The crystal structure of staphylococcal nuclease has been determined to 1.7 A resolution with a final R-factor of 16.2% using stereochemically restrained Hendrickson-Konnert least-squares refinement. The structure reveals a number of conformational changes relative to the structure of the ternary complex of staphylococcal nuclease 1,2 bound with deoxythymidine-3',5'-diphosphate and Ca2+. Tyr-113 and Tyr-115, which pack against the nucleotide base in the nuclease complex, are rotated outward creating a more open binding pocket in the absence of nucleotide. The side chains of Ca2+ ligands Asp-21 and Asp-40 shift as does Glu-43, the proposed general base in the hydrolysis of the 5'-phosphodiester bond. The significance of some changes in the catalytic site is uncertain due to the intrusion of a symmetry related Lys-70 side chain which hydrogen bonds to both Asp-21 and Glu-43. The position of a flexible loop centered around residue 50 is altered, most likely due to conformational changes propagated from the Ca2+ site. The side chains of Arg-35, Lys-84, Tyr-85, and Arg-87, which hydrogen bond to the 3'- and 5'-phosphates of the nucleotide in the nuclease complex, are unchanged in conformation, with packing interactions with adjacent protein side chains sufficient to fix the geometry in the absence of ligand. The nuclease structure presented here, in combination with the stereochemically restrained refinement of the nuclease complex structure at 1.65 A, provides a wealth of structural information for the increasing number of studies using staphylococcal nuclease as a model system of protein structure and function.

Multiple G protein signaling pathways operate in individual cells to maintain homeostasis and to bring about responses to external stimuli such as growth and differentiation. An important, but unresolved issue is how the specificity of these pathways is maintained among so much complexity. 23 G protein ␣ subunits, 5 ␤ subunits, and 12 ␥ subunits have been identified in mammals (1), which could give rise to more than 1300 combinations. However, inactivation of specific G protein subunits in vivo using antisense (2-6) and ribozyme (7,8) strategies has demonstrated a remarkable specificity of interaction between receptors, ␣␤␥ combinations, and effectors. For instance, ribozyme-mediated suppression of ␥ 7 in HEK-293 cells specifically reduces expression of ␤ 1 and disrupts activation of G s by ␤-adrenergic and D 1 dopamine receptors, but not by prostaglandin E 1 and D 5 dopamine receptors (8, 9). Moreover, knockout of ␥ 7 in the mouse results in behavioral changes and reductions in the level of ␣ olf in the striatum (10).One mechanism by which signaling specificity appears to be regulated is at the level of subcellular compartmentalization, which can facilitate or impair interactions between proteins expressed in the same cell (11,12). However, in the case of protein complexes such as G s , for which the localization patterns of the ␣ and ␤␥ subunits have been reported to change upon activation, it is not clear how specificity can be maintained. The G s subunits associate with the plasma membrane as a result of fatty acid modifications and association with each other. Targeting of ␤ subunits to the plasma membrane requires association with prenylated ␥ subunits (13) and is facilitated by association with ␣ subunits (14). Similarly, ␣ s attaches to the plasma membrane as a result of amino-terminal palmitoylation (15, 16) and association with ␤␥ (17). Activation of G s results in depalmitoylation of ␣ s (18), and studies using immunohistochemistry (19) and an ␣ s -GFP 1 fusion protein (20) have demonstrated activation-dependent movement of ␣ s from the plasma membrane to the cytoplasm. Activation-dependent changes in ␤␥ localization have not been imaged in cells, but subcellular fractionation indicated that ␤␥ redistributed from the plasma membrane to low density microsomes upon stimulation of ␤-adrenergic receptors (21). In the face of these localization changes, it is not clear how specific ␣ s ␤␥ combinations can be preserved throughout multiple signaling cycles.To address this issue, we have performed real time imaging of a G s heterotrimer, ␣ s ␤ 1 ␥ 7 , which mediates signaling from the ␤ 2 AR to adenylyl cyclase (7,8), in isoproterenol-stimulated HEK-293 cells. ␣ s was visualized using an internally tagged ␣ s -CFP fusion protein that has comparable activity to that of ␣ s , whereas ␤ 1 and ␥ 7 were imaged exclusively in the form of ␤ 1 ␥ 7 complexes using the strategy of BiFC (22). BiFC involves the production of a fluorescent signal by two nonfluorescent fragments of YFP when they are brought together by int...

The crystal structures of the inhibitor domain of Alzheimer's amyloid @-protein precursor (APPI) complexed to bovine chymotrypsin (C-APPI) and trypsin (T-APPI) and basic pancreatic trypsin inhibitor (BPTI) bound to chymotrypsin (C-BPTI) have been solved and analyzed at 2.1 A, 1.8 A, and 2.6 A resolution, respectively. APPI and BPTI belong to the Kunitz family of inhibitors, which is characterized by a distinctive tertiary fold with three conserved disulfide bonds. At the specificity-determining site of these inhibitors (PI), residue 15(I)' is an arginine in APPI and a lysine in BPTI, residue types that are counter to the chymotryptic hydrophobic specificity. In the chymotrypsin complexes, the Arg and Lys P1 side chains of the inhibitors adopt conformations that bend away from the bottom of the binding pocket to interact productively with elements of the binding pocket other than those observed for specificity-matched PI side chains. The stereochemistry of the nucleophilic hydroxyl of Ser 195 in chymotrypsin relative to the scissile P1 bond of the inhibitors is identical to that observed for these groups in the trypsin-APPI complex, where Arg 15(I) is an optimal side chain for tryptic specificity. To further evaluate the diversity of sequences that can be accommodated by one of these inhibitors, APPI, we used phage display to randomly mutate residues 11, 13, 15. 17, and 19, which are major binding determinants. Inhibitor variants were selected that bound to either trypsin or chymotrypsin. As expected, trypsin specificity was principally directed by having a basic side chain at P1 (position 15); however, the P1 residues that were selected for chymotrypsin binding were His and Asn, rather than the expected large hydrophobic types. This can be rationalized by modeling these hydrophilic side chains to have similar H-bonding interactions to those observed in the structures of the described complexes. The specificity, or lack thereof, for the other individual subsites is discussed in the context of the "allowed" residues determined from a phage display mutagenesis selection experiment.