In Saccharomyces cerevisiae, pheromone response requires Ste5 scaffold protein, which ensures efficient G-protein-dependent recruitment of mitogen-activated protein kinase (MAPK) cascade components Ste11 (MAPK kinase kinase), Ste7 (MAPK kinase), and Fus3 (MAPK) to the plasma membrane for activation by Ste20 protein kinase. Ste20, which phosphorylates Ste11 to initiate signaling, is activated by binding to Cdc42 GTPase (membrane anchored via its C-terminal geranylgeranylation). Less clear is how activated and membrane-localized Ste20 contacts Ste11 to trigger invasive growth signaling, which also requires Ste7 and the MAPK Kss1, but not Ste5. Ste50 protein associates constitutively via an N-terminal sterile-alpha motif domain with Ste11, and this interaction is required for optimal invasive growth and hyperosmotic stress (highosmolarity glycerol [HOG]) signaling but has a lesser role in pheromone response. We show that a conserved C-terminal, so-called "Ras association" (RA) domain in Ste50 is also essential for invasive growth and HOG signaling in vivo. In vitro the Ste50 RA domain is not able to associate with Ras2, but it does associate with Cdc42 and binds to a different face than does Ste20. RA domain function can be replaced by the nine C-terminal, plasma membrane-targeting residues (KKSKKCAIL) of Cdc42, and membrane-targeted Ste50 also suppresses the signaling deficiency of cdc42 alleles specifically defective in invasive growth. Thus, Ste50 serves as an adaptor to tether Ste11 to the plasma membrane and can do so via association with Cdc42, thereby permitting the encounter of Ste11 with activated Ste20.

Helix propagation of the S-peptide sequence (residues 1-19 of ribonuclease A) in 2,2,2-trifluoroethanol (TFE) solutions has been investigated with CD and nmr Overhauser effect spectroscopies. In this study, the S-peptide helix is covalently initiated at the N-terminus through disulfide bonds to a helix scaffold derived from the N-terminal sequence of the bee venom peptide apamin. The entire S-peptide sequence of this hybrid sequence peptide becomes helical at high proportions of TFE. Residues 14-19 of the S-peptide are not helical in the free peptide in TFE, nor are they helical in ribonuclease A. The "helix stop" signal encoded by the S-peptide sequence near residue 13 does not persist at high TFE with this hybrid sequence peptide. The helix-stabilizing effects of TFE are due at least in part to facilitated propagation of an extant helix. This stabilizing effect appears to be a general solvation effect and not due to specific interaction of the helical peptide with TFE. Specifically these data support the idea that TFE destabilizes the coil state by less effective hydrogen bonding of the peptide amide to the solvent.

The nucleases A produced by two strains of Staphylococcus aureus, which have different stabilities, differ only in the identity of the single amino acid at residue 124. The nuclease from the Foggi strain of S. aureus (by convention nuclease WT), which contains HisIz4, is 1.9 kcal.molp' less stable (at pH 5.5 and 20 "C) than the nuclease from the V8 strain (by convention nuclease H124L), which contains LeuIZ4. In addition, the population of the trans conformer at the Ly~'"-Pro''~ peptide bond, as observed by NMR spectroscopy, is different for the two variants: about 15% for nuclease WT and 9% for nuclease HI 24L. In order to improve our understanding of the origin of these differences, we compared the properties of WT and H124L with those of the H124A and HI241 variants. We discovered a correlation between effects of different residues at this position on protein stability and on stabilization of the cis configuration of the Lys'"-Pro'17 peptide bond. In terms of free energy, approximately 17% of the increase in protein stability manifests itself as stabilization of the cis configuration at Lys"h-Pro"7. This result implies that the differences in stability arise mainly from structural differences between the cis configurational isomers at Pro' l7 of the different variants at residue 124. We solved the X-ray structure of the cis form of the most stable variant, H 124L, and compared it with the published high-resolution X-ray structure of the cis form of the least stable variant, WT (Hynes TR, Fox RO, 1991, Proteins Struct Funct Genet 10:92-105). The two structures are identical within experimental error, except for the side chain at residue 124, which is exposed in the models of both variants. Thus, the increased stability and changes in the transkis equilibrium of the Lys'16-Pro'17 peptide bond observed in H124L relative to WT are due to subtle structural changes that are not observed by current structure determination techniques. Residue 124 is located in a helix. However, the stability changes are too large and follow the wrong order of stability to be explained simply by differences in helical propensity. A second site of conformational heterogeneity in native nuclease is found at the peptide bond, which is approximately 80% trans in both WT and H124L. Because proline to glycine substitutions at either residue 47 or 117 remove the structural heterogeneity at that position and increase protein stability, we determined the X-ray structures of H124L+P117G and H124L+P47G+P117G and the kinetic parameters of H124L, H124L+P47G, H124L+P117G, and H124L+P47G+P117G. The individual P117G and P47G mutations cause decreases in nuclease activity, with kc,, affected more than Km, and their effects are additive. The PI 17G mutation in nuclease H124L leads to the same local conformational rearrangement described for the PI 17G mutant of WT (Hynes TR, Hodel A, Fox RO, 1994, Biochemistry 335021-5030). In both P117G mutants, the loop formed by residues 112-1 17 is located closer to the adjacent loop formed by residues 77-...

Our recently reported pressure-jump relaxation kinetics experiments on staphylococcal nuclease folding and unfolding [Vidugiris et al. (1995) Biochemistry 34, 4909] demonstrated that both transitions exhibit positive activation volumes, with that of folding being much larger than that of unfolding. Thus high pressure denatures proteins by slowing the rate of folding more than that of unfolding. In the present work, we take advantage of the very slow folding and unfolding rates under pressure to examine the kinetics and volume changes along the reaction coordinate for protein folding-unfolding for an interesting set of mutants of staphylococcal nuclease: P42G, P47G, P117G, and the double mutant, P47G+P117G. Previous studies have shown that replacement of an individual proline residue at position 42, 47, or 117 by glycine leads to paradoxical protein stabilization against denaturation by guanidine chloride, high temperature, or high pressure. In order to observe unfolding over an attainable pressure range, guanidine hydrochloride was employed. Within experimental error, the activation volumes and equilibrium volume changes were independent of the concentration of this denaturant and our analysis of the rate constants is consistent with the generally accepted hypothesis that this denaturant acts both by increasing the rate of unfolding and decreasing the rate of folding. We show that the stabilization resulting from each of the proline-to-glycine substitutions arises primarily from a decrease in the unfolding rate, and to a small degree, from an increase in the folding rate. The changes in rate constants upon proline-to-glycine substitution can be modeled in terms of small stabilization of the unfolded state, a greater stabilization of the transition state, and a still greater stabilization of the folded state. Although the rates were found to change for all of the mutants in the set, no changes greater than experimental error were found in the corresponding equilibrium volume changes and activation volumes for folding and unfolding. At low pressures (well below the onset of unfolding) the pressure-jump relaxation profiles for wild type proteins (both Foggi and V8) showed kinetic complexity. Although the effect was attenuated somewhat in pressure-jump profiles of one proline-to-glycine mutant (P42G), its persistence in data from all the mutants studied leads us to conclude that its origin is not cis/trans peptide bond isomerization at proline 117, 47, or 42.

TBP-associated factor 4 (TAF4), an essential subunit of the TFIID complex acts as a coactivator for multiple transcriptional regulators, including Sp1 and CREB. However, little is known regarding the structural properties of the TAF4 subunit that lead to the coactivator function. Here, we report the crystal structure at 2.0-Å resolution of the human TAF4-TAFH domain, a conserved domain among all metazoan TAF4, TAF4b, and ETO family members. The hTAF4-TAFH structure adopts a completely helical fold with a large hydrophobic groove that forms a binding surface for TAF4 interacting factors. Using peptide phage display, we have characterized the binding preference of the hTAF4-TAFH domain for a hydrophobic motif, D⌿⌿ ⌿⌽, that is present in a number of nuclear factors, including several important transcriptional regulators with roles in activating, repressing, and modulating posttranslational modifications. A comparison of the hTAF4-TAFH structure with the homologous ETO-TAFH domain reveals several critical residues important for hTAF4-TAFH target specificity and suggests that TAF4 has evolved in response to the increased transcriptional complexity of metazoans.TFIID ͉ transcription ͉ x-ray crystallography ͉ TAFH domain T he general transcription factor TFIID, composed of the TATAbinding protein (TBP) and at least 14 additional TBPassociated factors (TAFs) (1), plays an important role in the regulation of gene transcription by RNA polymerase II. It contributes to a large number of activities necessary for regulated transcription, such as core promoter recognition and chromatin modification and recognition (2, 3). Individual TAFs are important for mediating distinct activator-specific transcriptional regulation in vivo (4). Studies of yeast strains having temperature-sensitive mutations indicate that Ϸ84% of yeast genes depend on one or more TAFs (5), indicating the importance of this factor in RNA Pol II transcription.The human TAF4 (hTAF II 130/dTAF II 110) subunit of the TFIID complex was the first of the TAF subunits demonstrated to possess coactivator activity for the glutamine-rich activators Sp1 (6), CREB (7), and nuclear receptors RAR and TR (8). More recently, TAF4 has been demonstrated to play a critical role in maintaining the stability of the TFIID complex (9). The hTAF4 sequence contains four glutamine-rich tracts mediating interactions with the activators Sp1 and CREB (7) and two highly conserved domains CI and CII [supporting information (SI) Fig. 5A]. The misregulation of Sp1 activation mediated by hTAF4 has been implicated in Huntington's disease and a number of other neurodegenerative diseases that result from polyglutamine expansions (10). A region (residues 870-911) of the CII domain has been shown to interact with the histone-fold motif of hTAF12 and form a novel histone-like pair (11). The CI region of hTAF4 is highly conserved among all metazoan TAF4, TAF4b, and ETO family members and is also known as the TAF homology (TAFH) domain (6, 12) (SI Fig. 5B). The ETO-TAFH domain has been demonstrated t...