include amino acids with α,α-disubstituted carbon atoms are described, including the discovery of our lead auristatin, PF-06380101. This modification of the peptide structure is unprecedented and led to analogues with excellent potencies in tumor cell proliferation assays and differential ADME properties when compared to other synthetic auristatin analogues that are used in the preparation of ADCs. In addition, auristatin cocrystal structures with tubulin are being presented that allow for the detailed examination of their binding modes. A surprising finding is that all analyzed analogues have a cis-configuration at the Val-Dil amide bond in their functionally relevant tubulin bound state, whereas in solution this bond is exclusively in the trans-configuration. This remarkable observation shines light onto the preferred binding mode of auristatins and serves as a valuable tool for structure-based drug design.