Application of automated NOE assignment to three-dimensional structure refinement of a 28 kDa single-chain T cell...

An automated procedure for NOE assignment and three-dimensional structure refinement is presented. The input to the procedure consists of (1) an ensemble of preliminary protein NMR structures, (2) partial sequence-specific assignments for the protein and (3) the positions and volumes of unassigned NOESY cross peaks. Chemical shifts for unassigned side chain protons are predicted from the preliminary structures. The chemical shifts and unassigned NOESY cross peaks are input to an automated procedure for NOE assignment and structure calculation (ARIA) [Nilges et al. (1997) J. Mol. Biol., 269, 408-422]. ARIA is optimized for the task of structure refinement of larger proteins. Errors are filtered to ensure that sequence-specific assignments are reliable. The procedure is applied to the 27.8 kDa single-chain T cell receptor (scTCR). Preliminary NMR structures, nearly complete backbone assignments, partial assignments of side chain protons and more than 1300 unassigned NOESY cross peaks are input. Using the procedure, the resonant frequencies of more than 40 additional side chain protons are assigned. Over 400 new NOE cross peaks are assigned unambiguously. Distances derived from the automatically assigned NOEs improve the precision and quality of calculated scTCR structures. In the refined structures, a hydrophobic cluster of side chains on the scTCR surface that binds major histocompatibility complex (MHC)/antigen is revealed. It is composed of the side chains of residues from three loops and stabilizes the conformation of residues that interact with MHC.