Prediction of the structure of a receptor-protein complex using a binary docking method.

To validate procedures of rational drug design, it is important to develop computational methods that predict binding sites between a protein and a ligand molecule. Many small molecules have been tested using such programs, but examination of protein-protein and peptide-protein interactions has been sparse. We were able to test such applications once the structures of both the maltose-binding protein (MBP) and the ligand-binding domain of the aspartate receptor, which binds MBP, became available. Here we predict the binding site of MBP to its receptor using a 'binary docking' technique in which two MBP octapeptide sequences containing mutations that eliminate maltose chemotaxis are independently docked to the receptor. The peptides in the docked solutions superimpose on their original positions in the structure of MBP and allow the formation of an MBP-receptor complex. The consistency of the computational and biological results supports this approach for predicting protein-protein and peptide-protein interactions.     

Computational redesign of endonuclease DNA binding and cleavage specificity

The reprogramming of DNA-binding specificity is an important challenge for computational protein design that tests current understanding of protein-DNA recognition, and has considerable practical relevance for biotechnology and medicine. Here we describe the computational redesign of the cleavage specificity of the intron-encoded homing endonuclease I-MsoI using a physically realistic atomic-level forcefield. Using an in silico screen, we identified single base-pair substitutions predicted to disrupt binding by the wild-type enzyme, and then optimized the identities and conformations of clusters of amino acids around each of these unfavourable substitutions using Monte Carlo sampling. A redesigned enzyme that was predicted to display altered target site specificity, while maintaining wild-type binding affinity, was experimentally characterized. The redesigned enzyme binds and cleaves the redesigned recognition site approximately 10,000 times more effectively than does the wild-type enzyme, with a level of target discrimination comparable to the original endonuclease. Determination of the structure of the redesigned nuclease-recognition site complex by X-ray crystallography confirms the accuracy of the computationally predicted interface. These results suggest that computational protein design methods can have an important role in the creation of novel highly specific endonucleases for gene therapy and other applications.     

Structure of the C2 domain of human factor VIII at 1.5 A resolution

Human factor VIII is a plasma glycoprotein that has a critical role in blood coagulation. Factor VIII circulates as a complex with von Willebrand factor. After cleavage by thrombin, factor VIIIa associates with factor IXa at the surface of activated platelets or endothelial cells. This complex activates factor X (refs 6, 7), which in turn converts prothrombin to thrombin in the presence of factor Va (refs 8, 9). The carboxyl-terminal C2 domain of factor VIII contains sites that are essential for its binding to von Willebrand factor and to negatively charged phospholipid surfaces. Here we report the structure of human factor VIII C2 domain at 1.5 A resolution. The structure reveals a beta-sandwich core, from which two beta-turns and a loop display a group of solvent-exposed hydrophobic residues. Behind the hydrophobic surface lies a ring of positively charged residues. This motif suggests a mechanism for membrane binding involving both hydrophobic and electrostatic interactions. The structure explains, in part, mutations in the C2 region of factor VIII that lead to bleeding disorders in haemophilia A.     

Homing endonucleases: structure, function and evolution

‘Homing’ is the lateral transfer of an intervening genetic sequence, either an intron or an intein, to a cognate allele that lacks that element. The end result of homing is the duplication of the intervening sequence. The process is initiated by site-specific endonucleases that are encoded by open reading frames within the mobile elements. Several features of these proteins make them attractive subjects for structural and functional studies. First, these endonucleases, while unique, may be contrasted with a variety of enzymes involved in nucleic acid strand breakage and rearrangement, particularly restriction endonucleases. Second, because they are encoded within the intervening sequence, there are interesting limitations on the position and length of their open reading frames, and therefore on their structures. Third, these enzymes display a unique strategy of flexible recognition of very long DNA target sites. This strategy allows these sequences to minimize nonspecific cleavage within the host genome, while maximizing the ability of the endonuclease to cleave closely related variants of the homing site. Recent studies explain a great deal about the biochemical and genetic mechanisms of homing, and also about the structure and function of several representative members of the homing endonuclease families. Received 6 January 1999; received after revision 24 February 1999; accepted 24 February 1999     

Choline activation of lithium transport

Zusammenfassung Nachweis einer Erhöhung des Li-Austausches in Rinder-Erythrozyten bei Anwesenheit von Cholinchlorid im Extrazellularraum.

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This work was supported in part by a grant from the National Institutes of Health, a National Institutes of Mental Health Special Fellowship to W.R.C., and a Public Health Service Career Development Award to D.F.M.     

Native plants dominate understory vegetation following ponderosa pine forest restoration treatments

Dense ponderosa pine forests in the southwestern United States inhibit understory production and diversity and are susceptible to high-severity wildfire. Restoration treatments involving overstory thinning and prescribed burning are being implemented to increase understory productivity and diversity and to reduce the risk of severe wildfire. However, disturbances associated with treatments may favor invasion of nonnative species, and the severity of the disturbance may be related to the level of nonnative species establishment. We examined understory community composition, species richness, and plant cover responses to 3 stand-scale replicates of 4 different tree-thinning intensities. Restoration treatments altered the composition of the understory community regardless of thinning intensity. Understory richness and cover were highly variable among experimental blocks, but we observed strong trends of increasing richness and cover in the treated stands. Immediately following restoration treatments, nonnative species cover comprised 6% of the total cover where treatment-induced disturbances were the greatest. However, the initial increase in nonnative species did not persist and was reduced by half 6 years after treatment. Plant community composition was still in flux by the sixth year after treatment, indicating that continued monitoring is necessary for evaluating whether restoration targets are maintained over time. Los densos bosques de pino ponderosa en el suroeste de los E.U.A. inhiben la producción y diversidad del sotobosque y son propensos a incendios severos. El raleo del dosel y la quema controlada se están implementando como tratamientos de restauración para incrementar la productividad y diversidad del sotobosque y para reducir el peligro de incendios severos. Sin embargo, las perturbaciones asociadas con estos tratamientos podrían favorecer la invasión por especies no nativas, y la severidad de la perturbación puede estar relacionada con el nivel de establecimiento de estas especies. Examinamos la reacción del sotobosque con respecto a la composición de sus comunidades, a la riqueza de especies y a la cobertura de la vegetación, a tres réplicas a nivel de rodal de cuatro distintas intensidades de raleo. Los tratamientos de restauración cambiaron la composición de la comunidad del sotobosque independientemente de la intensidad del raleo. La riqueza y la cobertura del sotobosque variaron mucho entre las áreas estudiadas, pero observamos marcadas tendencias de mayor riqueza y cobertura en los rodales que recibieron el tratamiento. Inmediatamente después de los tratamientos de restauración, la cobertura de especies invasoras constituía el 6% de la cobertura total donde las perturbaciones producidas por los tratamientos fueron más grandes. No obstante, el aumento inicial en la cobertura de especies invasoras no persistió, y se redujo a la mitad en un lapso de seis años después del tratamiento. La composición de las comunidades de plantas aun seguía cambiando en el sexto año después del tratamiento, lo cual indica que será preciso el monitoreo continuo para determinar si se lograron las metas de restauración.     

Predation enhances complexity in the evolution of electric fish signals.

Theories of sexual selection assume that predation is a restrictive, simplifying force in the evolution of animal display characters and many empirical studies have shown that predation opposes excessive elaboration of sexually selected traits. In an unexpected turnaround, I show here that predation pressure on neotropical, weakly electric fish (order Gymnotiformes) seems to have selected for greater signal complexity, by favouring characters that have enabled further signal elaboration by sexual selection. Most gymnotiform fish demonstrate adaptations that lower detectability of their electrolocation/communication signals by key predators. A second wave phase added to the ancestral monophasic signal shifts the emitted spectrum above the most sensitive frequencies of electroreceptive predators. By using playback trials with the predatory electric eel (Electrophorus electricus), I show that these biphasic signals are less detectable than the primitive monophasic signals. But sexually mature males of many species in the family Hypopomidae extend the duration of the second phase of their electric signal pulses and further amplify this sexual dimorphism nightly during the peak hours of reproduction. Thus a signal element that evolved for crypsis has itself been modified by sexual selection.