Protein domain


In molecular biology, a protein domain is a region of a protein's polypeptide chain that is self-stabilizing and that folds independently from the rest. Each domain forms a compact folded three-dimensional structure. Many proteins consist of several domains, and a domain may appear in a variety of different proteins. Molecular evolution uses domains as building blocks and these may be recombined in different arrangements to create proteins with different functions. In general, domains vary in length from between about 50 amino acids up to 250 amino acids in length. The shortest domains, such as zinc fingers, are stabilized by metal ions or disulfide bridges. Domains often form functional units, such as the calcium-binding EF hand domain of calmodulin. Because they are independently stable, domains can be "swapped" by genetic engineering between one protein and another to make chimeric proteins.

Background

The concept of the domain was first proposed in 1973 by Wetlaufer after X-ray
crystallographic studies of hen lysozyme and papain
and by limited proteolysis studies of immunoglobulins. Wetlaufer defined domains as stable units of protein structure that could fold autonomously. In the past domains have been described as units of:
  • compact structure
  • function and evolution
  • folding.
Each definition is valid and will often overlap, i.e. a compact structural domain that is found amongst diverse proteins is likely to fold independently within its structural environment. Nature often brings several domains together to form multidomain and multifunctional proteins with a vast number of possibilities. In a multidomain protein, each domain may fulfill its own function independently, or in a concerted manner with its neighbours. Domains can either serve as modules for building up large assemblies such as virus particles or muscle fibres, or can provide specific catalytic or binding sites as found in enzymes or regulatory proteins.

Example: Pyruvate kinase

An appropriate example is pyruvate kinase, a glycolytic enzyme that plays an important role in regulating the flux from fructose-1,6-biphosphate to pyruvate. It contains an all-β nucleotide-binding domain, an α/β-substrate binding domain and an α/β-regulatory domain, connected by several polypeptide linkers. Each domain in this protein occurs in diverse sets of protein families.
The central α/β-barrel substrate binding domain is one of the most common enzyme folds. It is seen in many different enzyme families catalysing completely unrelated reactions. The α/β-barrel is commonly called the TIM barrel named after triose phosphate isomerase, which was the first such structure to be solved. It is currently classified into 26 homologous families in the CATH domain database. The TIM barrel is formed from a sequence of β-α-β motifs closed by the first and last strand hydrogen bonding together, forming an eight stranded barrel. There is debate about the evolutionary origin of this domain. One study has suggested
that a single ancestral enzyme could have diverged into several families, while another suggests that a stable TIM-barrel structure has evolved
through convergent evolution.
The TIM-barrel in pyruvate kinase is 'discontinuous', meaning that more than one segment of the polypeptide is required to form the domain. This is likely to be the result of the insertion of one domain into another during the protein's evolution. It has been shown from known structures that about a quarter of structural domains are discontinuous. The inserted β-barrel regulatory domain is 'continuous', made up of a single stretch of polypeptide.

Units of protein structure

The primary structure of a protein ultimately encodes its uniquely folded three-dimensional conformation. The most important factor governing the folding of a protein into 3D structure is the distribution of polar and non-polar side chains. Folding is driven by the burial of hydrophobic side chains into the interior of the molecule so to avoid contact with the aqueous environment. Generally proteins have a core of hydrophobic residues surrounded by a shell of hydrophilic residues. Since the peptide bonds themselves are polar they are neutralised by hydrogen bonding with each other when in the hydrophobic environment. This gives rise to regions of the polypeptide that form regular 3D structural patterns called secondary structure. There are two main types of secondary structure: α-helices and β-sheets.
Some simple combinations of secondary structure elements have been found to frequently occur in protein structure and are referred to as supersecondary structure or motifs. For example, the β-hairpin motif consists of two adjacent antiparallel β-strands joined by a small loop. It is present in most antiparallel β structures both as an isolated ribbon and as part of more complex β-sheets. Another common super-secondary structure is the β-α-β motif, which is frequently used to connect two parallel β-strands. The central α-helix connects the C-termini of the first strand to the N-termini of the second strand, packing its side chains against the β-sheet and therefore shielding the hydrophobic residues of the β-strands from the surface.
Covalent association of two domains represents a functional and structural advantage since there is an increase in stability when compared with the same structures non-covalently associated. Other advantages are the protection of intermediates within inter-domain enzymatic clefts that may
otherwise be unstable in aqueous environments, and a fixed stoichiometric ratio of the enzymatic activity necessary for a sequential set of reactions.
Structural alignment is an important tool for determining domains.

Tertiary structure

Several motifs pack together to form compact, local, semi-independent units called domains.
The overall 3D structure of the polypeptide chain is referred to as the protein's tertiary structure. Domains are the fundamental units of tertiary structure, each domain containing an individual hydrophobic core built from secondary structural units connected by loop regions. The packing of the polypeptide is usually much tighter in the interior than the exterior of the domain producing a solid-like core and a fluid-like surface. Core residues are often conserved in a protein family, whereas the residues in loops are less conserved, unless they are involved in the protein's function. Protein tertiary structure can be divided into four main classes based on the secondary structural content of the domain.
  • All-α domains have a domain core built exclusively from α-helices. This class is dominated by small folds, many of which form a simple bundle with helices running up and down.
  • All-β domains have a core composed of antiparallel β-sheets, usually two sheets packed against each other. Various patterns can be identified in the arrangement of the strands, often giving rise to the identification of recurring motifs, for example the Greek key motif.
  • α+β domains are a mixture of all-α and all-β motifs. Classification of proteins into this class is difficult because of overlaps to the other three classes and therefore is not used in the CATH domain database.
  • α/β domains are made from a combination of β-α-β motifs that predominantly form a parallel β-sheet surrounded by amphipathic α-helices. The secondary structures are arranged in layers or barrels.

    Limits on size

Domains have limits on size. The size of individual structural domains varies from 36 residues in E-selectin to 692 residues in lipoxygenase-1, but the majority, 90%, have fewer than 200 residues with an average of approximately 100 residues. Very short domains, less than 40 residues, are often stabilised by metal ions or disulfide bonds. Larger domains, greater than 300 residues, are likely to consist of multiple hydrophobic cores.

Quaternary structure

Many proteins have a quaternary structure, which consists of several polypeptide chains that associate into an oligomeric molecule. Each polypeptide chain in such a protein is called a subunit. Hemoglobin, for example, consists of two α and two β subunits. Each of the four chains has an all-α globin fold with a heme pocket.

Domain swapping

Domain swapping is a mechanism for forming oligomeric assemblies. In domain swapping, a secondary or tertiary element of a monomeric protein is replaced by the same element of another protein. Domain swapping can range from secondary structure elements to whole structural domains. It also represents a model of evolution for functional adaptation by oligomerisation, e.g. oligomeric enzymes that have their active site at subunit interfaces.

Domains as evolutionary modules

Nature is a tinkerer and not an inventor, new sequences are adapted from pre-existing sequences rather than invented. Domains are the common material used by nature to generate new sequences; they can be thought of as genetically mobile units, referred to as 'modules'. Often, the C and N termini of domains are close together in space, allowing them to easily be "slotted into" parent structures during the process of evolution. Many domain families are found in all three forms of life, Archaea, Bacteria and Eukarya. Protein modules are a subset of protein domains which are found across a range of different proteins with a particularly versatile structure. Examples can be found among extracellular proteins associated with clotting, fibrinolysis, complement, the extracellular matrix, cell surface adhesion molecules and cytokine receptors. Four concrete examples of widespread protein modules are the following domains: SH2, immunoglobulin, fibronectin type 3 and the kringle.
Molecular evolution gives rise to families of related proteins with similar sequence and structure. However, sequence similarities can be extremely low between proteins that share the same structure. Protein structures may be similar because proteins have diverged from a common ancestor. Alternatively, some folds may be more favored than others as they represent stable arrangements of secondary structures and some proteins may converge towards these folds over the course of evolution. There are currently more than 200,000 experimentally determined protein 3D structures deposited within the Protein Data Bank. However, this set contains many identical or very similar structures. All proteins should be classified to structural families to understand their evolutionary relationships. Structural comparisons are best achieved at the domain level. For this reason many algorithms have been developed to automatically assign domains in proteins with known 3D structure.
The CATH domain database classifies domains into approximately 800 fold families; ten of these folds are highly populated and are referred to as 'super-folds'. Super-folds are defined as folds for which there are at least three structures without significant sequence similarity. The most populated is the α/β-barrel super-fold, as described previously.