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

Hiroyuki Takashima,Takuya Yoshida,Tetsuya Ishino,Katsumi Hasuda,Tadayasu Ohkubo,Yuji Kobayashi

doi:10.1074/jbc.m411579200

Hiroyuki Takashima, Takuya Yoshida + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m411579200

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Abstract

Holo-neocarzinostatin (holo-NCS) is a complex protein carrying the anti-tumor active enediyne ring chromophore by a scaffold consisting of an immunoglobulin-like seven-stranded anti-parallel beta-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 13C NMR methine relaxation experiments showed that the complex has a rigid structure for its loops as well as the beta-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.

Highlights

Holo-neocarzinostatin1 is a prominent member of the strongest anti-tumor reagent chromoprotein family (Fig. 1, B and D) (1)
We showed that the chromophore displayed a similar internal motion as the protein moiety
There was no clarification between conformational ambiguity and the internal motion, which should be investigated by relaxation data in NMR. 13C NMR relaxation studies were achieved for apo-NCS by Izadi-Pruneyre et al (24) and Mispelter et al (25), and yet there was no clear relation between the internal motion and the apo-NCS structural convergence (12, 26)

Summary

EXPERIMENTAL PROCEDURES

15N, 13C Uniform Label and Purification of Holo-NCS—To obtain stable isotope-labeled holo-NCS, S. carzinostaticus was grown in Waxman medium (0.05% meat extract, 0.05% peptone, 0.03% NaCl, and 0.1% D-glucose) containing algae 15N- and 15N/13C-labeled amino acid mixtures (Chlorella Industry Co., Ltd). The 15N and 13C HMQC spectrum indicated uniform labeling of the protein moiety, which was verified by multidimensional NMR experiments, but the chromophore was not uniformly labeled. NOE distance constraints were collected from the two three-dimensional NOESY-HMQC spectra (Table I). Because of non-uniform labeling of the chromophore, inter-molecular NOEs between the chromophore and the protein moiety elucidated by 1H homonuclear NOESY spectrum were used for structure calculations. NMR Data Handling and Structure Calculations—NOE intensities measured by 15N-edited NOESY-HMQC were corrected according to spin degeneracy and were converted to approximate inter-proton distances in inverse proportion to distance (r), 1/r3 and 1/r6, with proper normalization constants and a Ϯ0.5 Å margin of error. NOE intensities measured by 13C-edited NOESY-HMQC were treated in the same manner by normalization against intra-residual methylene protons or inter-residue C␣H-C␣H cross-peaks. The effective correlation time ␶e is related both to amplitude and rate of the internal dynamics

The effective correlation time for fast motion

RESULTS

Disallowed regions

DISCUSSION