Lesley H. Greene

The CATH domain structure database: new protocols and classification levels give a more comprehensive resource for exploring evolution

We report the latest release (version 3.0) of the CATH protein domain database (). There has been a 20% increase in the number of structural domains classified in CATH, up to 86 151 domains. Release 3.0 comprises 1110 fold groups and 2147 homologous superfamilies. To cope with the increases in diverse structural homologues being determined by the structural genomics initiatives, more sensitive methods have been developed for identifying boundaries in multi-domain proteins and for recognising homologues. The CATH classification update is now being driven by an integrated pipeline that links these automated procedures with validation steps, that have been made easier by the provision of information rich web pages summarising comparison scores and relevant links to external sites for each domain being classified. An analysis of the population of domains in the CATH hierarchy and several domain characteristics are presented for version 3.0. We also report an update of the CATH Dictionary of homologous structures (CATH-DHS) which now contains multiple structural alignments, consensus information and functional annotations for 1459 well populated superfamilies in CATH. CATH is directly linked to the Gene3D database which is a projection of CATH structural data onto ∼2 million sequences in completed genomes and UniProt.

Screening Transthyretin Amyloid Fibril Inhibitors: Characterization of Novel Multiprotein, Multiligand Complexes by Mass Spectrometry

Tetrameric transthyretin is involved in transport of thyroxine and, through its interactions with retinol binding protein, vitamin A. Dissociation of these structures is widely accepted as the first step in the formation of transthyretin amyloid fibrils. Using a mass spectrometric approach, we have examined a series of 18 ligands proposed as inhibitors of this process. The ligands were evaluated for their ability to bind to and stabilize the tetrameric structure, their cooperativity in binding, and their ability to compete with the natural ligand thyroxine. The observation of a novel ten-component complex containing six protein subunits, two vitamin molecules, and two synthetic ligands allows us to conclude that ligand binding does not inhibit association of transthyretin with holo retinol binding protein.

Sequence and Structural Analysis of the Chitinase Insertion Domain Reveals Two Conserved Motifs Involved in Chitin-Binding

Background Chitinases are prevalent in life and are found in species including archaea, bacteria, fungi, plants, and animals. They break down chitin, which is the second most abundant carbohydrate in nature after cellulose. Hence, they are important for maintaining a balance between carbon and nitrogen trapped as insoluble chitin in biomass. Chitinases are classified into two families, 18 and 19 glycoside hydrolases. In addition to a catalytic domain, which is a triosephosphate isomerase barrel, many family 18 chitinases contain another module, i.e., chitinase insertion domain. While numerous studies focus on the biological role of the catalytic domain in chitinase activity, the function of the chitinase insertion domain is not completely understood. Bioinformatics offers an important avenue in which to facilitate understanding the role of residues within the chitinase insertion domain in chitinase function. Results Twenty-seven chitinase insertion domain sequences, which include four experimentally determined structures and span five kingdoms, were aligned and analyzed using a modified sequence entropy parameter. Thirty-two positions with conserved residues were identified. The role of these conserved residues was explored by conducting a structural analysis of a number of holo-enzymes. Hydrogen bonding and van der Waals calculations revealed a distinct subset of four conserved residues constituting two sequence motifs that interact with oligosaccharides. The other conserved residues may be key to the structure, folding, and stability of this domain. Conclusions Sequence and structural studies of the chitinase insertion domains conducted within the framework of evolution identified four conserved residues which clearly interact with the substrates. Furthermore, evolutionary studies propose a link between the appearance of the chitinase insertion domain and the function of family 18 chitinases in the subfamily A.

Role of conserved residues in structure and stability: Tryptophans of human serum retinol-binding protein, a model for the lipocalin superfamily

Serum retinol binding protein (RBP) is a member of the lipocalin family, proteins with up‐and‐down β‐barrel folds, low levels of sequence identity, and diverse functions. Although tryptophan 24 of RBP is highly conserved among lipocalins, it does not play a direct role in activity. To determine if Trp24 and other conserved residues have roles in stability and/or folding, we investigated the effects of conservative substitutions for the four tryptophans and some adjacent residues on the structure, stability, and spectroscopic properties of apo‐RBP. Crystal structures of recombinant human apo‐RBP and of a mutant with substitutions for tryptophans 67 and 91 at 1.7 Å and 2.0 Å resolution, respectively, as well as stability measurements, indicate that these relatively exposed tryptophans have little influence on structure or stability. Although Trp105 is largely buried in the wall of the β‐barrel, it can be replaced with minor effects on stability to thermal and chemical unfolding. In contrast, substitutions of three different amino acids for Trp24 or replacement of Arg139, a conserved residue that interacts with Trp24, lead to similar large losses in stability and lower yields of native protein generated by in vitro folding. The results and the coordinated nature of natural substitutions at these sites support the idea that conserved residues in functionally divergent homologs have roles in stabilizing the native relative to misfolded structures. They also establish conditions for studies of the kinetics of folding and unfolding by ideying spectroscopic signals for monitoring the formation of different substructures.

The CATH Hierarchy Revisited—Structural Divergence in Domain Superfamilies and the Continuity of Fold Space

Summary This paper explores the structural continuum in CATH and the extent to which superfamilies adopt distinct folds. Although most superfamilies are structurally conserved, in some of the most highly populated superfamilies (4% of all superfamilies) there is considerable structural divergence. While relatives share a similar fold in the evolutionary conserved core, diverse elaborations to this core can result in significant differences in the global structures. Applying similar protocols to examine the extent to which structural overlaps occur between different fold groups, it appears this effect is confined to just a few architectures and is largely due to small, recurring super-secondary motifs (e.g., αβ-motifs, α-hairpins). Although 24% of superfamilies overlap with superfamilies having different folds, only 14% of nonredundant structures in CATH are involved in overlaps. Nevertheless, the existence of these overlaps suggests that, in some regions of structure space, the fold universe should be seen as more continuous.

Destruction of α-synuclein based amyloid fibrils by a low temperature plasma jet

Amyloid fibrils are ordered beta-sheet aggregates that are associated with a number of neurodegenerative diseases such as Alzheimer and Parkinson. At present, there is no cure for these progressive and debilitating diseases. Here we report initial studies that indicate that low temperature atmospheric pressure plasma can break amyloid fibrils into smaller units in vitro. The plasma was generated by the “plasma pencil,” a device capable of emitting a long, low temperature plasma plume/jet. This avenue of research may facilitate the development of a plasma-based medical treatment.

Conserved signature proposed for folding in the lipocalin superfamily

We systematically identify a group of evolutionarily conserved residues proposed for folding in a model β‐barrel superfamily, the lipocalins. The nature of conservation at the structural level is defined and we show that the conserved residues are involved in a network of interactions that form the core of the fold. Exploratory kinetic studies are conducted with a model superfamily member, human serum retinol‐binding protein, to examine their role. The present results, coupled with key experimental studies conducted with another lipocalin β‐lactoglobulin, suggest that the evolutionarily conserved regions fold on a faster folding time‐scale than the non‐conserved regions.

Protein structure networks

The application of the field of network science to the scientific disciplines of structural biology and biochemistry, have yielded important new insights into the nature and determinants of protein structures, function, dynamics and the folding process. Advancements in further understanding protein relationships through network science have also reshaped the way we view the connectivity of proteins in the protein universe. The canonical hierarchical classification can now be visualized for example, as a protein fold continuum. This review will survey several key advances in the expanding area of research being conducted to study protein structures and folding using network approaches.

Characterization of the molten globule state of retinol‐binding protein using a molecular dynamics simulation approach

Retinol‐binding protein transports retinol, and circulates in the plasma as a macromolecular complex with the protein transthyretin. Under acidic conditions retinol‐binding protein undergoes a transition to the molten globule state, and releases the bound retinol ligand. A biased molecular dynamics simulation method has been used to generate models for the ensemble of conformers populated within this molten globule state. Simulation conformers, with a radius of gyration at least 1.1 Å greater than that of the native state, contain on average 37%β‐sheet secondary structure. In these conformers the central regions of the two orthogonal β‐sheets that make up the β‐barrel in the native protein are highly persistent. However, there are sizable fluctuations for residues in the outer regions of the β‐sheets, and large variations in side chain packing even in the protein core. Significant conformational changes are seen in the simulation conformers for residues 85–104 (β‐strands E and F and the E‐F loop). These changes give an opening of the retinol‐binding site. Comparisons with experimental data suggest that the unfolding in this region may provide a mechanism by which the complex of retinol‐binding protein and transthyretin dissociates, and retinol is released at the cell surface.

Protein folding by ‘levels of separation’: A hypothesis

The protein folding process has been studied both computationally and experimentally for over 30 years. To date there is no detailed mechanism to explain the formation of long‐range interactions between the transition and native states. Long‐range interactions are the principle determinants of the tertiary structure. We present a theoretical model which proposes a mechanism for the acquisition of these interactions as they form in a modified version of ‘degrees of separation’, that we term ‘levels of separation’. It is based on the integration of network science and biochemistry.