Structure and malonyl CoA-ACP transacylase binding of streptomyces coelicolor fatty acid synthase acyl carrier protein.

Malonylation of an acyl carrier protein (ACP) by malonyl Coenzyme A-ACP transacylase (MCAT) is fundamental to bacterial fatty acid biosynthesis. Here, we report the structure of the Steptomyces coelicolor (Sc) fatty acid synthase (FAS) ACP and studies of its binding to MCAT. The carrier protein adopts an alpha-helical bundle structure common to other known carrier proteins. The Sc FAS ACP shows close structural homology with other fatty acid ACPs and less similarity with Sc actinorhodin (act) polyketide synthase (PKS) ACP where the orientation of helix I differs. NMR experiments were used to map the binding of ACP to MCAT. This data suggests that Sc FAS ACP interacts with MCAT through the negatively charged helix II of ACP, consistent with proposed models for ACP recognition by other FAS enzymes. Differential roles for residues at the interface are demonstrated using site-directed mutagenesis and in vitro assays. MCAT has been suggested, moreover, to participate in bacterial polyketide synthesis in vivo. We demonstrate that the affinity of the polyketide synthase ACP for MCAT is lower than that of the FAS ACP. Mutagenesis of homologous helix II residues on the polyketide synthase ACP suggests that the PKS ACP may bind to MCAT in a different manner than the FAS counterpart.

The malonyl transferase activity of type II polyketide synthase acyl carrier proteins.

Acyl carrier proteins (ACPs) play a fundamental role in directing intermediates among the enzyme active sites of fatty acid and polyketide synthases (PKSs). In this paper, we demonstrate that the Streptomyces coelicolor (S. coelicolor) actinorhodin (act) PKS ACP can catalyze transfer of malonate to type II S. coelicolor fatty acid synthase (FAS) and other PKS ACPs in vitro. The reciprocal transfer from S. coelicolor FAS ACP to a PKS ACP was not observed. Several mutations in both act ACP and S. coelicolor FAS ACP could be classified by their participation in either donation or acceptance of this malonyl group. These mutations indicated that self-malonylation and malonyl transfer could be completely decoupled, implying that they were separate processes and that a FAS ACP could be converted from a non-malonyl-transferring protein to one with malonyl transferase activity.

Self-malonylation is an intrinsic property of a chemically synthesized type II polyketide synthase acyl carrier protein.

During polyketide biosynthesis, malonyl groups are transferred to the acyl carrier protein (ACP) component of the polyketide synthase (PKS), and it has been shown that a number of type II polyketide ACPs undergo rapid self-acylation from malonyl-CoA in the absence of a malonyl-CoA:holo-acyl carrier protein transacylase (MCAT). More recently, however, the observation of self-malonylation has been ascribed to contamination with Escherichia coli MCAT (FabD) rather than an intrinsic property of the ACP. The wild-type apo-ACP from the actinorhodin (act) PKS of Streptomyces coelicolor (synthetic apo-ACP) has therefore been synthesized using solid-state peptide methods and refolded using the GroEL/ES chaperone system from E. coli. Correct folding of the act ACP has been confirmed by circular dichroism (CD) and 1H NMR. Synthetic apo-ACP was phosphopantetheinylated to 100% by S. coelicolor holo-acyl carrier protein synthase (ACPS), and the resultant holo-ACP underwent self-malonylation in the presence of malonyl-CoA. No malonylation of negative controls was observed, confirming that the use of ACPS and GroEL/ES did not introduce contamination with E. coli MCAT. This result proves unequivocally that self-malonylation is an inherent activity of this PKS ACP in vitro.

Structure of an allosteric inhibitor of LFA-1 bound to the I-domain studied by crystallography, NMR, and calorimetry.

LFA-1 (lymphocyte function-associated antigen-1) plays a role in intercellular adhesion and lymphocyte trafficking and activation and is an attractive anti-inflammatory drug target. The alpha-subunit of LFA-1, in common with several other integrins, has an N-terminally inserted domain (I-domain) of approximately 200 amino acids that plays a central role in regulating ligand binding to LFA-1. An additional region, termed the I-domain allosteric site (IDAS), has been identified exclusively within the LFA-1 I-domain and shown to regulate the function of this protein. The IDAS is occupied by small molecule LFA-1 inhibitors when cocrystallized or analyzed by (15)N-(1)H HSQC (heteronuclear single-quantum coherence) NMR (nuclear magnetic resonance) titration experiments. We report here a novel arylthio inhibitor that binds the I-domain with a K(d) of 18.3 nM as determined by isothermal titration calorimetry (ITC). This value is in close agreement with the IC(50) (10.9 nM) derived from a biochemical competition assay (DELFIA) that measures the level of inhibition of binding of whole LFA-1 to its ligand, ICAM-1. Having established the strong affinity of the arylthio inhibitor for the isolated I-domain, we have used a range of techniques to further characterize the binding, including ITC, NMR, and X-ray crystallography. We have first developed an effective ITC binding assay for use with low-solubility inhibitors that avoids the need for ELISA-based assays. In addition, we utilized a fast NMR-based assay for the generation of I-domain-inhibitor models. This is based around the collection of HCCH-TOCSY spectra of LFA-1 in the bound form and the identification of a subset of side chain methyl groups that give chemical shift changes upon binding of LFA-1 inhibitors. This subset was used in two-dimensional (13)C-(15)N and (15)N-filtered and -edited two-dimensional NMR experiments to identify a minimal set of intraligand and ligand-protein NOEs, respectively (nuclear Overhauser enhancements). Models from the NMR data were assessed by comparison to an X-ray crystallographic structure of the complex, confirming that the method correctly predicted the essential features of the bound ligand.

Solution structure and dynamics of oxytetracycline polyketide synthase acyl carrier protein from Streptomyces rimosus.

Type II polyketide synthases (PKSs) utilize a dedicated and essential acyl carrier protein (ACP) in the biosynthesis of a specific polyketide product. As part of our ongoing studies into the mechanisms and control of polyketide biosynthesis, we report the second structure of a polyketide synthase ACP. In this work, multidimensional, heteronuclear NMR was employed to investigate the structure and dynamics of the ACP involved in the biosynthesis of the commonly prescribed polyketide antibiotic, oxytetracycline (otc). An ensemble of 28 structures of the 95 amino acid otc ACP (9916Da) was computed by simulated annealing with the inclusion of 1132 experimental restraints. Atomic RMSDs about the mean structure for all 28 models is 0.66 A for backbone atoms, 1.15 A for all heavy atoms (both values calculated for the folded part of the protein (residues 3-80)), and 0.41 A for backbone atoms within secondary structure. Otc ACP adopts the typical right-handed, four-helix fold of currently known ACPs but with the addition of a 13-residue flexible C-terminus. A comparison of the global folds of all structurally characterized ACPs is described, illustrating that PKS ACPs show clear differences as well as similarities to FAS ACPs. (15)N relaxation experiments for the protein backbone also reveal that the long loop between helices I and II is flexible and helix II, a proposed site of protein-protein interactions, shows conformational exchange. The helices of the ACP form a rigid scaffold for the protein, but these are interspersed with an unusual proportion of flexible linker regions.

Evidence for plasticity and structural mimicry at the immunoglobulin light chain-protein L interface.

The multidomain bacterial surface protein L (PpL) is a virulence factor expressed by only 10% of Peptostreptococcus magnus strains, and its expression is correlated with bacterial vaginosis. The molecular basis for its ability to recognize 60% of mammalian immunoglobulin light chain variable regions (V(L)) has been described recently by x-ray crystallography, which suggested the presence of two V(L) binding sites on each protein L domain (Graille, M., Stura, E. A., Housden, N. G., Beckingham, J. A., Bottomley, S. P., Beale, D., Taussig, M. J., Sutton, B. J., Gore, M. G., and Charbonnier, J. (2001) Structure 9, 679-687). Here, we report the crystal structure at 2.1 A resolution of a protein L mutant complexed to an Fab' fragment with only 50% of the V(L) residues interacting with PpL site 1 conserved. Comparison of the site 1 interface from both structures shows how protein L is able to accommodate these sequence differences and therefore bind to a large repertoire of Ig. The x-ray structure and NMR results confirm the existence of two V(L) binding sites on a single protein L domain. These sites exhibit a remarkable structural mimicry of growth factors binding to their receptors. This could explain the protein L superantigenic activity on human B lymphocytes.