Dynamic interactions of proteins in complex networks: a more structured view.

Amelie Stein, Roland A Pache, Pau Bernadó, Miquel Pons, Patrick Aloy (FEBS Journal, 2009)

This recent mini-review by Stein et al. focuses on the mechanisms that enable dynamic, transient, short lived interactions in cellular networks. Of special interest are the always popular “motif recognition domain”-“short flexible peptide” interactions. However, post translational modifications and regulation by disorder are also discussed. We concise the review further to some basic/interesting/anecdotal/”pondering worthy” points.

  • For large molecular machines, it seems more important to achieve strong binding with a stable and relatively fixed position of the protein components, which serves to provide an optimal orientation of several active sites, thus increasing the global efficiency of the process.
  • The situation is radically different in signaling and regulatory networks that need to be extremely dynamic and versatile to be able to respond quickly to certain stimuli and to adapt this response over time. Thus, interactions in regulatory networks are often characterized by small interfaces, with only a few molecular contacts involved, in which a short peptide in one protein is bound by a recognition domain in another.
  • Around 50 of these protein recognition modules have already been described, and for many of them a high-resolution 3D structure is available. However, the mode of binding to other proteins has only been structurally characterized for about two-thirds of them (Stein et al.).
  • Using linear motifs is a strategy for nature to quickly explore a significant portion of the vast interaction space with a limited cost, in the sense that only one or a few residues in a protein need to change over the course of evolution to create a new interaction or to disable an existing one.
  • However, employing such a limited number of domains to mediate several thousands of interactions has brought into question the level of specificity of such interactions.
  • Motifs adopt a well-defined structure upon binding to domain. It has been repeatedly shown that isolated motifs are able to bind their domain with sufficient strength to establish a functional interaction (Kim et al.)
  • ELM: literature-curated, experimentally validated motifs involved in peptide-mediated interactions. Contains 81 motifs that bind to 51 different domains. There are structures covering 30 different of the recognition domains and 47 peptides.

Specificity issues:

  • Although these short recognition motifs theoretically match many sequences and can allow binding of many peptides, very high specificity is observed for known recognition domains such as: SH3 and PDZ. How then, is this specificity achieved ?
  • Contextual specificity provides at least part of the answer. At the first level, cellular location of the peptides and the recognition domains eliminate some of the possible interactions. At a finer level the residues in the motif that are not part of the consensus sequence (termed by Stein et al. – “context” residues) help to determine specificity.
  • Contextual residues in the motif contribute 20% of the binding energy (according to FoldX calculations). Sometime the context contains unfavorable interactions. The context uses sub-optimal interactions to achieve high specificity. Furthermore, motifs assume a well-formed structure upon binding, whereas the structure of the context is more variable.


  • Many peptide-recognition domains are modular in the sense that they fold independently and their N- and C-termini are close in space, which facilitates their integration into exposed regions of an existing protein.
  • Functional modularity: The domain spatial arrangement is negligible. e.g. Rewiring MAP kinase pathways using alternative scaffold assembly mechanisms.
  • A new connection in a cellular pathway can arise by the insertion of such a domain and mutations in a few residues, thus facilitating the evolution of new pathway or inter connection.

Post translational modifications (PTMs):

  • Peptide-mediated interactions often require specific PTMs to interact. Those PTMs are created by dedicated enzymes and are very often reversible (and fast), which gives them the ability to act as molecular switches. This provides a way to dynamically regulate complex cellular processes.
  • There are many different types of PTMs. Combination of different types of PTMs or of several instances of the same type on one protein results in different peptide-mediated interactions.
  • In cooperative interactions involving multisite PTMs, a signal is only generated after a given number of sites on the same protein have been modified. For example, Cdc4 binds to its target, Sic1, only when the target has been phosphorylated on at least six Ser/Thr residues.
  • Multiple PTMs on a protein can even be antagonistic if a PTM attached to one residue hinders the interaction with another modified residue.

Phosphorylation – the mother of PTMs:

  • Kinases, constitute one of the largest families of genes in eukaryotes, representing ~2% of the protein-coding genome.
  • In human, more than 510 protein kinases are known and it has been estimated that about one third of all human proteins may be phosphorylated.
  • Protein kinases can be roughly classified into two main groups: Ser/Thr-specific kinases – 80% of all kinases; and Tyr-specific kinases – the rest.
  • Databases of phosphorylation sites: Phospho.ELM, PHOSIDA and PhosphoSite.
  • For instance Phospho.ELM contains: 12,100 pSer, 2400 pThr and 2100 pTyr sites from the literature, covering almost 4100 different eukaryotic proteins.
  • Phospho3D combine knowledge about the position of phosphorylation sites with the 3D structure of the respective protein or a close homologue to show phosphorylation sites in their structural context.


  • It has been observed that most linear motifs in peptide-mediated interactions and residues targeted for PTMs are not embedded in globular protein domains but are rather found in linker regions or disordered regions.
  • More than half of eukaryotic proteins have long segments (> 30 residues) that are not structured in their native isolated forms. More than 20% of eukaryotic proteins are expected to be fully unfolded.
  • IUPs (Intrinsically unfolded proteins) are a recent acquisition in evolutionary terms and are much less common in Eubacteria and Archaea, which correlates well with the increase in regulatory complexity.
  • IUPs may offer a way to promote the evolution of protein interaction networks by providing an evolutionarily neutral environment in which substantial variability can be accumulated, from which in turn new interactions may appear.
  • IUPs adopt a folded conformation in the presence of a suitable partner. Two main mechanistic models were suggested for this process: The conformational selection model, and the induced folding model (see related post on “Incorporating flexibility into Docking using Normal Modes Analysis“)
  • Different binding modes and promiscuity can have important consequences in the thermodynamics of interactions. There is an entropic penalty associated with the disorder-to-order transition upon binding. This cost can be compensated for by a favourable enthalpy. The presence of preformed structured regions in the free-state precisely modulates the enthalpy–entropy balance and regulates the affinity of the interaction which is normally low.
  • The low affinity is very well suited for signal transduction and regulatory processes which require binding to initiate the signaling, but also dissociation when the signal is complete.

The authors conclude the review expressing a belief that (mind the pun) a more structured view  of transient protein interactions will ultimately lead to a better understanding of the molecular bases of cell regulatory networks.

Stein, A., Pache, R., Bernadó, P., Pons, M., & Aloy, P. (2009). Dynamic interactions of proteins in complex networks: a more structured view FEBS Journal, 276 (19), 5390-5405 DOI: 10.1111/j.1742-4658.2009.07251.x

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