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Structural and thermodynamical basis for molecular recognition between engineered binding proteins
KTH, School of Biotechnology (BIO), Molecular Biotechnology.
2006 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [en]

The structural determination of interacting proteins, both as individual proteins and in their complex, complemented by thermodynamical studies are vital in order to gain in-depth insights of the phenomena leading to the highly selective protein-protein interactions characteristic of numerous life processes. This thesis describes an investigation of the structural and thermodynamical basis for molecular recognition in two different protein-protein complexes, formed between so-called affibody proteins and their respective targets. Affibody proteins are a class of engineered binding proteins, which can be functionally selected for binding to a given target protein from large collections (libraries) constructed via combinatorial engineering of 13 surface-located positions of the 58-residue three-helix bundle Z domain derived from Staphylococcal protein (SPA).

In a first study, an affibody:target protein pair consisting of the ZSPA-1 affibody and the parental Z domain, with a dissociation constant (Kd) of approximately 1 µM, was investigated. ZSPA-1 was in its free state shown to display molten globule-like characteristics. The enthalpy change on binding between Z and ZSPA-1 as measured by isothermal titration calorimetry, was found to be a non-linear function of temperature. This nonlinearity was found to be due to the temperature dependent folded-unfolded equilibrium of ZSPA-1 upon binding to the Z domain and, the energetics of the unfolding equilibrium of the molten globule state of ZSPA-1 could be separated from the binding thermodynamics. Further dissection of the binding entropy revealed that a significant reduction in conformational entropy resulting from the stabilization of the molten globule state of ZSPA-1 upon complex formation could be a major reason for the moderate binding affinity.

A second studied affibody:target complex (Kd ~ 0.1 µM) consisted of the ZTaq affibody protein originally selected for binding to Taq DNA polymerase and the anti-ZTaq affibody protein, selected for selective binding to the ZTaq affibody protein, thus constituting an "anti-idiotypic" affinity protein pair. The structure of the ZTaq:anti-ZTaq affibody complex as well as the free state structures of ZTaq and anti-ZTaq were determined using NMR spectroscopy. Both ZTaq and anti-ZTaq are well defined three helix bundles in their free state and do not display the same molten globule-like behaviour of ZSPA-1. The interaction surface was found to involve all of the varied positions in helices 1 and 2 of the anti-ZTaq, the majority of the corresponding side chains in ZTaq, and also several non-mutated residues. The total buried surface area was determined to about 1670 Å2 which is well inside the range of what is typical for many protein-protein complexes, including antibody:antigen complexes. Structural rearrangements, primarily at the side chain level, were observed to take place upon binding. There are similarities between the ZTaq:anti-ZTaq and the Z:ZSPA-1 structure, for instance, the binding interface area in both complexes has a large fraction of non-polar content, the buried surface area is of similar size, and certain residues have the same positioning. However, the relative orientation between the subunits in ZTaq:anti-ZTaq is markedly different from that observed in Z:ZSPA-1. The thermodynamics of ZTaq:anti-ZTaq association were investigated by isothermal titration calorimetry. A dissection of the entropic contributions showed that a large and favourable desolvation entropy of non-polar surface is associated with the binding reaction which is in good agreement with hydrophobic nature of the binding interface, but as in the case for the Z:ZSPA-1 complex a significant loss in conformational entropy opposes complex formation.

A comparison with complexes involving affibody proteins or SPA domains suggests that affibody proteins inherit intrinsic binding properties from the original SPA surface. The structural and biophysical data suggest that although extensive mutations are carried out in the Z domain to obtain affibody proteins, this does not necessarily affect the structural integrity or lead to a significant destabilization.

Place, publisher, year, edition, pages
Stockholm: KTH , 2006. , 57 p.
Keyword [en]
protein structure, induced fit, binding thermodynamics, NMR spectroscopy, protein engineering, protein-protein interactions, protein stability, calorimetry
National Category
Other Industrial Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-4181ISBN: 91-7178-481-0 (print)OAI: oai:DiVA.org:kth-4181DiVA: diva2:11111
Public defence
2006-12-01, FR4, AlbaNova Universitetscentrum, Roslagstullsbacken 21, Stockholm, 13:00
Opponent
Supervisors
Note
QC 20110118Available from: 2006-11-22 Created: 2006-11-22 Last updated: 2011-12-08Bibliographically approved
List of papers
1. Biophysical characterization of ZSPA-1-A phage-display selected binder to protein A
Open this publication in new window or tab >>Biophysical characterization of ZSPA-1-A phage-display selected binder to protein A
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2004 (English)In: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 13, no 8, 2078-2088 p.Article in journal (Refereed) Published
Abstract [en]

Affibodies are a novel class of binding proteins selected from phagemid libraries of the Z domain from staphylococcal protein A. The Z(SPA-1) affibody was selected as a binder to protein A, and it binds the parental Z domain with micromolar affinity. In earlier work we determined the structure of the Z:Z(SPA-1) complex and noted that Z(SPA-1) in the free state exhibits several properties characteristic of a molten globule. Here we present a more detailed biophysical investigation of Z(SPA-1) and four Z(SPA-1) mutants with the objective to understand these properties. The characterization includes thermal and chemical denaturation profiles, ANS binding assays, size exclusion chromatography, isothermal titration calorimetry, and an investigation of structure and dynamics by NMR. The NMR characterization of Z(SPA-1) was facilitated by the finding that trimethylamine N-oxide (TMAO) stabilizes the molten globule conformation in favor of the fully unfolded state. All data taken together lead us to conclude the following: (1) The topology of the molten globule conformation of free Z(SPA-1) is similar to that of the fully folded structure in the Z-bound state; (2) the extensive mutations in helices 1 and 2 destabilize these without affecting the intrinsic stability of helix 3; (3) stabilization and reduced aggregation can be achieved by replacing mutated residues in Z(SPA-1) with the corresponding wild-type Z residues. This stabilization is better correlated to changes in helix propensity than to an expected increase in polar versus nonpolar surface area of the fully folded state.

Keyword
protein engineering, affibody, protein stability, osmolyte, NMR spectroscopy
National Category
Other Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-8643 (URN)10.1110/ps.04728604 (DOI)000222928800010 ()2-s2.0-3342879396 (Scopus ID)
Note

QC 20100924

Available from: 2005-11-08 Created: 2005-11-08 Last updated: 2017-06-14Bibliographically approved
2. Thermodynamics of folding, stabilization, and binding in an engineered protein-protein complex
Open this publication in new window or tab >>Thermodynamics of folding, stabilization, and binding in an engineered protein-protein complex
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2004 (English)In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 126, no 36, 11220-11230 p.Article in journal (Refereed) Published
Abstract [en]

We analyzed the thermodynamics of a complex protein-protein binding interaction using the (engineered) Z(SPA-1) affibody and it's Z domain binding partner as a model. Free Z(SPA-1) exists in an equilibrium between a molten-globule-like (MG) state and a completely unfolded state, wheras a well-ordered structure is observed in the Z:Z(SPA-1) complex. The thermodynamics of the MG state unfolding equilibrium can be separated from the thermodynamics of binding and stabilization by combined analysis of isothermal titration calorimetry data and a separate van't Hoff analysis of thermal unfolding. We find that (i) the unfolding equilibrium of free Z(SPA-1) has only a small influence on effective binding affinity, that (ii) the Z:Z(SPA-1) interface is inconspicuous and structure-based energetics calculations suggest that it should be capable of supporting strong binding, but that (iii) the conformational stabilization of the MG state to a well-ordered structure in the Z:Z(SPA-1) complex is associated with a large change in conformational entropy that opposes binding.

Keyword
combinatorial libraries, staphylococcus-aureus, molten globules, affibody, domain, entropy, dna, microcalorimetry, recognition, parameters
National Category
Other Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-6406 (URN)10.1021/ja047727y (DOI)000223799900035 ()2-s2.0-4544277195 (Scopus ID)
Note
QC 20101025 QC 20110916Available from: 2006-11-22 Created: 2006-11-22 Last updated: 2017-12-14Bibliographically approved
3. NMR assignments of the free and bound-state protein components of an anti-idiotypic affibody complex
Open this publication in new window or tab >>NMR assignments of the free and bound-state protein components of an anti-idiotypic affibody complex
2006 (English)In: Journal of Biomolecular NMR, ISSN 0925-2738, E-ISSN 1573-5001, Vol. 36, (Electronic publication ahead of print Feb. 6; doi:10.1007/s10858-005-5350-8) p.Article in journal (Refereed) Published
National Category
Other Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-6407 (URN)10.1007/s10858-005-5350-8 (DOI)000242836500013 ()2-s2.0-33746879057 (Scopus ID)
Note
QC 20101025Available from: 2006-11-22 Created: 2006-11-22 Last updated: 2017-12-14Bibliographically approved
4. Structural basis for molecular recognition in an affibody:affibody complex
Open this publication in new window or tab >>Structural basis for molecular recognition in an affibody:affibody complex
2006 (English)In: Asia-Pacific Journal of Molecular Biology and Biotechnology, ISSN 0128-7451, Vol. 359, no 5, 1293-1304 p.Article in journal (Refereed) Published
Abstract [en]

Affibody molecules constitute a class of engineered binding proteins based on the 58-residue three-helix bundle Z domain derived from staphylococcal protein A (SPA). Affibody proteins are selected as binders to target proteins by phage display of combinatorial libraries in which typically 13 side-chains on the surface of helices 1 and 2 in the Z domain have been randomized. The Z(Taq):anti-Z(Taq) affibody-affibody complex, consisting of Z(Taq), originally selected as a binder to Taq DNA polymerase, and anti-Z(Taq), selected as binder to Z(Taq), is formed with a dissociation constant K-d similar to 100 nM. We have determined high-precision solution structures of free Z(Taq) and anti-Z(Taq), and the Z(Taq):anti-Z(Taq) complex under identical experimental conditions (25 degrees C in 50 mM NaCl with 20 mM potassium phosphate buffer at pH 6.4). The complex is formed with helices 1 and 2 of anti-Z(Taq) in perpendicular contact with helices 1 and 2 of Z(Taq). The interaction surface is large (similar to 1670 angstrom(2)) and unusually non-polar (70 %) compared to other protein-protein complexes. It involves all varied residues on anti-Z(Taq), most corresponding (Taq DNA polymerase binding) side-chains on Z(Taq), and several additional side-chain and backbone contacts. Other notable features include a substantial rearrangement (induced fit) of aromatic side-chains in Z(Taq) upon binding, a close contact between glycine residues in the two subunits that might involve aliphatic glycine H alpha to backbone carbonyl hydrogen bonds, and four hydrogen bonds made by the two guanidinium (NH2)-H-eta groups of an arginine side-chain. Comparisons of the present structure with other data for affibody proteins and the Z domain suggest that intrinsic binding properties of the originating SPA surface might be inherited by the affibody binders. A thermodynamic characterization of Z(Taq) and anti-Z(Taq) is presented in an accompanying paper.

Keyword
protein-protein interactions, protein engineering, molecular recognition, NMR spectroscopy, induced fit
National Category
Other Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-6408 (URN)10.1016/j.jmb.2006.04.043 (DOI)000238988400011 ()2-s2.0-33746933474 (Scopus ID)
Note
QC 20101025Available from: 2006-11-22 Created: 2006-11-22 Last updated: 2017-12-14Bibliographically approved
5. Thermodynamics of folding and binding in an affibody:affibody complex
Open this publication in new window or tab >>Thermodynamics of folding and binding in an affibody:affibody complex
2006 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 359, no 5, 1305-1315 p.Article in journal (Refereed) Published
Abstract [en]

Affibody binding proteins are selected from phage-displayed libraries of variants of the 58 residue Z domain. Z(Taq) is an affibody originally selected as a binder to Taq DNA polymerase. The anti-Z(Taq) affibody was selected as a binder to Z(Taq) and the Z(Taq):anti-Z(Taq) complex is formed with a dissociation constant K-d = 0.1 mu M. We have determined the structure of the Z(Taq):anti-Z(Taq) complex as well as the free state structures of Z(Taq) and anti-Z(Taq) using NMR. Here we complement the structural data with thermodynamic studies of Z(Taq) and anti-Z(Taq) folding and complex formation. Both affibody proteins show cooperative two-state thermal denaturation at melting temperatures T-M similar to 56 degrees C. Z(Taq):anti-Z(Taq) complex formation at 25 degrees C in 50 mM NaCl and 20 mM phosphate buffer (pH 6.4) is enthalpy driven with Delta H degrees(bind) = -9.0(+/- 0.1) kcal mol(-1). The heat capacity change Delta C-P degrees,(bind) = -0.43(+/- 0.01) kcal mol(-1) K-1 is in accordance with the predominantly non-polar character of the binding surface, as judged from calculations based on changes in accessible surface areas. A further dissection of the small binding entropy at 25 degrees C (-T Delta S degrees(bind) = -0.6(+/- 0.1) kcal mol(-1)) suggests that a favourable desolvation of non-polar surface is almost completely balanced by unfavourable conformational entropy changes and loss of rotational and translational entropy. Such effects can therefore be limiting for strong binding also when interacting protein components are stable and homogeneously folded. The combined structure and thermodynamics data suggest that protein properties are not likely to be a serious limitation for the development of engineered binding proteins based on the Z domain.

Keyword
protein engineering, protein stability, molecular recognition, binding thermodynamics, calorimetry
National Category
Other Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-6409 (URN)10.1016/j.jmb.2006.04.041 (DOI)000238988400012 ()2-s2.0-33746927966 (Scopus ID)
Note
QC 20110118Available from: 2006-11-22 Created: 2006-11-22 Last updated: 2017-12-14Bibliographically approved

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