Crystal structure of the human fatty acid synthase enoyl-acyl carrier protein-reductase domain complexed with triclosan reveals allosteric protein-protein interface inhibition.

Human fatty acid synthase (FAS) is a large, multidomain protein that synthesizes long chain fatty acids. Because these fatty acids are primarily provided by diet, FAS is normally expressed at low levels; however, it is highly up-regulated in many cancers. Human enoyl-acyl carrier protein-reductase (hER) is one of the FAS catalytic domains, and its inhibition by drugs like triclosan (TCL) can increase cytotoxicity and decrease drug resistance in cancer cells. We have determined the structure of hER in the presence and absence of TCL. TCL was not bound in the active site, as predicted, but rather at the protein-protein interface (PPI). TCL binding induces a dimer orientation change that causes downstream structural rearrangement in critical active site residues. Kinetics studies indicate that TCL is capable of inhibiting the isolated hER domain with an IC50 of ∼ 55 µM. Given the hER-TCL structure and the inhibition observed in the hER domain, it seems likely that TCL is observed in the physiologically relevant binding site and that it acts as an allosteric PPI inhibitor. TCL may be a viable scaffold for the development of anti-cancer PPI FAS inhibitors.

Crystal structure of the cytoplasmic N-terminal domain of subunit I, a homolog of subunit a, of V-ATPase.

Subunit "a" is associated with the membrane-bound (V(O)) complex of eukaryotic vacuolar H(+)-ATPase acidification machinery. It has also been shown recently to be involved in diverse membrane fusion/secretory functions independent of acidification. Here, we report the crystal structure of the N-terminal cytosolic domain from the Meiothermus ruber subunit "I" homolog of subunit a. The structure is composed of a curved long central α-helix bundle capped on both ends by two lobes with similar α/ß architecture. Based on the structure, a reasonable model of its eukaryotic subunit a counterpart was obtained. The crystal structure and model fit well into reconstructions from electron microscopy of prokaryotic and eukaryotic vacuolar H(+)-ATPases, respectively, clarifying their orientations and interactions and revealing features that could enable subunit a to play a role in membrane fusion/secretion.

Structural insights into the dual activities of the nerve agent degrading organophosphate anhydrolase/prolidase.

The organophosphate acid anhydrolase (OPAA) is a member of a class of bimetalloenzymes that hydrolyze a variety of toxic acetylcholinesterase-inhibiting organophosphorus compounds, including fluorine-containing chemical nerve agents. It also belongs to a family of prolidases, with significant activity against various Xaa-Pro dipeptides. Here we report the X-ray structure determination of the native OPAA (58 kDa mass) from Alteromonas sp. strain JD6.5 and its cocrystal with the inhibitor mipafox [N,N'-diisopropyldiamidofluorophosphate (DDFP)], a close analogue of the nerve agent organophosphate substrate diisopropyl fluorophosphate (DFP). The OPAA structure is composed of two domains, amino and carboxy domains, with the latter exhibiting a "pita bread" architecture and harboring the active site with the binuclear Mn(2+) ions. The native OPAA structure revealed unexpectedly the presence of a well-defined nonproteinaceous density in the active site whose identity could not be definitively established but is suggestive of a bound glycolate, which is isosteric with a glycine (Xaa) product. All three glycolate oxygens coordinate the two Mn(2+) atoms. DDFP or more likely its hydrolysis product, N,N'-diisopropyldiamidophosphate (DDP), is present in the cocrystal structure and bound by coordinating the binuclear metals and forming hydrogen bonds and nonpolar interactions with active site residues. An unusual common feature of the binding of the two ligands is the involvement of only one oxygen atom of the glycolate carboxylate and the product DDP tetrahedral phosphate in bridging the two Mn(2+) ions. Both structures provide new understanding of ligand recognition and the prolidase and organophosphorus hydrolase catalytic activities of OPAA.

Normal mode refinement of anisotropic thermal parameters for a supramolecular complex at 3.42-A crystallographic resolution.

Here we report a normal-mode-based protocol for modeling anisotropic thermal motions of proteins in x-ray crystallographic refinement. The foundation for this protocol is a recently developed elastic normal mode analysis that produces much more accurate eigenvectors without the tip effect. The effectiveness of the procedure is demonstrated on the refinement of a 3.42-A structure of formiminotransferase cyclodeaminase, a 0.5-MDa homooctameric enzyme. Using an order of magnitude fewer adjustable thermal parameters than the conventional isotropic refinement, this protocol resulted in a decrease of the values of R(cryst) and R(free) and improvements of the density map. Several poorly resolved regions in the original isotropically refined structure became clearer so that missing side chains were fitted easily and mistraced backbone was corrected. Moreover, the distribution of anisotropic thermal ellipsoids revealed functionally important structure flexibility. This normal-mode-based refinement is an effective way of describing anisotropic thermal motions in x-ray structures and is particularly attractive for the refinement of very large and flexible supramolecular complexes at moderate resolutions.

Ligand-free and -bound structures of the binding protein (LivJ) of the Escherichia coli ABC leucine/isoleucine/valine transport system: trajectory and dynamics of the interdomain rotation and ligand specificity.

The leucine/isoleucine/valine-binding protein (LIVBP or LivJ) serves as the primary high-affinity receptor of the Escherichia coli ABC-type transporter for the three aliphatic amino acids. The first structure of LIVBP determined previously without bound ligand showed a molecule comprised of two domains which are far apart and bisected by a wide open, solvent-accessible cleft. Here we report the crystal structures of another ligand-free state and three complexes with the aliphatic amino acids. In the present ligand-free structure, the two domains are farther apart. In the three very similar complex structures, the two domains are in close proximity, and each desolvated ligand is completely engulfed in the cleft and bound by both domains. The two different ligand-free structures, combined with those of the very similar ligand-bound structures, indicate the trajectory and backbone torsion angle changes of the hinges that accompany domain closure and play crucial functional roles. The amino acids are bound by polar and nonpolar interactions, occurring predominantly in one domain. Consistent with the protein specificity, the aliphatic side chains of the ligands lie in a hydrophobic pocket fully formed following domain or cleft closure. Comparison of the structures of LIVBP with several different binding proteins indicates no correlations between the magnitudes of the hinge-bending angles and the protein masses, the ligand sizes, or the number of segments connecting the two domains. Results of normal-mode analysis and molecular dynamics simulations are consistent with the trajectory and intrinsic flexibility of the interdomain hinges and the dominance of one domain in ligand binding in the open state.

Structure of the bifunctional and Golgi-associated formiminotransferase cyclodeaminase octamer.

Mammalian formiminotransferase cyclodeaminase (FTCD), a 0.5 million Dalton homo-octameric enzyme, plays important roles in coupling histidine catabolism with folate metabolism and integrating the Golgi complex with the vimentin intermediate filament cytoskeleton. It is also linked to two human diseases, autoimmune hepatitis and glutamate formiminotransferase deficiency. Determination of the FTCD structure by X-ray crystallography and electron cryomicroscopy revealed that the eight subunits, each composed of distinct FT and CD domains, are arranged like a square doughnut. A key finding indicates that coupling of three subunits governs the octamer-dependent sequential enzyme activities, including channeling of intermediate and conformational change. The structure further shed light on the molecular nature of two strong antigenic determinants of FTCD recognized by autoantibodies from patients with autoimmune hepatitis and on the binding of thin vimentin filaments to the FTCD octamer.

Structural basis of peptide-carbohydrate mimicry in an antibody-combining site.

The structure of a complex between the Fab fragment of the antibody (SYA/J6) specific for the cell surface O-antigen polysaccharide of the pathogen Shigella flexneri Y and an octapeptide (Met-Asp-Trp-Asn-Met-His-Ala-Ala), a functional mimic of the O-antigen, has been determined at 1.8-A resolution. Comparison of the structure with that of the complex with the pentasaccharide antigen [-->2)-alpha-L-Rha-(1-->2)-alpha-L-Rha-(1-->3)-alpha-L-Rha-(1-->3)-beta-D-GlcNAc-(1-->2)-alpha-L-Rha-(1-->] reveals the molecular recognition process by which a peptide mimics a carbohydrate in binding to an antibody. The binding modes of the two ligands differ considerably. Octapeptide binding complements the shape of the combining site groove much better than pentasaccharide binding. Moreover, the peptide makes a much greater number of contacts (126), which are mostly van der Waals interactions, with the Fab than the saccharide (74). An unusual feature is also the involvement of 12 water molecules in mediating hydrogen bonds between residues within the peptide or of the peptide and Fab. Despite better shape complementarity and greater number of contacts, the octapeptide binds with an affinity (KA = 2.5 x 10(5) M-1, measured by calorimetry) only approximately 2-fold tighter than the pentasaccharide. The structural results are relevant to the design of peptide mimetics with improved affinity for use as vaccines.

Crystal structure of M tuberculosis ABC phosphate transport receptor: specificity and charge compensation dominated by ion-dipole interactions.

The 2.16 A structure of the phosphate-bound PstS-1, the primary extracellular receptor for the ABC phosphate transporter and immunodominant species-specific antigen of Mycobacterium tuberculosis, has been determined. The phosphate, completely engulfed in the cleft between two domains, is bound by 13 hydrogen bonds, 11 of which are formed with NH and OH dipolar donor groups. The further presence of two acidic residues, which serve as acceptors of the protonated phosphate, is key to conferring stringent specificity. The ion-dipole interactions between the phosphate and dipolar groups compensate the ligand's isolated negative charges. Moreover, the surprise finding that the electrostatic surface in and around the cleft is intensely negative demonstrates the power of ion-dipole interactions in anion binding and electrostatic balance. Additional functional features include both the flexible N-terminal segment that tethers PstS-1 on the cell surface and the hinge between the two domains, which should facilitate snaring the phosphate in the medium.

Molecular recognition of oligosaccharide epitopes by a monoclonal Fab specific for Shigella flexneri Y lipopolysaccharide: X-ray structures and thermodynamics.

The antigenic recognition of Shigella flexneri O-polysaccharide, which consists of a repeating unit ABCD [-->2)-alpha-L-Rhap-(1-->2)-alpha-L-Rhap-(1-->3)-alpha-L-Rhap-(1-->3)-beta-D-GlcpNAc-(1-->], by the monoclonal antibody SYA/J6 (IgG3, kappa) has been investigated by crystallographic analysis of the Fab domain and its two complexes with two antigen segments (a pentasaccharide Rha A-Rha B-Rha C-GlcNAc D-Rha A' and a modified trisaccharide Rha B-Rha C-GlcNAc D in which Rha C* is missing a C2-OH group). These complex structures, the first for a Fab specific for a periodic linear heteropolysaccharide, reveal a binding site groove (between the V(H) and V(L) domains) that makes polar and nonpolar contacts with all the sugar residues of the pentasaccharide. Both main-chain and side-chain atoms of the Fab are used in ligand binding. The charged side chain of Glu H50 of CDR H2 forms crucial hydrogen bonds to GlcNAc of the oligosaccharides. The modified trisaccharide is more buried and fits more snugly than the pentasaccharide. It also makes as many contacts (approximately 75) with the Fab as the pentasaccharide, including the same number of hydrogen bonds (eight, with four being identical). It is further engaged in more hydrophobic interactions than the pentasaccharide. These three features favorable to trisaccharide binding are consistent with the observation of a tighter complex with the trisaccharide than the pentasaccharide. Thermodynamic data demonstrate that the native tri- to pentasaccharides have free energies of binding in the range of 6.8-7.4 kcal mol(-1), and all but one of the hydrogen bonds to individual hydroxyl groups provide no more than approximately 0.7 kcal mol(-1). They further indicate that hydrophobic interactions make significant contributions to binding and, as the native epitope becomes larger across the tri-, tetra-, pentasaccharide series, entropy contributions to the free energy become dominant.