ARQUITECTURA

A number of conserved protein modules have been identified as important in signalling pathways by their ability to direct specific protein-protein interactions, and mediate activation induced changes in subceUular localisation of proteins. The prototype protein modules were first characterised from the Src family of tyrosine kinases namely the SH2 and SH3 domains, however a number of different domains have now been identified (Pawson, 1995; Cohen et al., 1995). SH2 and protein tyrosine binding (PTB) domains interact with pho sphotyro sine containing peptide targets, SH3 and WW domains direct interactions with polyproline motifs, and pleckstrin homology (PH) domains influence protein-phospholipid binding (Pawson, 1995).

Proteins with catalytic activity or those solely involved in generating multiprotein signalling complexes can contain multiple protein modules in tandem, indicating the complexity of signalling pathways. Protein modules that may provide a specific fimction in directing protein-protein interactions or securing protein structure and folding are found in different types of proteins including enzymes, adapter proteins, transcription factors and cytoskeletal proteins. For example the adapter protein Grb2 contains two SH3 domains and an SH2 domain and has been shown to link receptor tyrosine kinases to Ras signalling (Lowenstein et al., 1992). The SH2 domain of Grb2 interacts with phosphorylated tyrosine residues on the cytosolic domain of an activated receptor (e.g. the p-PDGF receptor). The SH3 domain of Grb2 interacts with a polyproline region in the Ras exchange factor Sos. These protein-protein interactions form a multiprotein complex which brings Sos to the membrane where it is able to interact with its substrate Ras, mediating exchange of the bound nucleotide on Ras for GTP and activation of downstream signalling events.

1.2 5.1. Src homology 3 (SH3) domains.

SH3 domains are highly conserved non-catalytic regions of 50-60 residues, which can mediate the direction of proteins to the membrane cytoskeleton (Shpetner et al., 1996; Bar-Sagi et ah, 1993), and appear to be required for the intermolecular interactions between the neutrophil NADPH oxidase components that mediate the assembly of the active NADPH enzyme complex (DeLeo and Quinn, 1996; see section 1.5).

The basic structure of an SH3 domain contains five anti-parallel p-sheets packed to form two perpendicular p-sheets. The ligand-binding site consists of a hydrophobic patch containing a cluster of conserved aromatic residues and is surrounded by two charged and variable loops. SH3 domains bind to pro line-rich peptides, with the minimal consensus P-X-X-P, which adopt a left-handed polyproline type II helix. An aliphatic residue often precedes each proline residue and these pairs (aliphatic-proline) bind to a hydrophobic pocket on the SH3 domain. In principal peptide targets can bind in either orientation. An additional non-pro line residue, frequently arginine, can form part of the binding core and contact the SH3 domain. Usually the afiSnity of such an interaction lies within the micromolar range. However, afiSnity and specificity can be markedly increased by tertiary interactions involving the loops within the SH3 domain (Lin et al., 1997a).

1.2.5.2. Tetratricopeptide repeat (TPR) motifs.

The tetratricopeptide repeat (TPR) motif is a degenerate 34 amino acid residue sequence identified in a wide variety of proteins. These motifs are found in tandem arrays of 3-16 motifs, and fold to form scaffolds that mediate protein-protein interactions and often the assembly of multiprotein complexes. The N-terminal of the NADPH oxidase component TPR motifs (Ponting, 1996). Alignment of TPR motif containing proteins has demonstrated that within the 34 amino acid sequence 8 residues are conserved in terms of their size, hydrophobicity and spacing, with the consensus as shown below.

Alignment o f the T PR m otifs (1-4) o f w ith the consensus (Lam b et al., 1995). C onsensus 1 XXXWXXLGXX | x x x x x x x x | x x x | x x | x x x XPXX 3-36 AIS^WNEGVL | aDKKDWKg| LDa| s a| qDP H 37-70 S R i i F N i | i c M | t i l k n m t e| e1ca| t r| i n r d k h l 71-104 AVA|FQRgML | yQTEKYDl| I Kd| k e| lIQ LRGN 121-154 c e v| y n i a f m | a k k e e w k k| e e q| a l| t s m k s e p

Each T PR m o tif folds into tw o alpha-helices o f the same length. Residues at positions 4, 7, 8 and 11 (show n in green) locate on one side o f the first alpha-helix and form a

hydrophobic pocket, or ‘hole’, w hereas positions 20, 24 and 27 (show n in pink) which locate to one surface o f the second alpha-helix form a bulky ‘k nob’ structure. It has been predicted that the tw o alpha-helical dom ains fit together w ith the bulky structure o f the second fitting into the hydrophobic pocket on the surface o f the first, by the so called *knob-hole’ m odel (Lam b et al., 1995). The lysine residue at position 58 and the glycine residue at position 78 o f p67^^^"^ deleted in form s o f autosom al CG D (Leusen et al.,

1996; de B oer et al., 1994) are show n above in low er case and bold type face. In protein phosphatase 5 (PP5) three T P R motifs are found in tandem array and adjacent TPR motifs pack together in a regular series o f anti-parallel alpha-helices (D as et al., 1998). A linker sequence may also exist betw een T P R m otifs within a polypeptide, for example betw een T P R 3 and 4 o f p67^^""" (Ponting, 1996).

1.2.5.3. WD repeat motifs.

W D repeat containing proteins are m ade up o f highly conserved repeating units which usually end w ith tryptophan-aspartic acid (W D ) residues. These proteins, w hich are only found in eukaryotes are generally thought to be involved in regulatory but not catalytic processes, and possess the potential to interact w ith other proteins through their repeats. WD repeats w ere first found in the p-subunits o f heterotrim eric G -proteins in which there a seven repeats. W D repeats have been identified in a num ber o f different proteins in which the conserved unit can recur 4 to 10 tim es, including the hum an coronin-like protein p57 w hich contains 5 repeats (Suzuki et al., 1995; see appendix A .7, for amino acid sequence and dom ain structure o f p57).

The W D repeat has a region o f variable length (X6.9 4), followed by a core o f m ore or less

constant length ( X 2 3 - 4 1 ) , which is bracketed by tw o characteristic elem ents; GH (glycine-

histidine) and WD; {X6.94- [GH-X23.41 - WD]} ^ ^. Analysis of the primary sequence of the WD repeat indicates that this motif may fold with a variable loop defined by the variable region which proceeds the GH, followed by P-strand-tum-p-strand-tum-P- strand ending with the WD element. That the predicted p-structure is small suggests that stabilisation by interaction with a ligand (such as a metal ion) or contact with other WD repeats may be required to maintain a stable folded structure. Determination of the structure of P-subunits of heterotrimeric G-proteins has shown that tandem WD repeat motifs fold together to form a propeller structure, with each of the seven propeller blades corresponding to a WD repeat motif (Neer et aL, 1996). WD repeats fimction to direct the formation of multiprotein complexes; for example Gp-subunits assist in the formation of the ternary complex of heterotrimeric G-protein components with receptor molecules. WD repeat containing proteins also play roles in assembling protein complexes required for mRNA splicing, mRNA modification, and transcription (reviewed by Neer et al.,

1994). WD repeats have also been shown to interact with PH domain ligands; for example the GPy dimer brings the p-adrenergic receptor kinase (P-ARK) to the receptor by binding p-ARK through its PH domain (Neer et al., 1994). PH domains direct protein-protein interactions and can simultaneously bind to phospholipids and hence may direct target proteins to the membrane (Lemmon et al., 1996; Shaw, 1996).