The molecular scaffold concept

The idea of constructing a new molecular recognition function through the engineering of a section of protein surface (i.e., a patch; Figure 5.1A) originates from the molecular architecture of immunoglobulins (Figure 5.1B). The immune system can produce antibodies against virtually any type of antigen by, to a first-order approximation, simply tuning the amino-acid sequences of a total of six short segments termed complementarity determining regions (CDRs). CDRs are surface-exposed loops located at one end of the immunoglobulin structure (Figure 5.1B). They collectively form a contiguous patch for molecular recognition. Sequence analysis revealed the presence of hypervariable regions that are primarily responsible for antigen binding (Wu et al. 1993). From these observations, the concept of molecular scaffold has been developed. This term usually refers to a protein framework that is essentially invariant and provides positions that can accommodate extensive sequence variations (substitution, insertion, and deletion) for the purpose of generating desired functions (Figure 5.1A). In the antibody example, the protein outside the CDRs can be considered as molecular scaffold that supports the CDRs, although it is well known that the surfaces outside the antigen-binding site are involved in many critical biological functions.

While the manifestation of the molecular scaffold concept is particularly evident in the immunoglobulins, it should be emphasized that this concept is perhaps a unifying theme in protein architecture. There are a number of protein folds, such as the triosephosphateisomerase (TIM) barrel and oligonucleotide/oligosaccharide binding (OB) fold (Nagano et al. 2002; Agrawal and Kishan 2003), that are used for recognition of a wide variety of ligands. These examples have established the "one fold-many functions" paradigm (Nagano et al. 2002). Small interaction domains are present in many eukaryotic proteins, and they are engaged in diverse molecular recognition functions. Comparisons of these domains uncover a set of positions that play major roles in target recognition (Figure 5.1C). While these proteins are not involved in adaptive immunity, their folds are clearly capable of supporting distinct amino-acid motifs for diverse functions, and thus conceptually they are molecular scaffolds

Protein Ligand Cartoon

FIGURE 5.1 Examples of natural protein-ligand interactions that illustrate the molecular scaffold concept. (A) A schematic representation of synthetic interface engineering. The oval at the top indicates an inert scaffold. The asterisks denote sequence diversity. The bottom is a binder-target complex with the optimized interface shown in black. (B) Antibody-antigen interaction (PDB: 1TZI) (Fellouse et al. 2004). The Fab is shown as a cartoon model with the heavy chain shown in darker gray. Only a portion of the antigen is shown as a surface model. The antigen-antibody interaction interface is shown expanded on the right, where the Ca positions of the CDR residues are marked with spheres. (C) PDZ domain-peptide interaction (PDB: 1MFG) (Birrane et al. 2003). The PDZ domain is shown as a cartoon model and the peptide is shown as spheres. The bottom figure shows the same complex in an approximately perpendicular view, with the spheres denoting the Ca positions of residues that are found to be important for ligand specificity (Chen et al. 2008). The molecular graphics were made with PyMOL (www.pymol.org).

FIGURE 5.1 Examples of natural protein-ligand interactions that illustrate the molecular scaffold concept. (A) A schematic representation of synthetic interface engineering. The oval at the top indicates an inert scaffold. The asterisks denote sequence diversity. The bottom is a binder-target complex with the optimized interface shown in black. (B) Antibody-antigen interaction (PDB: 1TZI) (Fellouse et al. 2004). The Fab is shown as a cartoon model with the heavy chain shown in darker gray. Only a portion of the antigen is shown as a surface model. The antigen-antibody interaction interface is shown expanded on the right, where the Ca positions of the CDR residues are marked with spheres. (C) PDZ domain-peptide interaction (PDB: 1MFG) (Birrane et al. 2003). The PDZ domain is shown as a cartoon model and the peptide is shown as spheres. The bottom figure shows the same complex in an approximately perpendicular view, with the spheres denoting the Ca positions of residues that are found to be important for ligand specificity (Chen et al. 2008). The molecular graphics were made with PyMOL (www.pymol.org).

just like immunoglobulins. The only difference is that, in these cases, new functions emerge through natural selection over a long time period, whereas the immune system has the capacity to perform directed evolution within days. Both of these examples demonstrate that natural proteins have the potential to evolve surfaces into distinct molecular recognition interfaces. Therefore, with suitable technologies, one should be able to create a series of new functions by evolving appropriate positions on a molecular scaffold.

Traditionally, molecular scaffolds have been classified into antibody fragments and nonantibody, "alternative" scaffolds. This distinction is based on the premise that only the antibody scaffolds (including T-cell receptors that have essentially the same binding site architecture) are used in nature to build a system that consistently produces highly functional recognition interfaces. Recent studies do not support such a narrow view. Perhaps the most illustrative case in point is the development of repeat proteins (e.g., leucine-rich repeats and ankyrin repeat proteins; see the next section) as "alternative" scaffolds by Pluckthun and colleagues (Forrer et al. 2003), which was followed by the discovery that leucine-rich repeats are used as the scaffold for adaptive immunity in the jawless fish (Pancer et al. 2004). Clearly, natural evolution is opportunistic and it is possible that other classes of protein could be capable scaffolds for supporting adaptive immunity.

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