Crystal Structures of the Binary Ca2+ and pdTp Complexes and the Ternary Complex of the Asp21 .fwdarw. Glu Mutant of Staphylococcal Nuclease. Implications for Catalysis and Ligand Binding

Abstract

The crystal structure of the Asp21-->Glu mutant (D21E) of staphylococcal nuclease (SNase) has been determined in three different complex forms. The structure of the D21E ternary complex in which D21E is bound to both Ca2+ and the transition-state analogue, thymidine 3',5'-diphosphate (pdTp), was determined to 1.95-A resolution. The structures of both binary complexes, D21E bound either to Ca2+ or pdTp, were determined to 2.15- and 2.05-A resolution, respectively. In the ternary structure, we find a 1.5-A movement of the Ca2+ in the active site, evidence of bidentate coordination of Ca2+ by Glu21 and inner-sphere coordination of the Ca2+ by Glu43. Comparison of the D21E binary structures with the ternary model shows large movements of active site side chains expected to play a direct role in catalysis. Glu43 moves in the binary nucleotide complex, whereas Arg35 is oriented differently in the binary metal complex. From these changes, we seek to explain the basis for the 1500-fold decrease in Vmax of D21E relative to wild-type SNase (WT). Furthermore, we describe direct structural evidence which explains the cooperativity of Ca2+ and pdTp binding in the ternary complex relative to that of the binary complexes.

Alterations in Carboxylate Ligation at the Active Site of Photosystem II

“…Other substantial changes in coordination of calcium occur, including a change in the number of coordinating water molecules and new ligation by glutamate 43, which is not coordinating in the wild-type protein. In addition, more long-range alterations in protein structure are also observed (46). This work provides a precedent for the interpretation of our vibrational spectra.…”

“…A change in inner-sphere coordination of a metal is also observed when an aspartate-to-glutamate mutation is introduced at a Ca 2ϩ ligand in staphylococcal nuclease (46). In this case, the substitution of glutamate for aspartate 21 results in a change from unidentate ligation by aspartate 21 to bidentate ligation by glutamate 21.…”

“…These additional changes in coordination may be rearrangements of bound water, which may not contribute to the spectral region investigated here (38), or additional carboxylate rearrangements, contributing to the spectra presented here. Although there is precedent for this interpretation (see discussion of staphylococcal nuclease above (46)), the possibility of long-range structural changes induced by the mutation must be considered. In this scenario, substitution of glutamate for aspartate 170 causes a structural change that results in the substitution of unidentate for bridging carboxylate ligands.…”

Sugar non-specific endonucleases

“…Through the crystal structure at 1.95 A î resolution of the D21E ternary complex, where D21E is bound to both Ca 2 and pdTp (transition state analogue), it was demonstrated that Ca 2 in the active site binds to Glu21 via bidentate coordination and to Glu43 through an inner sphere coordination. The cooperativity of the metal ion and the inhibitor (pdTp) in the ternary complex was also demonstrated [187].…”

P100, a transcriptional coactivator, is a human homologue of staphylococcal nuclease

“…inally proposed Glu-43 as acting as a general base in the reaction mechanism, yet more recent evidence (Wang et al, 1990;Judice et al, 1993;Libson et al, 1994) indicates otherwise. Clearly the catalytic mechanism for this enzyme is more complex than previously proposed Loll & Lattman, 1989), particularly given the requirement of not one but two equivalents of Ca2+ for maximal activity (Cuatrecasas et al, 1967b;Tucker et al, 1979a, Tucker et al, 1979b.…”