Mutations in the skeletal muscle alpha-actin gene in patients with actin myopathy and nemaline myopathy.

Muscle contraction results from the force generated between the thin filament protein actin and the thick filament protein myosin, which causes the thick and thin muscle filaments to slide past each other. There are skeletal muscle, cardiac muscle, smooth muscle and non-muscle isoforms of both actin and myosin. Inherited diseases in humans have been associated with defects in cardiac actin (dilated cardiomyopathy and hypertrophic cardiomyopathy), cardiac myosin (hypertrophic cardiomyopathy) and non-muscle myosin (deafness). Here we report that mutations in the human skeletal muscle alpha-actin gene (ACTA1) are associated with two different muscle diseases, 'congenital myopathy with excess of thin myofilaments' (actin myopathy) and nemaline myopathy. Both diseases are characterized by structural abnormalities of the muscle fibres and variable degrees of muscle weakness. We have identified 15 different missense mutations resulting in 14 different amino acid changes. The missense mutations in ACTA1 are distributed throughout all six coding exons, and some involve known functional domains of actin. Approximately half of the patients died within their first year, but two female patients have survived into their thirties and have children. We identified dominant mutations in all but 1 of 14 families, with the missense mutations being single and heterozygous. The only family showing dominant inheritance comprised a 33-year-old affected mother and her two affected and two unaffected children. In another family, the clinically unaffected father is a somatic mosaic for the mutation seen in both of his affected children. We identified recessive mutations in one family in which the two affected siblings had heterozygous mutations in two different exons, one paternally and the other maternally inherited. We also identified de novo mutations in seven sporadic probands for which it was possible to analyse parental DNA.

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.

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.

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.

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.

Crystal structure of a eukaryotic (pea seedling) copper-containing amine oxidase at 2.2 A resolution.

BACKGROUND: Copper-containing amine oxidases catalyze the oxidative deamination of primary amines to aldehydes, in a reaction that requires free radicals. These enzymes are important in many biological processes, including cell differentiation and growth, would healing, detoxification and signalling. The catalytic reaction requires a redox cofactor, topa quinone (TPQ), which is derived by post-translational modification of an invariant tyrosine residue. Both the biogenesis of the TPQ cofactor and the reaction catalyzed by the enzyme require the presence of a copper atom at the active site. The crystal structure of a prokaryotic copper amine oxidase from E. coli (ECAO) has recently been reported. RESULTS: The first structure of a eukaryotic (pea seedling) amine oxidase (PSAO) has been solved and refined at 2.2 A resolution. The crystallographic phases were derived from a single phosphotungstic acid derivative. The positions of the tungsten atoms in the W12 clusters were obtained by molecular replacement using E. coli amine oxidase as a search model. The methodology avoided bias from the search model, and provides an essentially independent view of a eukaryotic amine oxidase. The PSAO molecule is a homodimer; each subunit has three domains. The active site of each subunit lies near an edge of the beta-sandwich of the largest domain, but is not accessible from the solvent. The essential active-site copper atom is coordinated by three histidine side chains and two water molecules in an approximately square-pyramidal arrangement. All the atoms of the TPQ cofactor are unambiguously defined, the shortest distance to the copper atom being approximately 6 A. CONCLUSIONS: There is considerable structural homology between PSAO and ECAO. A combination of evidence from both structures indicates that the TPQ side chain is sufficiently flexible to permit the aromatic grouf to rotate about the Cbeta-Cgamma bond, and to move between bonding and non-bonding positions with respect to the Cu atom. Conformational flexibility is also required at the surface of the molecule to allow the substrates access to the active site, which is inaccessible to solvent, as expected for an enzyme that uses radical chemistry.

Crystallization, structural determination and analysis of a novel parasite vaccine candidate: Fasciola hepatica glutathione S-transferase.

Glutathione S-transferases (GSTs) represent the major class of detoxifying enzymes from parasitic helminths. As a result, they are candidates for chemotherapeutic and vaccine design. Indeed, GSTs from Fasciola hepatica have been found to be effective for vaccinating sheep and cattle against fasciolosis. This helminth contains at least seven GST isoforms, of which four have been cloned. The cloned isoforms (Fh51, Fh47, Fh7 and Fh1) all belong to the mu class of GSTs, share greater than 71% sequence identity, yet display distinct substrate specificities. Crystals of Fh47 were obtained using the hanging drop vapour diffusion technique. The crystals belong to space group I4122, with one monomer in the asymmetric unit, which corresponds to a very high solvent content of approximately 75%. The physiological dimer is generated via a crystallographic 2-fold rotation. The three-dimensional structure of Fh47 was solved by molecular replacement using the Schistosoma japonicum glutathione S-transferase (Sj26) crystal structure as a search model. The structure adopts the canonical GST fold comprising two domains: an N-terminal glutathione-binding domain, consisting of a four-stranded beta-sheet and three helices whilst the C-terminal domain is entirely alpha-helical. The presence of Phe19 in Fh47 results in a 6 degrees interdomain rotation in comparison to Sj26, where the equivalent residue is a leucine. Homology models of Fh51, Fh7 and Fh1, based on the Fh47 crystal structure, reveal critical differences in the residues lining the xenobiotic binding site, particularly at residue positions 9, 106 and 204. In addition, differences amongst the isoforms in the non-substrate binding site were noted, which may explain the observed differential binding of large ligands. The major immunogenic epitopes of Fh47 were surprisingly found not to reside on the most solvent-exposed regions of the molecule.

Crystallization and preliminary X-ray diffraction studies of a glutathione S-transferase from the Australian sheep blowfly, Lucilia cuprina.

Crystals of a glutathione S-transferase from the Australian sheep blowfly Lucilia cuprina have been grown from ammonium sulphate by the hanging drop vapour diffusion method. Successful crystallization required the presence of the inhibitor S-hexylglutathione. The crystals belong to the tetragonal space group P4(1)22 (or P4(3)22) with cell dimensions of a = b = 88.1 A and c = 66.9 A. They contain one monomer in the asymmetric unit and diffract beyond 2.8 A resolution.

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.

Crystallization and preliminary X-ray analysis of the auto-inhibited twitchin kinase.

An auto-inhibited fragment of twitchin kinase (residues 5890 to 6262) has been crystallized by vapor diffusion techniques using polyethylene glycol 4000 as the precipitant at pH 7.25 to 7.5 at 4 degrees C. We have found that MgSO4 and glycerol were essential for large crystal growth. The crystals belong to the orthorhombic space group P2(1)2(1)2, with unit cell dimensions of a = 144.1 A, b = 168.3 A and c = 60.6 A. They are suitable for X-ray analysis and diffract to a resolution of at least 2.8 A.

Structure and function of the bacterial mechanosensitive channel of large conductance.

Mechanosensation in bacteria involves transducing membrane stress into an electrochemical response. In Escherichia coli and other bacteria, this function is carried out by a number of proteins including MscL, the mechanosensitive channel of large conductance. MscL is the best characterized of all mechanosensitive channels. It has been the subject of numerous structural and functional investigations. The explosion in experimental data on MscL recently culminated in the solution of the three-dimensional structure of the MscL homologue from Mycobacterium tuberculosis. In this review, much of these data are united and interpreted in terms of the newly published M. tuberculosis MscL crystal structure.

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.

Luminal contents of benign and malignant prostatic glands: correspondence to altered secretory mechanisms.

Recent changes in tissue fixation strategy, using glutaraldehyde, have clarified the secretory mechanisms of the normal prostate identifying cytoplasmic prostatic secretory granules, structures not preserved by formalin fixation. This normal secretory mechanism was absent in most adenocarcinomas, depicting an important metabolic change in transformed prostate cells. The current study further investigates differences between benign and malignant prostate secretion and relates them to the production of corpora amylacea by benign glands and crystalloids or mucin by cancer. In all normal prostate cells examined (6 cases), prostate secretory granules (PSG) were approximately 1-microm, brightly eosinophilic granules filling the cytoplasm of secretory cells and released in packets by a specialized apocrine cell structure. After apocrine decapitation and luminal dispersal, some of the cytoplasmic and PSG remnants condensed to form eosinophilic bodies (EB) with a glycoprotein rim and central protein core. EB were observed adsorbing and layering onto the surface of prostatic corpora amylacea representing their chief mode of enlargement. Biochemical analysis and x-ray diffraction studies confirmed sulfated glycosaminoglycans of similar structure as the main constituent of both PSG and corpora amylacea. Peripheral zone amphiphilic "dark cell" carcinoma (9 cases) contained almost no PSG, and showed neither apical decapitation nor EB formation, but mucin secretion was frequently detected. Crystalloids that share the same staining characteristics and sulfur content as PSG and corpora amylacea were identified in 3 selected "clear cell" carcinomas, all of which showed at least focal PSG secretion. The recognition of these differing secretory mechanisms and their deviation from normal further defines the histological criteria and spectrum of prostate malignancy.

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.

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).

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.

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.

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.