Structural characterization of proteins with an attached ATCUN motif by paramagnetic relaxation enhancement NMR spectroscopy

The use of a short, three-residue Cu2+-binding sequence, the ATCUN motif, i s presented as an approach for extracting long-range distance restraints fr om relaxation enhancement NMR spectroscopy. The ATCUN motif is prepended to the N-termini of proteins and binds Cu2+ with a very high affinity. Relaxa tion rates of amide protons in ATCUN-tagged protein in the presence and abs ence of Cu2+ can be converted into distance restraints and used for structu re refinement by using a new routine, PMAG, that has been written for the s tructure calculation program CNS. The utility of the approach is demonstrat ed with an application to ATCUN-tagged ubiquitin. Excellent agreement betwe en measured relaxation rates and those calculated on the basis of the X-ray structure of the protein have been obtained.