Scoring functions for AutoDock.

Automated docking allows rapid screening of protein-ligand interactions. A scoring function composed of a force field and linear weights can be used to compute a binding energy from a docked atom configuration. For different force fields or types of molecules, it may be necessary to train a custom scoring function. This chapter describes the data and methods one must consider in developing a custom scoring function for use with AutoDock.

CHMP1A encodes an essential regulator of BMI1-INK4A in cerebellar development.

Charged multivesicular body protein 1A (CHMP1A; also known as chromatin-modifying protein 1A) is a member of the ESCRT-III (endosomal sorting complex required for transport-III) complex but is also suggested to localize to the nuclear matrix and regulate chromatin structure. Here, we show that loss-of-function mutations in human CHMP1A cause reduced cerebellar size (pontocerebellar hypoplasia) and reduced cerebral cortical size (microcephaly). CHMP1A-mutant cells show impaired proliferation, with increased expression of INK4A, a negative regulator of stem cell proliferation. Chromatin immunoprecipitation suggests loss of the normal INK4A repression by BMI in these cells. Morpholino-based knockdown of zebrafish chmp1a resulted in brain defects resembling those seen after bmi1a and bmi1b knockdown, which were partially rescued by INK4A ortholog knockdown, further supporting links between CHMP1A and BMI1-mediated regulation of INK4A. Our results suggest that CHMP1A serves as a critical link between cytoplasmic signals and BMI1-mediated chromatin modifications that regulate proliferation of central nervous system progenitor cells.

The cerebrospinal fluid provides a proliferative niche for neural progenitor cells.

Cortical development depends on the active integration of cell-autonomous and extrinsic cues, but the coordination of these processes is poorly understood. Here, we show that the apical complex protein Pals1 and Pten have opposing roles in localizing the Igf1R to the apical, ventricular domain of cerebral cortical progenitor cells. We found that the cerebrospinal fluid (CSF), which contacts this apical domain, has an age-dependent effect on proliferation, much of which is attributable to Igf2, but that CSF contains other signaling activities as well. CSF samples from patients with glioblastoma multiforme show elevated Igf2 and stimulate stem cell proliferation in an Igf2-dependent manner. Together, our findings demonstrate that the apical complex couples intrinsic and extrinsic signaling, enabling progenitors to sense and respond appropriately to diffusible CSF-borne signals distributed widely throughout the brain. The temporal control of CSF composition may have critical relevance to normal development and neuropathological conditions.

A truncating mutation of TRAPPC9 is associated with autosomal-recessive intellectual disability and postnatal microcephaly.

Although autosomal genes are increasingly recognized as important causes of intellectual disability, very few of them are known. We identified a genetic locus for autosomal-recessive nonsyndromic intellectual disability associated with variable postnatal microcephaly through homozygosity mapping of a consanguineous Israeli Arab family. Sequence analysis of genes in the candidate interval identified a nonsense nucleotide change in the gene that encodes TRAPPC9 (trafficking protein particle complex 9, also known as NIBP), which has been implicated in NF-kappaB activation and possibly in intracellular protein trafficking. TRAPPC9 is highly expressed in the postmitotic neurons of the cerebral cortex, and MRI analysis of affected patients shows defects in axonal connectivity. This suggests essential roles of TRAPPC9 in human brain development, possibly through its effect on NF-kappaB activation and protein trafficking in the postmitotic neurons of the cerebral cortex.

SynBioSS: the synthetic biology modeling suite.

UNLABELLED: SynBioSS (Synthetic Biology Software Suite) is a suite of software for the modeling and simulation of synthetic genetic constructs. SynBioSS utilizes the registry of standard biological parts, a database of kinetic parameters, and both graphical and command-line interfaces to multiscale simulation algorithms. AVAILABILITY: SynBioSS is available under the GNU General Public License at http://synbioss.sourceforge.net.

Computational analysis of glycoside hydrolase family 1 specificities.

Glycoside hydrolase family 1 consists of beta-glucosidases, beta-galactosidases, 6-phospho-beta-galactosidases, myrosinases, and other enzymes having similar primary and tertiary structures but diverse specificities. Among these enzymes, beta-glucosidases hydrolyze cellobiose to glucose, and therefore they are key players in any cellulose to glucose process. All family members attack beta-glycosidic bonds between a pyranosyl glycon and an aglycon, but most have little specificity for the aglycon or for the bond configuration. Furthermore, glycon specificity is not absolute. Sixteen family members (six beta-glucosidases, two cyanogenic beta-glucosidases, one 6-phospho-beta-galactosidase, two myrosinases, and five beta-glycosidases) have known tertiary structures. We have used automated docking to computationally bind disaccharides with allopyranosyl, galactopyranosyl, glucopyranosyl, mannopyranosyl, 6-phosphogalactopyranosyl, and 6-phosphoglucopyranosyl glycons, all linked by beta-(1,2), beta-(1,3), beta-(1,4), and beta-(1,6)-glycosidic bonds to beta-glucopyranoside aglycons, along with beta-(1,1-thio)-allopyranosyl, -galactopyranosyl, -glucopyranosyl, and -mannopyranosyl) beta-glucopyranosides, into all of these structures to investigate the structural determinants of their enzyme specificities. The following are the eight active-site residues: Glu191, Thr194, Phe205, Asn285, Arg336, Asn376, Trp378, and Trp465 (Zea mays beta-glucosidase numbering), that control a significant amount of glycon specificity.

Computational analyses of the conformational itinerary along the reaction pathway of GH94 cellobiose phosphorylase.

GH94 cellobiose phosphorylase (CBP) catalyzes the phosphorolysis of cellobiose into alpha-D-glucose 1-phosphate (G1P) and D-glucose with inversion of anomeric configuration. The complex crystal structure of CBP from Cellvibrio gilvus had previously been determined; glycerol, glucose, and phosphate are bound to subsites -1, +1, and the anion binding site, respectively. We performed computational analyses to elucidate the conformational itinerary along the reaction pathway of this enzyme. autodock was used to dock cellobiose with its glycon glucosyl residue in various conformations and with its aglycon glucosyl residue in the low-energy 4C1 conformer. An oxocarbenium ion-like glucose molecule mimicking the transition state was also docked. Based on the clustering analysis, docked energies, and comparison with the crystallographic ligands, we conclude that the reaction proceeds from 1S3 as the pre-transition state conformer (Michaelis complex) via E3 as the transition state candidate to 4C1 as the G1P product conformer. The predicted reaction pathway of the inverting phosphorylase is similar to that proposed for the first-half glycosylation reaction of retaining cellulases, but is different from those for inverting cellulases. NAMD was used to simulate molecular dynamics of the enzyme. The 1S3 pre-transition state conformer is highly stable compared with other conformers, and a conformational change from 4C1 to 1,4B was observed.

A Gibbs free energy correlation for automated docking of carbohydrates.

Thermodynamic information can be inferred from static atomic configurations. To model the thermodynamics of carbohydrate binding to proteins accurately, a large binding data set has been assembled from the literature. The data set contains information from 262 unique protein-carbohydrate crystal structures for which experimental binding information is known. Hydrogen atoms were added to the structures and training conformations were generated with the automated docking program AutoDock 3.06, resulting in a training set of 225,920 all-atom conformations. In all, 288 formulations of the AutoDock 3.0 free energy model were trained against the data set, testing each of four alternate methods of computing the van der Waals, solvation, and hydrogen-bonding energetic components. The van der Waals parameters from AutoDock 1 produced the lowest errors, and an entropic model derived from statistical mechanics produced the only models with five physically and statistically significant coefficients. Eight models predict the Gibbs free energy of binding with an error of less than 40% of the error of any similar models previously published.

Automated docking to explore subsite binding by glycoside hydrolase family 6 cellobiohydrolases and endoglucanases.

Cellooligosaccharides were computationally docked using AutoDock into the active sites of the glycoside hydrolase Family 6 enzymes Hypocrea jecorina (formerly Trichoderma reesei) cellobiohydrolase and Thermobifida fusca endoglucanase. Subsite -2 exerts the greatest intermolecular energy in binding beta-glucosyl residues, with energies progressively decreasing to either side. Cumulative forces imparting processivity exerted by these two enzymes are significantly less than by the equivalent glycoside hydrolase Family 7 enzymes studied previously. Putative subsites -4, -3, +3, and +4 exist in H. jecorina cellobiohydrolase, along with putative subsites -4, -3, and +3 in T. fusca endoglucanase, but they are less important than subsites -2, -1, +1, and +2. In general, binding adds 3-7 kcal/mol to ligand intramolecular energies because of twisting of scissile glycosidic bonds. Distortion of beta-glucosyl residues to the (2)S(O) conformation by binding in subsite -1 adds approximately 7 kcal/mol to substrate intramolecular energies.

A 2-Mb critical region implicated in the microcephaly associated with terminal 1q deletion syndrome.

Patients with distal deletions of chromosome 1q have a recognizable syndrome that includes microcephaly, hypoplasia or agenesis of the corpus callosum, and psychomotor retardation. Although these symptoms have been attributed to deletions of 1q42-1q44, the minimal chromosomal region involved has not been identified. Using microsatellite and single nucleotide polymorphism (SNP) markers, we have mapped the deleted regions in seven patients with terminal deletions of chromosome 1q to define a 2.0-Mb microcephaly critical region including the 1q43-1q44 boundary and no more than 11 genes.

Puckering coordinates of monocyclic rings by triangular decomposition.

We describe a new method of describing the pucker of an N-member monocyclic ring using N - 3 parameters. To accomplish this, three ring atoms define a reference plane, and the remainder of the ring is decomposed into triangular flaps. The angle of incidence for each flap upon the reference plane is then measured. The combination of these angles is characteristic of the ring's pucker. This puckering coordinate system is compared to existing reduced parameter systems to describe rings using a cyclohexane molecule. We show that this method has the same descriptive power of previous systems while offering advantages in molecular simulations.

Comparing programs for rigid-body multiple structural superposition of proteins.

Different programs and methods were employed to superimpose protein structures, using members of four very different protein families as test subjects, and the results of these efforts were compared. Algorithms based on human identification of key amino acid residues on which to base the superpositions were nearly always more successful than programs that used automated techniques to identify key residues. Among those programs automatically identifying key residues, MASS could not superimpose all members of some families, but was very efficient with other families. MODELLER, MultiProt, and STAMP had varying levels of success. A genetic algorithm program written for this project did not improve superpositions when results from neighbor-joining and pseudostar algorithms were used as its starting cases, but it always improved superpositions obained by MODELLER and STAMP. A program entitled PyMSS is presented that includes three superposition algorithms featuring human interaction.

Phylogenetic analysis of family 6 glycoside hydrolases.

Multiple sequence alignment separates members of glycoside hydrolase Family 6 into eight subfamilies: one of mainly actinobacterial endoglucanases (EGs), one of ascomycotal EGs, one of chytridiomycotal EGs and cellobiohydrolases (CBHs), one of actinobacterial and proteobacterial CBHs, one of chytridiomycotal CBHs, two of ascomycotal CBHs, and one of basidiomycotal CBHs. Each also has some proteins of unknown function. Multiple sequence alignment also extends to all of Family 6 the observation that lengths of loops that form the active-site tunnel in CBHs vary among subfamilies, and along with loop conformations, determine enzyme function.