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Crystal structure of a lead-dependent ribozyme revealing metal binding sites relevant to catalysis

Nature Structural Biology volume 6, pages 261–268 (1999)Cite this article

Abstract

The leadzyme is a small RNA motif that catalyzes a site-specific, Pb2+-dependent cleavage reaction. As such, it is an example of a metal-dependent RNA enzyme. Here we describe the X-ray crystallographic structure of the leadzyme, which reveals two independent molecules per asymmetric unit. Both molecules feature an internal loop in which a bulged purine base stack twists away from the helical stem. This kinks the backbone, rendering the phosphodiester bond susceptible to cleavage. The independent molecules have different conformations: one leadzyme copy coordinates Mg2+, whereas the other binds only Ba2+ or Pb2+. In the active site of the latter molecule, a single Ba2+ ion coordinates the 2´-OH nucleophile, and appears to mimic the binding of catalytic lead. These observations allow a bond cleavage reaction to be modeled, which reveals the minimal structural features necessary for catalysis by this small ribozyme.

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Figure 1: Sequence of the leadzyme, LZ4 (ref.13), used in the structural work.
Figure 2: Representative electron density and schematic drawings of the structure.
Figure 3: Stereo representation and schematic drawings comparing molecules 1 and 2.
Figure 4: Crystal packing interface of the bulged nucleotides from four internal loops.
Figure 5: Stereo representation and schematic drawings for the working model of the cleavage reaction.

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Acknowledgements

We thank C. Kielkopf, J. Puglisi, W. Weis, M. Sousa, and A. Kolatkar for helpful discussions, as well as expert technical advice and support; C. Trinh and the staff of SSRL for assistance with the X-ray data collection; also, we thank A. Pardi and C. Hoogstraten for discussions and sharing their unpublished results on the solution structure of the leadzyme. J.E.W. is a Burroughs Wellcome Fund Fellow of the Life Sciences Research Foundation (LSRF). This work was supported by funds from the LSRF and an NIH grant to D.B.M. Operation of SSRL beamlines 7-1 and 9-1 is supported by the DOE, Office of Basic Energy Sciences. The Biotechnology Program at SSRL is supported by the NIH, National Center for Research Resources Biomedical Technology Program and the DOE, Office of Biological and Environmental Research.

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  1. Department of Structural Biology, Stanford University School of Medicine, Stanford, 94305-5126, California, USA

    Joseph E. Wedekind & David B. McKay

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  1. Joseph E. Wedekind
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Correspondence to Joseph E. Wedekind or David B. McKay.

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Wedekind, J., McKay, D. Crystal structure of a lead-dependent ribozyme revealing metal binding sites relevant to catalysis. Nat Struct Mol Biol 6, 261–268 (1999). https://doi.org/10.1038/6700

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