Protein domains.
A protein domain is a part of protein sequence and structure that can evolve, function, and exist independently of the rest of the protein chain.  Each domain forms a compact three-dimensional structure and often can be independently stable and  folded. Many proteins consist of several structural domains.  One domain may appear in a variety of evolutionarily related proteins.  Domains vary in length from between about 25 amino acids up to 500 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 self-stable, domains can be "swapped" by genetic engineering between one protein and another to make chimera proteins.
Important tools in determining domains are structural alignment and sequence alignment.
The concept of the domain was first proposed in 1973 by Wetlaufer after X-ray
crystallographic studies of hen lysozyme (Phillips, 1966), papain (Drenth et al., 1968)
and by limited proteolysis studies of immunoglobulins (Porter, 1973; Edelman, 1973). 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 (Richardson, 1981);
- function and evolution (Bork, 1991);
- folding (Wetlaufer, 1973).
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 (Chothia, 1992). In a multidomain protein, each domain may fulfil 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.
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- regulatory domain, an /-substrate binding domain and an
/-nucleotide binding domain, connected by several polypeptide linkers (George and Heringa, 2002a) (see figure, right). 
Each domain in this protein occurs in diverse sets of protein families.
The central /-barrel substrate binding domain is one of the most common
enzyme folds. It is seen in many different enzyme families catalysing completely
unrelated reactions (Hegyi and Gerstein, 1999). The /-barrel is commonly called
the TIM barrel named after triose phosphate isomerase, which was the first such
structure to be solved (Banner et al., 1975). It is currently classified into 26
homologous families in the CATH domain database (Orengo et al., 1997). The
TIM barrel is formed from a sequence of -- 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 (Copley and
Bork, 2000), while another suggests that a stable TIM-barrel structure has evolved
through convergent evolution (Lesk et al., 1989).
The TIM-barrel in pyruvate kinase is 'discontinuous', meaning that more than
-ne 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 (Jones et al., 1998; Holm and Sander, 1994).
The inserted -barrel regulatory domain is 'continuous', made up of a single stretch
-f polypeptide.
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 (Ghelis and Yon, 1979). Other, advantages
are the protection of intermediates within inter-domain enzymatic clefts that may
-therwise be unstable in aqueous environments, and a fixed stoichiometric ratio of
the enzymatic activity necessary for a sequential set of reactions (Ostermeier and
Benkovic, 2000).

# Domains are units of protein structure

## Primary structure
The primary structure (string of amino acids) of a protein encodes its uniquely folded 3D 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.

## Secondary structure
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:
- -helices
- -sheet

## Secondary structure motifs
Some simple combinations of secondary structure elements have been found to frequently occur in protein structure and are referred to as 'super-secondary structure' or motifs. For example, the -hairpin motif consists of two adjacent antiparallel -strands joined by a small loop. It is present in most antiparallel  structures both as an isolated ribbon and as part of more complex -sheets. Another common super-secondary structure is the --  motif, which is frequently used to connect two parallel -strands. The central -helix connects the C-termini of the first strand to the N-termini of the second strand, packing its side chains against the -sheet and therefore shielding the hydrophobic residues of the -strands from the surface.

## 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. In fact, 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- domains have a domain core built exclusively from -helices. This class is dominated by small folds, many of which form a simple bundle with helices running up and down.
- All- domains have a core comprising of antiparallel -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.
- + domains are a mixture of all- and all- 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.
- / domains are made from a combination of -- motifs that predominantly form a parallel -sheet surrounded by amphipathic -helices. The secondary structures are arranged in layers or barrels.

## Domains have 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 less than 200 residues with an average of approximately 100 residues. Very short domains, less than 40 residues, are often stabilised by metal ions or disulphide bonds. Larger domains, greater than 300 residues, are likely to consist of multiple hydrophobic cores.

# Relationship between primary and tertiary structure

## 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'. Many domain families are found in all three forms of life, Archaea,
Bacteria and Eukarya. Domains that are repeatedly found in diverse proteins are
-ften referred to as modules, examples can be found among extracellular proteins
associated with clotting, fibrinolysis, complement, the extracellular matrix, cell surface
adhesion molecules and cytokine receptors.

## Protein families
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 about 45,000 experimentally determined protein 3D structures deposited within the Protein Data Bank (PDB). However this set contains a lot of 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, see 'Domain definition from structural co-ordinates'.

## Super-folds
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 /-barrel super-fold as described previously.

# Multidomain proteins
The majority of genomic proteins, two-thirds in unicellular organisms and
more than 80% in metazoa, are multidomain proteins created as a result of gene
duplication events. Many domains in multidomain structures
could have once existed as independent proteins. More and more domains in
eukaryotic multidomain proteins can be found as independent proteins in prokaryotes. For example, vertebrates have a multi-enzyme polypeptide
containing the  GAR synthetase, AIR synthetase and GAR transformylase modules
(GARs-AIRs-GARt; GAR: glycinamide ribonucleotide synthetase/transferase; AIR: aminoimidazole ribonucleotide synthetase). In insects, the polypeptide appears as GARs-(AIRs)2-GARt,
in yeast GARs-AIRs is encoded separately from GARt, and in bacteria each domain
is encoded separately.

## Origin
Multidomain proteins are likely to have emerged from a selective pressure during
evolution to create new functions. Various proteins have diverged from common
ancestors by different combinations and associations of domains. Modular units
frequently move about, within and between biological systems through mechanisms
-f genetic shuffling:
- transposition of mobile elements including horizontal transfers (between species);
- gross rearrangements such as inversions, translocations, deletions and duplications;
- homologous recombination;
- slippage of DNA polymerase during replication.

## Difference in proliferation
It is likely that all these mechanisms have contributed to the proliferation,
dispersal and loss of protein domains. Some domains are more promiscuous than
-thers, appearing in a diverse set of protein families and organisms. For example,
the ABC transporter domain constitutes one of the largest domain families that
appear in all organisms. Many other families that appear in
all organisms show much less proliferation. These include metabolic enzymes and
components of translational apparatus.

## Types of organisation
The simplest multidomain organisation seen in proteins is that of a single domain
repeated in tandem. The domains may interact with each other or remain isolated, like beads on string. The giant 30,000 residue muscle protein titin comprises about
120 fibronectin-III-type and Ig-type domains. In the serine proteases, a gene duplication event has led to the formation of a two -barrel domain
enzyme. The repeats have diverged so widely that there is no
-bvious sequence similarity between them. The active site is located at a cleft
between the two -barrel domains, in which functionally important residues are
contributed from each domain. Genetically engineered mutants of the chymotrypsin
serine protease were shown to have some proteinase activity even though their active
site residues were abolished and it has therefore been postulated that the duplication
event enhanced the enzyme's activity.

## Connectivity
Modules frequently display different connectivity relationships, as illustrated by
the kinesins and ABC transporters. The kinesin motor domain can be at either end
-f a polypeptide chain that includes a coiled-coil region and a cargo domain. ABC transporters are built with up to four domains consisting
-f two unrelated modules, ATP-binding cassette and an integral membrane module,
arranged in various combinations.

## Domain insertion
Not only do domains recombine, but there are many examples of a domain
having been inserted into another. Sequence or structural similarities to other
domains demonstrate that homologues of inserted and parent domains can exist
independently. An example is that of the 'fingers' inserted into the 'palm' domain
within the polymerases of the Pol I family.

### Difference between structural and evolutionary domain
Since a domain can be inserted into another, there should always be at least one continuous domain in a multidomain protein. This is the main difference between definitions of structural domains and evolutionary/functional domains. An evolutionary domain will be limited to one or two connections between domains, whereas structural domains can have unlimited
connections, within a given criterion of the existence of a common core. Several
structural domains could be assigned to an evolutionary domain.

# Domains are autonomous folding units

## Folding

### History
Protein folding - the unsolved problem
Since the seminal work of Anfinsen over forty years ago, the goal to completely understand the mechanism by which a polypeptide rapidly
folds into its stable native conformation remains elusive. Many experimental folding
studies have contributed much to our understanding, but the principles that govern
protein folding are still based on those discovered in the very first studies of folding.
Anfinsen showed that the native state of a protein is thermodynamically stable, the
conformation being at a global minimum of its free energy.

### Folding pathway
Folding is a directed search of conformational space allowing the protein to fold
-n a biologically feasible time scale. The Levinthal paradox states that if an averaged sized protein would sample all possible conformations before finding the one with
the lowest energy, the whole process would take billions of years.
Proteins typically fold within 0.1 and 1000 seconds, therefore the protein folding
process must be directed some way through a specific folding pathway. The forces
that direct this search are likely to be a combination of local and global influences whose effects are felt at various stages of the reaction.
Advances in experimental and theoretical studies have shown that folding can be
viewed in terms of energy landscapes,
where folding kinetics is considered as a progressive organisation of an ensemble
-f partially folded structures through which a protein passes on its way to the
folded structure. This has been described in terms of a folding funnel, in which
an unfolded protein has a large number of conformational states available and
there are fewer states available to the folded protein. A funnel implies that for
protein folding there is a decrease in energy and loss of entropy with increasing
tertiary structure formation. The local roughness of the funnel reflects kinetic traps,
corresponding to the accumulation of misfolded intermediates. A folding chain
progresses toward lower intra-chain free-energies by increasing its compactness. The
chains conformational options become increasingly narrowed ultimately toward one
native structure.

### Order of folding
Many experimental studies suggest that protein folding begins with the formation
-f secondary structure, followed by the co-operative assembly into tertiary structure
mainly driven by hydrophobic interactions. During the initial stages of folding, regions in the polypeptide will spontaneously form
elements of secondary structure stabilised by a combination of local and long
range interactions, both of which are primarily hydrophobic. Secondary and tertiary structure
are expected to appear simultaneously in a co-operative process, where largely pre-formed secondary structures are joined together in a cluster-induced collapse. This process of folding has been
termed 'nucleation-condensation'. There will be many possible folding
pathways because of the many different combinations of secondary structure, but
some pathways will be related since they involve the same secondary structural units.
Only the proper combination of secondary structural elements will condense to the
native structure.
The fact that algorithms used to predict secondary structure are at best 75%
accurate, suggests that some of the observed elements of secondary structure in
proteins are formed from non-local interactions. Also, the free
energy difference between -helical, -sheet and unstructured coil conformations
for most sequences is small enough that their structures can be interchangeable,
demonstrating that proteins have a structural plasticity that allows them to change
conformation readily. The tertiary interaction will make the final selection to the
actual native topology.

## Advantage of domains in protein folding
The organisation of large proteins by structural domains represents an advantage
for protein folding, with each domain being able to individually fold, accelerating the
folding process and reducing a potentially large combination of residue interactions. Furthermore, given the observed random distribution of hydrophobic residues in proteins, domain formation appears to be the optimal solution for a large protein to bury its hydrophobic residues while keeping the hydrophilic residues at the surface.
However, the role of inter-domain interactions in protein folding and in energetics
-f stabilisation of the native structure, probably differs for each protein. In T4
lysozyme, the influence of one domain on the other is so strong that the entire
molecule is resistant to proteolytic cleavage. In this case, folding is a sequential
process where the C-terminal domain is required to fold independently in an early
step, and the other domain requires the presence of the folded C-terminal domain
for folding and stabilisation.
It has been found that the folding of an isolated domain can take place at
the same rate or sometimes faster than that of the integrated domain. Suggesting that unfavourable interactions with the rest of the protein can occur during folding. Several arguments suggest that the slowest step in the folding of large proteins is the pairing of the folded domains. This is either because the domains are not folded entirely correctly or because the small adjustments required for their interaction are energetically unfavourable, such as the removal of water from the domain interface.

# Domains and quaternary structure

## About quaternary structures
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  and two 
subunits. Each of the four chains has an all- 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.
-ligomeric enzymes that have their active site at subunit interfaces.

# Domains and protein flexibility
The presence of multiple domains in proteins gives rise to a great deal of 
flexibility and mobility. One of the largest observed domain motions is the `swivelling'
mechanism in pyruvate phosphate dikinase. The phosphoinositide domain swivels
between two states in order to bring a phosphate group from the active site of the
nucleotide binding domain to that of the phosphoenolpyruvate/pyruvate domain. The phosphate group is moved over a distance of 45A
involving a domain motion of about 100 degrees around a single residue. Domain motions
are important for:
- catalysis;
- regulatory activity;
- transport of metabolites;
- formation of protein assemblies and
- cellular locomotion.
In enzymes, the closure of one domain onto another captures a substrate by an
induced fit, allowing the reaction to take place in a controlled way. Such motions
can be observed when two or more crystallographic 3D structures of a protein are
experimentally determined in alternate environments, or from the analysis of nuclear
magnetic resonance (NMR) derived structures. A detailed analysis by Gerstein et al. (1994) led to the classification of two basic types of domain motion; hinge and shear.
Only a relatively small portion of the chain, namely the inter-domain linker and
side chains undergo significant conformational changes upon domain rearrangement.

## Hinges by secondary structures
A study by Hayward found that the termini of -helices and
-sheets form hinges in a large number of cases. Many hinges were found to involve
two secondary structure elements acting like hinges of a door, allowing an opening
and closing motion to occur. This can arise when two neighbouring strands within
a -sheet situated in one domain, diverge apart as they join the other domain. The
two resulting termini then form the bending regions between the two domains. -
helices that preserve their hydrogen bonding network when bent are found to behave
as mechanical hinges, storing `elastic energy' that drives the closure of domains for
rapid capture of a substrate.

## Helical to extended conformation
The interconversion of helical and extended conformations at the site of a domain
boundary is not uncommon. In calmodulin, torsion angles change for five residues
in the middle of a domain linking -helix. The helix is split into two, almost
perpendicular, smaller helices separated by four residues of an extended strand.

## Shear motions
Shear motions involve a small sliding movement of domain interfaces, controlled
by the amino acid side chains within the interface. Proteins displaying shear motions
-ften have a layered architecture: stacking of secondary structures. The interdomain
linker has merely the role of keeping the domains in close proximity.

# Domain definition from structural co-ordinates
The importance of domains as structural building blocks and elements of evolution
has brought about many automated methods for their identification and classification
in proteins of known structure. Automatic procedures for reliable domain
assignment is essential for the generation of the domain databases, especially as the
number of protein structures is increasing. Although the boundaries of a domain
can be determined by visual inspection, construction of an automated method is not
straightforward. Problems occur when faced with domains that are discontinuous
-r highly associated. The fact that there is no
standard definition of what a domain really is has meant that domain assignments
have varied enormously, with each researcher using a unique set of criteria.
A structural domain is a compact, globular sub-structure with more interactions
within it than with the rest of the protein.
Therefore, a structural domain can be determined by two visual characteristics;
its compactness and its extent of isolation. Measures of local compactness in proteins have been used in many of the early methods
-f domain assignment and in several of the more recent methods.

## Considering proteins as small segments
One of the first algorithms used a C-C distance map together with a hierarchical clustering routine that considered proteins as several small segments, 10 residues in length. The initial segments were clustered one after another based
-n inter-segment distances; segments with the shortest distances were clustered and
considered as single segments thereafter. The stepwise clustering finally included
the full protein. Go (1978) also exploited the fact that inter-domain distances are
normally larger than intra-domain distances; all possible C-C distances were
represented as diagonal plots in which there were distinct patterns for helices,
extended strands and combinations of secondary structures.

## Sowdhamini and Blundells method
The method by Sowdhamini and Blundell (1995) clusters secondary structures in
a protein based on their C-C distances and identifies domains from the pattern in
their dendrograms. As the procedure does not consider the protein as a continuous
chain of amino acids there are no problems in treating discontinuous domains.
Specific nodes in these dendrograms are identified as tertiary structural clusters
-f the protein, these include both super-secondary structures and domains.
The DOMAK algorithm is used to create the 3Dee domain database. It calculates a 'split value' from the number of each type of
contact when the protein is divided arbitrarily into two parts. This split value is
large when the two parts of the structure are distinct.

## Method of Wodak and Janin
The method of Wodak and Janin (1981) was based on the calculated interface
areas between two chain segments repeatedly cleaved at various residue positions.
Interface areas were calculated by comparing surface areas of the cleaved segments
with that of the native structure. Potential domain boundaries can be identified at a
site where the interface area was at a minimum.
Other methods have used measures of solvent accessibility to calculate compactness.

## PUU algorithm
The PUU algorithm incorporates a harmonic model
used to approximate inter-domain dynamics. The underlying physical concept is
that many rigid interactions will occur within each domain and loose interactions
will occur between domains. This algorithm is used to define domains in the FSSP
domain database.

## DETECTIVE
Swindells (1995) developed a method, DETECTIVE, for identification of
domains in protein structures based on the idea that domains have a hydrophobic
interior. Deficiencies were found to occur when hydrophobic cores from different
domains continue through the interface region.

# Example domains
- Armadillo repeats. Named after the -catenin-like Armadillo protein of the fruit fly Drosophila.
- Basic Leucine zipper domain (bZIP domain) is found in many DNA-binding eukaryotic proteins. One part of the domain contains a region that mediates sequence-specific DNA-binding properties and the Leucine zipper that is required for the dimerization of two DNA-binding regions. The DNA-binding region comprises a number of basic aminoacids such as arginine and lysine
- Cadherin repeats. Cadherins function as Ca2+-dependent cell-cell adhesion proteins. Cadherin domains are extracellular regions which mediate cell-to-cell homophilic binding between cadherins on the surface of adjacent cells.
- Death effector domain (DED) allows protein-protein binding by homotypic interactions (DED-DED). Caspase proteases trigger apoptosis via proteolytic cascades. Pro-Caspase-8 and pro-caspase-9 bind to specific adaptor molecules via DED domains and this leads to autoactivation of caspases.
- EF hand, a helix-turn-helix structural motif found in each structural domain of the signaling protein calmodulin and in the muscle protein troponin-C.
- Immunoglobulin-like domains are found in proteins of the immunoglobulin superfamily (IgSF).  They contain about 70-110 amino acids and are classified into different categories (IgV, IgC1, IgC2 and IgI) according to their size and function. They possess a characteristic fold in which two beta sheets form a sandwich that is stabilized by interactions between conserved cysteines and other charged amino acids.  They are important for protein-to-protein interactions in processes of cell adhesion, cell activation, and molecular recognition. These domains are commonly found in molecules with roles in the immune system.
- Phosphotyrosine-binding domain (PTB). PTB domains usually bind to phosphorylated tyrosine residues. They are often found in signal transduction proteins. PTB-domain binding specificity is determined by residues to the amino-terminal side of the phosphotyrosine. Examples: the PTB domains of both SHC and IRS-1 bind to a NPXpY sequence. PTB-containing proteins such as SHC and IRS-1 are important for insulin responses of human cells.
- Pleckstrin homology domain (PH).  PH domains bind phosphoinositides with high affinity.  Specificity for PtdIns(3)P, PtdIns(4)P, PtdIns(3,4)P2, PtdIns(4,5)P2, and PtdIns(3,4,5)P3 have all been observed.  Given the fact that phosphoinositides are sequestered to various cell membranes (due to their long lipophilic tail) the PH domains usually causes recruitment of the protein in question to a membrane where the protein can exert a certain function in cell signalling, cytoskeletal reorganization or membrane trafficking.
- Src homology 2 domain (SH2). SH2 domains are often found in signal transduction proteins. SH2 domains confer binding to phosphorylated tyrosine (pTyr). Named after the phosphotyrosine binding domain of the src viral oncogene, which is itself a tyrosine kinase.  See also: SH3 domain.
- Zinc finger DNA binding domain (ZnF_GATA). ZnF_GATA domain-containing proteins are typically transcription factors that usually bind to the DNA sequence GATA of promoters.
The preceding text and figures originate from "Predicting Structural Domains in Proteins" George RA, 2002