Jacqueline Ellis

Domain motion in cytochrome P450 reductase: conformational equilibria revealed by NMR and small-angle x-ray scattering.

NADPH-cytochrome P450 reductase (CPR), a diflavin reductase, plays a key role in the mammalian P450 mono-oxygenase system. In its crystal structure, the two flavins are close together, positioned for interflavin electron transfer but not for electron transfer to cytochrome P450. A number of lines of evidence suggest that domain motion is important in the action of the enzyme. We report NMR and small-angle x-ray scattering experiments addressing directly the question of domain organization in human CPR. Comparison of the 1H-15N heteronuclear single quantum correlation spectrum of CPR with that of the isolated FMN domain permitted identification of residues in the FMN domain whose environment differs in the two situations. These include several residues that are solvent-exposed in the CPR crystal structure, indicating the existence of a second conformation in which the FMN domain is involved in a different interdomain interface. Small-angle x-ray scattering experiments showed that oxidized and NADPH-reduced CPRs have different overall shapes. The scattering curve of the reduced enzyme can be adequately explained by the crystal structure, whereas analysis of the data for the oxidized enzyme indicates that it exists as a mixture of approximately equal amounts of two conformations, one consistent with the crystal structure and one a more extended structure consistent with that inferred from the NMR data. The correlation between the effects of adenosine 2′,5′-bisphosphate and NADPH on the scattering curve and their effects on the rate of interflavin electron transfer suggests that this conformational equilibrium is physiologically relevant.

The crystal structures of chloramphenicol phosphotransferase reveal a novel inactivation mechanism

Chloramphenicol (Cm), produced by the soil bacterium Streptomyces venezuelae, is an inhibitor of bacterial ribosomal peptidyltransferase activity. The Cm‐producing streptomycete modifies the primary (C‐3) hydroxyl of the antibiotic by a novel Cm‐inactivating enzyme, chloramphenicol 3‐O‐phosphotransferase (CPT). Here we describe the crystal structures of CPT in the absence and presence of bound substrates. The enzyme is dimeric in a sulfate‐free solution and tetramerization is induced by ammonium sulfate, the crystallization precipitant. The tetrameric quaternary structure exhibits crystallographic 222 symmetry and has ATP binding pockets located at a crystallographic 2‐fold axis. Steric hindrance allows only one ATP to bind per dimer within the tetramer. In addition to active site binding by Cm, an electron‐dense feature resembling the enzymes product is found at the other subunit interface. The structures of CPT suggest that an aspartate acts as a general base to accept a proton from the 3‐hydroxyl of Cm, concurrent with nucleophilic attack of the resulting oxyanion on the γ‐phosphate of ATP. Comparison between liganded and substrate‐free CPT structures highlights side chain movements of the active sites Arg136 guanidinium group of >9 Å upon substrate binding.

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

Structural and Mechanistic Studies of Galactoside Acetyltransferase, the Escherichia coli LacA Gene Product

Escherichia coli galactoside acetyltransferase (GAT) is a member of a large family of acetyltransferases that O-acetylate dissimilar substrates but share limited sequence homology. Steady-state kinetic analysis of overexpressed GAT demonstrated that it accepted a range of substrates, including glucosides and lactosides which were acetylated at rates comparable to galactosides. GAT was shown to be a trimeric acetyltransferase by cross-linking with dimethyl suberimidate. Fluorometric analysis of coenzyme A binding showed that there is a fluorescence quench associated with acetyl-CoA binding whereas CoA has no effect. This difference was exploited to measure dissociation rates for both CoA and acetyl-CoA by stopped-flow fluorometry. The rate of dissociation of CoA (2500 s−1) is at least 170-fold faster than kcat for any substrate tested. The fluorescence response to acetyl-CoA binding is entirely due to Trp-139 since replacement by phenylalanine completely abolished the fluorescence quench. Treatment of GAT by [14C]iodoacetamide resulted in complete inactivation of the enzyme and the incorporation of label into histidyl and cysteinyl residues to approximately equal extents. Following replacement of His-115 by alanine, label was incorporated solely into cysteinyl residues. Furthermore, the substitution results in an 1800-fold decrease in kcat suggesting that His-115 has an important catalytic role in GAT.

Redox-Linked Domain Movements in the Catalytic Cycle of Cytochrome P450 Reductase

Summary NADPH-cytochrome P450 reductase is a key component of the P450 mono-oxygenase drug-metabolizing system. There is evidence for a conformational equilibrium involving large-scale domain motions in this enzyme. We now show, using small-angle X-ray scattering (SAXS) and small-angle neutron scattering, that delivery of two electrons to cytochrome P450 reductase leads to a shift in this equilibrium from a compact form, similar to the crystal structure, toward an extended form, while coenzyme binding favors the compact form. We present a model for the extended form of the enzyme based on nuclear magnetic resonance and SAXS data. Using the effects of changes in solution conditions and of site-directed mutagenesis, we demonstrate that the conversion to the extended form leads to an enhanced ability to transfer electrons to cytochrome c. This structural evidence shows that domain motion is linked closely to the individual steps of the catalytic cycle of cytochrome P450 reductase, and we propose a mechanism for this.

The kinetics of the interaction between the actin-binding domain of α-actinin and F-actin

Measurement of the binding equilibrium for the interaction of α‐actinin with F‐actin is complicated by secondary reactions involving cross‐linking and/or bundling of the actin filaments. To quantitate the initial binding event, we studied the interaction of the bacterially expressed actin‐binding domain (ABD) of chick smooth muscle α‐actinin with F‐actin. Stopped‐flow measurements revealed a quench in protein fluorescence and an enhancement in light scattering when ABD binds to F‐actin yielding second order rate constants for association of 2 × 105, 1.8 × 106 and 4 × 106 M−1 · s−1 at 5°C, 15°C and 25°C, respectively. At the latter two temperatures the dissociation rate constants were 1.5 and 9.6 s−1, giving equilibrium constants of 0.83 and 2.4 μM, respectively. Optical changes on mixing intact α‐actinin with F‐actin were dominated by secondary bundling events.

The last step in cephalosporin C formation revealed: crystal structures of deacetylcephalosporin C acetyltransferase from Acremonium chrysogenum in complexes with reaction intermediates.

Deacetylcephalosporin C acetyltransferase (DAC-AT) catalyses the last step in the biosynthesis of cephalosporin C, a broad-spectrum beta-lactam antibiotic of large clinical importance. The acetyl transfer step has been suggested to be limiting for cephalosporin C biosynthesis, but has so far escaped detailed structural analysis. We present here the crystal structures of DAC-AT in complexes with reaction intermediates, providing crystallographic snapshots of the reaction mechanism. The enzyme is found to belong to the alpha/beta hydrolase class of acetyltransferases, and the structures support previous observations of a double displacement mechanism for the acetyl transfer reaction in other members of this class of enzymes. The structures of DAC-AT reported here provide evidence of a stable acyl-enzyme complex, thus underpinning a mechanism involving acetylation of a catalytic serine residue by acetyl coenzyme A, followed by transfer of the acetyl group to deacetylcephalosporin C through a suggested tetrahedral transition state.

Cubic crystals of chloramphenicol phosphotransferase from Streptomyces venezuelae in complex with chloramphenicol

Chloramphenicol 3-O-phosphotransferase (CPT) from Streptomyces venezuelae ISP5230, a novel chloramphenicol-inactivating kinase, has been overexpressed and purified using Escherichia coli as the heterologous host. Crystals of CPT in complex with its substrate chloramphenicol (Cm) were obtained which were suitable for X-ray diffraction. The crystals belong to the cubic space group I4132 with unit-cell dimension a = 200.0 A. The initial CPT crystals diffracted to 3.5 A and the diffraction was improved significantly upon adding acetonitrile and Cm to the crystallization drop. The CPT-Cm crystals diffract to at least 2.8 A resolution.