Structural Analysis of an RNase T 1 Variant with an Altered Guanine Binding Segment Katja Ho È schler 1 , Helga Hoier 2 , Bernd Hubner 1 , Wolfram Saenger 2 Peter Orth 2 and Ulrich Hahn 1 * 1 Institut fu È r Biochemie Fakulta Èt fu È r Biowissenschaften Pharmazie und Psychologie Universita Èt Leipzig, Talstrasse 33, 04103 Leipzig, Germany 2 Institut fu È r Kristallographie Fachbereich Chemie, Freie Universita Èt Berlin, Takustrasse 6, 14195 Berlin, Germany The ribonuclease T 1 variant 9/5 with a guanine recognition segment, altered from the wild-type amino acid sequence 41-KYNNYE-46 to 41- EFRNWQ-46, has been cocrystallised with the speci®c inhibitor 2 0 -GMP. The crystal structure has been re®ned to a crystallographic R factor of 0.198 at 2.3 A Ê resolution. Despite a size reduction of the binding pocket, pushing the inhibitor outside by 1 A Ê ,2 0 -GMP is ®xed to the primary rec- ognition site due to increased aromatic stacking interactions. The phos- phate group of 2 0 -GMP is located about 4.2 A Ê apart from its position in wild-type ribonuclease T 1 -2 0 -GMP complexes, allowing a Ca 2 , coordinat- ing this phosphate group, to enter the binding pocket. The crystallo- graphic data can be aligned with the kinetic characterisation of the variant, showing a reduction of both, guanine af®nity and turnover rate. The presence of Ca 2 was shown to inhibit variant 9/5 and wild-type enzyme to nearly the same extent. # 1999 Academic Press Keywords: enzyme speci®city; mutagenesis; RNase T 1 ; substrate recognition; X-ray crystallography *Corresponding author Introduction Ribonuclease T 1 (RNase T 1 ; EC 3.1.27.3) from the mold fungus Aspergillus oryzae is a small globular protein composed of 104 amino acids with a rela- tive molecular mass of 11,085 Da. It cleaves the phosphodiester bond of single-stranded RNA speci®cally at the 3 0 -site of guanosine in a two step mechanism (Backmann et al., 1994; Takahashi, 1973) involving guanosine-2 0 ,3 0 -cyclic phosphate as intermediate reaction product. RNase T 1 is the key member of a family of microbial ribonucleases with bacterial and fungal origin. The amino acid alignment permits us to outline residues of functional and/or structural importance. The glutamate in position 46, an amino acid residue in the guanine recognition site of the enzyme, is strictly conserved within the RNase T 1 family. The residues 42 to 46, together with N98, form the nucleotide recognition site of RNase T 1 (Balaji et al., 1992; Kostrewa et al., 1989), where guanine is recognised by the enzyme through a number of main-chain and side-chain contacts (see Figure 1). Attempts to produce an RNase T 1 variant with altered nucleotide speci®city concentrated on those amino acid residues, involved in guanine binding via their side-chains. These are Y42 and Y45, which ®x the base through aromatic stacking, and E46. Replacement of the aromatic side-chains of the nucleotide binding site led to variants with partly higher catalytic activity than wild-type (wt) RNase T 1 such as Y45W/W59Y (Grunert et al., 1993), but also to enzymes with a strongly decreased turn- over number such as Y42W (Grunert et al., 1993; Loverix et al., 1997), but did not show an altered base preference. Regarding the speci®c nature of its side-chain interaction with the base, E46 became the main candidate for investigations of the base speci®city of RNase T 1 . Theoretical molecular dynamics and free-energy perturbation calculations on the var- iants E46Q and E46A in complex with 2 0 -guanylic acid (2 0 -GMP) and 2 0 -adenylic acid (2 0 -AMP), respectively, led to the suggestion, that ``both mutants have greater relative adenine af®nities than native RNase T 1 `` (Hirono & Kollman, 1991). Based on these predictions the two variants E46A and E46Q (Granzin et al., 1992; Steyaert et al., 1991) were constructed and biochemically charac- terised; both of them exhibited largely reduced activity. The crystallographic investigation of var- E-mail address of the corresponding author: uli.hahn@uni-leipzig.de Abbreviation used: wt, wild-type. Article No. jmbi.1999.3324 available online at http://www.idealibrary.com on J. Mol. Biol. (1999) 294, 1231±1238 0022-2836/99/501231±8 $30.00/0 # 1999 Academic Press