Structure, function and selective inhibition of bacterial acetyl-coa carboxylase.

Acetyl-CoA carboxylase (ACC) catalyses the first committed step in fatty acid biosynthesis: a metabolic pathway required for several important biological processes including the synthesis and maintenance of cellular membranes. ACC employs a covalently attached biotin moiety to bind a carboxyl anion and then transfer it to acetyl-CoA, yielding malonyl-CoA. These activities occur at two different subsites: the biotin carboxylase (BC) and carboxyltransferase (CT). Structural biology, together with small molecule inhibitor studies, has provided new insights into the molecular mechanisms that govern ACC catalysis, specifically the BC and CT subunits. Here, we review these recent findings and highlight key differences between the bacterial and eukaryotic isozymes with a view to establish those features that provide an opportunity for selective inhibition. Especially important are examples of highly selective small molecule inhibitors capable of differentiating between ACCs from different phyla. The implications for early stage antibiotic discovery projects, stemming from these studies, are discussed.

TB perspectives among a sample of Mexicans in the United States: results from an ethnographic study.

OBJECTIVE: A study was conducted to describe the sociocultural aspects of tuberculosis (TB) among Mexicans in the U.S. and to provide TB programs with practical recommendations for serving this population. METHODS: In-depth, structured, open-ended interviews were conducted with 50 persons from Mexico living in the U.S. Local bilingual, bicultural researchers conducted the interviews with respondents recruited from TB clinics and surrounding communities. Both qualitative and quantitative strategies were used to analyze the data. RESULTS: We found diverse TB perceptions and attitudes, but few were associated with specific participant characteristics. We detected widespread misperceptions about TB transmission and low perceptions of risk. Anticipated TB stigma among those with no history of disease was qualitatively greater than reported stigma among those who had TB disease. We also detected missed opportunities for TB education. Reported barriers to care included lack of transportation, limited clinic hours, cost of services, inconvenient clinic location, and communication problems with staff. CONCLUSIONS: To address the diverse needs of Mexican-born clients, we recommend that TB programs provide culturally-appropriate, patient-centered care. We suggest several strategies aimed at raising risk awareness and reducing stigma. Finally, we encourage programs to facilitate access by providing language-appropriate services, extending clinic hours, and facilitating transportation.

An asymmetric structure of the Bacillus subtilis replication terminator protein in complex with DNA.

In Bacillus subtilis, the termination of DNA replication via polar fork arrest is effected by a specific protein:DNA complex formed between the replication terminator protein (RTP) and DNA terminator sites. We report the crystal structure of a replication terminator protein homologue (RTP.C110S) of B. subtilis in complex with the high affinity component of one of its cognate DNA termination sites, known as the TerI B-site, refined at 2.5 A resolution. The 21 bp RTP:DNA complex displays marked structural asymmetry in both the homodimeric protein and the DNA. This is in contrast to the previously reported complex formed with a symmetrical TerI B-site homologue. The induced asymmetry is consistent with the complex's solution properties as determined using NMR spectroscopy. Concomitant with this asymmetry is variation in the protein:DNA binding pattern for each of the subunits of the RTP homodimer. It is proposed that the asymmetric "wing" positions, as well as other asymmetrical features of the RTP:DNA complex, are critical for the cooperative binding that underlies the mechanism of polar fork arrest at the complete terminator site.

Crystallization and preliminary X-ray diffraction analysis of the Bacillus subtilis replication termination protein in complex with the 37-base-pair TerI-binding site.

The replication terminator protein (RTP) of Bacillus subtilis binds to specific DNA sequences that halt the progression of the replisome in a polar manner. These terminator complexes flank a defined region of the chromosome into which they allow replication forks to enter but not exit. Forcing the fusion of replication forks in a specific zone is thought to allow the coordination of post-replicative processes. The functional terminator complex comprises two homodimers each of 29 kDa bound to overlapping binding sites. A preparation of RTP and a 37-base-pair TerI sequence (comprising two binding sites for RTP) has been purified and crystallized. A data set to 3.9 A resolution with 97.0% completeness and an R(sym) of 12% was collected from a single flash-cooled crystal using synchrotron radiation. The diffraction data are consistent with space group P622, with unit-cell parameters a = b = 118.8, c = 142.6 A.

Formation of an alphaCP1-KH3 complex with UC-rich RNA.

The alphaCP family of proteins [also known as poly(C)-binding or heterogeneous nuclear ribonucleoprotein E proteins] are involved in the regulation of messenger RNA (mRNA) stability and translational efficiency. They bind via their triple heterologous nuclear ribonucleoprotein K homology (KH) domain structures to C-rich mRNA, and are thought to interact with other mRNA-binding proteins as well as provide direct nuclease protection. In particular, alphaCP1 and alphaCP2 have been shown to bind to a specific region of androgen receptor (AR) mRNA, resulting in its increased stability. The roles of each of the KH motifs in the binding affinity and the specificity is not yet understood. We report the beginning of a systematic study of each of the alphaCP KH domains, with the cloning and expression of alphaCP1-KH2 and alphaCP1-KH3. We report the ability of alphaCP1-KH3, but not alphaCP1-KH2, to bind the target AR mRNA sequence using an RNA electrophoretic mobility gel shift assay. We also report the preparation of an alphaCP1-KH3/AR mRNA complex for structural studies. (1)H-(15)N heteronuclear single quantum correlation NMR spectra of (15)N-labelled alphaCP1-KH3 verified the integrity and good solution behaviour of the purified domain. The titration of the 11-nucleotide RNA target sequence from AR mRNA resulted in a rearrangement of the (1)H-(15)N correlations, demonstrating the complete binding of the protein to form a homogeneous protein/RNA complex suitable for future structural studies.

Structure and RNA binding of the third KH domain of poly(C)-binding protein 1.

Poly(C)-binding proteins (CPs) are important regulators of mRNA stability and translational regulation. They recognize C-rich RNA through their triple KH (hn RNP K homology) domain structures and are thought to carry out their function though direct protection of mRNA sites as well as through interactions with other RNA-binding proteins. We report the crystallographically derived structure of the third domain of alphaCP1 to 2.1 A resolution. alphaCP1-KH3 assumes a classical type I KH domain fold with a triple-stranded beta-sheet held against a three-helix cluster in a betaalphaalphabetabetaalpha configuration. Its binding affinity to an RNA sequence from the 3'-untranslated region (3'-UTR) of androgen receptor mRNA was determined using surface plasmon resonance, giving a K(d) of 4.37 microM, which is indicative of intermediate binding. A model of alphaCP1-KH3 with poly(C)-RNA was generated by homology to a recently reported RNA-bound KH domain structure and suggests the molecular basis for oligonucleotide binding and poly(C)-RNA specificity.

Contact tracing: comparing the approaches for sexually transmitted diseases and tuberculosis.

SETTING: Literature review for the process of contact tracing for sexually transmitted diseases (STD) and for tuberculosis (TB), focusing on articles that report results of studies or commentary. OBJECTIVE: To compare and contrast contact tracing in order to highlight emerging commonalities. DESIGN: A descriptive review, based on Medline search with augmentation from other published and unpublished sources. RESULTS: Contact tracing for STD and TB have some obvious differences because of differing routes of transmission, differing sensibilities required to work with the affected populations, a different potential for anonymous contacts, and a major difference in the epidemiologic value of biomarkers. Nonetheless, the convergence of these processes on disadvantaged populations where drug use and sexual activity are important social factors has engendered an increasing similarity. CONCLUSION: A broadened approach to both, with greater attention to how ancillary contacts and associates may be of use in interrupting deeply embedded endemic disease transmission, deserves further study. Some newer approaches in the use of network-informed methods to elicit contacts and investigate the community dynamics of transmission may be of particular value in TB case investigation. These strategies will be enhanced by the availability of DNA fingerprinting, a powerful biomarker of recent Mycobacterium tuberculosis transmission and case association (a technology not available for STD contact tracing).

Using the CDC framework for program evaluation in public health to assess tuberculosis contact investigation programs.

SETTING: In Massachusetts, despite the efforts of state and local health department tuberculosis (TB) programs, the rates of contact testing and follow-up remain below the state and national objectives. Changes in contact investigation practices are therefore needed to achieve these objectives. OBJECTIVE: To develop contact investigation self-evaluation tools in accordance with the Centers for Disease Control and Prevention's (CDC) Framework for Program Evaluation in Public Health. These tools will be used to assess state and local level contact investigation practices. DESIGN: The self-evaluation tools were developed using the CDC's framework and pilot-tested by public health nurse case managers in five city health departments. The tools were revised according to feedback received from the nurses. RESULTS: The Massachusetts TB Division conducted three of the six steps of the CDC's framework. Stakeholders of the evaluation were identified and engaged, logic models were created describing state and local TB program components, and self-evaluation tools were developed. CONCLUSION: The CDC's framework provided a useful methodology for beginning the assessment process for evaluating TB contact investigation programs. When the contact investigation self-evaluation tools are implemented statewide, the findings will be used to target areas in need of improvement and develop strategies to make noteworthy changes.

Factors associated with identifying tuberculosis contacts.

SETTING: Little is known about why some tuberculosis (TB) patients identify few or even no contacts. OBJECTIVES: To describe patient perceptions of the contact investigation interview and determine potential factors associated with identifying TB contacts. DESIGN: A total of 13 focus groups were conducted: 10 groups with previously smear-positive pulmonary TB patients born in the United States or Mexico, and three with program staff to discuss attitudes toward and perceptions of the contact investigation interview. Patients were recruited into separate groups by country of birth and number of contacts identified. RESULTS: The data indicated that patients-even those who identified few contacts-overwhelmingly reported identifying contacts easily and willingly. Understanding the purpose of the contact investigation and seriousness of TB facilitated naming contacts, while miscommunication and misconceptions about TB hindered the process. Patients felt strongly about informing their contacts before the health department contacted them. Staff respondents reported that education and effective communication were critical during contact investigation interviewing. CONCLUSION: Data indicated that patients, including those identifying few contacts, reported wanting to name their contacts. However, misconceptions may affect their understanding of who their contacts are, and hence the quantity and quality of the contacts identified. These findings underscore the need for effective communication and education.

The impact of single cysteine residue mutations on the replication terminator protein.

We report the structural and biophysical consequences of cysteine substitutions in the DNA-binding replication terminator protein (RTP) of Bacillus subtilis, that resulted in an optimised RTP mutant suitable for structural studies. The cysteine residue 110 was replaced with alanine, valine or serine. Protein secondary structure and stability (using circular dichroism spectropolarimetry), self-association (using analytical ultracentrifugation), and DNA-binding measurements revealed RTP.C110S to be the most similar mutant to wild-type RTP. The C110A and C110V.RTP mutants were less soluble, less stable and showed lower DNA-binding affinity. The structure of RTP.C110S, solved to 2.5A resolution using crystallographic methods, showed no major structural perturbation due to the mutation. Heteronuclear NMR spectroscopic studies revealed subtle differences in the electronic environment about the site of mutation. The study demonstrates the suitability of serine as a substitute for cysteine in RTP and the high sensitivity of protein behaviour to single amino acid substitutions.

The crystal structures of glutathione S-transferases isozymes 1-3 and 1-4 from Anopheles dirus species B.

Glutathione S-transferases (GSTs) are dimeric proteins that play an important role in cellular detoxification. Four GSTs from the mosquito Anopheles dirus species B (Ad), an important malaria vector in South East Asia, are produced by alternate splicing of a single transcription product and were previously shown to have detoxifying activity towards pesticides such as DDT. We have determined the crystal structures for two of these alternatively spliced proteins, AdGST1-3 (complexed with glutathione) and AdGST1-4 (apo form), at 1.75 and 2.45 A resolution, respectively. These GST isozymes show differences from the related GST from the Australian sheep blowfly Lucilia cuprina; in particular, the presence of a C-terminal helix forming part of the active site. This helix causes the active site of the Anopheles GSTs to be enclosed. The glutathione-binding helix alpha2 and flanking residues are disordered in the AdGST1-4 (apo) structure, yet ordered in the AdGST1-3 (GSH-bound) structure, suggesting that insect GSTs operate with an induced fit mechanism similar to that found in the plant phi- and human pi-class GSTs. Despite the high overall sequence identities, the active site residues of AdGST1-4 and AdGST1-3 have different conformations.

Structural biology and its applications to the health sciences.

Part of the decipherment of genomic information lies in understanding the structure and function of the protein products of these genes. Protein structure is of further importance because of the molecular basis of many diseases. Structural biology is the field of research focusing on the experimental determination of the structure of biological molecules. We review the field of structural biology and its application to medical research and drug discovery, and describe the structural results recently obtained in our laboratory for the detoxifying enzyme glutathione S-transferase from the Asian mosquito Anopheles dirus species B, an important malaria vector. These enzymes have detoxifying activity toward pesticides and thus contribute to pesticide resistance in insects.

Large conformational changes of the epsilon subunit in the bacterial F1F0 ATP synthase provide a ratchet action to regulate this rotary motor enzyme.

The F(1)F(0) ATP synthase is the smallest motor enzyme known. Previous studies had established that the central stalk, made of the gamma and epsilon subunits in the F(1) part and c subunit ring in the F(0) part, rotates relative to a stator composed of alpha(3)beta(3)deltaab(2) during ATP hydrolysis and synthesis. How this rotation is regulated has been less clear. Here, we show that the epsilon subunit plays a key role by acting as a switch of this motor. Two different arrangements of the epsilon subunit have been visualized recently. The first has been observed in beef heart mitochondrial F(1)-ATPase where the C-terminal portion is arranged as a two-alpha-helix hairpin structure that extends away from the alpha(3)beta(3) region, and toward the position of the c subunit ring in the intact F(1)F(0). The second arrangement was observed in a structure determination of a complex of the gamma and epsilon subunits of the Escherichia coli F(1)-ATPase. In this, the two C-terminal helices are apart and extend along the gamma to interact with the alpha and beta subunits in the intact complex. We have been able to trap these two arrangements by cross-linking after introducing appropriate Cys residues in E. coli F(1)F(0), confirming that both conformations of the epsilon subunit exist in the enzyme complex. With the C-terminal domain of epsilon toward the F(0), ATP hydrolysis is activated, but the enzyme is fully coupled in both ATP hydrolysis and synthesis. With the C-terminal domain toward the F(1) part, ATP hydrolysis is inhibited and yet the enzyme is fully functional in ATP synthesis; i.e., it works in one direction only. These results help explain the inhibitory action of the epsilon subunit in the F(1)F(0) complex and argue for a ratchet function of this subunit.

Crystallization of two glutathione S-transferases from an unusual gene family.

Two glutathione S-transferase isozymes from the mosquito Anopheles dirus (AdGST1-3 and AdGST1-4) from an alternately spliced gene family have been expressed, purified and crystallized. The isozymes share an N-terminal domain derived from a single exon and C-terminal domains from unique exons. Despite the high level of sequence identity (64% overall), the two isozymes crystallize in different space groups, the 1-3 isozyme in P3(1)21 or P3(2)21 (unit-cell parameters a = 49.9, c = 271.8 A at 100 K) and the 1-4 isozyme in P4(1) or P4(3) (unit-cell parameters a = 87.8, c = 166.1 at 100 K). Determination of these structures will advance our understanding of how these enzymes inactivate pesticides and the structural consequences of alternate splicing.

Crystallization and preliminary X-ray analysis of the complex of the epsilon-subunit and the central domain of the gamma-subunit from the Escherichia coli ATP synthase.

A complex of the epsilon-subunit and the central domain of the gamma-subunit from the ATP synthase of Escherichia coli has been purified and crystallized and preliminary X-ray analysis has been carried out. The crystals belong to space group C222(1), with unit-cell parameters a = 76.7, b = 176.1, c = 67.1 A (at 100 K). Determination of the structure of this protein complex promises to greatly improve the understanding of energy coupling between the F(0) and F(1) sectors within the enzyme complex.

Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest.

The coordinated termination of DNA replication is an important step in the life cycle of bacteria with circular chromosomes, but has only been defined at a molecular level in two systems to date. Here we report the structure of an engineered replication terminator protein (RTP) of Bacillus subtilis in complex with a 21 base pair DNA by X-ray crystallography at 2.5 A resolution. We also use NMR spectroscopic titration techniques. This work reveals a novel DNA interaction involving a dimeric 'winged helix' domain protein that differs from predictions. While the two recognition helices of RTP are in close contact with the B-form DNA major grooves, the 'wings' and N-termini of RTP do not form intimate contacts with the DNA. This structure provides insight into the molecular basis of polar replication fork arrest based on a model of cooperative binding and differential binding affinities of RTP to the two adjacent binding sites in the complete terminator.

Crystal structure of auracyanin, a "blue" copper protein from the green thermophilic photosynthetic bacterium Chloroflexus aurantiacus.

Auracyanin B, one of two similar blue copper proteins produced by the thermophilic green non-sulfur photosynthetic bacterium Chloroflexus aurantiacus, crystallizes in space group P6(4)22 (a=b=115.7 A, c=54.6 A). The structure was solved using multiple wavelength anomalous dispersion data recorded about the CuK absorption edge, and was refined at 1.55 A resolution. The molecular model comprises 139 amino acid residues, one Cu, 247 H(2)O molecules, one Cl(-) and two SO(4)(2-). The final residual and estimated standard uncertainties are R=0.198, ESU=0.076 A for atomic coordinates and ESU=0.05 A for Cu---ligand bond lengths, respectively. The auracyanin B molecule has a standard cupredoxin fold. With the exception of an additional N-terminal strand, the molecule is very similar to that of the bacterial cupredoxin, azurin. As in other cupredoxins, one of the Cu ligands lies on strand 4 of the polypeptide, and the other three lie along a large loop between strands 7 and 8. The Cu site geometry is discussed with reference to the amino acid spacing between the latter three ligands. The crystallographically characterized Cu-binding domain of auracyanin B is probably tethered to the periplasmic side of the cytoplasmic membrane by an N-terminal tail that exhibits significant sequence identity with known tethers in several other membrane-associated electron-transfer proteins.

Outcomes of contact investigations of infectious tuberculosis patients.

The objective of this study was to describe outcomes of tuberculosis (TB) contact investigations, factors correlated with those outcomes, and current successes and ways to improve TB contact investigations. We abstracted clinic records of a representative U.S. urban sample of 1,080 pulmonary, sputum-smear(+) TB patients reported to CDC July 1996 through June 1997 and the cohort of their 6,225 close contacts. We found a median of four close contacts per patient. Fewer contacts were identified for homeless patients. A visit to the patient's residence resulted in two additional (especially child) contacts identified. Eighty-eight percent of eligible contacts received tuberculin skin tests (TSTs). Recording the last exposure date to the infectious patient facilitated follow-up TST provision. Thirty-six percent of contacts were TST(+). Household contacts and contacts to highly smear(+) or cavitary TB patients were most likely to be TST(+). Seventy-four percent of TST(+) contacts started treatment for latent TB infection (LTBI), of whom 56% completed. Sites using public health nurses (PHNs) started more high-risk TST(-) contacts on presumptive treatment for LTBI. Using directly observed treatment (DOT) increased the likelihood of treatment completion. We documented outcomes of contact investigation efforts by urban TB programs. We identified several successful practices, as well as suggestions for improvements, that will help TB programs target policies and procedures to enhance contact investigation effectiveness.

Structure of the gamma-epsilon complex of ATP synthase.

ATP synthases (F(1)F(o)-ATPases) use energy released by the movement of protons down a transmembrane electrochemical gradient to drive the synthesis of ATP, the universal biological energy currency. Proton flow through F(o) drives rotation of a ring of c-subunits and a complex of the gamma and epsilon-subunits, causing cyclical conformational changes in F(1) that are required for catalysis. The crystal structure of a large portion of F(1) has been resolved. However, the structure of the central portion of the enzyme, through which conformational changes in F(o) are communicated to F(1), has until now remained elusive. Here we report the crystal structure of a complex of the epsilon-subunit and the central domain of the gamma-subunit refined at 2.1 A resolution. The structure reveals how rotation of these subunits causes large conformational changes in F(1), and thereby provides new insights into energy coupling between F(o) and F(1).

Macromolecular crystallography as a tool for investigating drug, enzyme and receptor interactions.

1. Protein crystallography is an essential tool for the discovery and investigation of pharmacological interactions at the molecular level. It allows investigators to directly visualize the three-dimensional structures of proteins, including enzymes, receptors and hormones. 2. Increasingly, knowledge of these interactions is being used in the drug-discovery process. This is popularly called structure-based drug design. The desired drug could be an enzyme inhibitor or an agonist that mimics endogenous transmitters or hormones. 3. Once the 3-D structure of a pharmacologically relevant target is known, computational processes can be used to search databases of compounds to identify ones that may interact strongly with the target. Lead compounds can be improved using the 3-D structure of the complex of the lead compound and its biological target. 4. The present review describes the processes involved in the determination of a structure by means of protein crystallography and the use of structures in the drug-discovery process. A number of successful examples of structure-based drug design are described. The limitations of the techniques are discussed.