Conserved part of a protein
A protein domain is a conserved part of a given protein sequence and tertiary 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 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.[1] 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.
The concept of the domain was first proposed in 1973 by Wetlaufer after X-raycrystallographic studies of hen lysozyme[2] and papain[3]and by limited proteolysis studies of immunoglobulins.[4][5] Wetlaufer defined domains as stable units of protein structure that could fold autonomously. In the past domains have been described as units of:
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.[9] 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.
An appropriate example is pyruvate kinase (see first figure), a glycolytic enzyme that plays an important role in regulating the flux from fructose-1,6-biphosphate to pyruvate. It contains an all- nucleotide binding domain (in blue), an /-substrate binding domain (in grey) and an /-regulatory domain (in olive green),[10] connected by several polypeptide linkers.[11] Each domain in this protein occurs in diverse sets of protein families.[12]
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.[13] The /-barrel is commonly called the TIM barrel named after triose phosphate isomerase, which was the first such structure to be solved.[14] It is currently classified into 26 homologous families in the CATH domain database.[15] 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 suggestedthat a single ancestral enzyme could have diverged into several families,[16] while another suggests that a stable TIM-barrel structure has evolvedthrough convergent evolution.[17]
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.[18][19] The inserted -barrel regulatory domain is 'continuous', made up of a single stretch of polypeptide.
The primary structure (string of amino acids) of a protein ultimately encodes its uniquely folded three-dimensional (3D) conformation.[20] The most important factor governing the folding of a protein into 3D structure is the distribution of polar and non-polar side chains.[21] 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: -helices and -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 -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.
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.[22] Other, advantages are the protection of intermediates within inter-domain enzymatic clefts that mayotherwise be unstable in aqueous environments, and a fixed stoichiometric ratio of the enzymatic activity necessary for a sequential set of reactions.[23]
Structural alignment is an important tool for determining domains.
Several motifs pack together to form compact, local, semi-independent units called domains.[6]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.[24] 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.[25]
Domains have limits on size.[27] The size of individual structural domains varies from 36 residues in E-selectin to 692 residues in lipoxygenase-1,[18] but the majority, 90%, have fewer than 200 residues[28] with an average of approximately 100 residues.[29] 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.[30]
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 is a mechanism for forming oligomeric assemblies.[31] 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.[32]
Nature is a tinkerer and not an inventor,[33] 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.[34] 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.[35] Four concrete examples of widespread protein modules are the following domains: SH2, immunoglobulin, fibronectin type 3 and the kringle.[36]
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 110,000 experimentally determined protein 3D structures deposited within the Protein Data Bank (PDB).[37] 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; see 'Domain definition from structural co-ordinates'.
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.[38] The most populated is the /-barrel super-fold, as described previously.
The majority of proteins, two-thirds in unicellular organisms and more than 80% in metazoa, are multidomain proteins.[39] However, other studies concluded that 40% of prokaryotic proteins consist of multiple domains while eukaryotes have approximately 65% multi-domain proteins.[40]
Many domains in eukaryotic multidomain proteins can be found as independent proteins in prokaryotes,[41] suggesting that domains in multidomain proteins have once existed as independent proteins. For example, vertebrates have a multi-enzyme polypeptide containing the GAR synthetase, AIR synthetase and GAR transformylase domains (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.[42]
Multidomain proteins are likely to have emerged from 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 of genetic shuffling:
The simplest multidomain organization seen in proteins is that of a single domain repeated in tandem.[46] The domains may interact with each other (domain-domain interaction) 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.[47] In the serine proteases, a gene duplication event has led to the formation of a two -barrel domain enzyme.[48] The repeats have diverged so widely that there is no obvious 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.[48]
Modules frequently display different connectivity relationships, as illustrated by the kinesins and ABC transporters. The kinesin motor domain can be at either end of a polypeptide chain that includes a coiled-coil region and a cargo domain.[49] ABC transporters are built with up to four domains consisting of two unrelated modules, ATP-binding cassette and an integral membrane module, arranged in various combinations.
Not only do domains recombine, but there are many examples of a domain having been inserted into another. Sequence or structural similarities to otherdomains 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.[50] 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.
A superdomain consists of two or more conserved domains of nominally independent origin, but subsequently inherited as a single structural/functional unit.[51] This combined superdomain can occur in diverse proteins that are not related by gene duplication alone. An example of a superdomain is the protein tyrosine phosphataseC2 domain pair in PTEN, tensin, auxilin and the membrane protein TPTE2. This superdomain is found in proteins in animals, plants and fungi. A key feature of the PTP-C2 superdomain is amino acid residue conservation in the domain interface.
Protein folding - the unsolved problem: Since the seminal work of Anfinsen in the early 1960s,[20] 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 is a directed search of conformational space allowing the protein to fold on 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.[52] 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 forcesthat direct this search are likely to be a combination of local and global influences whose effects are felt at various stages of the reaction.[53]
Advances in experimental and theoretical studies have shown that folding can be viewed in terms of energy landscapes,[54][55] where folding kinetics is considered as a progressive organisation of an ensemble of 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 chain's conformational options become increasingly narrowed ultimately toward one native structure.
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,[56] 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.[57][58]
However, the role of inter-domain interactions in protein folding and in energetics of 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.[59]
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,[60] 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.[30] This is either because the domains are not folded entirely correctly or because the small adjustments required for their interaction are energetically unfavourable,[61] such as the removal of water from the domain interface.
Protein domain dynamics play a key role in a multitude of molecular recognition and signaling processes.Protein domains, connected by intrinsically disordered flexible linker domains, induce long-range allostery via protein domain dynamics.The resultant dynamic modes cannot be generally predicted from static structures of either the entire protein or individual domains. They can however be inferred by comparing different structures of a protein (as in Database of Molecular Motions). They can also be suggested by sampling in extensive molecular dynamics trajectories[62] and principal component analysis,[63] or they can be directly observed using spectra[64][65]measured by neutron spin echo spectroscopy.
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 known 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 or highly associated.[66] 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.[67]
A structural domain is a compact, globular sub-structure with more interactions within it than with the rest of the protein.[68]Therefore, a structural domain can be determined by two visual characteristics: its compactness and its extent of isolation.[69] Measures of local compactness in proteins have been used in many of the early methods of domain assignment[70][71][72][73] and in several of the more recent methods.[28][74][75][76][77]
One of the first algorithms[70] 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 on 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[73] 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.
The method by Sowdhamini and Blundell clusters secondary structures in a protein based on their C-C distances and identifies domains from the pattern intheir dendrograms.[66] 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 of the protein, these include both super-secondary structures and domains. The DOMAK algorithm is used to create the 3Dee domain database.[75] 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 islarge when the two parts of the structure are distinct.
The method of Wodak and Janin[78] 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.[28][79][80]
The PUU algorithm[19] 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.[74]
Swindells (1995) developed a method, DETECTIVE, for identification of domains in protein structures based on the idea that domains have a hydrophobicinterior. Deficiencies were found to occur when hydrophobic cores from different domains continue through the interface region.
RigidFinder is a novel method for identification of protein rigid blocks (domains and loops) from two different conformations. Rigid blocks are defined as blocks where all inter residue distances are conserved across conformations.
The method RIBFIND developed by Pandurangan and Topf identifies rigid bodies in protein structures by performing spacial clustering of secondary structural elements in proteins.[81] The RIBFIND rigid bodies have been used to flexibly fit protein structures into cryo electron microscopy density maps.[82]
A general method to identify dynamical domains, that is proteinregions that behave approximately as rigid units in the course ofstructural fluctuations, has been introduced by Potestio et al.[62] and, among other applications was also usedto compare the consistency of the dynamics-based domainsubdivisions with standard structure-based ones. The method,termed PiSQRD, is publicly available in the form of a webserver.[83] The latter allows users to optimally subdivide single-chainor multimeric proteins into quasi-rigid domains[62][83] based on the collective modes of fluctuation of the system. By default thelatter are calculated through an elastic network model;[84]alternatively pre-calculated essential dynamical spaces can beuploaded by the user.
A large fraction of domains are of unknown function. Adomain of unknown function(DUF) is aprotein domainthat has no characterized function. These families have been collected together in thePfamdatabase using the prefix DUF followed by a number, with examples beingDUF2992andDUF1220. There are now over 3,000 DUF families within the Pfam database representing over 20% of known families.[86]
This article incorporates text and figures from George, R. A. (2002) "Predicting Structural Domains in Proteins" Thesis, University College London, which were contributed by its author.
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Protein domain - Wikipedia
