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

Published online by Cambridge University Press:  01 April 1999

ULRIKE LANGHORST ,
REMY LORIS ,
VLADIMIR P. DENISOV ,
JAN DOUMEN ,
PATRICE ROOSE ,
DOMINIQUE MAES ,
BERTIL HALLE  and
JAN STEYAERT
ULRIKE LANGHORST
Affiliation:
Dienst Ultrastructuur, Vlaams Interuniversitair instituut voor Biotechnologie, Vrije Universiteit Brussel, Paardenstraat 65, B-1640 Sint-Genesius-Rode, Belgium
REMY LORIS
Affiliation:
Dienst Ultrastructuur, Vlaams Interuniversitair instituut voor Biotechnologie, Vrije Universiteit Brussel, Paardenstraat 65, B-1640 Sint-Genesius-Rode, Belgium
VLADIMIR P. DENISOV
Affiliation:
Condensed Matter Magnetic Resonance Group, Department of Chemistry, Lund University, P.O. Box 124, S-22100 Lund, Sweden
JAN DOUMEN
Affiliation:
Dienst Ultrastructuur, Vlaams Interuniversitair instituut voor Biotechnologie, Vrije Universiteit Brussel, Paardenstraat 65, B-1640 Sint-Genesius-Rode, Belgium
PATRICE ROOSE
Affiliation:
Dienst Magnetische Resonantie, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussel, Belgium
DOMINIQUE MAES
Affiliation:
Dienst Ultrastructuur, Vlaams Interuniversitair instituut voor Biotechnologie, Vrije Universiteit Brussel, Paardenstraat 65, B-1640 Sint-Genesius-Rode, Belgium
BERTIL HALLE
Affiliation:
Condensed Matter Magnetic Resonance Group, Department of Chemistry, Lund University, P.O. Box 124, S-22100 Lund, Sweden
JAN STEYAERT
Affiliation:
Dienst Ultrastructuur, Vlaams Interuniversitair instituut voor Biotechnologie, Vrije Universiteit Brussel, Paardenstraat 65, B-1640 Sint-Genesius-Rode, Belgium
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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 17O and 2H 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 Gln93Nε2 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 17O NMRD profiles from wild-type and Thr93Gln RNase T1 yield a mean residence time of 7 ns at 27 °C 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 waterhydrogen bond networkNMRDprotein hydrationresidence timeRNase T1
Type
Research Article
Information
Protein Science , Volume 8 , Issue 4 , April 1999 , pp. 722 - 730
Copyright
© 1999 The Protein Society

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