Structure of a bound peptide phosphonate reveals the mechanism of nocardicin bifunctional thioesterase epimerase-hydrolase half-reactions.

Nonribosomal peptide synthetases (NRPSs) underlie the biosynthesis of many natural products that have important medicinal utility. Protection of the NRPS peptide products from proteolysis is critical to these pathways and is often achieved by structural modification, principally the introduction of D-amino acid residues into the elongating peptide. These amino acids are generally formed in situ from their L-stereoisomers by epimerization domains or dual-function condensation/epimerization domains. In singular contrast, the thioesterase domain of nocardicin biosynthesis mediates both the effectively complete L- to D-epimerization of its C-terminal amino acid residue (≥100:1) and hydrolytic product release. We report herein high-resolution crystal structures of the nocardicin thioesterase domain in ligand-free form and reacted with a structurally precise fluorophosphonate substrate mimic that identify the complete peptide binding pocket to accommodate both stereoisomers. These structures combined with additional functional studies provide detailed mechanistic insight into this unique dual-function NRPS domain.

Crystal structure of human cyclophilin A bound to the amino-terminal domain of HIV-1 capsid.

The HIV-1 capsid protein forms the conical core structure at the center of the mature virion. Capsid also binds the human peptidyl prolyl isomerase, cyclophilin A, thereby packaging the enzyme into the virion. Cyclophilin A subsequently performs an essential function in HIV-1 replication, possibly helping to disassemble the capsid core upon infection. We report the 2.36 A crystal structure of the N-terminal domain of HIV-1 capsid (residues 1-151) in complex with human cyclophilin A. A single exposed capsid loop (residues 85-93) binds in the enzyme's active site, and Pro-90 adopts an unprecedented trans conformation. The structure suggests how cyclophilin A can act as a sequence-specific binding protein and a nonspecific prolyl isomerase. In the crystal lattice, capsid molecules assemble into continuous planar strips. Side by side association of these strips may allow capsid to form the surface of the viral core. Cyclophilin A could then function by weakening the association between capsid strips, thereby promoting disassembly of the viral core.

Preliminary analysis of multiple crystal forms of the bovine cyclophilin-cyclosporin A complex.

The immunosuppressant cyclosporin-A (CsA) has been crystallized in complex with the bovine form of its major receptor protein cyclophilin (CyP) in three different forms by the hanging-drop vapor diffusion method. A hexagonal crystal form (P6(1)22 or P6(5)22; a = b = 110 A, c = 440 A) diffracts to 4 A resolution. Orthorhombic (P2(1)2(1)2(1) or P2(1)2(1)2; a = 154.3 A, b = 163.3 A, c = 94.7 A), and monoclinic (P2(1); a = 70.0 A, b = 162.5 A, c = 94.7, beta = 100.0 degrees) forms diffract to 2.2 A resolution. Self-rotation function analysis of the orthorhombic and monoclinic forms shows 52 point group local symmetry for both. A previously reported tetragonal crystal form of the complex also shares this local symmetry, suggesting that the observed motif may pre-exist in solution.

Identification of the immunophilins capable of mediating inhibition of signal transduction by cyclosporin A and FK506: roles of calcineurin binding and cellular location.

The immunosuppressants cyclosporin A (CsA) and FK506 appear to block T-cell function by inhibiting the calcium-regulated phosphatase calcineurin. While multiple distinct intracellular receptors for these drugs (cyclophilins and FKBPs, collectively immunophilins) have been characterized, the functionally active ones have not been discerned. We found that overexpression of cyclophilin A or B or FKBP12 increased T-cell sensitivity to CsA or FK506, respectively, demonstrating that they are able to mediate the inhibitory effects of their respective immunosuppressants in vivo. In contrast, cyclophilin C, FKBP13, and FKBP25 had no effect. Direct comparison of the Ki of each drug-immunophilin complex for calcineurin in vitro revealed that although calcineurin binding was clearly necessary, it was not sufficient to explain the in vivo activity of the immunophilin. Subcellular localization was shown also to play a role, since gene deletions of cyclophilins B and C which changed their intracellular locations altered their activities significantly. Cyclophilin B has been shown previously to be located within calcium-containing intracellular vesicles; its ability to mediate CsA inhibition implies that certain components of the signal transduction machinery are also spatially restricted within the cell.

NMR studies of [U-13C]cyclosporin A bound to human cyclophilin B.

NMR data (1H and 13C chemical shifts, NOEs) on [U-13C]cyclosporin A bound to cyclophilin B were compared to previously published data on the [U-13C]CsA/CyPA complex [Fesik et al., (1991) Biochemistry 30, 6574-6583]. Despite only 64% sequence identity between CyPA and CyPB, the conformation and active site environment of CsA when bound to CyPA and CyPB are nearly identical as judged by the similarity of the NMR data.