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 us ing an increased number of NOE constraints obtained from a C-13,N-15-labele d protein sample and by applying a recently developed triple-resonance cros s-correlated relaxation experiment for the determination of the backbone di hedral angle ip. Additionally, the relative orientation of the N-15(i)-H-1( N)(i) dipole and the (CO)-C-13(i) CSA tensor, which is related to both back bone 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 cha racterized by proton-detected C-13 ' (i-1)-N-15(i) and N-15(i)-H-1(N)(i) mu ltiple-quantum relaxation, respectively. We show that regions having cross- correlated time modulation of backbone isotropic chemical shifts on the mil lisecond to microsecond time scale correlate with residues that are structu rally altered in the mutant protein CRP2(LIM2)RI22A (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-bi nding sites. This indicates the presence of long-range collective motions i n the two zinc-binding subdomains. The conformational plasticity of the LIM domain may be of functional relevance for this important protein recogniti on motif.