Interaction Modules and Their Importance in Signaling Pathways

As we have seen in the previous paragraph, proteins associate to form either stable or transient multi-protein complexes. The assembly of stable macromolecular complexes often requires high-affinity interactions, involving large portions of the protein surfaces in the formation of the contact area. Such high-energy bonds are not easily disrupted and are thus well suited to confer stability on molecular machines, such as the centrosome, the proteasome and others (Brooijmans et al. 2002). However, when flexibility and responsiveness are needed, e.g., in signaling pathways, such great stability would be a limitation. In fact, in highly dynamic contexts protein-protein interactions frequently involve a modular protein domain binding to a short amino acid sequence, termed a "motif' (Pawson et al. 2002).

A motif is a sequence of 3-12 amino acids that acts as a recognition element for a modular protein domain that binds to it. Since these motifs are thought to be unstructured in their unbound form, they are often referred to as "linear" motifs: nonetheless, they may acquire a well-defined three-dimensional structure upon binding (for example, the proline-rich motifs recognized by SH3 domains adopt a left-handed poly-proline II helix conformation). Short linear motifs often lie within disordered regions of the protein; for example, they may reside in exposed flexible loops.

Binding motifs are recognized by a set of conserved protein interaction modules playing a major role in many biological processes: as an example, SH3, SH2, WW, EVH1 and PDZ domains are involved in protein trafficking and degradation, cytoskel-etal organization, cell-cycle progression, cell survival and regulation of gene expression; plus, they can mediate the assembly of multi-protein complexes (PDZ domain proteins often act as scaffolds). Graphical representations of these and other interaction modules are illustrated in Fig. 2, and their properties are summarized in Table 2.

Each family of interaction modules has a defined conserved structure forming one or more "recognition pockets" to which ligands bind. A small number of highly conserved amino acids have their side chains protruding outward in the recognition

Fig. 2 Three-dimensional structures of six protein interaction modules. (a) A 14-3-3 Zeta/Delta dimer in complex with phospho-peptides (PDB code: 1QJB). (b) EVH1 domain of Homer protein homolog 1 in complex with peptide TPPSPF derived from metabotropic glutamate receptor 5 (PDB code: 1DDV). (c) The third PDZ domain from the synaptic protein Psd-95 in complex with a C-terminal peptide derived from CRIPT (PDB code: 1BE9). (d) Src SH2 domain bound to doubly phosphorylated peptide PQpYEpYIPI (PDB code: 1NZL). (e) C-terminal Sem-5 SH3 domain bound to a proline-rich peptide from mSos (Ac-PPPVPPRRR) (PDB code: 1SEM). (f) Yap65 (L30K mutant) WW domain in complex with GTPPPPYTVG peptide (PDB code: 1JMQ)

Fig. 2 Three-dimensional structures of six protein interaction modules. (a) A 14-3-3 Zeta/Delta dimer in complex with phospho-peptides (PDB code: 1QJB). (b) EVH1 domain of Homer protein homolog 1 in complex with peptide TPPSPF derived from metabotropic glutamate receptor 5 (PDB code: 1DDV). (c) The third PDZ domain from the synaptic protein Psd-95 in complex with a C-terminal peptide derived from CRIPT (PDB code: 1BE9). (d) Src SH2 domain bound to doubly phosphorylated peptide PQpYEpYIPI (PDB code: 1NZL). (e) C-terminal Sem-5 SH3 domain bound to a proline-rich peptide from mSos (Ac-PPPVPPRRR) (PDB code: 1SEM). (f) Yap65 (L30K mutant) WW domain in complex with GTPPPPYTVG peptide (PDB code: 1JMQ)

Table 2 The table summarizes the properties of six of the most relevant and well-studied interaction modules

Domain Size

Structure

Target motifs

Binding pocket Function

14-3-3 ~30 kDa proteins

EVH1

-110

80-90

Nine anti-parallel Phosphoth-

amino acids amino acids helices forming an L-shaped structure

Compact parallel betasandwich, closed along one edge by a long alpha-helix

Six beta-strands and two alpha-helices compactly arranged in a globular structure reonine or phos-phoserine motifs consensus: RSxpSxP

Proline-rich peptides consensus: E/DFPPPPXD/E

Four helices containing hydro-phobic residues form a concave amphipatic groove Highly conserved cluster of three surface-exposed aromatic side-chains

C-terminal motifs several PDZ domains bind to phosphoinositide PIP2

Elongated surface groove forms as an antiparallel beta-strand, interacts with the betaB strand and the B helix

-100 amino acids

A central hydro-phobic anti-parallel beta-sheet, flanked by two short alpha-helices forming a compact flattened hemisphere

Phosphotyrosine motifs consensus: p-Yxx^

~50 amino Five or six acids beta-strands arranged as two tightly packed antiparallel beta sheets

Proline-rich motifs consensus: PxxP

Ligand binds perpendicular to the beta-sheet and interacts with the loop between strands 2 and 3 and a hydro-phobic binding pocket that interacts with a pY+3 side chain Flat, hydrophobic pocket consisting of three shallow grooves defined by conservative aromatic residues, ligand adopts an extended left-handed helical arrangement

Regulation of many pathways (e.g., apoptosis, cell cycle)

Scaffolding, signaling, nuclear transport and cytoskeletal organization

Scaffolding, localization of proteins to the plasma membrane, regulation of intracellular signaling

Regulation of intracellular signaling cascades found in adaptor proteins and non-receptor tyrosine kinases

Signaling, cytoskeletal organization, assembly of macromo-lecular complexes found in adaptor proteins

(continued)

Table 2 (continued)

Domain

Size

Structure

Target motifs

Binding pocket

Function

WW

~40

Stable, triple

Phosphoserine-

WW or WWP

Involved in

amino

stranded

phosphothreo-

name takes

a variety

acids

beta-sheet

nine motifs, proline-rich motifs, consensus:

PPxY

after the residues responsible for binding:

two tryptophan residues that are spaced 20-23 amino acids apart and a conserved proline

of signal transduc-tion processes

The descriptions are mainly taken from InterPro version 15.1 (Mulder et al., 2007). Y indicates a hydrophobic amino acid, x indicates any amino acid, and p- indicates phosphorylation

The descriptions are mainly taken from InterPro version 15.1 (Mulder et al., 2007). Y indicates a hydrophobic amino acid, x indicates any amino acid, and p- indicates phosphorylation pocket. These mediate recognition of a specific signature in the ligand molecule, a short "core" motif of a few amino acids whose side-chain chemical groups are complementary to those in the domain recognition pocket. Within a single domain family, the broad specificity encoded in the core motif is further refined by a few flanking residues. Thus, the residues surrounding the core motif in the target peptide contribute to defining which domains it will preferentially attach to and modulate the binding affinity. Domain-motif interactions involve a small portion of the proteins' surface area and are characterized by relatively low affinities, ranging from 0.1 to 100 ||M. This characteristic confers on domain-motif interactions a dynamic nature and explains their ubiquitous presence in signaling pathways (Bhattacharyya et al. 2006), where protein complexes associate and disassociate in response to any arbitrarily varying stimulus. Some interaction modules bind their ligands dependently of some type of covalent modification, the most common being phosphorylation: for instance, SH2 domains bind with high affinity phospho-tyrosine-containing peptides, while 14-3-3 proteins prefer phospho-serine-contain-ing motifs.

The widespread distribution of proteins containing interaction modules and the specificity of the interaction mechanism, closely resembling a key-lock model, has led scientists to postulate the existence of a "protein recognition code" analogous to the genetic code (Sudol 1998). Such a code would be composed of a set of rules that may be encoded by relatively simple regular expressions, determining how protein interactions mediated by interaction modules can occur. However, caution should be taken in following this analogy too strictly to avoid overlooking some relevant differences between the two. Perhaps the most noteworthy features of the genetic code are its universality and its absoluteness: a three-letter codon encodes a specific amino acid always and anywhere (or at least with very few exceptions), independently of the organism and the cellular context. Even though protein interaction modules are present in a wide range of species, from unicellular to multi-cellular organisms, from plants to animals, their behavior is by no means universal or context-independent. The in vivo binding of a potential ligand peptide is always conditioned to local concentrations, subcellular localizations and, in some cases, to the coordinated action of other interaction modules (e.g., adaptor proteins, Pawson 2007). Furthermore, domains belonging to the same family frequently share a considerable number of cognate ligands (Castagnoli et al. 2004), and a certain degree of overlap also exists between the target recognition rules of different families: this indicates that the protein recognition code presents a high level of degeneracy. This is known to be also an important feature of the genetic code (even if the level of redundancy in the genetic code is certainly lower) and may help confer robustness on the system by resisting the detrimental effect of mutations.

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