Structure-guided selection of puromycin N-acetyltransferase mutants with enhanced selection stringency for deriving mammalian cell lines expressing recombinant proteins.

Puromycin and the Streptomyces alboniger-derived puromycin N-acetyltransferase (PAC) enzyme form a commonly used system for selecting stably transfected cultured cells. The crystal structure of PAC has been solved using X-ray crystallography, revealing it to be a member of the GCN5-related N-acetyltransferase (GNAT) family of acetyltransferases. Based on structures in complex with acetyl-CoA or the reaction products CoA and acetylated puromycin, four classes of mutations in and around the catalytic site were designed and tested for activity. Single-residue mutations were identified that displayed a range of enzymatic activities, from complete ablation to enhanced activity relative to wild-type (WT) PAC. Cell pools of stably transfected HEK293 cells derived using two PAC mutants with attenuated activity, Y30F and A142D, were found to secrete up to three-fold higher levels of a soluble, recombinant target protein than corresponding pools derived with the WT enzyme. A third mutant, Y171F, appeared to stabilise the intracellular turnover of PAC, resulting in an apparent loss of selection stringency. Our results indicate that the structure-guided manipulation of PAC function can be utilised to enhance selection stringency for the derivation of mammalian cell lines secreting elevated levels of recombinant proteins.

Structural and biochemical analyses of an aminoglycoside 2'-N-acetyltransferase from Mycolicibacterium smegmatis.

The expression of aminoglycoside-modifying enzymes represents a survival strategy of antibiotic-resistant bacteria. Aminoglycoside 2'-N-acetyltransferase [AAC(2')] neutralizes aminoglycoside drugs by acetylation of their 2' amino groups in an acetyl coenzyme A (CoA)-dependent manner. To understand the structural features and molecular mechanism underlying AAC(2') activity, we overexpressed, purified, and crystallized AAC(2') from Mycolicibacterium smegmatis [AAC(2')-Id] and determined the crystal structures of its apo-form and ternary complexes with CoA and four different aminoglycosides (gentamicin, sisomicin, neomycin, and paromomycin). These AAC(2')-Id structures unraveled the binding modes of different aminoglycosides, explaining the broad substrate specificity of the enzyme. Comparative structural analysis showed that the α4-helix and ß8-ß9 loop region undergo major conformational changes upon CoA and substrate binding. Additionally, structural comparison between the present paromomycin-bound AAC(2')-Id structure and the previously reported paromomycin-bound AAC(6')-Ib and 30S ribosome structures revealed the structural features of paromomycin that are responsible for its antibiotic activity and AAC binding. Taken together, these results provide useful information for designing AAC(2') inhibitors and for the chemical modification of aminoglycosides.

Distinct effects on mRNA export factor GANP underlie neurological disease phenotypes and alter gene expression depending on intron content.

Defects in the mRNA export scaffold protein GANP, encoded by the MCM3AP gene, cause autosomal recessive early-onset peripheral neuropathy with or without intellectual disability. We extend here the phenotypic range associated with MCM3AP variants, by describing a severely hypotonic child and a sibling pair with a progressive encephalopathic syndrome. In addition, our analysis of skin fibroblasts from affected individuals from seven unrelated families indicates that disease variants result in depletion of GANP except when they alter critical residues in the Sac3 mRNA binding domain. GANP depletion was associated with more severe phenotypes compared with the Sac3 variants. Patient fibroblasts showed transcriptome alterations that suggested intron content-dependent regulation of gene expression. For example, all differentially expressed intronless genes were downregulated, including ATXN7L3B, which couples mRNA export to transcription activation by association with the TREX-2 and SAGA complexes. Our results provide insight into the molecular basis behind genotype-phenotype correlations in MCM3AP-associated disease and suggest mechanisms by which GANP defects might alter RNA metabolism.

Crystal structure of the aminoglycosides N-acetyltransferase Eis2 from Mycobacterium abscessus.

Mycobacterium abscessus is an emerging human pathogen that is notorious for being one of the most drug-resistant species of Mycobacterium. It has developed numerous strategies to overcome the antibiotic stress response, limiting treatment options and leading to frequent therapeutic failure. The panel of aminoglycosides (AG) usually used in the treatment of M. abscessus pulmonary infections is restricted by chemical modification of the drugs by the N-acetyltransferase Eis2 protein (Mabs_Eis2). This enzyme acetylates the primary amine of AGs, preventing these antibiotics from binding ribosomal RNA and thereby impairing their activity. In this study, the high-resolution crystal structures of Mabs_Eis2 in its apo- and cofactor-bound forms were solved. The structural analysis of Mabs_Eis2, supported by the kinetic characterization of the enzyme, highlights the large substrate specificity of the enzyme. Furthermore, in silico docking and biochemical approaches attest that Mabs_Eis2 modifies clinically relevant drugs such as kanamycin and amikacin, with a better efficacy for the latter. In line with previous biochemical and in vivo studies, our work suggests that Mabs_Eis2 represents an attractive pharmacological target to be further explored. The high-resolution crystal structures presented here may pave the way to the design of Eis2-specific inhibitors with the potential to counteract the intrinsic resistance levels of M. abscessus to an important class of clinically important antibiotics. DATABASE: Structural data are available in the PDB database under the accession numbers: 6RFY, 6RFX and 6RFT.

Crystal structure of Pseudomonas aeruginosa N-acetyltransferase PA4534.

The GCN5-related N-acetyltransferase (GNAT) superfamily includes a large and diverse group of enzymes that catalyzes the transfer of an acetyl group from acetyl coenzyme A (Ac-CoA) to the amine group of a substrate. Substrates include protein N-terminus, lysine of histone tails, and other small molecules such as aminoglycoside, serotonin, and glucose-6-phosphate. GNAT superfamily of proteins is involved in many physiologically important reactions in eukaryotes and prokaryotes. However, functions of many GNATs remain unknown and PA4534 is one of those uncharacterized GNAT proteins from Pseudomonas aeruginosa. To investigate functions of the PA4534, we determined the apo and Ac-CoA bound PA4534 structures. Our structures showed that PA4534 shared common characteristic structures with other GNAT family N-acetyltransferases and contained a potential substrate binding tunnel close to the bound Ac-CoA.

Biophysical analysis of the putative acetyltransferase SACOL2570 from methicillin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of a myriad of insidious and intractable infections in humans, especially in patients with compromised immune systems and children. Here, we report the apo- and CoA-bound crystal structures of a member of the galactoside acetyltransferase superfamily from methicillin-resistant S. aureus SACOL2570 which was recently shown to be down regulated in S. aureus grown in the presence of fusidic acid, an antibiotic used to treat MRSA infections. SACOL2570 forms a homotrimer in solution, as confirmed by small-angle X-ray scattering and dynamic light scattering. The protein subunit consists of an N-terminal alpha-helical domain connected to a C-terminal LßH domain. CoA binds in the active site formed by the residues from adjacent LßH domains. After determination of CoA-bound structure, molecular dynamics simulations were performed to model the binding of AcCoA. Binding of both AcCoA and CoA to SACOL2570 was verified by isothermal titration calorimetry. SACOL2570 most likely acts as an acetyltransferase, using AcCoA as an acetyl group donor and an as-yet-undetermined chemical moiety as an acceptor. SACOL2570 was recently used as a scaffold for mutations that lead the generation of cage-like assemblies, and has the potential to be used for the generation of more complex nanostructures.

Changing the charge distribution of beta-helical-based nanostructures can provide the conditions for charge transfer.

In this work we present a computational approach to the design of nanostructures made of structural motifs taken from left-handed beta-helical proteins. Previously, we suggested a structural model based on the self-assembly of motifs taken from Escherichia coli galactoside acetyltransferase (Protein Data Bank 1krr, chain A, residues 131-165, denoted krr1), which produced a very stable nanotube in molecular dynamics simulations. Here we modify this model by changing the charge distribution in the inner core of the system and testing the effect of this change on the structural arrangement of the construct. Our results demonstrate that it is possible to generate the proper conditions for charge transfer inside nanotubes based on assemblies of krr1 segment. The electronic transfer would be achieved by introducing different histidine ionization states in selected positions of the internal core of the construct, in addition to specific mutations with charged amino acids that altogether will allow the formation of coherent networks of aromatic ring stacking, salt-bridges, and hydrogen bonds.

Characterization of the native and fibrillar conformation of the human Nalpha-acetyltransferase ARD1.

ARD1 (arrest-defective protein 1), together with NAT1 (N-acetyltransferase protein 1), is part of the major N(alpha)-acetyltransferase complex in eukaryotes responsible for alpha-acetylation of proteins and peptides. Protein acetylation has been implicated in gene expression regulation and protein-protein interaction. We characterized the native folded and misfolded conformation of hARD1. Structural characterization of native hARD1 using size exclusion chromatography, circular dichroism, and fluorescence spectroscopy shows the protein consists of a compact globular region comprising two thirds of the protein and a flexible unstructured C terminus. In addition, hARD1 forms protofilaments under physiological conditions of pH and temperature, as judged by electron microscopy and staining with the dyes Congo red and thioflavin T. The process is accelerated by thermal denaturation and high protein concentrations. Limited proteolysis of aggregated hARD1 revealed a resistant fragment whose sequence matched a region contained within the acetyl transferase domain.

Optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy.

A computational procedure is described for assigning the absolute hand of the structure of a protein or assembly determined by single-particle electron microscopy. The procedure requires a pair of micrographs of the same particle field recorded at two tilt angles of a single tilt-axis specimen holder together with the three-dimensional map whose hand is being determined. For orientations determined from particles on one micrograph using the map, the agreement (average phase residual) between particle images on the second micrograph and map projections is determined for all possible choices of tilt angle and axis. Whether the agreement is better at the known tilt angle and axis of the microscope or its inverse indicates whether the map is of correct or incorrect hand. An increased discrimination of correct from incorrect hand (free hand difference), as well as accurate identification of the known values for the tilt angle and axis, can be used as targets for rapidly optimizing the search or refinement procedures used to determine particle orientations. Optimized refinement reduces the tendency for the model to match noise in a single image, thus improving the accuracy of the orientation determination and therefore the quality of the resulting map. The hand determination and refinement optimization procedure is applied to image pairs of the dihydrolipoyl acetyltransferase (E2) catalytic core of the pyruvate dehydrogenase complex from Bacillus stearothermophilus taken by low-dose electron cryomicroscopy. Structure factor amplitudes of a three-dimensional map of the E2 catalytic core obtained by averaging untilted images of 3667 icosahedral particles are compared to a scattering reference using a Guinier plot. A noise-dependent structure factor weight is derived and used in conjunction with a temperature factor (B=-1000A(2)) to restore high-resolution contrast without amplifying noise and to visualize molecular features to 8.7A resolution, according to a new objective criterion for resolution assessment proposed here.

Direct evidence for the size and conformational variability of the pyruvate dehydrogenase complex revealed by three-dimensional electron microscopy. The "breathing" core and its functional relationship to protein dynamics.

Structural studies by three-dimensional electron microscopy of the Saccharomyces cerevisiae truncated dihydrolipoamide acetyltransferase (tE(2)) component of the pyruvate dehydrogenase complex reveal an extraordinary example of protein dynamics. The tE(2) forms a 60-subunit core with the morphology of a pentagonal dodecahedron and consists of 20 cone-shaped trimers interconnected by 30 bridges. Frozen-hydrated and stained molecules of tE(2) in the same field vary in size approximately 20%. Analyses of the data show that the size distribution is bell-shaped, and there is an approximately 40-A difference in the diameter of the smallest and largest structures that corresponds to approximately 14 A of variation in the length of the bridge between interconnected trimers. Companion studies of mature E(2) show that the complex of the intact subunit exhibits a similar size variation. The x-ray structure of Bacillus stearothermophilus tE(2) shows that there is an approximately 10-A gap between adjacent trimers and that the trimers are interconnected by the potentially flexible C-terminal ends of two adjacent subunits. We propose that this springlike feature is involved in a thermally driven expansion and contraction of the core and, since it appears to be a common feature in the phylogeny of pyruvate dehydrogenase complexes, protein dynamics is an integral component of the function of these multienzyme complexes.

Serine acetyltransferase from Escherichia coli is a dimer of trimers.

Equilibrium sedimentation studies show that the serine acetyltransferase (SAT) of Escherichia coli is a hexamer. The results of velocity sedimentation and quasi-elastic light scattering experiments suggest that the identical subunits are loosely packed and/or arranged in an ellipsoidal fashion. Chemical cross-linking studies indicate that the fundamental unit of quaternary structure is a trimer. The likelihood, therefore, is that in solution SAT exists as an open arrangement of paired trimers. Crystals of SAT have 32 symmetry, consistent with such an arrangement, and the cell density function is that expected for a hexamer. Electron microscopy with negative staining provides further evidence that SAT has an ellipsoidal subunit organization, the dimensions of the particles consistent with the proposed paired trimeric subunit arrangement. A bead model analysis supports the view that SAT has a low packing density and, furthermore, indicates that the monomers may have an ellipsoidal shape. Such a view is in keeping with the ellipsoidal subunit shape of trimeric LpxA, an acyltransferase with which SAT shares contiguous repeats of a hexapeptide motif.

The catalytic domain of dihydrolipoyl acetyltransferase from the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus. Expression, purification and reversible denaturation.

A sub-gene encoding the catalytic (acetyltransferase) domain (E2pCD) comprising residues 173-427 of the dihydrolipoyl acetyltransferase (E2p) chain of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus was expressed in Escherichia coli. The product assembled to form the characteristic icosahedral (60-mer) core structure with full catalytic activity. The Km values for dihydrolipoamide and acetyl-CoA were 1.2 mM and 13 microM, respectively. Dissociation of the icosahedral E2pCD into monomers by exposure to guanidine hydrochloride and the subsequent reassociation by gradual removal of the denaturing agent demonstrated the ability of the polypeptide chain to fold and reassemble in the absence of chaperonins.

The high-resolution structure of the peripheral subunit-binding domain of dihydrolipoamide acetyltransferase from the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus.

The three-dimensional structure of a 43-residue active, synthetic peptide encompassing the peripheral subunit-binding domain of dihydrolipoamide acetyltransferase from the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus has been determined by means of a multi-cooling dynamical simulated annealing protocol using restraints derived from 1H nuclear magnetic resonance spectroscopy. A total of 442 experimentally derived restraints including 13 dihedral angle (phi, chi 1) restraints were used. A final set of 35 structures was calculated with a root-mean-square deviation from the mean co-ordinates of 0.36 A for the backbone atoms and 0.96 A when side-chain heavy atoms were included for the well-defined region comprising residues Val7 to Leu39. Although assignments were made and sequential connectivities observed for the N-terminal six and C-terminal four residues, the absence of long-range NOEs suggests that the terminal regions are largely unstructured. The binding domain contains two short parallel alpha-helices (residues Val7 to Lys14 and Lys32 to Leu39), a3(10)-helix (residues Asp17 to Val21) and a structured loop made up of overlapping beta-turns (residues Gln22 to Leu31), which enclose a close-packed hydrophobic core. The loop is stabilized to a large extent by Asp34. This residue is conserved in all peripheral subunit-binding domains and its carboxylate side-chain forms a set of side-chain-main-chain hydrogen bonds with the main-chain amide protons of Gly23, Thr24, Gly25 and Leu31 and a side-chain-side-chain hydrogen bond with the hydroxyl group of Thr24. We propose that a peripheral subunit-binding site may be located in the loop region, which contains a series of highly conserved residues and provides a number of potential recognition sites. The structured region of the binding domain, comprising 33 residues, represents an exceptionally short amino acid sequence with defined tertiary structure that has no disulphide bond, ligand or cofactor to stabilize the fold. It may be approaching the lower size limit for a three-dimensional structure possessing features characteristic of larger structures, including a close-packed, non-polar interior. The organization of the side-chains in the hydrophobic core may have implications for de novo protein design.

Three-dimensional structure of the truncated core of the Saccharomyces cerevisiae pyruvate dehydrogenase complex determined from negative stain and cryoelectron microscopy images.

Dihydrolipoamide acyltransferase (E2), a catalytic and structural component of the three functional classes of multienzyme complexes that catalyze the oxidative decarboxylation of alpha-keto acids, forms the central core to which the other components are attached. We have imaged by negative stain and cryoelectron microscopy the truncated dihydrolipoamide acetyltransferase core (60 subunits; M(r) = 2.7 x 10(6)) of the Saccharomyces cerevisiae pyruvate dehydrogenase complex. Using icosahedral particle reconstruction techniques, we determined its structure to 25 A resolution. Although the model derived from the negative stain reconstruction was approximately 20% smaller than the model derived from the frozen-hydrated data, when corrected for the effects of the electron microscope contrast transfer functions, the reconstructions showed excellent correspondence. The pentagonal dodecahedron-shaped macromolecule has a maximum diameter, as measured along the 3-fold axis, of approximately 226 A (frozen-hydrated value), and 12 large openings (approximately 63 A in diameter) on the 5-fold axes that lead into a large solvent-accessible cavity (approximately 76-140 A diameter). The 20 vertices consist of cone-shaped trimers, each with a flattened base on the outside of the structure and an apex directed toward the center. The trimers are interconnected by 20 A thick "bridges" on the 2-fold axes. These studies also show that the highest resolution features apparent in the frozen-hydrated reconstruction are revealed in a filtered reconstruction of the stained molecule.

The serine acetyltransferase from Escherichia coli. Over-expression, purification and preliminary crystallographic analysis.

An expression vector has been constructed which increases the expression of serine acetyltransferase (SAT) from E. coli to 17% of the soluble cell protein. A novel purification procedure, using dye-affinity chromatography, allows purification of SAT to homogeneity. The enzyme has been crystallised from polyethylene glycol, in the presence of L-cysteine (an inhibitor of SAT). The crystals which diffract to beyond 3.0 A resolution are of the tetragonal spacegroup P4(1)2(1)2 (or P4(3)2(1)2) with cell dimensions a = b = 123 A, c = 79 A. Since ultracentrifugation and gel-filtration experiment indicate that purified SAT is a tetramer, there appears to be one-half tetramer in the asymmetric unit (Vm = 2.55 A3/Da).