<p>Proteins with highly specific binding properties constitute the basis for many important applications in biotechnology and medicine. Immunoglobulins have so far been the obvious choice but recent advances in protein engineering have provided several novel constructs that indeed challenge antibodies. One class of such binding proteins is based on the 58 residues three-helix bundle Z domain from staphylococcal protein A (SPA). These so-called affibodies are selected from libraries containing Z domain variants with 13 randomised positions at the immunoglobulin Fc-binding surface. This thesis aims to describe the principles for molecular recognition in two protein-protein complexes involving affibody proteins. The first complex is formed by the Z<sub>SPA-1</sub> affibody binding to its own ancestor, the Z domain (Kd ~1 μM). The second complex consists of two affibodies: Z<sub>Taq</sub>, originally selected to bind Taq DNA polymerase, and anti-Z<sub>Taq</sub>, an anti-idiotypic binder to Z<sub>Taq</sub> with a Kd ~0.1 μM. The basis for the study is the determination of the three-dimensional structures using NMR spectroscopy supported by biophysical characterization of the uncomplexed proteins and investigation of binding thermodynamics using isothermal titration calorimetry. The free Z<sub>SPA-1</sub> affibody is a molten globule-like protein with reduced stability compared to the original scaffold. However, upon target binding it folds into a well-defined structure with an interface topology resembling that displayed by the immunoglobulin Fc fragment when bound to the Z domain. At the same time, structural rearrangements occur in the Z domain in a similar way as in the Fc-binding process. The complex interface buries 1632 Å<sup>2</sup> total surface area and 10 out of 13 varied residues in Z<sub>SPA-1</sub> are directly involved in inter-molecular contacts. Further characterization of the molten globule state of Z<sub>SPA-1</sub> revealed a native-like overall structure with increased dynamics in the randomised regions (helices 1 and 2). These features were reduced when replacing some of the mutated residues with the corresponding wild-type Z domain residues. The nature of the free Z<sub>SPA-1</sub> affects the thermodynamics of the complex formation. The contribution from the unfolding equilibrium of the molten globule was successfully separated from the binding thermodynamics. Further decomposition of the binding entropy suggests that the conformational entropy penalty associated with stabilizing the molten globule state of Z<sub>SPA-1</sub> upon binding seriously reduces the binding affinity. The Z<sub>Taq</sub>:anti-Z<sub>Taq</sub> complex buries in total 1672 Å<sup>2</sup> surface area and all varied positions in anti-Z<sub>Taq</sub> are directly involved in binding. The main differences between the Z:Z<sub>SPA-1</sub> and the Z<sub>Taq:</sub>anti-Z<sub>Taq</sub> complexes are the relative subunit orientation and certain specific interactions. However, there are also similarities, such as the hydrophobic interface character and the role of certain key residues, which are also found in the SPA:Fc interaction. Structural rearrangements upon binding are also common features of these complexes. Even though neither Z<sub>Taq</sub> nor anti-Z<sub>Taq</sub> shows the molten globule behaviour seen for Z<sub>SPA-1</sub>, there are indications of dynamic events that might affect the binding affinity. This study provides not only a molecular basis for affibody-target recognition, but also contributions to the understanding of the mechanisms regulating protein stability and protein-protein interactions in general.</p>
| Identifer | oai:union.ndltd.org:UPSALLA/oai:DiVA.org:kth-480 |
| Date | January 2005 |
| Creators | Lendel, Christofer |
| Publisher | KTH, School of Biotechnology (BIO) |
| Source Sets | DiVA Archive at Upsalla University |
| Language | English |
| Detected Language | English |
| Type | Doctoral thesis, comprehensive summary, text |
Page generated in 0.0021 seconds