C.E. Nichols

A procedure for setting up high-throughput nanolitre crystallization experiments. II. Crystallization results

An initial tranche of results from day-to-day use of a robotic system for setting up 100 nl-scale vapour-diffusion sitting-drop protein crystallizations has been surveyed. The database of over 50 unrelated samples represents a snapshot of projects currently at the stage of crystallization trials in Oxford research groups and as such encompasses a broad range of proteins. The results indicate that the nanolitre-scale methodology consistently identifies more crystallization conditions than traditional hand-pipetting-style methods; however, in a number of cases successful scale-up is then problematic. Crystals grown in the initial 100 nl-scale drops have in the majority of cases allowed useful characterization of x-ray diffraction, either in-house or at synchrotron beamlines. For a significant number of projects, full x-ray diffraction data sets have been collected to 3 A resolution or better (either in-house or at the synchrotron) from crystals grown at the 100 nl scale. To date, five structures have been determined by molecular replacement directly from such data and a further three from scale-up of conditions established at the nanolitre scale.

The structure of the negative transcriptional regulator NmrA reveals a structural superfamily which includes the short‐chain dehydrogenase/reductases

NmrA is a negative transcriptional regulator involved in the post‐translational modulation of the GATA‐type transcription factor AreA, forming part of a system controlling nitrogen metabolite repression in various fungi. X‐ray structures of two NmrA crystal forms, both to 1.8 Å resolution, show NmrA consists of two domains, including a Rossmann fold. NmrA shows an unexpected similarity to the short‐chain dehydrogenase/reductase (SDR) family, with the closest relationship to UDP‐galactose 4‐epimerase. We show that NAD binds to NmrA, a previously unreported nucleotide binding property for this protein. NmrA is unlikely to be an active dehydrogenase, however, as the conserved catalytic tyrosine in SDRs is absent in NmrA, and thus the nucleotide binding to NmrA could have a regulatory function. Our results suggest that other transcription factors possess the SDR fold with functions including RNA binding. The SDR fold appears to have been adapted for other roles including non‐enzymatic control functions such as transcriptional regulation and is likely to be more widespread than previously recognized.

Crystal structures of Zidovudine- or Lamivudine-resistant human immunodeficiency virus type 1 reverse transcriptases containing mutations at codons 41, 184, and 215.

ABSTRACT Six structures of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) containing combinations of resistance mutations for zidovudine (AZT) (M41L and T215Y) or lamivudine (M184V) have been determined as inhibitor complexes. Minimal conformational changes in the polymerase or nonnucleoside RT inhibitor sites compared to the mutant RTMC (D67N, K70R, T215F, and K219N) are observed, indicating that such changes may occur only with certain combinations of mutations. Model building M41L and T215Y into HIV-1 RT-DNA and docking in ATP that is utilized in the pyrophosphorolysis reaction for AZT resistance indicates that some conformational rearrangement appears necessary in RT for ATP to interact simultaneously with the M41L and T215Y mutations.

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.

Characterization of Salmonella typhimurium YegS, a putative lipid kinase homologous to eukaryotic sphingosine and diacylglycerol kinases

Salmonella typhimurium YegS is a protein conserved in many prokaryotes. Although the function of YegS is not definitively known, it has been annotated as a potential diacylglycerol or sphingosine kinase based on sequence similarity with eukaryotic enzymes of known function. To further characterize YegS, we report its purification, biochemical analysis, crystallization, and structure determination. The crystal structure of YegS reveals a two‐domain fold related to bacterial polyphosphate/ATP NAD kinases, comprising a central cleft between an N‐terminal α/β domain and a C‐terminal two‐layer β‐sandwich domain; conserved structural features are consistent with nucleotide binding within the cleft. The N‐terminal and C‐ terminal domains of YegS are however counter‐rotated, relative to the polyphosphate/ATP NAD kinase archetype, such that the potential nucleotide binding site is blocked. There are also two Ca2+ binding sites and two hydrophobic clefts, one in each domain of YegS. Analysis of mutagenesis data from eukaryotic homologues of YegS suggest that the N‐terminal cleft may bind activating lipids while the C‐terminal cleft may bind the lipid substrate. Microcalorimetry experiments showed interaction between recombinant YegS and Mg2+, Ca2+, and Mn2+ ions, with a weaker interaction also observed with polyphosphates and ATP. However, biochemical assays showed that recombinant YegS is endogenously neither an active diacylglycerol nor sphingosine kinase. Thus although the bioinformatics analysis and structure of YegS indicate that many of the ligand recognition determinants for lipid kinase activity are present, the absence of such activity may be due to specificity for a different lipid substrate or the requirement for activation by an, as yet, undetermined mechanism. In this regard the specific interaction of YegS with the periplasmic chaperone OmpH, which we demonstrate from pulldown experiments, may be of significance. Such an interaction suggests that YegS can be translocated to the periplasm and directed to the outer‐membrane, an environment that may be required for enzyme activity. Proteins 2007.

Structure of the ribosomal interacting GTPase YjeQ from the enterobacterial species Salmonella typhimurium

The YjeQ class of P-loop GTPases assist in ribosome biogenesis and also bind to the 30S subunit of mature ribosomes. YjeQ ribosomal binding is GTP-dependent and thought to specifically direct protein synthesis, although the nature of the upstream signal causing this event in vivo is as yet unknown. The attenuating effect of YjeQ mutants on bacterial growth in Escherichia coli makes it a potential target for novel antimicrobial agents. In order to further explore the structure and function of YjeQ, the isolation, crystallization and structure determination of YjeQ from the enterobacterial species Salmonella typhimurium (StYjeQ) is reported. Whilst the overall StYjeQ fold is similar to those of the previously reported Thematoga maritima and Bacillus subtilis orthologues, particularly the GTPase domain, there are larger differences in the three OB folds. Although the zinc-finger secondary structure is conserved, significant sequence differences alter the nature of the external surface in each case and may reflect varying signalling pathways. Therefore, it may be easier to develop YjeQ-specific inhibitors that target the N- and C-terminal regions, disrupting the metabolic connectivity rather than the GTPase activity. The availability of coordinates for StYjeQ will provide a significantly improved basis for threading Gram-negative orthologue sequences and in silico compound-screening studies, with the potential for the development of species-selective drugs.

Crystal structures of Staphylococcus aureus type I dehydroquinase from enzyme turnover experiments.

Introduction. Staphylococcus aureus is a major nosocomial pathogen which causes a range of diseases, including endocarditis, osteomyelitis, pneumonia, toxic-shock syndrome, food poisoning, carbuncles, and boils. The acquisition and spread of a variety of resistance genes has led to the emergence of endemic S. aureus populations resistant to all current antibiotics, even vancomycin. A promising approach in the search for new antimicrobial drugs to combat resistant bacteria is the structural characterization of biosynthetic enzymes common to bacteria and microbial eukaryotes, with a view to rational design. Shikimate pathway enzymes make excellent potential targets as they have no mammalian homologues and pathogenic bacteria mutant in this pathway are attenuated for virulence. The enzyme 3-dehydroquinate dehydratase (DHQase; EC 4.2.1.10) catalyses the third step of the shikimate pathway, the dehydration of 3-dehydroquinate (DHQ) to 3-dehydroshikimate (DHS), a step also common to the catabolic quinate pathway. DHQases can be divided into two classes according to mechanism of action, stereochemistry, overall structure, and sequence homology. Type I enzymes, which are generally only involved in biosynthesis, occur either as homodimers of subunit Mw 27 KDa or as one domain of the multifunctional AROM complex. Type I DHQases catalyse a syn elimination of water with the loss of the pro-R hydrogen from C2 via an imine intermediate. By contrast, type II enzymes occur as homo-dodecamers with subunit Mw 16 KDa, catalyse an anti elimination reaction with the loss of the more acidic axial pro-S hydrogen from C2, via an enolate intermediate and may function anabolically in the shikimate pathway and/or catabolically in the quinic acid pathway. Structures have been solved for both type-I and type-II enzymes. Structures of Salmonella typhi type-I DHQase have a typical ( / )8 (TIM barrel) fold, with an additional “N”terminal anti-parallel -sheet region blocking substrate ingress from one side and a flexible loop region that folds in to close off the other end when substrate binds. In this paper, we report the crystallization and structure determination of an additional type-I DHQase from S. aureus (SaDHQase) in unliganded and liganded forms, both resulting from enzyme turnover experiments. Addition of substrate was essential for growing usable crystals. Different substrate concentrations yielded both an apo enzyme and a binary product complex with DHS still covalently linked to the catalytic lysine (Lys160) allowing a comparative analysis of the active site structure and the conformational changes associated with the bound ligand.

Biophysical and kinetic analysis of wild-type and site-directed mutants of the isolated and native dehydroquinate synthase domain of the AROM protein

Dehydroquinate synthase (DHQS) is the N‐terminal domain of the pentafunctional AROM protein that catalyses steps 2 to 7 in the shikimate pathway in microbial eukaryotes. DHQS converts 3‐deoxy‐D‐arabino‐heptulosonate‐7‐phosphate (DAHP) to dehydroquinate in a reaction that includes alcohol oxidation, phosphate β‐elimination, carbonyl reduction, ring opening, and intramolecular aldol condensation. Kinetic analysis of the isolated DHQS domains with the AROM protein showed that for the substrate DAHP the difference in Km is less than a factor of 3, that the turnover numbers differed by 24%, and that the Km for NAD+ differs by a factor of 3. Isothermal titration calorimetry revealed that a second (inhibitory) site for divalent metal binding has an approximately 4000‐fold increase in KD compared to the catalytic binding site. Inhibitor studies have suggested the enzyme could act as a simple oxidoreductase with several of the reactions occurring spontaneously, whereas structural studies have implied that DHQS participates in all steps of the reaction. Analysis of site‐directed mutants experimentally test and support this latter hypothesis. Differential scanning calorimetry, circular dichroism spectroscopy, and molecular exclusion chromatography demonstrate that the mutant DHQS retain their secondary and quaternary structures and their ligand binding capacity. R130K has a 135‐fold reduction in specific activity with DAHP and a greater than 1100‐fold decrease in the kcat/Km ratio, whereas R130A is inactive.

Expression, purification and crystallization of Aspergillus nidulans NmrA, a negative regulatory protein involved in nitrogen-metabolite repression

The NmrA repressor protein of Aspergillus nidulans was overproduced in Escherichia coli and purified to homogeneity. Gel-exclusion chromatography showed that NmrA was monomeric in solution under the buffer conditions used. The protein was crystallized in three forms, belonging to trigonal, monoclinic and hexagonal space groups. Two of these crystal forms (A and B) diffract to high resolution and thus appear suitable for structure determination. Crystal form A belongs to space group P3((1))21, with unit-cell parameters a = b = 76.8, c = 104.9 A. Crystal form B belongs to space group C2, with unit-cell parameters a = 148.8, b = 64.3, c = 110.2 A, beta = 121.8 degrees.

Identification of many crystal forms of Aspergillus nidulans dehydroquinate synthase

Extensive crystallization trials of Aspergillus nidulans dehydroquinate synthase, a potential novel target for antimicrobial drugs, in complexes with different ligands have resulted in the identification of nine crystal forms. Crystals of unliganded DHQS, binary complexes with either the substrate analogue, carbaphosphonate or the cofactor NADH, as well as the ternary DHQS-carbaphosphonate-cofactor complex, were obtained. The ternary complex crystallizes from ammonium sulfate and CoCl(2) in space group P2(1)2(1)2, with unit-cell parameters a = 133.8, b = 86.6, c = 74.9 A. The binary carbaphosphonate complex crystallizes from PEG 6000 in space group P2(1)2(1)2(1), with a = 70.0, b = 64.0, c = 197.6 A, and the binary cofactor complex crystallizes from PEG 3350 and sodium potassium tartrate in space group P2(1), with a = 83.7, b = 70.4, c = 144.3 A, beta = 89.2 degrees. DHQS in the absence of ligands crystallizes in space group P2(1), with a = 41.0, b = 68.9, c = 137.7 A, beta = 94.8 degrees. Each of these crystal forms are suitable for high-resolution structure determination. Structures of a range of DHQS-ligand complexes will be of value in the structure-based design of novel antimicrobial drugs.