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Conformational Stabilization of an Engineered Binding Protein
KTH, School of Biotechnology (BIO).
2006 (English)In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 128, no 23, 7651-7660 p.Article in journal (Refereed) Published
Abstract [en]

We analyzed the thermodynamic basis for improvement of a binding protein by disulfide engineering. The Z(SPA-1) affibody binds to its Z domain binding partner with a dissociation constant K-d = 1.6 mu M, and previous analyses suggested that the moderate \affinity is due to the conformational heterogeneity of free Z(SPA-1) rather than to a suboptimal binding interface. Studies of five stabilized Z(SPA-1) double cystein mutants show that it is possible to improve the affinity by an order of magnitude to K-d = 130 nM, which is close to the range (20 to 70 nM) observed with natural Z domain binders, without altering the protein-protein interface obtained by phage display. Analysis of the binding thermodynamics reveals a balance between conformational entropy and desolvation entropy: the expected and favorable reduction of conformational entropy in the best-binding Z(SPA-1) mutant is completely compensated by an unfavorable loss of desolvation entropy. This is consistent with a restriction of possible conformations in the disulfide-containing mutant and a reduction of average water-exposed nonpolar surface area in the free state, resulting in a smaller conformational entropy penalty, but also a smaller change in surface area, for binding of mutant compared to wild-type Z(SPA-1). Instead, higher Z domain binding affinity in a group of eight Z(SPA-1) variants correlates with more favorable binding enthalpy and enthalpy- entropy compensation. These results suggest that protein-protein binding affinity can be improved by stabilizing conformations in which enthalpic effects can be fully explored.

Place, publisher, year, edition, pages
2006. Vol. 128, no 23, 7651-7660 p.
Keyword [en]
disulfide bonds, combinatorial libraries, molten globules, stability, domain, affibody, complex, thermodynamics, hydration, lysozyme
National Category
Other Industrial Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-6611DOI: 10.1021/ja060933gISI: 000238099500055Scopus ID: 2-s2.0-33745100912OAI: oai:DiVA.org:kth-6611DiVA: diva2:11366
Note
QC 20100924Available from: 2006-12-12 Created: 2006-12-12 Last updated: 2017-12-14Bibliographically approved
In thesis
1. Structure determination and thermodynamic stabilization of an engineered protein-protein complex
Open this publication in new window or tab >>Structure determination and thermodynamic stabilization of an engineered protein-protein complex
2006 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [en]

The interaction between two 6 kDa proteins has been investigated. The studied complex of micromolar affinity (Kd) consists of the Z domain derived from staphylococcal protein A and the related protein ZSPA-1, belonging to a group of binding proteins denoted affibody molecules generated via combinatorial engineering of the Z domain. Affibody-target protein complexes are good model systems for structural and thermodynamic studies of protein-protein interactions. With the Z:ZSPA-1 pair as a starting point, we determined the solution structure of the complex and carried out a preliminary characterization of ZSPA-1. We found that the complex contains a rather large (ca. 1600 Å2) interaction interface with tight steric and polar/nonpolar complementarity. The structure of ZSPA-1 in the complex is well-ordered in a conformation that is very similar to that of the Z domain. However, the conformation of the free ZSPA-1 is best characterized by comparisons with protein molten globules. It shows a reduced secondary structure content, aggregation propensity, poor thermal stability, and binds the hydrophobic dye ANS. This molten globule state of ZSPA-1 is the native state in the absence of the Z domain, and the ordered state is only adopted following a stabilization that occurs upon binding. A more extensive characterization of ZSPA-1 suggested that the average topology of the Z domain is retained in the molten globule state but that it is represented by a multitude of conformations. Furthermore, the molten globule state is only marginally stable, and a significant fraction of ZSPA-1 exists in a completely unfolded state at room temperature. A complete thermodynamic characterization of the Z:ZSPA-1 pair suggests that the stabilization of the molten globule state to an ordered three helix structure in the complex is associated with a significant conformational entropy penalty that might influence the binding affinity negatively and result in an intermediate-affinity (µM) binding protein. This can be compared to a dissociation constant of 20-70 nM for the complex Z:Fc of IgG where Z uses the same binding surface as in Z:ZSPA-1. Structure analyses of Z in the free and bound state reveal an induced fit response upon complex formation with ZSPA-1 where a conformational change of several side chains in the binding surface increases the accessible surface area with almost 400 Å2 i.e. almost half of the total interaction surface in the complex. Two cysteine residues were introduced at specific positions in ZSPA-1 for five mutants in order to stabilize the conformation of ZSPA-1 by disulfide bridge formation. The mutants were thermodynamically characterized and the binding affinity of one mutant showed an improvement by more than a factor of ten. The improvement of the introduced cysteine bridge correlates with an increase in binding enthalpy rather than with entropy. Further analysis of the binding entropy suggests that the conformational entropy change in fact is reduced but its favorable contribution is opposed by a less favorable desolvation enthalpy change. These studies illustrate the structural and thermodynamic complexity of protein-protein interactions, but also that this complexity can be dissected and understood. In this study, a comprehensive characterization of the ZSPA-1 affibody has gained insight into the intricate mechanisms involved in complex formation. These theories were supported by the design of a ZSPA-1 mutant with improved binding affinity.

Place, publisher, year, edition, pages
Stockholm: KTH, 2006. vii, 67 p.
Keyword
affibody, protein structure, coupled folding, NMR spectroscopy, protein stability, protein-protein interactions, binding thermodynamics, isothermal titration calorimetry, protein engineering
National Category
Other Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-4230 (URN)91-7178-457-8 (ISBN)
Public defence
2006-12-15, FD5, AlbaNova, Roslagstullsbacken 21, Stockholm, 13:30
Opponent
Supervisors
Note
QC 20100924Available from: 2006-12-12 Created: 2006-12-12 Last updated: 2011-12-08Bibliographically approved

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