Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
An affibody in complex with a target protein: Structure and coupled folding.
KTH, Superseded Departments, Biotechnology.
KTH, Superseded Departments, Biotechnology.ORCID iD: 0000-0001-9238-7246
KTH, Superseded Departments, Biotechnology.
KTH, Superseded Departments, Biotechnology.
Show others and affiliations
2003 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 100, no 6, 3185-3190 p.Article in journal (Refereed) Published
Abstract [en]

Combinatorial protein engineering provides powerful means for functional selection of novel binding proteins. One class of engineered binding proteins, denoted affibodies, is based on the three-helix scaffold of the Z domain derived from staphylococcal protein A. The Z(SPA-1) affibody has been selected from a phage-displayed library as a binder to protein A. Z(SPA-1) also binds with micromolar affinity to its own ancestor, the Z domain. We have characterized the Z(SPA-1) affibody in its uncomplexed state and determined the solution structure of a Z:Z(SPA-1) protein-protein complex. Uncomplexed Z(SPA-1) behaves as an aggregation-prone molten globule, but folding occurs on binding, and the original (Z) three-helix bundle scaffold is fully formed in the complex. The structural basis for selection and strong binding is a large interaction interface with tight steric and polar/nonpolar complementarity that directly involves 10 of 13 mutated amino acid residues on Z(SPA-1). We also note similarities in how the surface of the Z domain responds by induced fit to binding of Z(SPA-1) and Ig Fc, respectively, suggesting that the Z(SPA-1) affibody is capable of mimicking the morphology of the natural binding partner for the Z domain.

Place, publisher, year, edition, pages
2003. Vol. 100, no 6, 3185-3190 p.
Keyword [en]
protein engineering, protein-protein interactions, molecular recognition, NMR spectroscopy, induced fit
National Category
Other Industrial Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-8642DOI: 10.1073/pnas.0436086100ISI: 000181675200039OAI: oai:DiVA.org:kth-8642DiVA: diva2:14020
Note
Uppdaterad från "In press" till published: 20100924. QC 20100924Available from: 2005-11-08 Created: 2005-11-08 Last updated: 2017-12-14Bibliographically approved
In thesis
1. Molecular principles of protein stability and protein-protein interactions
Open this publication in new window or tab >>Molecular principles of protein stability and protein-protein interactions
2005 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [en]

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 ZSPA-1 affibody binding to its own ancestor, the Z domain (Kd ~1 μM). The second complex consists of two affibodies: ZTaq, originally selected to bind Taq DNA polymerase, and anti-ZTaq, an anti-idiotypic binder to ZTaq 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 ZSPA-1 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 Å2 total surface area and 10 out of 13 varied residues in ZSPA-1 are directly involved in inter-molecular contacts. Further characterization of the molten globule state of ZSPA-1 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 ZSPA-1 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 ZSPA-1 upon binding seriously reduces the binding affinity. The ZTaq:anti-ZTaq complex buries in total 1672 Å2 surface area and all varied positions in anti-ZTaq are directly involved in binding. The main differences between the Z:ZSPA-1 and the ZTaq:anti-ZTaq 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 ZTaq nor anti-ZTaq shows the molten globule behaviour seen for ZSPA-1, 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.

Place, publisher, year, edition, pages
Stockholm: KTH, 2005. x, 77 p.
Keyword
affibody, binding thermodynamics, induced fit, molten globule, NMR spectroscopy, protein engineering, protein-protein interactions, protein stability, protein structure.
National Category
Other Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-480 (URN)91-7178-189-7 (ISBN)
Public defence
2005-11-25, E1, Lindstedtvägen 3, Stockholm, 13:00
Opponent
Supervisors
Note
QC 20101025Available from: 2005-11-08 Created: 2005-11-08 Last updated: 2011-12-08Bibliographically approved
2. 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

Open Access in DiVA

No full text

Other links

Publisher's full text

Authority records BETA

Lendel, ChristoferNygren, Per-Åke

Search in DiVA

By author/editor
Wahlberg, ElisabetLendel, ChristoferHelgstrand, MagnusAllard, PeterDincbas-Renqvist, VildanBerglund, HelenaNygren, Per-ÅkeHärd, Torleif
By organisation
Biotechnology
In the same journal
Proceedings of the National Academy of Sciences of the United States of America
Other Industrial Biotechnology

Search outside of DiVA

GoogleGoogle Scholar

doi
urn-nbn

Altmetric score

doi
urn-nbn
Total: 78 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf