An RNA aptamer possessing a novel monovalent cation-mediated fold inhibits lysozyme catalysis by inhibiting the binding of long natural substrates

Camille S. Padlan, Vladimir N. Malashkevich, Steven C. Almo, Matthew Levy, Michael D. Brenowitz, Mark E. Girvin

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

RNA aptamers are being developed as inhibitors of macromolecular and cellular function, diagnostic tools, and potential therapeutics. Our understanding of the physical nature of this emerging class of nucleic acid-protein complexes is limited; few atomic resolution structures have been reported for aptamers bound to their protein target. Guided by chemical mapping, we systematically minimized an RNA aptamer (Lys1) selected against hen egg white lysozyme. The resultant 59-nucleotide compact aptamer (Lys1.2minE) retains nanomolar binding affinity and the ability to inhibit lysozyme's catalytic activity. Our 2.0-Å crystal structure of the aptamer-protein complex reveals a helical stem stabilizing two loops to form a protein binding platform that binds lysozyme distal to the catalytic cleft. This structure along with complementary solution analyses illuminate a novel protein-nucleic acid interface; (1) only 410 Å2 of solvent accessible surface are buried by aptamer binding; (2) an unusually small fraction (~18%) of the RNA-protein interaction is electrostatic, consistent with the limited protein phosphate backbone contacts observed in the structure; (3) a single Na+ stabilizes the loops that constitute the protein-binding platform, and consistent with this observation, Lys1.2minE-lysozyme complex formation takes up rather than displaces cations at low ionic strength; (4) Lys1.2minE inhibits catalysis of large cell wall substrates but not catalysis of small model substrates; and (5) the helical stem of Lys1.2minE can be shortened to four base pairs (Lys1.2minF) without compromising binding affinity, yielding a 45-nucleotide aptamer whose structure may be an adaptable protein binding platform.

Original languageEnglish (US)
Pages (from-to)447-461
Number of pages15
JournalRNA
Volume20
Issue number4
DOIs
StatePublished - 2014

Fingerprint

Nucleotide Aptamers
Monovalent Cations
Muramidase
Catalysis
Protein Binding
Proteins
Nucleic Acids
Egg White
Static Electricity
Base Pairing
Osmolar Concentration
Cell Wall
Cations
Phosphates
RNA

Keywords

  • Aptamer
  • Catalysis
  • Crystal structure
  • Footprinting
  • Inhibition
  • Lysozyme

ASJC Scopus subject areas

  • Molecular Biology

Cite this

An RNA aptamer possessing a novel monovalent cation-mediated fold inhibits lysozyme catalysis by inhibiting the binding of long natural substrates. / Padlan, Camille S.; Malashkevich, Vladimir N.; Almo, Steven C.; Levy, Matthew; Brenowitz, Michael D.; Girvin, Mark E.

In: RNA, Vol. 20, No. 4, 2014, p. 447-461.

Research output: Contribution to journalArticle

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