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Rigidity of the extracellular part of HER2: evidence from engineering subdomain interfaces and shared-helix DARPin-DARPin fusions


Jost, Christian; Stüber, Jakob C; Honegger, Annemarie; Wu, Yufan; Batyuk, Alexander; Plückthun, Andreas (2017). Rigidity of the extracellular part of HER2: evidence from engineering subdomain interfaces and shared-helix DARPin-DARPin fusions. Protein Science, 26(9):1796-1806.

Abstract

The second member of the human ErbB family of receptor tyrosine kinases, HER2/hErbB2, is regarded as an exceptional case: The four extracellular subdomains could so far only be found in one fixed overall conformation, designated "open" and resembling the ligand-bound form of the other ErbB receptors. It thus appears to be different from the extracellular domains of the other family members that show inter-subdomain flexibility and exist in a "tethered" form in the absence of ligand. For HER2, there was so far no direct evidence for such a tethered conformation on the cell surface. Nonetheless, alternative conformations of HER2 in vivo could so far not be excluded. We now demonstrate the rigidity of HER2 on the surface of tumor cells by employing two orthogonal approaches of protein engineering: To directly test the potential of the extracellular domain of HER2 to adopt a pseudo-tethered conformation on the cell surface, we first designed HER2 variants with a destabilized interface between extracellular subdomains I and III that would favor deviation from the "open" conformation. Secondly, we used differently shaped versions of a Designed Ankyrin Repeat Protein (DARPin) fusion, recognizing subdomain I of HER2, devised to work as probes for a putative pseudo-tethered extracellular domain of HER2. Combining our approaches, we exclude, on live cells and in vitro, that significant proportions of HER2 deviate from the "open" conformation.

Abstract

The second member of the human ErbB family of receptor tyrosine kinases, HER2/hErbB2, is regarded as an exceptional case: The four extracellular subdomains could so far only be found in one fixed overall conformation, designated "open" and resembling the ligand-bound form of the other ErbB receptors. It thus appears to be different from the extracellular domains of the other family members that show inter-subdomain flexibility and exist in a "tethered" form in the absence of ligand. For HER2, there was so far no direct evidence for such a tethered conformation on the cell surface. Nonetheless, alternative conformations of HER2 in vivo could so far not be excluded. We now demonstrate the rigidity of HER2 on the surface of tumor cells by employing two orthogonal approaches of protein engineering: To directly test the potential of the extracellular domain of HER2 to adopt a pseudo-tethered conformation on the cell surface, we first designed HER2 variants with a destabilized interface between extracellular subdomains I and III that would favor deviation from the "open" conformation. Secondly, we used differently shaped versions of a Designed Ankyrin Repeat Protein (DARPin) fusion, recognizing subdomain I of HER2, devised to work as probes for a putative pseudo-tethered extracellular domain of HER2. Combining our approaches, we exclude, on live cells and in vitro, that significant proportions of HER2 deviate from the "open" conformation.

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > Department of Biochemistry
07 Faculty of Science > Department of Biochemistry
Dewey Decimal Classification:570 Life sciences; biology
610 Medicine & health
Uncontrolled Keywords:DARPins; ErbB receptors; HER2; conformational probe; protein engineering
Language:English
Date:September 2017
Deposited On:26 Sep 2017 14:29
Last Modified:26 Sep 2017 14:29
Publisher:Wiley-Blackwell Publishing, Inc.
ISSN:0961-8368
Free access at:Publisher DOI. An embargo period may apply.
Publisher DOI:https://doi.org/10.1002/pro.3216
PubMed ID:28639341

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