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

An automated procedure for NOE assignment and three-dimensional structure r efinement is presented. The input to the procedure consists of (1) an ensem ble of preliminary protein NMR structures, (2) partial sequence-specific as signments for the protein and (3) the positions and volumes of unassigned N OESY cross peaks. Chemical shifts for unassigned side chain protons are pre dicted from the preliminary structures. The chemical shifts and unassigned NOESY cross peaks are input to an automated procedure for NOE assignment an d structure calculation (ARIA) [Nilges et al. (1997) J. Mol. Biol., 269, 40 8-422]. ARIA is optimized for the task of structure refinement of larger pr oteins. Errors are filtered to ensure that sequence-specific assignments ar e reliable. The procedure is applied to the 27.8 kDa single-chain T cell re ceptor (scTCR). Preliminary NMR structures, nearly complete backbone assign ments, partial assignments of side chain protons and more than 1300 unassig ned NOESY cross peaks are input. Using the procedure, the resonant frequenc ies of more than 40 additional side chain protons are assigned. Over 400 ne w NOE cross peaks are assigned unambiguously. Distances derived from the au tomatically assigned NOEs improve the precision and quality of calculated s cTCR structures. In the refined structures, a hydrophobic cluster of side c hains on the scTCR surface that binds major histocompatibility complex (MHC )/antigen is revealed. It is composed of the side chains of residues from t hree loops and stabilizes the conformation of residues that interact with M HC.