A minimalist approach toward protein recognition by epitope transfer from functionally evolved β-sheet surfaces

Srivats Rajagopal, Scott C. Meyer, Aaron Goldman, Min Zhou, Indraneel Ghosh

Research output: Contribution to journalArticle

12 Scopus citations

Abstract

New approaches for identifying small molecules that specifically target protein surfaces as opposed to active site clefts are of much current interest. Toward this goal, we describe a three-step methodology: in step one, we target a protein of interest by directed evolution of a small β-sheet scaffold; in step two, we identify residues on the scaffold that are implicated in binding; and in step three, we transfer the chemical information from the β-sheet to a small molecule mimic. As a case study, we targeted the proteolytic enzyme thrombin, involved in blood coagulation, utilizing a library of β-sheet epitopes displayed on phage that were previously selected for conservation of structure. We found that the thrombin-binding, β-sheet displaying mini-proteins retained their structure and stability while inhibiting thrombin at low micromolar inhibition constants. A conserved dityrosine recognition motif separated by 9.2 Å was found to be common among the mini-protein inhibitors and was further verified by alanine scanning. A molecule containing two tyrosine residues separated by a linker that matched the spacing on the β-sheet scaffold inhibited thrombin, whereas a similar dityrosine molecule separated by a shorter 6 Å linker could not. Moreover, kinetic analysis revealed that both the mini-protein as well as its minimalist mimic with only two functional residues exhibited noncompetitive inhibition of thrombin. Thus, this reductionist approach affords a simple methodology for transferring information from structured protein scaffolds to yield small molecule leads for targeting protein surfaces with novel mechanisms of action.

Original languageEnglish (US)
Pages (from-to)14356-14363
Number of pages8
JournalJournal of the American Chemical Society
Volume128
Issue number44
DOIs
StatePublished - Nov 8 2006

ASJC Scopus subject areas

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry

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