Matthew Jenner

Detection of a Protein Conformational Equilibrium by Electrospray Ionisation‐Ion Mobility‐Mass Spectrometry

Ion mobility spectrometry (IMS) is emerging as a promising technique for providing low-resolution protein-structure information, particularly in combination with electrospray ionization (ESI) and mass spectrometry (MS). There is currently much debate on the structure of protein ions in the gas phase, as summarized by Breuker and McLafferty. Whilst it appears probable that some structural collapse occurs within picoseconds of dehydration, the onset of gross structural rearrangement may require tens of milliseconds. This time provides a potential window for the observation of “near-native” structures that may retain some elements of the solution structure, with the ability to provide biologically relevant information. A number of recent applications have used IMS to study the conformation and stoichiometry of proteins and their complexes. Structural changes to amyloid and prion proteins, as well as steps in amyloid fibril assembly, have been detected by using this approach, and the calcium-dependent conformational change in calmodulin has been probed by IM-MS, as has the relationship between tertiary structure and chemotactic activity in antibacterial peptides. Insights into the structures of large multiprotein complexes, such as the RNA-binding TRAP protein, GroEL, and the 20S proteasome have also been provided by ion mobility measurements. We postulated that IM-MS may be used to study the dynamic equilibrium between well-characterized conformations of a monomeric multidomain protein. To test this hypothesis, we have examined NADPH-cytochrome P450 reductase (CPR) using ESI-IM-MS. CPR is a 76 kDa membrane bound flavoprotein that catalyses the transfer of electrons from NADPH to a number of oxygenase enzymes. CPR consists of three folded domains, an FADand NADPH-binding domain, an FMN-binding domain, and a linker domain which may serve to orient the other two domains. The FMN-binding domain is connected to the rest of the protein by a 14-residue “hinge”, thus providing the flexibility that is thought to be important for the function of the protein. An N-terminal 57 amino acid peptide is responsible for anchoring CPR to the endoplasmic reticulum membrane; recombinant CPR, which lacks this N-terminal peptide, is both soluble and functional, thus facilitating detailed structure–activity studies. The CPR-mediated electron transfer from NADPH to cytochrome P450 proceeds in a stepwise fashion: NADPH! FAD!FMN!P450. Interflavin electron transfer requires spatial proximity of the two prosthetic groups, and the X-ray crystal structure of CPR (PDB file: 1AMO) confirms this is the case (closest approach of the FAD and FMN methyl groups: 3.85 (C–C)). However, in this compact or “closed” conformation, the FMN cofactor appears to be inaccessible to the large cytochrome P450 molecule, and so the need for domain movement as an essential part of the catalytic cycle has been widely assumed. Recently, NMR spectroscopy, small-angle X-ray scattering (SAXS), and crystallographic evidence for this movement has been obtained, thus suggesting that in solution, CPR exists in an equilibrium between a compact conformation appropriate for interflavin electron transfer and an extended conformation appropriate for electron transfer to P450 (Figure 1). Herein we show that two major conformations of wildtype CPR are present in the gas phase, and that their relative abundance can be influenced by the ionic strength of the solution from which they are electrosprayed, by removal of key intramolecular ionic interactions, and, crucially, by the redox state of the flavin groups. This study demonstrates the ability of ESI-IMS-MS to detect a protein conformational

A Close Look at a Ketosynthase from a Trans-Acyltransferase Modular Polyketide Synthase

The recently discovered trans-acyltransferase modular polyketide synthases catalyze the biosynthesis of a wide range of bioactive natural products in bacteria. Here we report the structure of the second ketosynthase from the bacillaene trans-acyltransferase polyketide synthase. This 1.95 Å resolution structure provides the highest resolution view available of a modular polyketide synthase ketosynthase and reveals a flanking subdomain that is homologous to an ordered linker in cis-acyltransferase modular polyketide synthases. The structure of the cysteine-to-serine mutant of the ketosynthase acylated by its natural substrate provides high-resolution details of how a native polyketide intermediate is bound and helps explain the basis of ketosynthase substrate specificity. The substrate range of the ketosynthase was further investigated by mass spectrometry.

Mechanism‐Based Inhibition of Quinone Reductase 2 (NQO2): Selectivity for NQO2 over NQO1 and Structural Basis for Flavoprotein Inhibition

A role for the flavoprotein NRH:quinone oxidoreductase 2 (NQO2, QR2) in human diseases such as malaria, leukemia and neurodegeneration has been proposed. In order to explore the potential of NQO2 as a therapeutic target, we have developed potent and selective mechanism‐based inhibitors centered on the indolequinone pharmacophore. The compounds show remarkable selectivity for NQO2 over the closely related flavoprotein NQO1, with small structural changes defining selectivity. Biochemical studies confirmed the mechanism‐based inhibition, whereas X‐ray crystallography and mass spectrometry revealed the nature of the inhibitor interaction with the protein. These indolequinones represent the first mechanism‐based inhibitors of NQO2, and their novel mode of action involving alkylation of the flavin cofactor, provides significant advantages over existing competitive inhibitors in terms of potency and irreversibility, and will open new opportunities to define the role of NQO2 in disease.

Discovery and Biosynthesis of Gladiolin: A Burkholderia gladioli Antibiotic with Promising Activity against Mycobacterium tuberculosis

An antimicrobial activity screen of Burkholderia gladioli BCC0238, a clinical isolate from a cystic fibrosis patient, led to the discovery of gladiolin, a novel macrolide antibiotic with potent activity against Mycobacterium tuberculosis H37Rv. Gladiolin is structurally related to etnangien, a highly unstable antibiotic from Sorangium cellulosum that is also active against Mycobacteria. Like etnangien, gladiolin was found to inhibit RNA polymerase, a validated drug target in M. tuberculosis. However, gladiolin lacks the highly labile hexaene moiety of etnangien and was thus found to possess significantly increased chemical stability. Moreover, gladiolin displayed low mammalian cytotoxicity and good activity against several M. tuberculosis clinical isolates, including four that are resistant to isoniazid and one that is resistant to both isoniazid and rifampicin. Overall, these data suggest that gladiolin may represent a useful starting point for the development of novel drugs to tackle multidrug-resistant tuberculosis. The B. gladioli BCC0238 genome was sequenced using Single Molecule Real Time (SMRT) technology. This resulted in four contiguous sequences: two large circular chromosomes and two smaller putative plasmids. Analysis of the chromosome sequences identified 49 putative specialized metabolite biosynthetic gene clusters. One such gene cluster, located on the smaller of the two chromosomes, encodes a trans-acyltransferase (trans-AT) polyketide synthase (PKS) multienzyme that was hypothesized to assemble gladiolin. Insertional inactivation of a gene in this cluster encoding one of the PKS subunits abrogated gladiolin production, confirming that the gene cluster is responsible for biosynthesis of the antibiotic. Comparison of the PKSs responsible for the assembly of gladiolin and etnangien showed that they possess a remarkably similar architecture, obfuscating the biosynthetic mechanisms responsible for most of the structural differences between the two metabolites.

Acyl-chain elongation drives ketosynthase substrate selectivity in trans-acyltransferase polyketide synthases.

Type I modular polyketide synthases (PKSs), which are responsible for the biosynthesis of many biologically active agents, possess a ketosynthase (KS) domain within each module to catalyze chain elongation. Acylation of the KS active site Cys residue is followed by transfer to malonyl-ACP to yield an extended β-ketoacyl chain (ACP = acyl carrier protein). To date, the precise contribution of KS selectivity in controlling product fidelity has been unclear. Six KS domains from trans-acyltransferase (trans-AT) PKSs were subjected to a mass spectrometry based elongation assay, and higher substrate selectivity was identified for the elongating step than in preceding acylation. A close correspondence between the observed KS selectivity and that predicted by phylogenetic analysis was seen. These findings provide insights into the mechanism of KS selectivity in this important group of PKSs, can serve as guidance for engineering, and show that targeted mutagenesis can be used to expand the repertoire of acceptable substrates.

SilE is an intrinsically disordered periplasmic 'molecular sponge' involved in bacterial silver resistance.

Ag+ resistance was initially found on the Salmonella enetrica serovar Typhimurium multi‐resistance plasmid pMG101 from burns patients in 1975. The putative model of Ag+ resistance, encoded by the sil operon from pMG101, involves export of Ag+ via an ATPase (SilP), an effluxer complex (SilCFBA) and a periplasmic chaperon of Ag+ (SilE). SilE is predicted to be intrinsically disordered. We tested this hypothesis using structural and biophysical studies and show that SilE is an intrinsically disordered protein in its free apo‐form but folds to a compact structure upon optimal binding to six Ag+ ions in its holo‐form. Sequence analyses and site‐directed mutagenesis established the importance of histidine and methionine containing motifs for Ag+‐binding, and identified a nucleation core that initiates Ag+‐mediated folding of SilE. We conclude that SilE is a molecular sponge for absorbing metal ions.

Amino acid -accepting ketosynthase domain from a trans -AT polyketide synthase exhibits high selectivity for predicted intermediate

The trans-acyltransferase (AT) polyketide synthases are a recently recognised group of bacterial enzymes that generate complex polyketides. A prerequisite for re-engineering these poorly studied systems is knowledge about the substrate specificity of their components. In this work, KS domain 1 from the bacillaene polyketide synthase has been shown to possess high specificity towards 2-amidoacetyl intermediates, which are derived from incorporation of alpha amino acids into the polyketide chain. N-Acetylcysteamine (SNAC) analogues of full-length substrates were synthesised and incubated with the KS1 domain. The natural glycine-derived acyl–SNAC was found to acylate KS1 with highest efficiency, as evidenced by mass spectrometry (MS). An alanine variant was also incorporated, but its valine equivalent was not, which indicated limited tolerance of substitution at the α-position. Substrate analogues without an amine or amide nitrogen substituted on the 2-position were not accepted by KS1 at the standard assay concentration of 0.5 mM. Moreover, removal of Asn-206 from the active site of KS1 by site-directed mutagenesis reduced kcat/Km by a factor of approx. 2. This residue is conserved in most known 2-amidoacetyl-accepting KS domains from trans-AT PKSs and we postulate an important interaction between Asn-206 and the amide nitrogen of the substrate.

Mechanism of intersubunit ketosynthase–dehydratase interaction in polyketide synthases

Modular polyketide synthases (PKSs) produce numerous structurally complex natural products that have diverse applications in medicine and agriculture. PKSs typically consist of several multienzyme subunits that utilize structurally defined docking domains (DDs) at their N and C termini to ensure correct assembly into functional multiprotein complexes. Here we report a fundamentally different mechanism for subunit assembly in trans-acyltransferase (trans-AT) modular PKSs at the junction between ketosynthase (KS) and dehydratase (DH) domains. This mechanism involves direct interaction of a largely unstructured docking domain (DD) at the C terminus of the KS with the surface of the downstream DH. Acyl transfer assays and mechanism-based crosslinking established that the DD is required for the KS to communicate with the acyl carrier protein appended to the DH. Two distinct regions for binding of the DD to the DH were identified using NMR spectroscopy, carbene footprinting, and mutagenesis, providing a foundation for future elucidation of the molecular basis for interaction specificity.

Substrate Specificity of Ketosynthase Domains Part I: β-Branched Acyl Chains

Recent phylogenetic work has shown that KS domains from trans-AT PKSs correlate, at the sequence level, with their predicted biosynthetic intermediates (Nguyen et al., Nat. Biotechnol. 26:225–233, 2008, [1]). The extent of this evolution-based specificity is believed to reach as far as the β-position for each given biosynthetic intermediate, as detailed in section “KS Specificity-Based Assignment of trans-AT PKSs”. Compared to textbook colinearity rules, employed for cis-AT PKS systems, a KS specificity-based approach works remarkably well as a predictive method for assigning biosynthetic intermediates to their PKS, however it lacks functional testing. Herein, the substrate specificity of BaeL KS5 from the bacillaene cluster, and KS1, KS2 and KS30 from the psymberin PKS are reported. Using the data from these assays, in conjunction with homology modelling, the molecular rules dictating the ability of KS domains to accept carbon based β-branched substrates is revealed.

Substrate Specificity of Ketosynthase Domains Part II: Amino Acid-Containing Acyl Chains

In this chapter, the first study of a KS domain immediately downstream of a NRPS module is reported. Using full-length acyl precursors the substrate specificity of BaeJ KS1 from the bacillaene trans-AT PKS was examined. BaeJ KS1 is the first PKS module in the biosynthesis of bacillaene, and is believed to accept a glycine-derived intermediate incorporated by the previous NRPS module. KS1 is positioned in a phylogenetic clade with other amino-acid accepting KS domains, of which the majority accept a glycine intermediate.