1H NMR assignments of apo calcyclin and comparative structural analysis with calbindin d(9k) and s 100β

Research output: Contribution to journalArticle

Abstract

The homodimeric S100 protein calcyclin has been studied in the apo state by two-dimensional 1H NMR spectroscopy. Using a combination of scalar correlation and NOE experiments, sequence-specific 1H NMR assignments were obtained for all but one backbone and >90% of the side-chain resonances. To our knowledge, the 2 x 90 residue (20 kDa) calcyclin dimer is the largest protein system for which such complete assignments have been made by purely homonuclear methods. Sequential and medium-range NOEs and slowly exchanging backbone amide protons identified directly the four helices and the short antiparallel β-type interaction between the two binding loops that comprise each subunit of the dimer. Further analysis of NOEs enabled the unambiguous assignment of 556 intrasubunit distance constraints, 24 intrasubunit hydrogen bonding constraints, and 2 x 26 intersubunit distance constraints. The conformation of the monomer subunit was refined by distance geometry and restrained molecular dynamics calculations using the intrasubunit constraints only. Calculation of the dimer structure starting from this conformational ensemble has been reported elsewhere. The extent of structural homology among the apo calcyclin subunit, the monomer subunit of apo S100β, and monomeric apo calbindin D(9k) has been examined in detail by comparing 1H NMR chemical shifts and secondary structures. This analysis was extended to a comprehensive comparison of the three-dimensional structures of the calcyclin monomer subunit and calbindin D(9k), which revealed greater similarity in the packing of their hydrophobic cores than was anticipated previously. Together, these results support the hypothesis that all members of the S100 family have similar core structures and similar modes of dimerization. Analysis of the amphiphilicity of Helix IV is used to explain why calbindin D(9k) is monomeric, but full-length S100 proteins form homodimers.

Details

Authors
  • Barbara C.M. Potts
  • Göran Carlström
  • Katsuo Okazaki
  • Hiroyoshi Hidaka
  • Walter J. Chazin
Organisations
External organisations
  • Scripps Research Institute
  • Nagoya University
Research areas and keywords

Subject classification (UKÄ) – MANDATORY

  • Biochemistry and Molecular Biology

Keywords

  • calcyclin, chemical shift, EF-hand calcium-binding protein, homology, hydrophobic core, S100 protein
Original languageEnglish
Pages (from-to)2162-2174
Number of pages13
JournalProtein Science
Volume5
Issue number11
Publication statusPublished - 1996 Jan 1
Publication categoryResearch
Peer-reviewedYes