Solution NMR Structure Investigation for Releasing Mechanism of Neocarzinostatin Chromophore from the Holoprotein

Abstract

Holo-neocarzinostatin (holo-NCS) is a complex protein carrying the anti-tumor active enediyne ring chromophore by a scaffold consisting of an immunoglobulinlike seven-stranded anti-parallel ␤-barrel. Because of the labile chromophore reflecting its extremely strong DNA cleavage activity and complete stabilization in the complex, holo-NCS has attracted much attention in clinical use as well as for drug delivery systems. Despite many structural analyses for holo-NCS, the chromophore-releasing mechanism to trigger prompt attacks on the target DNA is still unclear. We determined the three-dimensional structure of the protein and the internal motion by multinuclear NMR to investigate the releasing mechanism. The internal motion studied by 13 C NMR methine relaxation experiments showed that the complex has a rigid structure for its loops as well as the ␤-barrel in aqueous solution. This agrees with the refined NMR solution structure, which has good convergence in the loop regions. We also showed that the chromophore displayed a similar internal motion as the protein moiety. The structural comparison between the refined solution structure and x-ray crystal structure indicated characteristic differences. Based on the findings, we proposed the chromophore-releasing mechanism by a three-state equilibrium, which sufficiently describes both the strong binding and the prompt releasing of the chromophore. We demonstrated that we could bridge the dynamic properties and the static structure features with simple kinetic assumptions to solve the biochemical function.Holo-neocarzinostatin (holo-NCS) 1 (molecular mass 12 kDa, 113 amino acid residues ϩ chromophore) is a prominent member of the strongest anti-tumor reagent chromoprotein family (Fig. 1, B and D) (1). It is a non-covalent complex of an enediyne ring chromophore and its carrier protein isolated from Streptomyces carzinostaticus (2-4). The chromophore, which has a selective bulged DNA-cleaving activity (5), is easily inactivated by light, heat, and molecular oxygen because of its labile structure. Despite its intrinsic instability in the free state, the chromophore in the complex is stable even in vivo. It is thought that holo-NCS penetrates into the target cell carrying the chromophore and promptly releases it at the cell nucleus to kill the cell. Therefore, the holo-NCS family has attracted much attention in clinical use as well as for use in targeting drug delivery systems (6 -14). The three-dimensional structures of holo-NCS and the chromoprotein family have been investigated by solution NMR (12, 15-19) and x-ray diffraction (20), elucidating an immunoglobulin-like sevenstranded anti-parallel ␤-barrel structure (Fig. 1B) (21).Based on the previous NMR and x-ray studies for holo-NCS, several mechanisms of the chromophore stabilization have been proposed. These mechanisms were dominated by hydrophobic interactions with the binding pocket. However, the mechanism of releasing and binding of the chromophore remains unclear, even though the process has attracted much atte...

A New Model for Ligand Release

“…Although Phe 78 appears to be quite dynamic from these NMR data, a newer three-dimensional NMR study (48) suggests that there is no significant internal motion for the loop 75-84, and the side chain of Phe 78 has a rigid conformation. Despite some contradictions, Phe 78 has been consistently considered to be one of the critical residues responsible for NCS-C stabilization.…”

“…Whether any major structural changes are involved in NCS action in vivo is not yet known. A "sliding" model is proposed recently for chromophore-releasing mechanism, which involves certain backbone conformational changes in protein (48). Lately, we find that apoNCS is highly resistant to denaturants (49).…”

Role of Steric Effects in Protein‐Directed Enediyne Cycloaromatization of Neocarzinostatin

“…In accordance with the reported 15 H NMR chemical shifts; the changes were measured by comparing the spectrum of the apoprotein to that of the holoprotein ( Figure 6 e and f). More than 40 amide resonances were highly shifted (Dd > 0.10 ppm) in the spectrum of both proteins after binding to NCS-C, and many of those perturbed residues have been reported to exhibit an intermolecular NOE on the protein-bound NCS-C. [42] Whereas both WT and C37S/C47S proteins were considerably perturbed by binding to NCS-C, interestingly, the characteristics of perturbation distribution were very similar for the two proteins. The highly shifted residues in the WT were also found to be highly shifted in the C37S/C47S mutant.…”

“…The number of transients in the 1 H-dimension was eight. The cross-peaks of amino acid residues in apo-and holoNCSs were identified based on the reported assignments (BMRB entries 5343 and 5969) [41,42] using Tyr 32 or Thr 65 as an internal reference. All spectra were processed using VARIAN VNMR and Sparky software.…”

Preparation of Neocarzinostatin Apoprotein Mutants and the Randomized Library on the Chromophore-Binding Cavity

“…The Phe 78 of apo-NCS closely associate with the amino-sugar of NCS-chr and has a rigid conformation in the complex in the aqueous solution. 28) So, the slight decrease in affinity for F78W to EtdBr in previous our report should be related to a local steric hindrance induced by mutation, not protein stabilization. 10) The binding properties of a chromophore mimic EtdBr to newly prepared mutants were evaluated by the changes in total fluorescence intensity and fluorescence polarization.…”