Ruiying Wu

Automation of protein purification for structural genomics.

AbstractA critical issue in structural genomics, and in structural biology in general, is the availability of high-quality samples. The additional challenge in structural genomics is the need to produce high numbers of proteins with low sequence similarities and poorly characterized or unknown properties. ‘Structural-biology-grade’ proteins must be generated in a quantity and quality suitable for structure determination experiments using X-ray crystallography or nuclear magnetic resonance (NMR). The choice of protein purification and handling procedures plays a critical role in obtaining high-quality protein samples. The purification procedure must yield a homogeneous protein and must be highly reproducible in order to supply milligram quantities of protein and/or its derivative containing marker atom(s). At the Midwest Center for Structural Genomics we have developed protocols for high-throughput protein purification. These protocols have been implemented on AKTA EXPLORER 3D and AKTA FPLC 3D workstations capable of performing multidimensional chromatography. The automated chromatography has been successfully applied to many soluble proteins of microbial origin. Various MCSG purification strategies, their implementation, and their success rates are discussed in this paper. abbreviations MCSG — Midwest Center for Structural Genomics; IMAC — immobilized metal affinity chromatography; TEV — tobacco etch virus; —β-ME —β-mercaptoethanol; DTT — dithiothreitol; EDTA — ethylenediaminetetraacetate; SDS-PAGE — polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate.

Activation of Inhibitors by Sortase Triggers Irreversible Modification of the Active Site

Sortases anchor surface proteins to the cell wall of Gram-positive pathogens through recognition of specific motif sequences. Loss of sortase leads to large reductions in virulence, which identifies sortase as a target for the development of antibacterials. By screening 135,625 small molecules for inhibition, we report here that aryl (β-amino)ethyl ketones inhibit sortase enzymes from staphylococci and bacilli. Inhibition of sortases occurs through an irreversible, covalent modification of their active site cysteine. Sortases specifically activate this class of molecules via β-elimination, generating a reactive olefin intermediate that covalently modifies the cysteine thiol. Analysis of the three-dimensional structure of Bacillus anthracis sortase B with and without inhibitor provides insights into the mechanism of inhibition and reveals binding pockets that can be exploited for drug discovery.

Large-scale evaluation of protein reductive methylation for improving protein crystallization

Large-scale evaluation of protein reductive methylation for improving protein crystallization

Insight into the sporulation phosphorelay: Crystal structure of the sensor domain of Bacillus subtilis histidine kinase, KinD†‡

The Bacillus subtilis KinD signal‐transducing histidine kinase is a part of the sporulation phosphorelay known to regulate important developmental decisions such as sporulation and biofilm formation. We have determined crystal structures of the extracytoplasmic sensing domain of KinD, which was copurified and crystallized with a pyruvate ligand. The structure of a ligand‐binding site mutant was also determined; it was copurified and crystallized with an acetate ligand. The structure of the KinD extracytoplasmic segment is similar to that of several other sensing domains of signal transduction proteins and is composed of tandem Per‐Arnt‐Sim (PAS)‐like domains. The KinD ligand‐binding site is located on the membrane distal PAS‐like domain and appears to be highly selective; a single mutation, R131A, abolishes pyruvate binding and the mutant binds acetate instead. Differential scanning fluorimetry, using a variety of monocarboxylic and dicarboxylic acids, identified pyruvate, propionate, and butyrate but not lactate, acetate, or malate as KinD ligands. A recent report found that malate induces biofilm formation in a KinD‐dependent manner. It was suggested that malate might induce a metabolic shift and increased secretion of the KinD ligand of unknown identity. The structure and binding assays now suggests that this ligand is pyruvate and/or other small monocarboxylic acids. In summary, this study gives a first insight into the identity of a molecular ligand for one of the five phosphorelay kinases of B. subtilis.

Crystal Structure of Bacillus anthracis Transpeptidase Enzyme CapD

Bacillus anthracis elaborates a poly-γ-d-glutamic acid capsule that protects bacilli from phagocytic killing during infection. The enzyme CapD generates amide bonds with peptidoglycan cross-bridges to anchor capsular material within the cell wall envelope of B. anthracis. The capsular biosynthetic pathway is essential for virulence during anthrax infections and can be targeted for anti-infective inhibition with small molecules. Here, we present the crystal structures of the γ-glutamyltranspeptidase CapD with and without α-l-Glu-l-Glu dipeptide, a non-hydrolyzable analog of poly-γ-d-glutamic acid, in the active site. Purified CapD displays transpeptidation activity in vitro, and its structure reveals an active site broadly accessible for poly-γ-glutamate binding and processing. Using structural and biochemical information, we derive a mechanistic model for CapD catalysis whereby Pro427, Gly428, and Gly429 activate the catalytic residue of the enzyme, Thr352, and stabilize an oxyanion hole via main chain amide hydrogen bonds.

Biochemical and Structural Analysis of an Eis Family Aminoglycoside Acetyltransferase from Bacillus anthracis.

Proteins from the enhanced intracellular survival (Eis) family are versatile acetyltransferases that acetylate amines at multiple positions of several aminoglycosides (AGs). Their upregulation confers drug resistance. Homologues of Eis are present in diverse bacteria, including many pathogens. Eis from Mycobacterium tuberculosis (Eis_Mtb) has been well characterized. In this study, we explored the AG specificity and catalytic efficiency of the Eis family protein from Bacillus anthracis (Eis_Ban). Kinetic analysis of specificity and catalytic efficiency of acetylation of six AGs indicates that Eis_Ban displays significant differences from Eis_Mtb in both substrate binding and catalytic efficiency. The number of acetylated amines was also different for several AGs, indicating a distinct regiospecificity of Eis_Ban. Furthermore, most recently identified inhibitors of Eis_Mtb did not inhibit Eis_Ban, underscoring the differences between these two enzymes. To explain these differences, we determined an Eis_Ban crystal structure. The comparison of the crystal structures of Eis_Ban and Eis_Mtb demonstrates that critical residues lining their respective substrate binding pockets differ substantially, explaining their distinct specificities. Our results suggest that acetyltransferases of the Eis family evolved divergently to garner distinct specificities while conserving catalytic efficiency, possibly to counter distinct chemical challenges. The unique specificity features of these enzymes can be utilized as tools for developing AGs with novel modifications and help guide specific AG treatments to avoid Eis-mediated resistance.

Crystal structure of Bacillus subtilis YdaF protein: a putative ribosomal N-acetyltransferase.

Comparative sequence analysis suggests that the ydaF gene encodes a protein (YdaF) that functions as an N-acetyltransferase, more specifically, a ribosomal N-acetyltransferase. Sequence analysis using basic local alignment search tool (BLAST) suggests that YdaF belongs to a large family of proteins (199 proteins found in 88 unique species of bacteria, archaea, and eukaryotes). YdaF also belongs to the COG1670, which includes the Escherichia coli RimL protein that is known to acetylate ribosomal protein L12. N-acetylation (NAT) has been found in all kingdoms. NAT enzymes catalyze the transfer of an acetyl group from acetyl-CoA (AcCoA) to a primary amino group. For example, NATs can acetylate the N-terminal {alpha}-amino group, the {epsilon}-amino group of lysine residues, aminoglycoside antibiotics, spermine/speridine, or arylalkylamines such as serotonin. The crystal structure of the alleged ribosomal NAT protein, YdaF, from Bacillus subtilis presented here was determined as a part of the Midwest Center for Structural Genomics. The structure maintains the conserved tertiary structure of other known NATs and a high sequence similarity in the presumed AcCoA binding pocket in spite of a very low overall level of sequence identity to other NATs of known structure.

Crystal Structures of the F and pSLT Plasmid TraJ N-Terminal Regions Reveal Similar Homodimeric PAS Folds with Functional Interchangeability

In the F family of conjugative plasmids, TraJ is an essential transcriptional activator of the tra operon that encodes most of the proteins required for conjugation. Here we report for the first time the X-ray crystal structures of the TraJ N-terminal domains from the prototypic F plasmid (TraJF11–130) and from the Salmonella virulence plasmid pSLT (TraJpSLT1–128). Both structures contain similar Per-ARNT-Sim (PAS) folds, which further homodimerize through the N-terminal helix and the structurally conserved β-sheet of the PAS fold from each protomer. Mutational analysis reveals that the observed dimeric interface is critical for TraJF transcriptional activation, indicating that dimerization of TraJ is required for its in vivo function. TraJ is specific in activating its cognate tra operon promoter; however, heterologous PAS domains from pSLT and R100 TraJ can functionally replace the TraJF PAS domain, suggesting that the allelic specificity of TraJ is solely mediated by the region C-terminal to the PAS domain.

Insights into PG-Binding, Conformational Change, and Dimerization of the OmpA C-terminal Domains from Salmonella enterica serovar Typhimurium and Borrelia burgdorferi

Salmonella enterica serovar Typhimurium can induce both humoral and cell‐mediated responses when establishing itself in the host. These responses are primarily stimulated against the lipopolysaccharide and major outer membrane (OM) proteins. OmpA is one of these major OM proteins. It comprises a N‐terminal eight‐stranded β‐barrel transmembrane domain and a C‐terminal domain (OmpACTD). The OmpACTD and its homologs are believed to bind to peptidoglycan (PG) within the periplasm, maintaining bacterial osmotic homeostasis and modulating the permeability and integrity of the OM. Here we present the first crystal structures of the OmpACTD from two pathogens: S. typhimurium (STOmpACTD) in open and closed forms and causative agent of Lyme Disease Borrelia burgdorferi (BbOmpACTD), in closed form. In the open form of STOmpACTD, an aspartate residue from a long β2‐α3 loop points into the binding pocket, suggesting that an anion group such as a carboxylate group from PG is favored at the binding site. In the closed form of STOmpACTD and in the structure of BbOmpACTD, a sulfate group from the crystallization buffer is tightly bound at the binding site. The differences between the closed and open forms of STOmpACTD, suggest a large conformational change that includes an extension of α3 helix by ordering a part of β2‐α3 loop. We propose that the sulfate anion observed in these structures mimics the carboxylate group of PG when bound to STOmpACTD suggesting PG‐anchoring mechanism. In addition, the binding of PG or a ligand mimic may enhance dimerization of STOmpACTD, or possibly that of full length STOmpA.

Molecular control of gene expression by Brucella BaaR, an IclR-family repressor

The Brucella abortus general stress response sigma factor, σE1, directly and indirectly regulates the transcription of dozens of genes that influence stress survival and host infection. Characterizing the functions of σE1 regulated genes therefore contributes to understanding of B. abortus physiology and infection biology. Transcription of the IclR family regulator, Bab2_0215, is indirectly activated by σE1 but its function remains undefined. We present a structural and functional characterization of Bab2_0215, which we have named Brucella adipic acid activated regulator (BaaR). BaaR adopts a classic IclR-family fold and directly regulates the transcription of two operons with predicted roles in carboxylic acid oxidation. BaaR binds two sites on chromosome II between baaR and a divergently transcribed hydratase/dehydrogenase (acaD2), and represses transcription. We identified three carboxylic acids (adipic acid tetradecanedioic acid, ε-aminocaproic acid) and a lactone (ε-caprolactone) that enhance transcription from the baaR and acaD2 promoters. However, neither the activating acids nor caprolactone enhance transcription by binding directly to BaaR. Induction of baaR transcription by adipic acid requires the gene bab2_0213, which encodes a major facilitator superfamily transporter, suggesting that Bab2_0213 transports adipic acid across the inner membrane. We conclude that a set of structurally related organic molecules activate transcription of genes repressed by BaaR. Our study provides molecular-level understanding of a gene expression program regulated downstream of σE1.