Fragment-linking peptide design yields a high-affinity ligand for microtubule-based transport

Jessica A Cross, Magda S Chegkazi, Roberto A Steiner*, Derek N Woolfson*, Mark P Dodding*

*Corresponding author for this work

Research output: Contribution to journalArticle (Academic Journal)peer-review

Abstract

Synthetic peptides are attractive candidates to manipulate protein-protein interactions inside the cell as they mimic natural interactions to compete for binding. However, protein-peptide interactions are often dynamic and weak. A challenge is to design peptides which make improved interactions with the target. Here, we devise a fragment-linking strategy - ‘mash-up’ design - to deliver a high-affinity ligand, KinTag, for the kinesin-1 motor. Using structural insights from natural micromolaraffinity cargo-adaptor ligands, we have identified and combined key binding features in a single, high-affinity ligand. An X-ray crystal structure demonstrates interactions as designed and reveals only a modest increase in interface area. Moreover, when genetically encoded, KinTag promotes transport of lysosomes with higher efficiency than natural sequences, revealing a direct link between motor-adaptor binding affinity and organelle transport. Together, these data demonstrate a fragment-linking strategy for peptide design and its application in a synthetic motor ligand to direct cellular cargo transport.
Original languageEnglish
Number of pages15
JournalCell Chemical Biology
Early online date9 Apr 2021
DOIs
Publication statusE-pub ahead of print - 9 Apr 2021

Bibliographical note

Funding Information:
J.A.C. is supported by the Engineering and Physical Sciences Research Council (EPSRC) Bristol Centre for Doctoral Training in Chemical Synthesis. M.P.D. is a Lister Institute for Preventative Medicine Research Prize Fellow and this work is supported by Biotechnology and Biological Sciences Research Council (BBSRC) grant BB/S000917/1. D.N.W. held a Royal Society Wolfson Research Merit Award (WM140008). M.S.C. and R.A.S. are supported by BBSRC grant BB/S000828/1. We also thank the University of Bristol School of Chemistry Mass Spectrometry Facility for use of the EPSRC-funded Bruker Ultraflex MALDI-TOF/TOF instrument (EP/K03927X/1), and the BBSRC/EPSRC-funded BrisSynBio (BB/L01386X1) for access to its peptide synthesizers and plate reader. The authors gratefully acknowledge the Wolfson Bioimaging Facility and the Proteomics Facility at the University of Bristol for their support and assistance in this work. The X-ray crystallographic work was conducted at beamline I03 of Diamond Light Source. The authors also thank Dr Kirsty McMillan and Dr Kevin Wilkinson for donation of rat hippocampal neurons and antibodies and assistance with experimental protocols. M.P.D. D.N.W. and J.A.C. conceived the project and designed the peptides. J.A.C. synthesized the peptides and completed biophysical and cellular experiments. R.A.S. and M.S.C. designed the construct used for crystallographic studies and solved the X-ray crystal structure of the complex. The manuscript was written by J.A.C. M.P.D. D.N.W. and R.A.S. and commented on by all authors. The authors declare no competing interests.

Funding Information:
J.A.C. is supported by the Engineering and Physical Sciences Research Council (EPSRC) Bristol Centre for Doctoral Training in Chemical Synthesis. M.P.D. is a Lister Institute for Preventative Medicine Research Prize Fellow and this work is supported by Biotechnology and Biological Sciences Research Council ( BBSRC ) grant BB/S000917/1 . D.N.W. held a Royal Society Wolfson Research Merit Award (WM140008). M.S.C. and R.A.S. are supported by BBSRC grant BB/S000828/1 . We also thank the University of Bristol School of Chemistry Mass Spectrometry Facility for use of the EPSRC -funded Bruker Ultraflex MALDI-TOF/TOF instrument ( EP/K03927X/1 ), and the BBSRC /EPSRC-funded BrisSynBio ( BB/L01386X1 ) for access to its peptide synthesizers and plate reader. The authors gratefully acknowledge the Wolfson Bioimaging Facility and the Proteomics Facility at the University of Bristol for their support and assistance in this work. The X-ray crystallographic work was conducted at beamline I03 of Diamond Light Source. The authors also thank Dr Kirsty McMillan and Dr Kevin Wilkinson for donation of rat hippocampal neurons and antibodies and assistance with experimental protocols.

Publisher Copyright:
© 2021 Elsevier Ltd

Structured keywords

  • BCS and TECS CDTs

Keywords

  • peptide design
  • TPR domain
  • kinesin-1
  • intracellular transport
  • short linear motif
  • SLiM
  • microtubule transport

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