Andrew P. Leech

Purification of the Escherichia coli ammonium transporter AmtB reveals a trimeric stoichiometry.

The Amt family of high-affinity ammonium transporters is a family of integral membrane proteins that are found in archaea, bacteria, fungi, plants and animals. Furthermore, the family has recently been extended to humans with the recognition that both the erythroid and non-erythroid Rhesus proteins are also ammonium transporters. The Escherichia coli AmtB protein offers a good model system for the Amt family and in order to address questions relating to both its structure and function we have overproduced a histidine-tagged form of the protein (AmtB6H) and purified it to homogeneity. We examined the quaternary structure of AmtB6H (which is active in vivo) by SDS/PAGE, gel-filtration chromatography, dynamic light scattering and sedimentation ultracentrifugation. The protein was resistant to dissociation by SDS and behaved as a stable oligomer on SDS/PAGE. By equilibrium desorption chromatography we determined the mass ratio of dodecyl beta-D-maltoside to AmtB in the detergent-solubilized complex to be 1.03+/-0.03, and this allowed us to calculate, from analytical-ultracentrifugation data, that AmtB purifies as a trimer.

Mutagenesis of Active Site Residues in Type I Dehydroquinase from Escherichia coli STALLED CATALYSIS IN A HISTIDINE TO ALANINE MUTANT

Chemical modification experiments have previously implicated four amino acid residues in the mechanism of type I dehydroquinase from Escherichia coli. To further test their importance, these residues were mutated, and the resulting mutants were expressed, purified, and characterized. When the highly conserved, Schiff base-forming lysine residue was mutated (K170A) the resulting enzyme showed a 10-fold reduction in catalytic activity, but was still able to bind both substrate and product, as shown by a novel fluorescence-based ligand-binding assay. This is consistent with Lys-170 playing a central role in catalysis and shows that, although forming a covalent bond with the substrate, it is not essential for ground state binding of substrate or product. Conversely, substituting leucine for the conserved, iodoacetate-reactive methionine residue (M205L) had little effect on k or K. Diethylpyrocarbonate experiments had previously implicated either His-143 or His-146 as the putative active site general base. Substituting alanine for each shows that H146A retains full catalytic activity while H143A shows a 10-fold loss of activity. As with the K170A mutant, H143A can bind ligand, and in addition to the predicted role of this residue as the proton-abstracting general base, our data suggest that it is also involved in the formation and breakdown of Schiff base intermediates. Isoelectric focusing, electrospray ionization mass spectrometry, and fluorescence spectroscopy show that the H143A mutant preferentially stabilizes the formation of the product Schiff base, and that this results in burst kinetics reminiscent of p-nitrophenyl acetate hydrolysis by chymotrypsin. The most striking illustration of this stabilization is the fact that the H143A mutant is isolated from overexpressing cells with a significant proportion of the enzyme monomers covalently bound to the product, 3-dehydroshikimate, via a Schiff base linkage. Our data suggest that the H143A mutant is able to slowly transform substrate to product but that the hydrolytic release of the product is stalled. The proposed dual role of His-143 in the mechanism of type I dehydroquinase may explain why the elimination reaction catalyzed by this enzyme proceeds with syn stereochemistry.

PrrC from Rhodobacter sphaeroides, a homologue of eukaryotic Sco proteins, is a copper-binding protein and may have a thiol-disulfide oxidoreductase activity

PrrC from Rhodobacter sphaeroides provides the signal input to a two‐component signal transduction system that senses changes in oxygen tension and regulates expression of genes involved in photosynthesis (Eraso, J.M. and Kaplan, S. (2000) Biochemistry 39, 2052‐2062; Oh, J.‐I. and Kaplan, S. (2000) EMBO J. 19, 4237‐4247). It is also a homologue of eukaryotic Sco proteins and each has a C‐x‐x‐x‐C‐P sequence. In mitochondrial Sco proteins these cysteines appear to be essential for the biogenesis of the CuA centre of respiratory cytochrome oxidase. Overexpression and purification of a water‐soluble and monomeric form of PrrC has provided sufficient material for a chemical and spectroscopic study of the properties of the four cysteine residues of PrrC, and its ability to bind divalent cations, including copper. PrrC expressed in the cytoplasm of Escherichia coli binds Ni2+ tightly and the data are consistent with a mononuclear metal site. Following removal of Ni2+ and formation of renatured metal‐free rPrrC (apo‐PrrC), Cu2+ could be loaded into the reduced form of PrrC to generate a protein with a distinctive UV‐visible spectrum, having absorbance with a λ max of 360 nm. The copper:PrrC ratio is consistent with the presence of a mononuclear metal centre. The cysteines of metal‐free PrrC oxidise in the presence of air to form two intramolecular disulfide bonds, with one pair being extremely reactive. The cysteine thiols with extreme O2 sensitivity are involved in copper binding in reduced PrrC since the same copper‐loaded protein could not be generated using oxidised PrrC. Thus, it appears that PrrC, and probably Sco proteins in general, could have both a thiol‐disulfide oxidoreductase function and a copper‐binding role.

Staphylococcal biofilm-forming protein has a contiguous rod-like structure

Staphylococcus aureus and Staphylococcus epidermidis form communities (called biofilms) on inserted medical devices, leading to infections that affect many millions of patients worldwide and cause substantial morbidity and mortality. As biofilms are resistant to antibiotics, device removal is often required to resolve the infection. Thus, there is a need for new therapeutic strategies and molecular data that might assist their development. Surface proteins S. aureus surface protein G (SasG) and accumulation-associated protein (S. epidermidis) promote biofilm formation through their “B” regions. B regions contain tandemly arrayed G5 domains interspersed with approximately 50 residue sequences (herein called E) and have been proposed to mediate intercellular accumulation through Zn2+-mediated homodimerization. Although E regions are predicted to be unstructured, SasG and accumulation-associated protein form extended fibrils on the bacterial surface. Here we report structures of E–G5 and G5–E–G5 from SasG and biophysical characteristics of single and multidomain fragments. E sequences fold cooperatively and form interlocking interfaces with G5 domains in a head-to-tail fashion, resulting in a contiguous, elongated, monomeric structure. E and G5 domains lack a compact hydrophobic core, and yet G5 domain and multidomain constructs have thermodynamic stabilities only slightly lower than globular proteins of similar size. Zn2+ does not cause SasG domains to form dimers. The work reveals a paradigm for formation of fibrils on the 100-nm scale and suggests that biofilm accumulation occurs through a mechanism distinct from the “zinc zipper.” Finally, formation of two domains by each repeat (as in SasG) might reduce misfolding in proteins when the tandem arrangement of highly similar sequences is advantageous.

Copper-mediated dimerization of CopZ, a predicted copper chaperone from Bacillus subtilis.

Understanding the metal-binding properties and solution states of metallo-chaperones is a key step in understanding how they function in metal ion transfer. Using spectroscopic, bioanalytical and biochemical methods, we have investigated the copper-binding properties and association states of the putative copper chaperone of Bacillus subtilis, CopZ, and a variant of the protein lacking the two cysteine residues of the MXCXXC copper-binding motif. We show that copper-free CopZ exists as a monomer, but that addition of copper(I) causes the protein to associate into homodimers. The nature of the copper(I)-CopZ complex is dependent on the level of copper loading, and we report the detection of three distinct forms, containing 0.5, 1.0 and 1.5 copper(I) ions per protein. The presence of excess dithiothreitol has a significant effect on copper(I) binding to CopZ, such that, in its presence, copper(I)-CopZ occurs mainly as a monomer species. Data for copper binding to the double-cysteine variant of CopZ are consistent with an essential role for these residues in tight copper binding in the wild-type protein. We conclude that the complex nature of copper(I) binding to CopZ may underpin mechanisms of protein-to-protein copper(I) transfer.

Sialic Acid Mutarotation Is Catalyzed by the Escherichia coli β-Propeller Protein YjhT

The acquisition of host-derived sialic acid is an important virulence factor for some bacterial pathogens, but in vivo this sugar acid is sequestered in sialoconjugates as the α-anomer. In solution, however, sialic acid is present mainly as the β-anomer, formed by a slow spontaneous mutarotation. We studied the Escherichia coli protein YjhT as a member of a family of uncharacterized proteins present in many sialic acid-utilizing pathogens. This protein is able to accelerate the equilibration of the α- and β-anomers of the sialic acid N-acetylneuraminic acid, thus describing a novel sialic acid mutarotase activity. The structure of this periplasmic protein, solved to 1.5Å resolution, reveals a dimeric 6-bladed unclosed β-propeller, the first of a bacterial Kelch domain protein. Mutagenesis of conserved residues in YjhT demonstrated an important role for Glu-209 and Arg-215 in mutarotase activity. We also present data suggesting that the ability to utilize α-N-acetylneuraminic acid released from complex sialoconjugates in vivo provides a physiological advantage to bacteria containing YjhT.

Structural and Functional Analysis of the Tandem β-Zipper Interaction of a Streptococcal Protein with Human Fibronectin

Bacterial fibronectin-binding proteins (FnBPs) contain a large intrinsically disordered region (IDR) that mediates adhesion of bacteria to host tissues, and invasion of host cells, through binding to fibronectin (Fn). These FnBP IDRs consist of Fn-binding repeats (FnBRs) that form a highly extended tandem β-zipper interaction on binding to the N-terminal domain of Fn. Several FnBR residues are highly conserved across bacterial species, and here we investigate their contribution to the interaction. Mutation of these residues to alanine in SfbI-5 (a disordered FnBR from the human pathogen Streptococcus pyogenes) reduced binding, but for each residue the change in free energy of binding was <2 kcal/mol. The structure of an SfbI-5 peptide in complex with the second and third F1 modules from Fn confirms that the conserved FnBR residues play equivalent functional roles across bacterial species. Thus, in SfbI-5, the binding energy for the tandem β-zipper interaction with Fn is distributed across the interface rather than concentrated in a small number of “hot spot” residues that are frequently observed in the interactions of folded proteins. We propose that this might be a common feature of the interactions of IDRs and is likely to pose a challenge for the development of small molecule inhibitors of FnBP-mediated adhesion to and invasion of host cells.

Dimer-induced signal propagation in Spo0A

Spo0A, the response regulator protein controlling the initiation of sporulation in Bacillus, has two distinct domains, an N‐terminal phosphoacceptor (or receiver) domain and a C‐terminal DNA‐binding (or effector) domain. The phosphoacceptor domain mediates dimerization of Spo0A on phosphorylation. A comparison of the crystal structures of phosphorylated and unphosphorylated response regulators suggests a mechanism of activation in which structural changes originating at the phosphorylatable aspartate extend to the α4β5α5 surface of the protein. In particular, the data show an important role in downstream signalling for a conserved aromatic residue (Phe‐105 in Spo0A), the conformation of which alters upon phosphorylation. In this study, we have prepared a Phe‐105 to Ala mutant to probe the contribution of this residue to Spo0A function. We have also made an alanine substitution of the neighbouring residue Tyr‐104 that is absolutely conserved in the Spo0As of spore‐forming Bacilli. The spo0A(Y104A) and spo0A(F105A) alleles severely impair sporulation in vivo. In vitro phosphorylation of the purified proteins by phosphoramidate is unaffected, but dimerization and DNA binding are abolished by the mutations. We have identified intragenic suppressor mutations of spo0A(F105A) and shown that these second‐site mutations in the purified proteins restore phosphorylation‐dependent dimer formation. Our data support a model in which dimerization and signal transduction between the two domains of Spo0A are mediated principally by the α4β5α5 signalling surface in the receiver domain.

Mutations in subdomain B of the minichromosome maintenance (MCM) helicase affect DNA binding and modulate conformational transitions.

Minichromosome maintenance (MCM) proteins are believed to provide the replicative helicase activity in eukaryotes and archaea. The single MCM orthologue from Methanothermobacter thermautotrophicus (MthMCM) has been extensively characterized as a model of the eukaryotic heterohexameric MCM complex. MthMCM forms high molecular weight complexes in solution consistent with a dodecamer. Visualization of this complex by electron microscopy suggests that single and double heptameric or hexameric rings can form. We have mutated two arginine residues (Arg-137, Arg-160) in the N-terminal subdomain B of MthMCM based on their apparent potential to form inter-ring hydrogen bonds. Both the single R137A and the double R137A,R160A mutants were characterized by a combination of biophysical, biochemical, and electron microscopy techniques. Biophysical analysis coupled with electron microscopy studies shows that the R137A mutant forms a double heptameric ring, whereas the R137A,R160A protein assembles as a single heptamer. They both show a defect in DNA binding and a concomitant conformational change in subdomain A, with the double mutant displaying significant defects in helicase activity as well. We propose a model in which MCM loading and the subsequent activation of the helicase activity involve a conformational transition that is connected to a DNA binding event.

Consequences of Inducing Intrinsic Disorder in a High-Affinity Protein–Protein Interaction

The kinetic and thermodynamic consequences of intrinsic disorder in protein-protein recognition are controversial. We address this by inducing one partner of the high-affinity colicin E3 rRNase domain-Im3 complex (K(d) ≈ 10(-12) M) to become an intrinsically disordered protein (IDP). Through a variety of biophysical measurements, we show that a single alanine mutation at Tyr507 within the hydrophobic core of the isolated colicin E3 rRNase domain causes the enzyme to become an IDP (E3 rRNase(IDP)). E3 rRNase(IDP) binds stoichiometrically to Im3 and forms a structure that is essentially identical to the wild-type complex. However, binding of E3 rRNase(IDP) to Im3 is 4 orders of magnitude weaker than that of the folded rRNase, with thermodynamic parameters reflecting the disorder-to-order transition on forming the complex. Critically, pre-steady-state kinetic analysis of the E3 rRNase(IDP)-Im3 complex demonstrates that the decrease in affinity is mostly accounted for by a drop in the electrostatically steered association rate. Our study shows that, notwithstanding the advantages intrinsic disorder brings to biological systems, this can come at severe kinetic and thermodynamic cost.