Catalytic Sites Of Multi Domain Proteins example essay topic

Describe the Nature, Structure, and Function of Domains in Proteins Domains " Within a single subunit [polypeptide chain], contiguous portions of the polypeptide chain frequently fold into compact, local semi-independent units called domains. ' - Richardson, 1981 In the hierarchical organisation of proteins, domains are found at the highest level of tertiary structure. Since the term was first used by Wetlaufer (1973) a number of definitions exist reflecting author bias, however all of the definitions agree that domains are independently folding compact units. Domains are frequently coded by eons and therefore have specific functionality. Among the many descriptions of protein domains the two most striking and simple are ' Protein evolutionary units' and 'Basic currency of Proteins'. Domains may be considered to be connected units, which are to varying extents independent in terms of their structure, function and folding behaviour.

Each domain can be described by its fold. While some proteins consist of a single domain, others consist of several or many. A number of globular protein chains consist of two or three domains appearing as 'lobes'. In other cases the domains may be of very different nature- for example some proteins located in cell membranes have a globular intracellular or extracellular domain distinct from that which spans the membrane. Protein domains occur in large polypeptides, (proteins that have more than 200 residues).

These proteins have two or more globular clusters which in turn have domains composed of 100-200 amino acids. Thus many domains are structurally independent units that have the characteristics of small globular proteins. If we examine the detailed structures of many trans membrane proteins, we see that they often have three different domains, two hydrophilic and one hydrophobic. (fig 1&2) A hydrophilic domain (consisting of hydrophilic amino acids) at the N-terminus pokes out in the extracellular medium, a hydrophobic domain in the middle of the amino acid chain, often only 20-30 amino acids long, is threaded through the plasma membrane, and a hydrophilic domain at the C-terminus protrudes into the cytoplasm. The trans membrane domain, because it is made of amino acids having hydrophobic side chains, exists comfortably in the hydrophobic inner layers of the plasma membrane. Because these trans membrane domains anchor many proteins in the lipid bi layer, these proteins are not free-floating and cannot be isolated and purified biochemically without first dissolving away the lipid bi layer with detergents. (Indeed, much of the washing we do in our lives is necessitated by the need to solubilize proteins that are embedded in lipid membranes using detergents!) Mosaic proteins are those which consist of many repeated copies of one or a few domains, all within one polypeptide chain.

Many extracellular proteins are of this nature. The domains in question are termed modules and are sometimes relatively small. However this term is often applied to sequences, whose structures may not be known for certain. The tertiary structure of a protein describes the association of units within domains, but tertiary structure also includes the way in which domains fit together. The domain can perhaps be considered the unit of tertiary structure (c. f. helices and sheets, the units of secondary structure. However super secondary forces or motifs occur in many unrelated globular proteins.

Beta-alpha-beta motif: (fig 3) is the most common form of super secondary structure. The fig clearly shows how the right-handed cross-over connection between the two consecutive parallel strands of a beta sheet consists of an alpha helix. Beta hair-pin motif: is due to an antiparallel beta sheet formed by sequential segments of polypeptide chain that are connected by tight reverse turns. Alpha-alpha motif: (fig 4) occurs when two successive antiparallel alpha helices pack against each other with their axis inclined so that they can allow energetically favourable inter meshing of the contacting side chains. Beta barrels: (fig 5) are formed by extended beta sheets rolling up together.

I am now going to give a brief description of the different types of roles of a multi- domain protein architecture. These have been viewed in the perspective of the advantages they confer to a protein and ultimately of the evolutionary advantages, for a species, of proteins with a multi-domain organisation. The definition of protein domains adopted; is that of compactly folded structures with their own hydrophobic core. substrate binding Substrate binding sites or catalytic sites of multi-domain proteins are often situated within a cleft between two domains. Relative, rigid body movements between the two adjacent domains allow an induced-fit binding of the substrate and the creation of a catalytic environment that is isolated from the solvent. Calmodulin, (free and bound to a polypeptide) (fig 6) segregation of functions Several multi-domain enzymes show segregation of their functions, such as substrate binding and catalysis, to separate domains.

The NAD (adenine di nucleotide) dependent dehydrogenase's, such as lactate dehydrogenase, are classical examples of this. These enzymes contain a di nucleotide-binding domain, with an alpha / beta doubly wound topology known as the Rossman fold, and a substrate-binding domain. Phosphofructokinase is another case where different functions are seen to be carried by different domains. One subunit of consists of two domains one of which carries the active site and the other, which carries the effector, site where both activators and inhibitors bind. It is important to note that the effect is transmitted through changes in the inter-subunit interfaces and not inter-domain interfaces. substrate-binding and NAD-binding domains of D- -3-phosphate dehydrogenase. catalytic and effector domains of one subunit of. (fig 7&8 respectively) specificity or function swapping The segregation of different functionalities to different domains allows the swapping of functionalities trough the swapping of domains. The NAD (adenine di nucleotide) dependant dehydrogenase's provide a nice example of this.

The di nucleotide binding domains of these enzymes, which have a alpha / beta doubly-wound topology known as the Rossman fold, are similar but their substrate binding domains are different allowing differences in specificity. NAD-dependant dehydrogenase's (fig 9) multi-functional proteins E. coli DNA polymerase I, which is involved in DNA replication and repair, possess three distinct activities. The first of these activities is a DNA polymerase activity while the two other are activities, one in the 3'-5' direction and one in the 5'-3' direction. The 3'-5' activity allows incorrect nucleotides added by the DNA polymerase activity to be removed. The 5'-3' activity allows removal of the old RNA primers. The three distinct enzymatic functions of DNA pol I are segregated to the three domains.

This multi-domain organisation insures that the three activities involved in DNA replication can be carried out efficiently, in the required order and that they are present in equivalent quantities in the cell. The Kle now fragment produced from DNA pol I by proteolytic cleavage corresponds to the two C-terminal domains which have 3'-5' and DNA polymerase activities. domain repeats A multi-domain architecture offers the potential of reuse trough domain repeats. Domain duplication is essential for the formation of the active sites of enzymes such as the proteinases, the serine proteinases and deaminate or the binding sites of transport proteins such as, transferring and sulfate binding protein. Domain repeats can also allow duplication of active sites or binding sites such as the calcium binding sites in or the 9 DNA-binding zinc fingers of the transcription factor TFI IID. The two lobes of, each being composed of two similar domains (fig 10) spacer domains Several protein domains are thought to have a purely structural role, allowing functional domains to be advantageously situated or providing the overall protein with the necessary flexibility. But beware, a domain may be considered to have a purely structural role be simply because of the lack of knowledge about its true function.

The immunoglobulin domains family provides examples of this such as the constant heavy 1 and 2 domains and the constant light domain of immunoglobulin. an immunoglobulin Fab fragment which contains two 'spacer' ig constant domains and two 'functional' ig variable domains (fig 11) folding Although I am not aware of any experimental evidence, in any of the resources that I have looked in, the idea that a multi-domain architecture makes the folding of large proteins more efficient seems viable. Thus I can conclude in reinstating that domains are 'Protein evolutionary units' and the 'Basic currency of Proteins. They are extremely specific and provide adaptations and functions that speed up various biological processes. However there is still a lot left to be learnt and understand.