Application of cross-correlated NMR spin relaxation to the zinc-finger protein CRP2(LIM2): evidence for collective motions in LIM domains.
The solution structure of quail CRP2(LIM2) was significantly improved by using an increased number of NOE constraints obtained from a 13C,15N-labeled protein sample and by applying a recently developed triple-resonance cross-correlated relaxation experiment for the determination of the backbone dihedral angle psi. Additionally, the relative orientation of the 15N(i)-1HN(i) dipole and the 13CO(i) CSA tensor, which is related to both backbone angles phi and psi, was probed by nitrogen-carbonyl multiple-quantum relaxation and used as an additional constraint for the refinement of the local geometry of the metal-coordination sites in CRP2(LIM2). The backbone dynamics of residues located in the folded part of CRP2(LIM2) have been characterized by proton-detected 13C'(i-1)-15N(i) and 15N(i)-1HN(i) multiple-quantum relaxation, respectively. We show that regions having cross-correlated time modulation of backbone isotropic chemical shifts on the millisecond to microsecond time scale correlate with residues that are structurally altered in the mutant protein CRP2(LIM2)R122A (disruption of the CCHC zinc-finger stabilizing side-chain hydrogen bond) and that these residues are part of an extended hydrogen-bonding network connecting the two zinc-binding sites. This indicates the presence of long-range collective motions in the two zinc-binding subdomains. The conformational plasticity of the LIM domain may be of functional relevance for this important protein recognition motif.
Schüler
W
W
Institute of Organic Chemistry and Institute of Biochemistry, University of Innsbruck, Austria.
Kloiber
K
K
Matt
T
T
Bister
K
K
Konrat
R
R
D000595:Amino Acid Sequence; D000818:Animals; D030161:Avian Proteins; D006860:Hydrogen Bonding; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000737:chemistry*; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D017434:Protein Structure, Tertiary; D016271:Proto-Oncogene Proteins c-myc / Q000737:chemistry*; D011784:Quail; D016335:Zinc Fingers
D030161:Avian Proteins; D009124:Muscle Proteins; D016271:Proto-Oncogene Proteins c-myc; C081447:cysteine-rich protein, avian