Genomewide studies of histone deacetylase function in yeast.

The trichostatin A (TSA)-sensitive histone deacetylase (HDAC) Rpd3p exists in a complex with Sin3p and Sap30p in yeast that is recruited to target promoters by transcription factors including Ume6p. Sir2p is a TSA-resistant HDAC that mediates yeast silencing. The transcription profile of rpd3 is similar to the profiles of sin3, sap30, ume6, and TSA-treated wild-type yeast. A Ume6p-binding site was identified in the promoters of genes up-regulated in the sin3 strain. Two genes appear to participate in feedback loops that modulate HDAC activity: ZRT1 encodes a zinc transporter and is repressed by RPD3 (Rpd3p is zinc-dependent); BNA1 encodes a nicotinamide adenine dinucleotide (NAD)-biosynthesis enzyme and is repressed by SIR2 (Sir2p is NAD-dependent). Although HDACs are transcriptional repressors, deletion of RPD3 down-regulates certain genes. Many of these are down-regulated rapidly by TSA, indicating that Rpd3p may also activate transcription. Deletion of RPD3 previously has been shown to repress ("silence") reporter genes inserted near telomeres. The profiles demonstrate that 40% of endogenous genes located within 20 kb of telomeres are down-regulated by RPD3 deletion. Rpd3p appears to activate telomeric genes sensitive to histone depletion indirectly by repressing transcription of histone genes. Rpd3p also appears to activate telomeric genes repressed by the silent information regulator (SIR) proteins directly, possibly by deacetylating lysine 12 of histone H4. Finally, bioinformatic analyses indicate that the yeast HDACs RPD3, SIR2, and HDA1 play distinct roles in regulating genes involved in cell cycle progression, amino acid biosynthesis, and carbohydrate transport and utilization, respectively.

A bisubstrate analog induces unexpected conformational changes in phosphoglycerate kinase from Trypanosoma brucei.

The glycolytic enzyme phosphoglycerate kinase (PGK) catalyzes phosphoryl transfer between 1,3-bis-phosphoglycerate and ADP to form 3-phosphoglycerate and ATP. During catalysis, a major hinge bending motion occurs which brings the N and C-terminal enzyme domains and their bound substrates together and in-line for phosphoryl transfer. We have crystallized Trypanosoma brucei PGK in the presence of the bisubstrate analog, adenylyl 1,1,5,5-tetrafluoropentane-1, 5-bisphosphonate, and solved the structure of this complex in two different crystal forms at 1.6 and 2.0 A resolution, obtained from PEG 8000 and ammonium phosphate solutions, respectively. These high resolution structures of PGK:inhibitor complexes are of particular interest for drug design since Trypanosoma brucei, the causative agent of African sleeping sickness, relies on glycolysis as its sole energy source. In both structures, the inhibitor is bound in a fully extended conformation with its adenosine moiety assuming exactly the same position as in ADP:PGK complexes and with its 5' phosphonate group occupying part of the 1,3-bis-phosphoglycerate binding site. The bisubstrate analog forces PGK to assume a novel, "inhibited" conformation, intermediate in hinge angle between the native structures of open and closed form PGK. These structures of enzyme-inhibitor complexes demonstrate that PGK has two distinct hinge points that can each be independently activated. In the "PEG" structure, the C-terminal hinge is partially activated while the N-terminal hinge point remains in an open state. In the "phosphate" structure, closure of the N-terminal hinge point is also evident. Finally and most unexpectedly, both complex structures also contain a 3 A shift of a helix that lies outside the flexible hinge region. We propose that a transient shift of this helix is a required element of PGK hinge closure during catalysis.

The importance of dynamic light scattering in obtaining multiple crystal forms of Trypanosoma brucei PGK.

Phosphoglycerate kinase (PGK) catalyzes the phosphoryl transfer between 1,3 bis-phosphoglycerate and ADP to form 3-phosphoglycerate and ATP, undergoing significant conformational changes during catalysis. To more precisely document this reaction and the corresponding conformational changes, we have crystallized Trypanosoma brucei PGK in several crystal forms: (1) in the presence of 3-phosphoglycerate and MgADP, PGK crystallizes with four molecules in the asymmetric unit; (2) in the presence of the ATP analog, AMP-PNP, PGK crystallizes in a similar form; (3) in the presence of the bisubstrate analog, adenylyl 1,1,5,5-tetrafluoropentane-1,5-bisphosphonate, PGK crystals grow with one molecule in the asymmetric unit. Large scale expression and purification of T. brucei PGK from an E. coli overexpression system was required to obtain sufficient enzyme yields. Results from dynamic light scattering experiments allowed us to identify substrates and analogs which were amenable for crystallization. Ease of crystal growth and diffraction quality for a particular PGK-ligand complex is highly consistent with the apparent monodispersity of the complex in solution as judged by dynamic light scattering. The three-dimensional structures of the various enzyme-ligand complexes are currently being exploited to obtain a better understanding of PGK catalysis, as well as for structure based design of enzyme inhibitors to be used in the development of anti-trypanosomal agents.

Crystal structures of substrates and products bound to the phosphoglycerate kinase active site reveal the catalytic mechanism.

Phosphoglycerate kinase (PGK) catalyzes the reversible phosphoryl transfer between 1,3-bisphosphoglycerate and ADP to form 3-phosphoglycerate and ATP in the presence of magnesium. The detailed positions of the substrates during catalysis have been a long-standing puzzle due to the major conformational changes required for active site formation. Here we report the refined closed form Trypanosoma brucei PGK ternary complex at an improved resolution of 2.5 A, together with the crystal structure of closed form T. brucei PGK in complex with the nucleotide analogue AMP-PNP. In the 180 000 Da asymmetric unit of the ternary complex, four closed form PGK molecules appear to be arranged as two asymmetric dimers. Quite surprisingly, each dimer is comprised of one 3-phosphoglycerate. MgADP.PGK ternary complex and one Pi.MgADP.PGK pseudoternary complex. The substrates in the ternary complex are bound in a fashion nearly identical to that in open form PGK, but a 30 degrees hinge bending conformational change has brought them together and in-line for catalysis. The pseudoternary complex subunits exhibit a similar hinge closure but contain, instead of 3-phosphoglycerate, a single phosphate molecule bound in the active site. This phosphate binds to a site expected for the 1-position phosphate of 1,3-bisphosphoglycerate, hence providing information for the binding mode for this chemically unstable substrate. The structure of the binary PGK.MgAMP-PNP complex indicates the binding mode for MgATP. An examination of the interactions made by the transferring phosphate groups of the substrate, 1, 3-bisphosphoglycerate, and the product, ATP, reveals that in each case only two of the three nonbridging phosphate oxygens are stabilized by hydrogen bonds. In contrast, a model of the transition state phosphoryl group based on all available structural data reveals active site stabilization of all three negatively charged phosphoryl oxygens. These structural models provide insight into the nature of the phosphoryl-transfer reaction catalyzed by PGK and related enzymes.

A general method of domain closure is applied to phosphoglycerate kinase and the result compared with the crystal structure of a closed conformation of the enzyme.

The occurrence of large domain motions associated with the mechanism of action of many proteins is well established. We present a general method of predicting domain closure applicable to proteins containing domains separated by an apparent hinge. The method attempts to allow for natural directional bias within the closing protein by repeatedly applying a weak pulling force over a short distance between pairs of atoms chosen at random in the two domains in question. Appropriate parameters governing the pulling function were determined empirically. The method was applied to the bi-lobal protein PGK and a closed-form activated ternary complex generated for Bacillus stearothermophilus PGK. This model was compared with the recently determined crystal structure of closed-form Trypanosoma brucei PGK. The model predicts the correct hinge regions, although the magnitude of movement at one hinge point was overestimated, and provides a reasonable representation of the closed-form ternary complex.

Synergistic effects of substrate-induced conformational changes in phosphoglycerate kinase activation.

Phosphoglycerate kinase (PGK), a key enzyme in glycolysis, catalyses the transfer of a phosphoryl-group from 1,3-bisphosphoglycerate to ADP to form 3-phosphoglycerate and ATP. Despite extensive kinetic and structural investigations over more than two decades, the conformation assumed by this enzyme during catalysis remained unknown. Here we present the 2.8 A crystal structure of a ternary complex of PGK from Trypanosoma brucei, the causative agent of sleeping sickness. This structure determination relied on a procedure in which fragments containing less than 10% of the scattering mass were successively positioned in the unit cell to obtain phases. The PGK ternary complex exhibits a dramatic closing of the large cleft between the two domains seen in all previous studies, thereby bringing the two ligands, 3-phosphoglycerate and ADP into close proximity. Our results demonstrate that PGK is a hinge-bending enzyme, reveal a novel mechanism in which substrate-induced effects combine synergistically to induce major conformational changes and, to our knowledge, afford the first observation of the PGK active site in a catalytic conformation.

Probing the limits of the molecular replacement method: the case of Trypanosoma brucei phosphoglycerate kinase.

It is of general interest to explore the limits of the molecular replacement method. Described here are the specifics of a successful molecular replacement structure determination in a difficult case: the X-ray crystal structure of a Trypanosoma brucei phosphoglycerate kinase (PGK) ternary complex. This ternary complex crystallizes with four 45 kDa subunits in the asymmetric unit, whereas the available search models were all monomers consisting of two distinct domains. Initial molecular replacement attempts using complete subunits were unsuccessful. Attributing this failure to a presumed change in the relative orientations of the N- and C-terminal phosphoglycerate kinase domains, a second attempt was made using each domain as an independent search model. In this way, two N-terminal and then two C-terminal domains could be oriented and positioned in the unit cell. On the basis of this result, a new search model containing both domains in the correct mutual orientation was created and used to identify the two remaining phosphoglycerate kinase subunits. The ability to successfully orient and position an N-terminal domain containing 8.4% of the scattering mass in the asymmetric unit was the key to this structure determination. Further investigations show that a truncated version of this search model containing 5.8% of the scattering mass would have been sufficient for this purpose. A retrospective analysis suggests that the effectiveness of this probe is enhanced by structural conservation, retained temperature factors and a disparity in the degree of order among the various subunits in the T. brucei PGK asymmetric unit. Based on these observations, 231 well ordered Calpha atoms were selected from a single refined T. brucei PGK subunit and it was demonstrated that this collection of atoms, representing just 1.6% of the scattering mass, could be correctly oriented and positioned in the unit cell.

Structure of a histidine-X4-histidine zinc finger domain: insights into ADR1-UAS1 protein-DNA recognition.

The solution structure for a mutant zinc finger peptide based on the sequence of the C-terminal ADR1 finger has been determined by two-dimensional NMR spectroscopy. The mutant peptide, called PAPA, has both proline residues from the wild-type sequence replaced with alanines. A nonessential cysteine was also replaced with alanine. The behavior of PAPA in solution implicates the prolines in the conformational heterogeneity reported earlier for the wild-type peptide [Xu, R. X., Horvath, S. J., & Klevit, R. E. (1991) Biochemistry 30, 3365-3371]. The solution structure of PAPA reveals several interesting features of the zinc finger motif. The residue immediately following the second cysteine ligand adopts a positive phi angle, which we propose is a common feature of this class of zinc fingers, regardless of whether this residue is a glycine. The NMR spectrum and resulting solution structure of PAPA suggest that a side-chain to side-chain hydrogen bond involving an arginine and an aspartic acid analogous to one observed in the Zif268 protein-DNA cocrystal structure exists in solution in the absence of DNA [Pavletich, N. P., & Pabo, C. O. (1991) Science 252, 809-817]. A model for the interaction between the two ADR1 zinc fingers and their DNA binding sites was built by superpositioning the refined solution structures of PAPA and ADR1b onto the Zif268 structure. This model offers structural explanations for a variety of mutations to the ADR1 zinc finger domains that have been shown to affect DNA-binding affinity or specificity.

Malpractice: future shock of the 1980s.

Once the therapeutic relationship is established, a client acquires certain rights and the social worker certain duties. If these duties are breached due to negligence, and the client is damaged in any way, the social worker may be financially liable, and this may lead to the cause of action called malpractice.

Lawyer and social worker as a team: preparing for trial in neglect cases.

Effective presentation of evidence at a court hearing in a child neglect case calls for thorough preparation. This can be best achieved by lawyer and social worker operating as an interdisciplinary team.

Legal needs of the ill: the social worker's role on an interdisciplinary team.

The social worker in a medical setting is in a unique position to become aware of a patient's legal needs. The worker must therefore be able to furnish information about the patient's right to die a natural death, the donation of the body to science, powers of attorney, joint bank accounts, and the importance of a comprehensive estate plan. The author discusses how the worker can bring together professionals to best serve patients' needs.

A potential peril of pastoral care: Malpractice.

The pastoral counselor may be a full-time counselor trained in pastoral care or a clergyman with a congregation, who from time to time serves his flock as a counselor, ministering to the personal needs of his congregation in a manner designated "pastoral care" or perhaps, more appropriately, "pastoral psychotherapy." Once a therapeutic relationship is established, a congregant acquires certain rights and the clergyman, duties. If these duties are breached due to the negligence of the pastoral counselor and this causes the congregant to be damaged in any way, the pastoral counselor may be liable and may be called upon to respond in damages. The cause of action is called malpractice. This paper isolates areas of actual and potential malpractice.

Lawyer and counselor as an interdisciplinary team serving the terminally ill.

Mental health professionals consult with the terminally ill and provide therapy to the client, spouse, and family. Some clients have been forewarned and have a carefully drafted estate plan that incorporates all the nuances of property and personal planning. In addition, the client may have an interest in donating his or her body or its parts as an anatomical gift or may inquire concerning the right to die a natural death. He or she may further be concerned with the appropriateness of a power of attorney or a living trust. Whatever the inquiry, the mental health professional should be ready, willing, and able to offer informed data regarding questions in these areas and then to serve with the attorney as an interdisciplinary team to respond quickly and effectively to these legitimate personal concerns.

Malpractice: an ogre on the horizon.

Although social workers are sued for malpractice only infrequently, the possibility exists nonetheless. Drawing analogies with areas of liability that exist in other professions, particularly psychiatry, the author calls attention to ways in which the social worker can protect himself against malpractice suits.

Lawyer and social worker as collaborators in the medical setting.

Part of the role of the social worker in a medical setting should be to determine if a patient has reviewed his legal status concerning wills, insurance, and estate planning. If not, the worker should encourage the patient and his family to seek legal advice so that the patient can receive medical treatment free of legal and financial concerns.