A thermophilic mini-chaperonin contains a conserved polypeptide-binding surface: Combined crystallographic and NMR studies of the GroEL apical domainwith implications for substrate interactions

A homologue of the Escherichia coli GroEL apical domain was obtained from t hermophilic eubacterium Thermus thermophilus. The domains share 70 % sequen ce identity (101 out of 145 residues). The thermal stability of the T. ther mophilus apical domain (T-m > 100 degreesC as evaluated by circular dichroi sm) is at least 35 degreesC greater than that of the E. coli apical domain (T-m = 65 degreesC). The crystal structure of a selenomethione-substituted apical domain from T. thermophilus was determined to a resolution of 1.78 A ngstrom using multiwavelength-anomalous-diffraction phasing. The structure is similar to that of the E. coli apical domain (root-mean-square deviation 0.45 Angstrom based on main-chain atoms). The thermophilic structure conta ins seven additional salt bridges of which four contain charge-stabilized h ydrogen bonds. Only one of the additional salt bridges would face the "Anfi nsen cage" in GroEL. High temperatures were exploited to map sites of inter actions between the apical domain and molten globules. NMR footprints of ap ical domain-protein complexes were obtained at elevated temperature using N -15-H-1 correlation spectra of N-15-labeled apical domain. Footprints emplo ying two polypeptides unrelated in sequence or structure (an insulin monome r and the SRY high-mobility-group box, each partially unfolded at 50 degree sC) are essentially the same and consistent with the peptide-binding surfac e previously defined in E. coli GroEL and its epical domain-peptide complex es. An additional part of this surface comprising a short N-terminal alpha -helix is observed. The extended footprint rationalizes mutagenesis studies of intact GroEL in which point mutations affecting substrate binding were found outside the "classical" peptide-binding site. Our results demonstrate structural conservation of the apical domain among GroEL homologues and co nservation of an extended non-polar surface recognizing diverse polypeptide s. (C) 2001 Academic Press.