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Contents
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Protein-Protein Interactions in a Bottom-up Systems Biology Approach
Recently, we proposed that protein-protein binding sites have preferred organizations (Keskin O et al. Phys Biol 2: S24–S35, 2005; Keskin O et al. J Mol Biol 345: 1281–94, 2005). Binding sites can be divided into independent modules or “hot regions,” which consist of spatially adjacent residues that are tightly packed. They typically contain clusters of “hot spot” residues, that is, residues that have either been shown experimentally to contribute significantly (more than 2.0 Kcal/mol) to the binding free energy or found to be conserved in a multiple-structure (or sequence) alignment. Tight packing leads to high conservation because it is difficult to accommodate mutations without either steric clashes or “hole” formation. Such an organization suggests that hot spots located within a hot region contribute cooperatively to the stability of the complex. However, the contributions of separate, independent hot regions are additive. Accounting for this cooperativity has led to landmark experimental and computational investigations of the mechanisms and pathways of protein folding, seeking to answer the question of how the protein chain searches the immense number of possible nonlocal interactions to yield the hydrophobic core in the protein interior. To understand cooperativity, we need to think of the system as a cohesive unit. The overall behavior is the outcome of the properties of the entire system, rather than the sum of the properties of its components. Hence, we argue that the thermodynamic stability of the protein-protein complex is not a summation of the individual, independent contributions of the residues; rather, residues in spatial contact influence the stability of the association in a non-additive manner. When a residue is tightly packed with others, its substitution could affect the structure and interactions of its neighboring residues. If this residue and its neighbors contribute significantly to the stability of the complex, its mutation might affect that stability, not only through a change in its own interactions, but also through changes in the interactions of its neighbors. This would affect stability beyond the more direct effects of the altered interactions of the mutated residue. On the other hand, if the protein-protein interface were to consist of separate units, the impact of mutations in any of these would be independent, that is, non-cooperative. Our proposition was recently corroborated experimentally by using the TEM1–β-lactamase and β-lactamase inhibitor protein (BLIP) system (Reichmann D et al. Proc Natl Acad Sci U S A 102: 57–62, 2005). The authors have shown that within a module, mutations cause complex energetic and structural consequences; on the other hand, the structural and energetic consequences resulting from the removal of entire modules are small. A bottom-up, structure-based systems biology approach focuses on proteins. It aims to put the proteins together to create a structure-based map of interactions. The availability of maps should not be looked at only as a mere enumeration of static interactions. Rather, a structural map of the macromolecular interaction network may allow comprehension of the dynamics of the system. This is the essence of control mechanisms and of functional switches. Static maps of protein interactions tell us which proteins interact; however, they do not tell us under which conditions different paths dominate, how they dominate, or which intermolecular interactions overlap and which can coexist. To understand the dynamics of the system on the molecular level, we need to know not only which proteins interact but how they interact. This implies that we need to have at our disposal the structures of the proteins and the structures of their associations. Most highly connected proteins are among those that perform the same function for many of their partners. The kinase CDK1, the second most highly connected protein in yeast, phosphorylates more than 200 proteins in the progression of the cell cycle. Importin, the third most highly connected protein, plays a role in translocating proteins from the cytoplasm to the nucleus. These proteins have a single, promiscuous interface, creating an economical way of executing a multitude of functions through the various proteins needed for them. Interestingly, importin’s interface also contains (at least) two hot regions, as exemplified in its partners’ nuclear localization sequence.
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e propose a bottom-up, structure-based approach to systems biology. A bottom-up strategy starts from specific contacts between molecules and builds the system up to create a map. In contrast, a top-down strategy begins from the overall organization; it seeks the molecular components and the specific contacts that take place at each organizational level. A bottom-up approach, however, aims to predict which proteins will interact and how the interactions will take place. Within the systems biology framework, such predictions would assist in assigning function, providing clues to the system dynamics, and yielding information on system robustness.