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.

To investigate the role of subcellular localization in regulating the specificity of G protein ␤␥ signaling, we have applied the strategy of bimolecular fluorescence complementation (BiFC) to visualize ␤␥ dimers in vivo. We fused an amino-terminal yellow fluorescent protein fragment to ␤ and a carboxyl-terminal yellow fluorescent protein fragment to ␥. When expressed together, these two proteins produced a fluorescent signal in human embryonic kidney 293 cells that was not obtained with either subunit alone. Fluorescence was dependent on ␤␥ assembly in that it was not obtained using ␤ 2 and ␥ 1 , which do not form a functional dimer. In addition to assembly, BiFC ␤␥ complexes were functional as demonstrated by more specific plasma membrane labeling than was obtained with individually tagged fluorescent ␤ and ␥ subunits and by their abilities to potentiate activation of adenylyl cyclase by ␣ s in COS-7 cells. To investigate isoform-dependent targeting specificity, the localization patterns of dimers formed by pair-wise combinations of three different ␤ subunits with three different ␥ subunits were compared. BiFC ␤␥ complexes containing either ␤ 1 or ␤ 2 localized to the plasma membrane, whereas those containing ␤ 5 accumulated in the cytosol or on intracellular membranes. These results indicate that the ␤ subunit can direct trafficking of the ␥ subunit. Taken together with previous observations, these results show that the G protein ␣, ␤, and ␥ subunits all play roles in targeting each other. This method of specifically visualizing ␤␥ dimers will have many applications in sorting out roles for particular ␤␥ complexes in a wide variety of cell types.More than a thousand G protein-coupled receptors play roles in a vast range of biological processes. An important but poorly understood issue is how signaling specificity is maintained in vivo. Most combinations of the 5 G protein ␤ subunits and 12 ␥ subunits that have been identified in mammals (1) can form dimers in vitro that exhibit similar abilities to modulate the activities of effectors such as adenylyl cyclase (2), phospholipase C (3), and G protein-gated inwardly rectifying K ϩ channels (4). However, emerging evidence suggests that the specificity of receptor-G protein signaling is determined by specific ␣␤␥ combinations (5). Inactivation of specific G protein subunits in vivo using antisense (6 -10) and ribozyme (11,12) 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, but not prostaglandin E 1 receptors (11, 12). Knockout of ␥ 7 in the mouse results in behavioral changes and reductions in the level of ␣ olf in the striatum (13).Reconstitution experiments indicate clear differences in the ␣␤␥ combinations that are preferred by particular receptors (14 -18). However, these differences generally do not appear to be great enough to account f...

We have identified the binding site of a new class of allosteric HLGP inhibitors. The crystal structure revealed the details of inhibitor binding, led to the design of a new class of compounds, and should accelerate efforts to develop therapeutically relevant molecules for the treatment of diabetes.

Alzheimer's amyloid beta-protein precursor contains a Kunitz protease inhibitor domain (APPI) potentially involved in proteolytic events leading to cerebral amyloid deposition. To facilitate the identification of the physiological target of the inhibitor, the crystal structure of APPI has been determined and refined to 1.5-A resolution. Sequences in the inhibitor-protease interface of the correct protease target will reflect the molecular details of the APPI structure. While the overall tertiary fold of APPI is very similar to that of the Kunitz inhibitor BPTI, a significant rearrangement occurs in the backbone conformation of one of the two protease binding loops. A number of Kunitz inhibitors have similar loop sequences, indicating the structural alteration is conserved and potentially an important determinant of inhibitor specificity. In a separate region of the protease binding loops, APPI side chains Met-17 and Phe-34 create an exposed hydrophobic surface in place of Arg-17 and Val-34 in BPTI. The restriction this change places on protease target sequences is seen when the structure of APPI is superimposed on BPTI complexed to serine proteases, where the hydrophobic surface of APPI faces a complementary group of nonpolar side chains on kallikrein A versus polar side chains on trypsin.

Four-residue beta-turns and larger loop structures represent a significant fraction of globular protein surfaces and play an important role in determining the conformation and specificity of enzyme active sites and antibody-combining sites. Turns are an attractive starting point to develop protein design methods, as they involve a small number of consecutive residues, adopt a limited number of defined conformations and are minimally constrained by packing interactions with the remainder of the protein. The ability to substitute one beta-turn geometry for another will extend protein engineering beyond the redecoration of fixed backbone conformations to include local restructuring and the repositioning of surface side chains. To determine the feasibility and to examine the effect of such a structural modification on the fold and thermodynamic stability of a globular protein, we have substituted a five-residue turn sequence from concanavalin A for a type I' beta-turn in staphylococcal nuclease. The resulting hybrid protein is folded and has full nuclease enzymatic activity but reduced thermodynamic stability. The crystal structure of the hybrid protein reveals that the guest turn sequence retains the conformation of the parent concanavalin A structure when substituted in the nuclease host.

The G protein ␤ 5 subunit differs from other ␤ subunits in having divergent sequence and subcellular localization patterns. Although ␤ 5 ␥ 2 modulates effectors, ␤ 5 associates with R7 family regulators of G protein signaling (RGS) proteins when purified from tissues. To investigate ␤ 5 complex formation in vivo, we used multicolor bimolecular fluorescence complementation in human embryonic kidney 293 cells to compare the abilities of 7 ␥ subunits and RGS7 to compete for interaction with ␤ 5 . Among the ␥ subunits, ␤ 5 interacted preferentially with ␥ 2 , followed by ␥ 7 , and efficacy of phospholipase C-␤2 activation correlated with amount of ␤ 5 ␥ complex formation. ␤ 5 also slightly preferred ␥ 2 over RGS7. In the presence of coexpressed R7 family binding protein (R7BP), ␤ 5 interacted similarly with ␥ 2 and RGS7. Moreover, ␥ 2 interacted preferentially with ␤ 1 rather than ␤ 5 . These results suggest that multiple coexpressed proteins influence ␤ 5 complex formation. Fluorescent ␤ 5 ␥ 2 labeled discrete intracellular structures including the endoplasmic reticulum and Golgi apparatus, whereas ␤ 5 RGS7 stained the cytoplasm diffusely. Coexpression of ␣ o targeted both ␤ 5 complexes to the plasma membrane, and ␣ q also targeted ␤ 5 ␥ 2 to the plasma membrane. The constitutively activated ␣ o mutant, ␣ o R179C, produced greater targeting of ␤ 5 RGS7 and less of ␤ 5 ␥ 2 than did ␣ o . These results suggest that ␣ o may cycle between interactions with ␤ 5 ␥ 2 or other ␤␥ complexes when inactive, and ␤ 5 RGS7 when active. Moreover, the ability of ␤ 5 ␥ 2 to be targeted to the plasma membrane by ␣ subunits suggests that functional ␤ 5 ␥ 2 complexes can form in intact cells and mediate signaling by G protein-coupled receptors.