Polyketides are secondary metabolites which display both valuable pharmaceutical and agrochemical properties. Biosynthesis is performed by polyketide synthases (PKSs), and the acyl carrier protein (ACP), a small acidic protein, that transports the growing polyketide chain and is essential for activity. Here we report the synthesis of two aromatic probes and a linear octaketide mimic that have been tethered to actinorhodin ACP. These experiments were aimed at probing the ACP's capacity to sequester a non-polar versus a phenolic aromatic ring (that more closely mimics a polyketide intermediate) as well as investigations with extended polyketide chain surrogates. The binding of these mimics has been assessed using high-resolution solution NMR studies and high-resolution structure determination. These results reveal that surprisingly a PKS ACP is able to bind and sequester a bulky non-polar substrate containing an aromatic ring in a fatty acid type binding mode, but the introduction of even a small degree of polarity favours a markedly different association at a surface site that is distinct from that employed by fatty acid ACPs.

Among the 15 extracellular domains of the mannose 6-phosphate/insulin-like growth factor-2 receptor (M6P/IGF2R), domain 11 has evolved a binding site for IGF2 to negatively regulate ligand bioavailability and mammalian growth. Despite the highly evolved structural loops of the IGF2:domain 11 binding site, affinity-enhancing AB loop mutations suggest that binding is modifiable. Here we examine the extent to which IGF2:domain 11 affinity, and its specificity over IGF1, can be enhanced, and we examine the structural basis of the mechanistic and functional consequences. Domain 11 binding loop mutants were selected by yeast surface display combined with high-resolution structure-based predictions, and validated by surface plasmon resonance. We discovered previously unidentified mutations in the ligand-interacting surface binding loops (AB, CD, FG, and HI). Five combined mutations increased rigidity of the AB loop, as confirmed by NMR. When added to three independently identified CD and FG loop mutations that reduced the koff value by twofold, these mutations resulted in an overall selective 100-fold improvement in affinity. The structural basis of the evolved affinity was improved shape complementarity established by interloop (AB-CD) and intraloop (FG-FG) side chain interactions. The high affinity of the combinatorial domain 11 Fc fusion proteins functioned as ligand-soluble antagonists or traps that depleted pathological IGF2 isoforms from serum and abrogated IGF2-dependent signaling in vivo. An evolved and reengineered high-specificity M6P/IGF2R domain 11 binding site for IGF2 may improve therapeutic targeting of the frequent IGF2 gain of function observed in human cancer.

The tyrosine kinase A (TrkA) receptor is a validated therapeutic intervention point for a wide range of conditions. TrkA activation by nerve growth factor (NGF) binding the second extracellular immunoglobulin (TrkAIg2) domain triggers intracellular signaling cascades. In the periphery, this promotes the pain phenotype and, in the brain, cell survival or differentiation. Reproducible structural information and detailed validation of protein–ligand interactions aid drug discovery. However, the isolated TrkAIg2 domain crystallizes as a β-strand-swapped dimer in the absence of NGF, occluding the binding surface. Here we report the design and structural validation by nuclear magnetic resonance spectroscopy of the first stable, biologically active construct of the TrkAIg2 domain for binding site confirmation. Our structure closely mimics the wild-type fold of TrkAIg2 in complex with NGF (1WWW.pdb), and the 1H–15N correlation spectra confirm that both NGF and a competing small molecule interact at the known binding interface in solution.

Atropisomerism of pharmaceutical compounds is a challenging area for drug discovery programs ( Angew. Chem., Int. Ed. 2009, 48, 6398−6401). Strategies for dealing with these compounds include raising the energy barrier to atropisomerization in order to develop the drug as a single isomer ( Tetrahedron 2004, 60, 4337−4347) or reducing the barrier to rotation and developing a mixture of rapidly interconverting isomers ( Chirality 1996, 8, 364−371). Commonly, however, the atropisomers will be differentiated in terms of their affinity for a given protein target, and it is therefore important to rapidly identify the most active component prior to further compound development. We present equilibrium dialysis and saturation transfer difference NMR (STD-NMR) as techniques for assessing relative affinities of an atropisomeric mixture against antiapoptotic protein targets Bcl-2 and Bcl-xL. These techniques require no prior separation of the mixture of compounds and are therefore rapid and simple approaches. We also explore the use of noncovalent mass spectrometry for determining KD values of individual atropisomers separated from the equilibrium mixture and compare the results to solution-phase measurements. Results from equilibrium dialysis, STD-NMR, and noncovalent mass spectrometry are all in excellent agreement and provide complementary information on differential binding, amplification of the strongest binders, and KD values.

Identifying protein–ligand binding interactions is a key step during early-stage drug discovery. Existing screening techniques are often associated with drawbacks such as low throughput, high sample consumption, and dynamic range limitations. The increasing use of fragment-based drug discovery (FBDD) demands that these techniques also detect very weak interactions (mM KD values). This paper presents the development and validation of a fully automated screen by mass spectrometry, capable of detecting fragment binding into the millimolar KD range. Low sample consumption, high throughput, and wide dynamic range make this a highly attractive, orthogonal approach. The method was applied to screen 157 compounds in 6 h against the anti-apoptotic protein target Bcl-xL. Mass spectrometry results were validated using STD-NMR, HSQC-NMR, and ITC experiments. Agreement between techniques suggests that mass spectrometry offers a powerful, complementary approach for screening.

Parental genetic conflict may have exploited changes in the coding of a protein loop in a growth factor receptor.

The transfer of the phosphopantetheine chain from coenzyme A (CoA) to the acyl carrier protein (ACP), a key protein in both fatty acid and polyketide synthesis, is catalyzed by ACP synthase (AcpS). Streptomyces coelicolor AcpS is a doubly promiscuous enzyme capable of activation of ACPs from both fatty acid and polyketide synthesis and catalyzes the transfer of modified CoA substrates. Five crystal structures have been determined, including those of ligand-free AcpS, complexes with CoA and acetyl-CoA, and two of the active site mutants, His110Ala and Asp111Ala. All five structures are trimeric and provide further insight into the mechanism of catalysis, revealing the first detailed structure of a group I active site with the essential magnesium in place. Modeling of ACP binding supported by mutational analysis suggests an explanation for the promiscuity in terms of both ACP partner and modified CoA substrates.

It remains unclear whether in a bacterial fatty acid synthase (FAS) acyl chain transfer is a programmed or diffusion controlled and random action. Acyl carrier protein (ACP), which delivers all intermediates and interacts with all synthase enzymes, is the key player in this process. High-resolution structures of intermediates covalently bound to an ACP representing each step in fatty acid biosynthesis have been solved by solution NMR. These include hexanoyl-, 3-oxooctanyl-, 3R-hydroxyoctanoyl-, 2-octenoyl-, and octanoyl-ACP from Streptomyces coelicolor FAS. The high-resolution structures reveal that the ACP adopts a unique conformation for each intermediate driven by changes in the internal fatty acid binding pocket. The binding of each intermediate shows conserved structural features that may ensure effective molecular recognition over subsequent rounds of fatty acid biosynthesis.Graphical AbstractFigure optionsDownload full-size imageDownload high-quality image (204 K)Download as PowerPoint slideHighlights► We present NMR solution structures of five fatty acid intermediate derivatized ACPs ► ACP noncovalently binds each intermediate over a complete biosynthetic cycle ► The burial of the intermediate is modulated by its functionality ► The structure of the ACP changes with the chemistry of the bound intermediate

Acyl (peptidyl) carrier protein (ACP or PCP) is a crucial component involved in the transfer of thiol ester-bound intermediates during the biosynthesis of primary and secondary metabolites such as fatty acids, polyketides, and nonribosomal peptides. Although many carrier protein three-dimensional structures have been determined, to date there is no model available for a fungal type I polyketide synthase ACP. Here we report the solution structure of the norsolorinic acid synthase (NSAS) holo ACP domain that has been excised from the full-length multifunctional enzyme. NSAS ACP shows similarities in three-dimensional structure with other type I and type II ACPs, consisting of a four-helix bundle with helices I, II, and IV arranged in parallel. The N-terminus of helix III, however, is unusually hydrophobic, and Phe1768 and Leu1770 pack well with the core of the protein. The result is that unlike other carrier proteins, helix III lies almost perpendicular to the three major helices. Helix III is well-defined by numerous NMR-derived distance restraints and may be less flexible than counterparts in type II FAS and PKS ACPs. When the holo ACP is derivatized with a hexanoyl group, only minor changes are observed between the HSQC spectra of the two ACP species and no NOEs are observed for this hydrophobic acyl group. Along with the mammalian type I FAS, this further strengthens the view that type I ACPs do not show any significant affinity for hydrophobic (nonpolar) chain assembly intermediates attached via the 4′-phosphopantetheine prosthetic group.

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 α-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.

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.

Acyl carrier proteins (ACPs) are essential to both fatty acid synthase (FAS) and polyketide synthase (PKS) biosynthetic pathways, yet relatively little is known about how they function at a molecular level. Seven thiol ester and thiol ether derivatives of the actinorhodin (act) PKS ACP from Streptomyces coelicolor have been prepared and structurally characterised by NMR to gain insight into ACP–intermediate interactions. Holo ACP synthase has been used to prepare early-stage ACP intermediates of polyketide biosynthesis (holo ACP, acetyl ACP, and malonyl ACP) from the respective coenzyme A derivatives. A synthetic route to stabilised thiol ether ACPs was developed and applied to the preparation of stable 3-oxobutyl and 3,5-dioxohexyl ACP as diketide and triketide analogues. No interaction between the protein and the acyl phosphopantetheine moieties of acetyl, malonyl, or 3-oxobutyl ACP was detected. Analysis of 1H–15N heteronuclear single quantum coherence and nuclear Overhauser enhancement spectroscopy spectra for the triketide ACP revealed exchange between a major (‘Tri’, 85%) and a minor protein conformer in which the polyketide interacts with the protein (‘Tri⁎’, 15%). Act ACP was also derivatised with butyryl, hexanoyl, and octanoyl groups. The corresponding NMR spectra showed large chemical shift perturbations centred on helices II and III, indicative of acyl chain binding and significant structural rearrangement. Unexpectedly, butyryl act ACP showed almost identical backbone 1H–15N chemical shifts to Tri⁎, suggesting comparable structural changes that might provide insight into the structurally uncharacterised polyketide bound form. Furthermore, butyryl ACP itself underwent slow conformational exchange with a second minor conformer (But⁎) with almost identical backbone chemical shifts to octanoyl act ACP. High-resolution NMR structures of these acylated forms revealed that act ACP was able to undergo dramatic conformational changes that exceed those seen in FAS ACPs. When compared to E. coli FAS ACP, the substrate binding pocket of the act PKS ACP has three specific amino acid substitutions (Thr39/Leu45, Ala68/Leu74, and Leu42/Thr48) that alter the size, shape, and location of this cavity. These conformational changes may play a role in protein–protein recognition and assist the binding of bulky polyketide intermediates.

The insulin-like growth factor II/mannose-6-phosphate receptor (IGF2R) mediates trafficking of mannose-6-phosphate (M6P)-containing proteins and the mitogenic hormone IGF2. IGF2R also plays an important role as a tumor suppressor, as mutation is frequently associated with human carcinogenesis. IGF2 binds to domain 11, one of 15 extracellular domains on IGF2R. The crystal structure of domain 11 and the solution structure of IGF2 have been reported, but, to date, there has been limited success when using crystallography to study the interaction of IGFs with their binding partners. As an approach to investigate the interaction between IGF2 and IGF2R, we have used heteronuclear NMR in combination with existing mutagenesis data to derive models of the domain 11-IGF2 complex by using the program HADDOCK. The models reveal that the molecular interaction is driven by critical hydrophobic residues on IGF2 and IGF2R, while a ring of flexible, charged residues on IGF2R may modulate binding.

Polyketides are secondary metabolites which display both valuable pharmaceutical and agrochemical properties. Biosynthesis is performed by polyketide synthases (PKSs), and the acyl carrier protein (ACP), a small acidic protein, that transports the growing polyketide chain and is essential for activity. Here we report the synthesis of two aromatic probes and a linear octaketide mimic that have been tethered to actinorhodin ACP. These experiments were aimed at probing the ACP's capacity to sequester a non-polar versus a phenolic aromatic ring (that more closely mimics a polyketide intermediate) as well as investigations with extended polyketide chain surrogates. The binding of these mimics has been assessed using high-resolution solution NMR studies and high-resolution structure determination. These results reveal that surprisingly a PKS ACP is able to bind and sequester a bulky non-polar substrate containing an aromatic ring in a fatty acid type binding mode, but the introduction of even a small degree of polarity favours a markedly different association at a surface site that is distinct from that employed by fatty acid ACPs.