Advanced Molecular Genetics-Biology 566 Prokaryotic - Polymerase Activation, Promoter Activation, Silencing
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Structurally conserved protein modules

Many proteins involved in cellular signalling have regions that show conserved 3d structure, these are referred to as "conserved structural domains" or just "structural domains". The amino acid sequence of these domains tends not to be conserved in amino acid sequence and therefore they are hard to identify by amino acid sequence alone. Structural domains are usually 40-100 amino acids long and are held together in a compact structure by a hydrophobic core. Structural domains are entirely contained within a single exon and their distribution between different receptors may result from exon shuffling and sequence divergence.

It is common for signalling proteins to have multiple structural domains.

Domains that bind oligopeptide motifs

SH2 Domains

SH2 is short for Src homology region 2. They are present in all non-receptor Protein Tyrosine Kinases PTKs and are generally located immediately N-terminal tothe kinase domain. They are also present in a large number of other proteins. The structural domain is formed by a cental aniparallel b-sheet flanked by two a-helicies.

The target is a phosphorylated tyrosine residue and three adjacent amino acids, the fourth being hydrophobic, like pYEEI. Each bind a pocket on either side of the b-sheet (See figure 18.1).

Five classes of SH2 domains are distinguishable by their binding specificities to residues in the target.

An SH2-containing protein may also have catalytic activity like Scr or in the case of an adapter like Grb2 it may not have catalytic activity but two different structural domains.

PTB/PID Domains

Phosphotyrosine binding domane PTB or alternatively Phosphotyrosine Interaction Domain, PID, recognizes phosphotyrosine in a motif NPXpY where additional members of the motif are N-terminal of the phosphotyrosine.

The structural domain is made up of a beta-barrel with an a-helix closing off one end (Fig. 18.3).

SH3 Domains

SH3 (Src homology 3) domains are 55-75 amino acids long and form twisted b-barrel structures that bind to proline-rich structures that are 8-10 residues long and form left-handed helicies with three residues per turn. The binding site is lined with aromatic amino acids that form screw-like threads that fit into the grooves of the proline-rich helix. About three turns of the proline-rich residue is involved in binding at the consensus RXLPPLPXX or XXXPPLPXR giving rise to two classes of SH3 domain binding proteins. Such bindings are weak and there are usually more than 1 SH3 domains present in signalling proteins.

Domains that bind proteins and lipids

PH Domain

PH (pleckstrin homology) domains are about 100 amino acids long and is made up of seven antiparallel b-sheets that form a barrel. The sequence is more variable than SH2 and SH3 domains.

PH may mediate protein-protein or protein-lipid interactions.Table 18.3 shows some physiological ligands of PH domains.

Domains that bind Ca2+

The EF-Hand motif

A helix-turn-helix that occurs in pairs and binds Ca2+ in the 12 amino acid loop via the oxygen atoms in aspartate and glutamate R-groups.

C2 Domains

C2 domains consists of 130 amino acids arranged in a rigid, eight-stranded, antiparallel beta sandwich. Ca2+ is bound by oxygens from five aspartate residues located on two of the outer loops which can hold up to three Ca2+ atoms.

C2 domains can also bind negatively charged phospholipids after being partially charged with calcium ions. Therefore, proteins which are normally soluble in an aqueous environment can become associated with membranes upon binding calcium that reaches micromolar levels.

Protein Kinases

Protein kinases transfer a phosphate group from ATP to the hydroxyl group of either a serine or threonine, or to the hydroxyl group of tyrosine (two classes of kinases). In both classes of kinase the catalytic domain is housed in a cleft between a small beta-structure and a larger alpha-helical structure. A helix holds the two domains together and is aided by a myristol group attached to the N-terminus. The small lobe binds ATP and the large lobe binds the substrate and enables catalysis. In the large lobe a 24 residue segment controls activation through phosphorylation of a critical threonine in the activation loop. There are also contact groups that stabilize Ca2+ and two Mg2+ residues.

Not all protein kinases are regulated by phosphorylation.