International
Tables for
Crystallography
Volume F
Crystallography of biological macromolecules
Edited by M. G. Rossmann and E. Arnold

International Tables for Crystallography (2006). Vol. F. ch. 23.4, p. 635   | 1 | 2 |

Section 23.4.4.2.3. Ribonuclease T1

C. Mattosa* and D. Ringeb

aDepartment of Molecular and Structural Biochemistry, North Carolina State University, 128 Polk Hall, Raleigh, NC 02795, USA, and  bRosenstiel Basic Medical Sciences Research Center, Brandeis University, 415 South St, Waltham, MA 02254, USA
Correspondence e-mail:  mattos@bchserver.bch.ncsu.edu

23.4.4.2.3. Ribonuclease T1

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A group of four crystal structures of ribonuclease T1 in complex with guanosine, guanosine-2′-phosphate, guanylyl-2′,5′-guanosine and vanadate were used for an analysis of conserved water positions that contribute to the structural stabilization of the protein (Malin et al., 1991[link]). The four structures were obtained from isomorphous crystals and ranged in resolution from 1.7 to 1.9 Å. Conserved water molecules were considered to be those found within a sphere of 1 Å from each other in all four structures. All other water molecules were excluded from the analysis. 30 water molecules were found to be conserved. Of these, ten were observed near crystal contacts, although only one appears to be dictated by the crystal contact itself, making a single hydrogen bond with each of the symmetry-related protein molecules. Ten other water molecules form a channel that brings together an α-helix and a hairpin-like loop structure and then go on to wrap around the calcium ion, providing half of its coordination sphere. The first five of these water molecules are completely buried, holding together the two secondary-structure elements, which would otherwise collapse (Malin et al., 1991[link]). Two water molecules are found to stabilize the N and C termini, which are brought together by a disulfide bond. The remaining eight conserved water molecules hold together various elements of secondary structure or are located in the active site.

An interesting extension to this study included four additional structures: the E58A mutant in complex with guanosine-2′-monophosphate, the H92A mutant crystallized under two different conditions and wild-type RNase T1 in complex with guanosine-3′,5′-biphosphate. Two of these crystal forms were not isomorphous with the native protein crystals or with each other. Thus a total of eight structures solved in three different space groups were analysed (Pletinckx et al., 1994[link]). Although the effect of crystal packing on the three-dimensional structure of the protein is minimal, there are some significant differences in the solvent structure. In particular, there is no evidence of the calcium-binding site and its coordinating water structure in any crystal forms other than the canonical wild type. Instead, the E58A mutant has a sodium-binding site at a different position, along with three previously unobserved water molecules. It is clear that the presence of the metal ions is fortuitous and linked to the crystallization conditions.

There are 25 water molecules structurally conserved throughout the different packing arrangements studied. Ten of these are single sites, there are three clusters of two water molecules and a larger cluster originally described by Malin et al. (1991)[link] to hold together the core of the protein. As was observed for the study on T4 lysozyme (Zhang & Matthews, 1994[link]), the strictly conserved water-binding sites present in crystal structures solved across different space groups are involved in bridging protein secondary-structure elements and seem to be crucial for the integrity of the protein structure.

References

First citation Malin, R., Zielenkiewicz, P. & Saenger, W. (1991). Structurally conserved water molecules in ribonuclease T1. J. Biol. Chem. 266, 4848–4852.Google Scholar
First citation Pletinckx, J., Steyaert, J., Zegers, I., Choe, H.-W., Heinemann, U. & Wyns, L. (1994). Crystallographic study of Glu58Ala RNase T1-2′-guanosine monophosphate at 1.9 Å resolution. Biochemistry, 33, 1654–1662.Google Scholar
First citation Zhang, X.-J. & Matthews, B. W. (1994). Conservation of solvent-binding sites in 10 crystal forms of T4 lysozyme. Protein Sci. 3, 1031–1039.Google Scholar








































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