Suhail A. Islam

Analysis of the relationship between side-chain conformation and secondary structure in globular proteins.

The relationship between the preferred side-chain dihedral angles and the secondary structure of a residue was examined. The structures of 61 proteins solved to a resolution of 2.0 A (1 A = 0.1 nm) or better were analysed using a relational database to store the information. The strongest feature observed was that the chi 1 distribution for most side-chains in an alpha-helix showed an absence of the g- conformation and a shift towards the t conformation when compared to the non-alpha/beta structures. The exceptions to this tendency were for short polar side-chains that form hydrogen bonds with the main-chain which prefer g+. Shifts in the chi 1 preferences for residues in the beta-sheet were observed. Other side-chain dihedral angles (chi 2, chi 3, chi 4) were found to be influenced by the main-chain. This paper presents more accurate distributions for the side-chain dihedral angles which were obtained from the increased number of proteins determined to high resolution. The means and standard deviations for chi 1 and chi 2 angles are presented for all residues according to the secondary structure of the main-chain. The means and standard deviations are given for the most popular conformations for side-chains in which chi 3 and chi 4 rotations affect the position of C atoms.

Mutations in a Sar1 GTPase of COPII vesicles are associated with lipid absorption disorders.

Dietary fat is an important source of nutrition. Here we identify eight mutations in SARA2 that are associated with three severe disorders of fat malabsorption. The Sar1 family of proteins initiates the intracellular transport of proteins in COPII (coat protein)-coated vesicles. Our data suggest that chylomicrons, which vastly exceed the size of typical COPII vesicles, are selectively recruited by the COPII machinery for transport through the secretory pathways of the cell.

Promyelocytic leukemia nuclear bodies associate with transcriptionally active genomic regions

The promyelocytic leukemia (PML) protein is aggregated into nuclear bodies that are associated with diverse nuclear processes. Here, we report that the distance between a locus and its nearest PML body correlates with the transcriptional activity and gene density around the locus. Genes on the active X chromosome are more significantly associated with PML bodies than their silenced homologues on the inactive X chromosome. We also found that a histone-encoding gene cluster, which is transcribed only in S-phase, is more strongly associated with PML bodies in S-phase than in G0/G1 phase of the cell cycle. However, visualization of specific RNA transcripts for several genes showed that PML bodies were not themselves sites of transcription for these genes. Furthermore, knock-down of PML bodies by RNA interference did not preferentially change the expression of genes closely associated with PML bodies. We propose that PML bodies form in nuclear compartments of high transcriptional activity, but they do not directly regulate transcription of genes in these compartments.

Application of the diffusion-collision model to the folding of three-helix bundle proteins

The diffusion-collision model has been successful in explaining many features of protein folding kinetics, particularly for helical proteins. In the model the folding reaction is described in terms of coupled chemical kinetic (Master) equations of coarse grained entities, called microdomains. Here, the diffusion-collision model is applied to compute the folding kinetics of four three-helix bundle proteins, all of which fold on a time scale of tens of microseconds and appear to have two-state folding. The native structure and the stability of the helical microdomains are used to determine the parameters of the model. The formulation allows computation of the overall rate and determination of the importance of kinetic intermediates. The proteins considered are the B domain of protein A (1BDC), the Engrailed Homeodomain (1ENH), the peripheral sub-unit-binding domain (1EBD C-chain) and the villin headpiece subdomain (1VII). The results for the folding time of protein A, the Engrailed Homeodomain, and 1EBD C-chain are in agreement with experiment, while 1VII is not stable in the present model. In the three proteins that are stable, two-state folding is predicted by the diffusion-collision model. This disagrees with published assertions that multistate kinetics would be obtained from the model. The contact order prediction agrees with experiment for protein A, but yields values that are a factor of 40, 30 and 15 too slow for 1ENH, 1EBD C-chain and 1VII. The effect of mutants on folding is described for protein A and it is demonstrated that significant intermediate concentrations (i.e. deviation from two-state folding) can occur if the stability of some of the helical microdomains is increased. A linear relationship between folding time and the length of the loop between helices B and C in protein A is demonstrated; this is not evident in the contact order description.

Protein–protein docking using 3D-Dock in rounds 3, 4, and 5 of CAPRI

In rounds 3–5 of CAPRI, the community‐wide experiment on the comparative evaluation of protein–protein docking for structure prediction, we applied the 3D‐Dock software package to predict the atomic structures of nine biophysical interactions. This approach starts with an initial grid‐based shape complementarity search. The product of this is a large number of potential interacting conformations that are subsequently ranked by interface residue propensities and interaction energies. Refinement through detailed energetics and optimization of side‐chain positions using a rotamer library is also performed. For rounds 3, 4, and 5 of the CAPRI evaluation, where possible, we clustered functional residues on the surfaces of the monomers as an indication of binding sites, using sequence based evolutionary conservations. In certain targets this provided a very useful tool for identifying the areas of interaction. During round 5, we also applied the techniques of side‐chain trimming and geometrical clustering described in the literature. Of the nine target complexes in rounds 3–5, we predicted conformations that contained at least some correct contact residues for seven of these systems. For two of the targets, we submitted predictions that were considered as medium‐quality. These were a nidogen–laminin complex for target 8 (T08) and a serine‐threonine phosphatase bound to a targeting subunit (T14). For a further three target systems, we produced models that were rated as acceptable predictions. Proteins 2005;60:281–288.

HAD, a Data Bank of Heavy-Atom Binding Sites in Protein Crystals: a Resource for Use in Multiple Isomorphous Replacement and Anomalous Scattering

Information on the preparation and characterization of heavy-atom derivatives of protein crystals has been collected, either from the literature or directly from protein crystallographers, and assembled in the form of a heavy-atom data bank (HAD). The data bank contains coordinate data for the heavy-atom positions in a form that is compatible with the crystallographic data in the Brookhaven Protein Data Bank, together with a wealth of information on the crystallization conditions, the nature of the heavy-atom reagent and references to relevant publications. Some statistical information derived from the data bank, such as the most popular heavy-atom derivatives, is also included. The information can be directly accessed and should be useful to protein crystallographers seeking to improve their success in preparing heavy-atom derivatives for the methods of isomorphous replacement and anomalous dispersion. The World Wide Web address of HAD is http://www.icnet.uk/bmm/had.

The Transcriptional Regulator CBP Has Defined Spatial Associations within Interphase Nuclei

It is becoming increasingly clear that nuclear macromolecules and macromolecular complexes are compartmentalized through binding interactions into an apparent three-dimensionally ordered structure. This ordering, however, does not appear to be deterministic to the extent that chromatin and nonchromatin structures maintain a strict 3-D arrangement. Rather, spatial ordering within the cell nucleus appears to conform to stochastic rather than deterministic spatial relationships. The stochastic nature of organization becomes particularly problematic when any attempt is made to describe the spatial relationship between proteins involved in the regulation of the genome. The CREB–binding protein (CBP) is one such transcriptional regulator that, when visualised by confocal microscopy, reveals a highly punctate staining pattern comprising several hundred individual foci distributed within the nuclear volume. Markers for euchromatic sequences have similar patterns. Surprisingly, in most cases, the predicted one-to-one relationship between transcription factor and chromatin sequence is not observed. Consequently, to understand whether spatial relationships that are not coincident are nonrandom and potentially biologically important, it is necessary to develop statistical approaches. In this study, we report on the development of such an approach and apply it to understanding the role of CBP in mediating chromatin modification and transcriptional regulation. We have used nearest-neighbor distance measurements and probability analyses to study the spatial relationship between CBP and other nuclear subcompartments enriched in transcription factors, chromatin, and splicing factors. Our results demonstrate that CBP has an order of spatial association with other nuclear subcompartments. We observe closer associations between CBP and RNA polymerase II–enriched foci and SC35 speckles than nascent RNA or specific acetylated histones. Furthermore, we find that CBP has a significantly higher probability of being close to its known in vivo substrate histone H4 lysine 5 compared with the closely related H4 lysine 12. This study demonstrates that complex relationships not described by colocalization exist in the interphase nucleus and can be characterized and quantified. The subnuclear distribution of CBP is difficult to reconcile with a model where chromatin organization is the sole determinant of the nuclear organization of proteins that regulate transcription but is consistent with a close link between spatial associations and nuclear functions.

The proteome: structure, function and evolution.

This paper reports two studies to model the inter-relationships between protein sequence, structure and function. First, an automated pipeline to provide a structural annotation of proteomes in the major genomes is described. The results are stored in a database at Imperial College, London (3D-GENOMICS) that can be accessed at www.sbg.bio.ic.ac.uk. Analysis of the assignments to structural superfamilies provides evolutionary insights. 3D-GENOMICS is being integrated with related proteome annotation data at University College London and the European Bioinformatics Institute in a project known as e-protein (http://www.e-protein.org/). The second topic is motivated by the developments in structural genomics projects in which the structure of a protein is determined prior to knowledge of its function. We have developed a new approach PHUNCTIONER that uses the gene ontology (GO) classification to supervise the extraction of the sequence signal responsible for protein function from a structure-based sequence alignment. Using GO we can obtain profiles for a range of specificities described in the ontology. In the region of low sequence similarity (around 15%), our method is more accurate than assignment from the closest structural homologue. The method is also able to identify the specific residues associated with the function of the protein family.

Nucleic acid binding drugs. VII. Molecular-mechanics studies on the conformational properties of the anti-cancer drug daunomycin: some observations on the use of differing potential-energy functions

The conformation of the anti-cancer drug daunomycin has been investigated in detail by potential-energy calculations. The flexibility around the ether linkage, connecting the anthracycline chromophore and the amino sugar group, has been evaluated using several types of potential-energy function. The results largely support the hypothesis that the crystallographically observed conformation is the most stable one, although considerable detailed variation with respect to potential function was found.

The discovery of novel 10,11-dihydro-5H-dibenz[b,f]azepine SIRT2 inhibitors

Isoform selective inhibitors of the sirtuins (NAD+-dependent histone deacetylases) should enable an in depth study of the molecular biology underpinning these targets and how they are deregulated in diseases such as cancer and neurodegeneration. Herein, we present the discovery of structurally novel SIRT2 inhibitors. Hit molecule 8 was discovered through the chemical synthesis and biological characterization of a small-molecule compound library based around the 10,11-dihydro-5H-dibenz[b,f]azepine scaffold. In vitro screening assays revealed compound 8 to have an IC50 of 18 μM against SIRT2 and to exhibit more than 30-fold selectivity compared to SIRT1. Cellular assays, performed on MCF-7 cells, confirmed the in vitro selectivity and showed hit 8 to have antiproliferative activity at a concentration of 30 μM. Computational studies were performed to predict the SIRT2 binding mode and to rationalise the observed selectivity.