Dissection of the structural and functional role of a conserved hydration site in RNase T1

Abstract

The reoccurrence of water molecules in crystal structures of RNase T1 was investigated. Five waters were found to be invariant in RNase T1 as well as in six other related fungal RNases. The structural, dynamical, and functional characteristics of one of these conserved hydration sites~WAT1! were analyzed by protein engineering, X-ray crystallography, and 17 O and 2 H nuclear magnetic relaxation dispersion~NMRD!. The position of WAT1 and its surrounding hydrogen bond network are unaffected by deletions of two neighboring side chains. In the mutant Thr93Gln, the Gln93NE2 nitrogen replaces WAT1 and participates in a similar hydrogen bond network involving Cys6, Asn9, Asp76, and Thr91. The ability of WAT1 to form four hydrogen bonds may explain why evolution has preserved a water molecule, rather than a side-chain atom, at the center of this intricate hydrogen bond network. Comparison of the 17 O NMRD profiles from wild-type and Thr93Gln RNase T1 yield a mean residence time of 7 ns at 27 8C and an orientational order parameter of 0.45. The effects of mutations around WAT1 on the kinetic parameters of RNase T1 are small but significant and probably relate to the dynamics of the active site.Keywords: conserved water; hydrogen bond network; NMRD; protein hydration; residence time; RNase T1The structure and function of proteins and other biological macromolecules depend on their interaction with the surrounding aqueous solvent via the hydrophobic effect, dielectric screening, and specific hydrogen bonds. Most globular proteins contain a few water molecules buried in cavities. Such internal water molecules heal packing defects and extend the intramolecular hydrogen bond framework, thus contributing importantly to protein structure and stability, and are sometimes directly involved in enzyme catalysis Meyer, 1992!. Because they are conserved among homologous proteins to the same extent as amino acid residues~Baker, 1995!, internal water molecules are clearly an integral part of the protein structure. The residence times of these internal water molecules are typically in the range 10 Ϫ8 -10 Ϫ6 s at ambient temperature~Den-isov & Halle, 1996!, but may be as long as 200 ms at 27 8C Denisov et al., 1996!.The several hundred water molecules that make contact with the surface of a small protein are considered much more mobile~Den-isov & Halle, 1996!, and the relationship between hydration in solution and the water sites that are found in macromolecular crystal structures is poorly understood. In the present study, we analyze the reoccurrence of water molecules in the different crystal structures of RNase T1 and the related RNases F1, Ms, Ap, Pb, Th, and U2. Based on the results of this comparative study, a single water molecule is chosen~referred to as WAT1!, of which the structural, dynamical, and functional properties are investigated using site-directed mutagenesis and X-ray crystallography. From the analysis of NMRD profiles, the mean residence time and orientational order parameter of WAT1 are calculated. This is t...

A “structural” water molecule in the family of fatty acid binding proteins

“…Designed replacement of a buried water molecule by a serine side chain increased stability of the neutral protease of Bacillus stearothermophilus~Vriend et al, 1991!. A comparative study of hydration sites in the family of microbial ribonucleases has been reported recently~Loris et al, 1999!, and one conserved water molecule was found to have a residence time of 7 ns and an orientational order parameter of 0.45 Langhorst et al, 1999!. Despite these important advances, we still lack a general, quantitative understanding of the dynamics and energetics associated with the interaction of internal water molecules with their host proteins. This is illustrated, for example, by difficulties in predicting and explaining the effects of mutations that displace a single interior water molecule in cytochrome c~Lett Hickey et al, 1999!. In different proteins, internal water molecules are likely to play different roles, and the molecular mechanisms that underlie exchange of such water molecules are largely unknown.…”

Thermodynamics of Ligand-Protein Interactions: Implications for Molecular Design

“…Tightly bound water molecules are often conserved across multiple crystal structures of ligand-protein complexes (Poornima and Dean, 1995c). Often, those water molecules play an important role in tuning the biological activity of the protein, as in the case of many enzymes (Langhorst et al, 1999, Nagendra et al, 1998, Poornima and Dean, 1995a. Those water molecules may be regarded as part of the protein structure.…”

Minimizing frustration by folding in an aqueous environment

“…In fact, the B-factors of crystallographically visible buried water molecules typically are similar to those of the protein atoms with which they interact, demonstrating that buried water molecules observed in crystal structures are generally well ordered. RNase T1, for example, contains a buried and evolutionarily conserved water molecule (Wat1) that forms four hydrogen bonds to several side chains in the protein [118]. These interactions may affect the dynamics of the active site, so evolution has chosen to preserve the water molecule rather than use an additional amino acid side-chain to contribute to the hydrogen bonding network [118,119].…”

“…RNase T1, for example, contains a buried and evolutionarily conserved water molecule (Wat1) that forms four hydrogen bonds to several side chains in the protein [118]. These interactions may affect the dynamics of the active site, so evolution has chosen to preserve the water molecule rather than use an additional amino acid side-chain to contribute to the hydrogen bonding network [118,119].…”

“…Although the structural database suggests that buried water molecules should help stabilize protein structure, the limited experimental database suggests that stabilization or destabilization is context or position dependent, so no general principles have been established from those studies. For RNase T1, as well as other enzymes, the contributions of buried water molecules on preserving enzyme activity through affecting the protein dynamics may be a larger consideration than protein stability [118,119].…”