Annealing in crystallography: a powerful optimization tool.
Explore the source record for details and available documents.
Crystallography, X-Ray↗
Biomedical subjects
P D Adams
Publications and source records attributed to P D Adams.
Retinoblastoma protein contains a C-terminal motif that targets it for phosphorylation by cyclin-cdk complexes.
Stable association of certain proteins, such as E2F1 and p21, with cyclin-cdk2 complexes is dependent upon a conserved cyclin-cdk2 binding motif that contains the core sequence ZRXL, where Z and X are usually basic. In vitro phosphorylation of the retinoblastoma tumor suppressor protein, pRB, by cyclin A-cdk2 and cyclin E-cdk2 was inhibited by a short peptide spanning the cyclin-cdk2 binding motif present in E2F1. Examination of the pRB C terminus revealed that it contained sequence elements related to ZRXL. Site-directed mutagenesis of one of these sequences, beginning at residue 870, impaired the phosphorylation of pRB in vitro. A synthetic peptide spanning this sequence also inhibited the phosphorylation of pRB in vitro. pRB C-terminal truncation mutants lacking this sequence were hypophosphorylated in vitro and in vivo despite the presence of intact cyclin-cdk phosphoacceptor sites. Phosphorylation of such mutants was restored by fusion to the ZRXL-like motif derived from pRB or to the ZRXL motifs from E2F1 or p21. Phospho-site-specific antibodies revealed that certain phosphoacceptor sites strictly required a C-terminal ZRXL motif whereas at least one site did not. Furthermore, this residual phosphorylation was sufficient to inactivate pRB in vivo, implying that there are additional mechanisms for directing cyclin-cdk complexes to pRB. Thus, the C terminus of pRB contains a cyclin-cdk interaction motif of the type found in E2F1 and p21 that enables it to be recognized and phosphorylated by cyclin-cdk complexes.
Amino Acid Sequence↗
Models for the transmembrane region of the phospholamban pentamer: which is correct?
Phospholamban is a 52-amino acid protein that assembles into a pentamer in the membranes of the sarcoplasmic reticulum. Pentamer formation is driven in large part by interactions of the transmembrane regions of the protein, which are thought to be arranged as interacting alpha-helices. The structural properties of phospholamban have been studied by mutagenesis and optical spectroscopy, resulting in a large database. In this discussion, we present advances in computational modeling, which identifies two probable structures for the transmembrane pentamer. A new approach to mutagenesis is described, which should lead to a clear distinction between the two possible models.
Amino Acid Sequence↗
Crystallography & NMR system: A new software suite for macromolecular structure determination.
A new software suite, called Crystallography & NMR System (CNS), has been developed for macromolecular structure determination by X-ray crystallography or solution nuclear magnetic resonance (NMR) spectroscopy. In contrast to existing structure-determination programs, the architecture of CNS is highly flexible, allowing for extension to other structure-determination methods, such as electron microscopy and solid-state NMR spectroscopy. CNS has a hierarchical structure: a high-level hypertext markup language (HTML) user interface, task-oriented user input files, module files, a symbolic structure-determination language (CNS language), and low-level source code. Each layer is accessible to the user. The novice user may just use the HTML interface, while the more advanced user may use any of the other layers. The source code will be distributed, thus source-code modification is possible. The CNS language is sufficiently powerful and flexible that many new algorithms can be easily implemented in the CNS language without changes to the source code. The CNS language allows the user to perform operations on data structures, such as structure factors, electron-density maps, and atomic properties. The power of the CNS language has been demonstrated by the implementation of a comprehensive set of crystallographic procedures for phasing, density modification and refinement. User-friendly task-oriented input files are available for nearly all aspects of macromolecular structure determination by X-ray crystallography and solution NMR.
Computer Simulation↗
Negative control elements of the cell cycle in human tumors.
The retinoblastoma protein and p53 are both cell-cycle regulators and are, directly or indirectly, inactivated in the majority of human tumors. Recent studies have provided new mechanistic insights into how these proteins regulate cell growth in response to various intracellular and extracellular signals.
Cell Cycle↗
Recent developments for the efficient crystallographic refinement of macromolecular structures.
Macromolecular crystallographic refinement has recently been made more efficient by the use of cross-validated maximum likelihood targets and torsion-angle molecular dynamics simulated annealing. In combination with automated model building methods, the amount of manual intervention required to complete and refine a structure is greatly reduced.
Crystallography, X-Ray↗
Deregulated expression of E2F-1 induces cyclin A- and E-associated kinase activities independently from cell cycle position.
The transcription factor E2F activates genes required for S phase, such as cyclin E and cyclin A. We show that, contrary to long term effects of E2F-1 overexpression, short ectopic overexpression of this transcription factor in logarithmically growing cells does neither affect the cell cycle distribution nor the cell size, but heavily induces cyclin E and A expression as well as cyclin E- and A-dependent kinase activities. We further separated logarithmically growing E2F-1-overexpressing cells according to their different cell cycle phases by centrifugal elutriation. These experiments revealed that deregulated E2F-1 expression triggers high levels of cyclin E and A expression and kinase activities in small early G1 cells, normally not exhibiting these activities. These effects on the regulation of cyclin E- and A-associated kinases are not accompanied by any detectable alteration in the rate of progression through the cell cycle, suggesting that these changes are independent of any mitogenic properties of E2F-1.
Animals↗
Cross-validated maximum likelihood enhances crystallographic simulated annealing refinement.
Recently, the target function for crystallographic refinement has been improved through a maximum likelihood analysis, which makes proper allowance for the effects of data quality, model errors, and incompleteness. The maximum likelihood target reduces the significance of false local minima during the refinement process, but it does not completely eliminate them, necessitating the use of stochastic optimization methods such as simulated annealing for poor initial models. It is shown that the combination of maximum likelihood with cross-validation, which reduces overfitting, and simulated annealing by torsion angle molecular dynamics, which simplifies the conformational search problem, results in a major improvement of the radius of convergence of refinement and the accuracy of the refined structure. Torsion angle molecular dynamics and the maximum likelihood target function interact synergistically, the combination of both methods being significantly more powerful than each method individually. This is demonstrated in realistic test cases at two typical minimum Bragg spacings (dmin = 2.0 and 2.8 A, respectively), illustrating the broad applicability of the combined method. In an application to the refinement of a new crystal structure, the combined method automatically corrected a mistraced loop in a poor initial model, moving the backbone by 4 A.
Amylases↗
New applications of simulated annealing in X-ray crystallography and solution NMR.
Explore the source record for details and available documents.
Chemical Phenomena↗
Structure of the transmembrane cysteine residues in phospholamban.
Phospholamban, a 52-residue membrane protein, associates to form a pentameric complex of five long alpha-helices traversing the sarcoplasmic reticulum membrane of cardiac muscle cells. The transmembrane domain of the protein is largely hydrophobic, with only three cysteine residues having polar side chains, yet it functions as a Ca2+-selective ion channel. In this report, infrared spectroscopy is used to probe the conformation of the three cysteine side chains and to establish whether the free S-H groups form intrahelical hydrogen bonds in the pentameric complex. Vibrational spectra of a transmembrane peptide were obtained which corresponded to the transmembrane domain of wild-type phospholamban and three peptides each containing a cysteine --> alanine substitution. The observed S-H frequencies argue that each of the sulfhydryl groups is hydrogen-bonded to an i-4 backbone carbonyl oxygen. Electrostatic calculations on a model of phospholamban based on molecular dynamics and mutagenesis studies, show that the sulfhydryl groups may significantly contribute to the electrostatic potential field of the protein.
Adenosine Triphosphatases↗
Fluorescence-activated cell sorting of transfected cells.
Explore the source record for details and available documents.
Animals↗
Structural perspectives of phospholamban, a helical transmembrane pentamer.
Phospholamban is a 52-amino-acid protein that assembles into a pentamer in sarcoplasmic reticulum membranes. The protein has a role in the regulation of the resident calcium ATPase through an inhibitory association that can be reversed by phosphorylation. The phosphorylation of phospholamban is initiated by beta-adrenergic stimulation, identifying phospholamban as an important component in the stimulation of cardiac activity by beta-agonists. In this role of phospholamban that has motivated studies in recent decades. There is evidence that phospholamban may also function as a Ca(2+)-selective ion channel. The structural properties of phospholamban have been studied by mutagenesis, modeling, and spectroscopy, resulting in a new view of the organization of this key molecule in membranes.
Amino Acid Sequence↗
Cloning and characterization of phorbol ester differentiation-resistant U937 cell variants.
Differentiation-resistant U937 cells were derived from parental U937 human promonocytic leukemia cells by selecting for a nonadherent phenotype in cell cultures continuously exposed to 12-O-tetradecanoylphorbol-13-acetate (TPA). Subsequent analysis indicated no differences between wildtype (wt) and resistant U937 cells with respect to protein kinase C (PKC) isozyme expression, activation, or down-modulation. The subcellular localization of PKCs is identical in wt and resistant cells with the exception of PKC beta 2, which no longer colocalizes with microtubules in the TPA-resistant cell lines. In contrast to wt-U937 cells, the resistant cells do not express beta 2-integrin adhesion molecules, cd11b and cd11c, on the cell surface following TPA treatment but do express cd11b and cd11c in intracellular vesicles. TPA stimulates the translocation of these vesicles to the cell surface in wt U937 cells but not in the resistant cells. These results suggest that events downstream of PKC activation may mediate cytoskeletal reorganization and beta 2-integrin transport to the cell surface in wt-U937 cells but not in the differentiation-resistant cells.
CD18 Antigens↗
Improved prediction for the structure of the dimeric transmembrane domain of glycophorin A obtained through global searching.
A more global search method, using fewer assumptions, has been used to predict the structure of the dimeric transmembrane region of the protein glycophorin A. The resulting model significantly differs from that previously determined. In particular, the arrangement between the two transmembrane helices is now more symmetric resulting in improved interaction energies and an increased buried surface area. An increase in the van der Waals interaction energy due to tighter packing compensates for the loss of the interhelical hydrogen bond observed between Thr-87 of each helix in the previous model.
Computer Simulation↗
The cellular effects of E2F overexpression.
The product of the retinoblastoma tumor-suppressor gene (RB) is a ubiquitously expressed, 105-kDa nuclear phosphoprotein (pRB). The pRB protein negatively regulates the cellular G1/S phase transition, and it is at this point in the cell cycle that it is thought to play its role as a tumor suppressor. The growth-inhibitory effects of pRB are exerted, at least in part, through the E2F family of transcription factors. This chapter reviews the insights into the mechanism of action of the E2F family members that have been obtained through overexpression studies. Studies in RB-/- SAOS-2 cells have provided evidence in support of the hypothesis that the E2F family members are negatively regulated by pRB and the related protein p130. In particular, the results obtained are consistent with the earlier biochemical data which suggested that E2F1 is regulated primarily by pRB, and E2F4 by p130. Results relating to p107 are also discussed. Consistent with the proposed role of pRB and E2F1 as coregulators of entry into S phase, experiments have demonstrated that overexpression of E2F1 is sufficient to override the cell cycle arrests caused by serum deprivation of fibroblasts or transforming growth factor-beta (TGF-beta) treatment of mink lung epithelial cells. However, at least in the case of the serum deprivation induced arrest, the ultimate result of E2F1 overexpression is death by p53-dependent apoptosis. In light of this and other data, a model is discussed as to how functional inactivation of pRB and p53 might cooperate to promote tumorigenesis. A number of studies have demonstrated the oncogenic potential of E2F family members, at least under certain conditions. This is, again, in keeping with the notion that these proteins play a critical role in controlling proliferation.
Animals↗
Identification of a cyclin-cdk2 recognition motif present in substrates and p21-like cyclin-dependent kinase inhibitors.
Understanding how cyclin-cdk complexes recognize their substrates is a central problem in cell cycle biology. We identified an E2F1-derived eight-residue peptide which blocked the binding of cyclin A and E-cdk2 complexes to E2F1 and p21. Short peptides spanning similar sequences in p107, p130, and p21-like cdk inhibitors likewise bound to cyclin A-cdk2 and cyclin E-cdk2. In addition, these peptides promoted formation of stable cyclin A-cdk2 complexes in vitro but inhibited the phosphorylation of the retinoblastoma protein by cyclin A- but not cyclin B-associated kinases. Mutation of the cyclin-cdk2 binding motifs in p107 and E2F1 likewise prevented their phosphorylation by cyclin A-associated kinases in vitro. The cdk inhibitor p21 was found to contain two functional copies of this recognition motif, as determined by in vitro kinase binding/inhibition assays and in vivo growth suppression assays. Thus, these studies have identified a cyclin A- and E-cdk2 substrate recognition motif. Furthermore, these data suggest that p21-like cdk inhibitors function, at least in part, by blocking the interaction of substrates with cyclin-cdk2 complexes.
Amino Acid Sequence↗
Transcriptional control by E2F.
Considerable evidence suggests that the E2F/DRTF1 family of transcription factors (hereafter referred to as 'E2F') plays a critical role in cell growth control. For example, the ability of several small DNA tumour viruses, such as SV40, adenovirus and human papillomavirus, to transform certain cells is tightly linked to their ability to deregulate E2F. Furthermore, E2F appears to directly regulate the transcription of a diverse set of genes implicated in DNA replication and cell growth control. Finally, a number of known cell cycle regulators, some of which are commonly mutated in human tumours, appear to exert their effects, at least in part, by altering E2F activity. Among these are pRb, p53, cdk-2, cdk-4 and certain cyclins.
Animals↗
Computational searching and mutagenesis suggest a structure for the pentameric transmembrane domain of phospholamban.
Structural and environmental constraints greatly simplify the folding problem for membrane proteins. Computational methods can be used in a global search to find a small number of chemically reasonable models within these constraints, such that a modest set of experimental data can distinguish among them. We show that, for phospholamban, the global search can be further simplified by reducing the problem to two-body, rather than many-body, interactions. This method of a constrained global search combined with experimental mutagenesis data yields a three-dimensional structure for this pentameric ion channel. The model is a left-handed symmetric homopentamer of alpha-helices with a well-defined channel, lined solely by hydrophobic residues.
Amino Acid Sequence↗