An ultra-high affinity protein–protein interface displaying sequence-robustness
Møller, Marie Sofie LU ; Olesen, Sita Vaag and André, Ingemar LU
(2021)
In Protein Science
30(6).
p.1144-1156
- Abstract
Protein–protein interactions are crucial in biology and play roles in for example, the immune system, signaling pathways, and enzyme regulation. Ultra-high affinity interactions (Kd <0.1 nM) occur in these systems, however, structures and energetics behind stability of ultra-high affinity protein–protein complexes are not well understood. Regulation of the starch debranching barley limit dextrinase (LD) and its endogenous cereal type inhibitor (LDI) exemplifies an ultra-high affinity complex (Kd of 42 pM). In this study the LD–LDI complex is investigated to unveil how robust the ultra-high affinity is to LDI sequence variation at the protein–protein interface and whether alternative sequences can retain the... (More)
Protein–protein interactions are crucial in biology and play roles in for example, the immune system, signaling pathways, and enzyme regulation. Ultra-high affinity interactions (Kd <0.1 nM) occur in these systems, however, structures and energetics behind stability of ultra-high affinity protein–protein complexes are not well understood. Regulation of the starch debranching barley limit dextrinase (LD) and its endogenous cereal type inhibitor (LDI) exemplifies an ultra-high affinity complex (Kd of 42 pM). In this study the LD–LDI complex is investigated to unveil how robust the ultra-high affinity is to LDI sequence variation at the protein–protein interface and whether alternative sequences can retain the ultra-high binding affinity. The interface of LD–LDI was engineered using computational protein redesign aiming at identifying LDI variants predicted to retain ultra-high binding affinity. These variants present a very diverse set of mutations going beyond conservative and alanine substitutions typically used to probe interfaces. Surface plasmon resonance analysis of the LDI variants revealed that high affinity of LD–LDI requires interactions of several residues at the rim of the protein interface, unlike the classical hotspot arrangement where key residues are found at the center of the interface. Notably, substitution of interface residues in LDI, including amino acids with functional groups different from the wild-type, could occur without loss of affinity. This demonstrates that ultra-high binding affinity can be conferred without hotspot residues, thus making complexes more robust to mutational drift in evolution. The present mutational analysis also demonstrates how energetic coupling can emerge between residues at large distances at the interface.
(Less)
- author
- Møller, Marie Sofie
LU
; Olesen, Sita Vaag
and André, Ingemar
LU
- organization
- publishing date
- 2021-06-01
- type
- Contribution to journal
- publication status
- published
- subject
- keywords
- computational protein redesign, non-hotspot-centric interactions, proteinaceous inhibitor, protein–protein complex, surface plasmon resonance
- in
- Protein Science
- volume
- 30
- issue
- 6
- pages
- 13 pages
- publisher
- The Protein Society
- external identifiers
-
- scopus:85104524672
- pmid:33837990
- ISSN
- 0961-8368
- DOI
- 10.1002/pro.4080
- language
- English
- LU publication?
- yes
- additional info
- Funding Information: Professor Birte Svensson (Technical University of Denmark, DTU) is thanked for many valuable discussions and proof reading. Science Director Carsten Andersen (Novozymes) is thanked for his support of the study. Karina Jansen (DTU) is thanked for help with protein production and purification. This work was supported by Independent Research Fund Denmark|Technology and Production Sciences (FTP; DFF–1337‐00158 and DFF–1335‐00769 to Marie Sofie Møller). Independent Research Fund Denmark|Natural Sciences supported the purchase of the Biacore T100 instrument (DFF‐272‐06‐0050). Publisher Copyright: © 2021 The Protein Society. Copyright: Copyright 2021 Elsevier B.V., All rights reserved.
- id
- 7d6e7fd1-3d5d-47d0-84bd-348b23946439
- date added to LUP
- 2021-05-03 15:59:25
- date last changed
- 2024-06-15 10:45:54
@article{7d6e7fd1-3d5d-47d0-84bd-348b23946439,
abstract = {{<p>Protein–protein interactions are crucial in biology and play roles in for example, the immune system, signaling pathways, and enzyme regulation. Ultra-high affinity interactions (K<sub>d</sub> <0.1 nM) occur in these systems, however, structures and energetics behind stability of ultra-high affinity protein–protein complexes are not well understood. Regulation of the starch debranching barley limit dextrinase (LD) and its endogenous cereal type inhibitor (LDI) exemplifies an ultra-high affinity complex (K<sub>d</sub> of 42 pM). In this study the LD–LDI complex is investigated to unveil how robust the ultra-high affinity is to LDI sequence variation at the protein–protein interface and whether alternative sequences can retain the ultra-high binding affinity. The interface of LD–LDI was engineered using computational protein redesign aiming at identifying LDI variants predicted to retain ultra-high binding affinity. These variants present a very diverse set of mutations going beyond conservative and alanine substitutions typically used to probe interfaces. Surface plasmon resonance analysis of the LDI variants revealed that high affinity of LD–LDI requires interactions of several residues at the rim of the protein interface, unlike the classical hotspot arrangement where key residues are found at the center of the interface. Notably, substitution of interface residues in LDI, including amino acids with functional groups different from the wild-type, could occur without loss of affinity. This demonstrates that ultra-high binding affinity can be conferred without hotspot residues, thus making complexes more robust to mutational drift in evolution. The present mutational analysis also demonstrates how energetic coupling can emerge between residues at large distances at the interface.</p>}},
author = {{Møller, Marie Sofie and Olesen, Sita Vaag and André, Ingemar}},
issn = {{0961-8368}},
keywords = {{computational protein redesign; non-hotspot-centric interactions; proteinaceous inhibitor; protein–protein complex; surface plasmon resonance}},
language = {{eng}},
month = {{06}},
number = {{6}},
pages = {{1144--1156}},
publisher = {{The Protein Society}},
series = {{Protein Science}},
title = {{An ultra-high affinity protein–protein interface displaying sequence-robustness}},
url = {{http://dx.doi.org/10.1002/pro.4080}},
doi = {{10.1002/pro.4080}},
volume = {{30}},
year = {{2021}},
}