Michael Lockyer

Processing of the precursor to the major merozoite surface antigens of Plasmodium falciparum

Specific sequences derived from the gene for the precursor to the major merozoite surface antigens (PMMSA) of Plasmodium falciparum have been expressed in Escherichia coli and the products have been used to produce antibodies. These antibodies, together with monoclonal antibodies, have been used to investigate the form of the PMMSA protein associated with merozoites. Polypeptide fragments derived by processing from the PMMSA protein have been detected in extracts of merozoites and assigned to locations within the PMMSA coding sequence.

Structures of R- and T-state Escherichia coli Aspartokinase III MECHANISMS OF THE ALLOSTERIC TRANSITION AND INHIBITION BY LYSINE

Aspartokinase III (AKIII) from Escherichia coli catalyzes an initial commitment step of the aspartate pathway, giving biosynthesis of certain amino acids including lysine. We report crystal structures of AKIII in the inactive T-state with bound feedback allosteric inhibitor lysine and in the R-state with aspartate and ADP. The structures reveal an unusual configuration for the regulatory ACT domains, in which ACT2 is inserted into ACT1 rather than the expected tandem repeat. Comparison of R- and T-state AKIII indicates that binding of lysine to the regulatory ACT1 domain in R-state AKIII instigates a series of changes that release a “latch”, the β15-αK loop, from the catalytic domain, which in turn undergoes large rotational rearrangements, promoting tetramer formation and completion of the transition to the T-state. Lysine-induced allosteric transition in AKIII involves both destabilizing the R-state and stabilizing the T-state tetramer. Rearrangement of the catalytic domain blocks the ATP-binding site, which is therefore the structural basis for allosteric inhibition of AKIII by lysine.

Identification of the gene for a Plasmodium yoelii rhoptry protein. Multiple copies in the parasite genome

Serum from mice hyperimmune to Plasmodium yoelii was used to screen a P. yoelii genomic DNA library. Antibodies selected from hyperimmune serum by lambda gt11 clone J7 or raised against a specific fusion protein or peptide produced a punctate pattern of immunofluorescence on fixed smears of parasitised erythrocytes and immunoprecipitated a 235-kDa protein apparently identical to a rhoptry protein previously implicated in red cell invasion. The cloned DNA hybridised to at least seven RsaI fragments of P. yoelii genomic DNA and to three DraI fragments of similar but not identical sequence. These results suggest that the gene encoding the 235-kDa rhoptry protein may be represented more than once in the P. yoelii genome.

GTP Cyclohydrolase II Structure and Mechanism.

GTP cyclohydrolase II converts GTP to 2,5-diamino-6-β-ribosyl-4(3H)-pyrimidinone 5′-phosphate, formate and pyrophosphate, the first step in riboflavin biosynthesis. The essential role of riboflavin in metabolism and the absence of GTP cyclohydrolase II in higher eukaryotes makes it a potential novel selective antimicrobial drug target. GTP cyclohydrolase II catalyzes a distinctive overall reaction from GTP cyclohydrolase I; the latter converts GTP to dihydroneopterin triphosphate, utilized in folate and tetrahydrobiopterin biosynthesis. The structure of GTP cyclohydrolase II determined at 1.54-Å resolution reveals both a different protein fold to GTP cyclohydrolase I and distinctive molecular recognition determinants for GTP; although in both enzymes there is a bound catalytic zinc. The GTP cyclohydrolase II·GMPCPP complex structure shows Arg128 interacting with the α-phosphonate, and thus in the case of GTP, Arg128 is positioned to act as the nucleophile for pyrophosphate release and formation of the proposed covalent guanylyl-GTP cyclohydrolase II intermediate. Tyr105 is identified as playing a key role in GTP ring opening; it is hydrogen-bonded to the zinc-activated water molecule, the latter being positioned for nucleophilic attack on the guanine C-8 atom. Although GTP cyclohydrolase I and GTP cyclohydrolase II both use a zinc ion for the GTP ring opening and formate release, different residues are utilized in each case to catalyze this reaction step.

Structural diversity of the major surface antigen of Plasmodium falciparum merozoites.

The structures of the major merozoite surface antigen of Plasmodium falciparum and the gene encoding it were indistinguishable for the Wellcome strain and the Thai clone T9/94 but different for clones T9/96, T9/98, and T9/101. The central portion of the gene is subject to the greatest variation in structure. The protein from all five lines was found to be posttranslationally modified by covalent addition of both carbohydrate and fatty acid.

Functional analysis of the GTPases EngA and YhbZ encoded by Salmonella typhimurium

The S. typhimurium genome encodes proteins, designated EngA and YhbZ, which have a high sequence identity with the GTPases EngA/Der and ObgE/CgtAE of Escherichia coli. The wild‐type activity of the E. coli proteins is essential for normal ribosome maturation and cell viability. In order to characterize the potential involvement of the Salmonella typhimurium EngA and YhbZ proteins in ribosome biology, we used high stringency affinity chromatography experiments to identify strongly binding ribosomal partner proteins. A combination of biochemical and microcalorimetric analysis was then used to characterize these protein:protein interactions and quantify nucleotide binding affinities. These experiments show that YhbZ specifically interacts with the pseudouridine synthase RluD (KD = 2 μM and 1:1 stoichiometry), and we show for the first time that EngA can interact with the ribosomal structural protein S7. Thermodynamic analysis shows both EngA and YhbZ bind GDP with a higher affinity than GTP (20‐fold difference for EngA and 3.8‐fold for YhbZ), and that the two nucleotide binding sites in EngA show a 5.3‐fold difference in affinity for GDP. We report a fluorescence assay for nucleotide binding to EngA and YhbZ, which is suitable for identifying inhibitors specific for this ligand‐binding site, which would potentially inhibit their biological functions. The interactions of YhbZ with ribosome structural proteins that we identify may demonstrate a previously unreported additional function for this class of GTPase: that of ensuring delivery of rRNA modifying enzymes to the appropriate region of the ribosome.

Structural characterization of Salmonella typhimurium YeaZ, an M22 O-sialoglycoprotein endopeptidase homolog

The Salmonella typhimurium “yeaZ” gene (StyeaZ) encodes an essential protein of unknown function (StYeaZ), which has previously been annotated as a putative homolog of the Pasteurella haemolytica M22 O‐sialoglycoprotein endopeptidase Gcp. YeaZ has also recently been reported as the first example of an RPF from a gram‐negative bacterial species. To further characterize the properties of StYeaZ and the widely occurring MK‐M22 family, we describe the purification, biochemical analysis, crystallization, and structure determination of StYeaZ. The crystal structure of StYeaZ reveals a classic two‐lobed actin‐like fold with structural features consistent with nucleotide binding. However, microcalorimetry experiments indicated that StYeaZ neither binds polyphosphates nor a wide range of nucleotides. Additionally, biochemical assays show that YeaZ is not an active O‐sialoglycoprotein endopeptidase, consistent with the lack of the critical zinc binding motif. We present a detailed comparison of YeaZ with available structural homologs, the first reported structural analysis of an MK‐M22 family member. The analysis indicates that StYeaZ has an unusual orientation of the A and B lobes which may require substantial relative movement or interaction with a partner protein in order to bind ligands. Comparison of the fold of YeaZ with that of a known RPF domain from a gram‐positive species shows significant structural differences and therefore potentially distinctive RPF mechanisms for these two bacterial classes. Proteins 2006.

Cloning and characterisation of the rabbit 5-HT1Dα and 5-HT1Dβ receptors

The genes encoding the rabbit 5HT1Dα and 5HT1Dβ receptors have been cloned. The deduced amino acid sequence of these receptors shows 91–92% amino acid sequence identity with their human homologues, and similar high sequence identity with homologues from other species. The receptors were transiently expressed in COS‐7 cells and exhibit a pharmacological profile closely resembling their human homologues, including a higher affinity of ketanserin for the 5‐HT1Dα subtype. However, sumatriptan had a lower affinity for both the rabbit receptors compared to their human counterparts. This may be accounted for by differences between the primary amino acid sequences of these species homologues.

Crystallographic studies of shikimate binding and induced conformational changes in Mycobacterium tuberculosis shikimate kinase

The X‐ray crystal structure of Mycobacterium tuberculosis shikimate kinase (SK) with bound shikimate and adenosine diphosphate (ADP) has been determined to a resolution of 2.15 Å. The binding of shikimate in a shikimate kinase crystal structure has not previously been reported. The substrate binds in a pocket lined with hydrophobic residues and interacts with several highly conserved charged residues including Asp34, Arg58, Glu61 and Arg136 which project into the cavity. Comparisons of our ternary SK–ADP–shikimate complex with an earlier binary SK–ADP complex show that conformational changes occur on shikimate binding with the substrate‐binding domain rotating by 10°. Detailed knowledge of shikimate binding is an important step in the design of inhibitors of SK, which have potential as novel anti‐tuberculosis agents.

Structures of S. aureus thymidylate kinase reveal an atypical active site configuration and an intermediate conformational state upon substrate binding

Methicillin‐resistant Staphylococcus aureus (MRSA) poses a major threat to human health, particularly through hospital acquired infection. The spread of MRSA means that novel targets are required to develop potential inhibitors to combat infections caused by such drug‐resistant bacteria. Thymidylate kinase (TMK) is attractive as an antibacterial target as it is essential for providing components for DNA synthesis. Here, we report crystal structures of unliganded and thymidylate‐bound forms of S. aureus thymidylate kinase (SaTMK). His‐tagged and untagged SaTMK crystallize with differing lattice packing and show variations in conformational states for unliganded and thymidylate (TMP) bound forms. In addition to open and closed forms of SaTMK, an intermediate conformation in TMP binding is observed, in which the site is partially closed. Analysis of these structures indicates a sequence of events upon TMP binding, with helix α3 shifting position initially, followed by movement of α2 to close the substrate site. In addition, we observe significant conformational differences in the TMP‐binding site in SaTMK as compared to available TMK structures from other bacterial species, Escherichia coli and Mycobacterium tuberculosis as well as human TMK. In SaTMK, Arg 48 is situated at the base of the TMP‐binding site, close to the thymine ring, whereas a cis‐proline occupies the equivalent position in other TMKs. The observed TMK structural differences mean that design of compounds highly specific for the S. aureus enzyme looks possible; such inhibitors could minimize the transfer of drug resistance between different bacterial species.