The importance of structural transitions of the switch II region for the functions of elongation factor Tu on the ribosome

Elongation factor Tu (EF-Tu) undergoes a large conformational transition when switching from the GTP to GDP forms. Structural changes in the switch I and II regions in the G domain are particularly important for this rearrangement. In the switch II region, helix alpha2 is flanked by two glycine residues: Gly(83) in the consensus element DXXG at the N terminus and Gly(94) at the C terminus. The role of helix alpha2 was studied by pre-steady-state kinetic experiments using Escherichia coli EF-Tu mutants where either Gly(83), Gly(94), or both were replaced with alanine. The G83A mutation slows down the association of the ternary complex EF-Tu.GTP.aminoacyl-tRNA with the ribosome and abolishes the ribosome-induced GTPase activity of EF-Tu. The G94A mutation strongly impairs the conformational change of EF-Tu from the GTP- to the GDP-bound form and decelerates the dissociation of EF-Tu.GDP from the ribosome. The behavior of the double mutant is dominated by the G83A mutation. The results directly relate structural transitions in the switch II region to specific functions of EF-Tu on the ribosome.     

Probing the mechanism of rhodopsin-catalyzed transducin activation

An agonist-bound G protein-coupled receptor (GPCR) induces a GDP/GTP exchange on the G protein alpha-subunit (G alpha) followed by the release of G alpha GTP and G beta gamma which, subsequently, activate their targets. The C-terminal regions of G alpha subunits constitute a major receptor recognition domain. In this study, we tested the hypothesis that the GPCR-induced conformational change is communicated from the G alpha C-terminus, via the alpha 5 helix, to the nucleotide-binding beta 6/alpha 5 loop causing GDP release. Mutants of the visual G protein, transducin, with a modified junction of the C-terminus were generated and analyzed for interaction with photoexcited rhodopsin (R*). A flexible linker composed of five glycine residues or a rigid three-turn alpha-helical segment was inserted between the 11 C-terminal residues and the alpha 5 helix of G alpha(t)-like chimeric G alpha, G alpha(ti). The mutant G alpha subunits with the Gly-loop (G alpha(ti)L) and the extended alpha 5 helix (G alpha(ti)H) retained intact interactions with G beta gamma(t), and displayed modestly reduced binding to R*. G alpha(ti)H was capable of efficient activation by R*. In contrast, R* failed to activate G alpha(ti)L, suggesting that the Gly-loop absorbs a conformational change at the C-terminus and blocks G protein activation. Our results provide evidence for the role of G alpha C-terminus/alpha 5 helix/beta 6/alpha 5 loop route as a dominant channel for transmission of the GPCR-induced conformational change leading to G protein activation.     

Biochemical and genetic analysis of dominant-negative mutations affecting a yeast G-protein gamma subunit.

Heterotrimeric guanine nucleotide-binding proteins (G proteins) consisting of alpha, beta, and gamma subunits mediate signalling between cell surface receptors and intracellular effectors in eukaryotic cells. To define signalling functions of G gamma subunits (STE18 gene product) involved in pheromone response and mating in the yeast Saccharomyces cerevisiae, we isolated and characterized dominant-negative STE18 alleles. We obtained dominant-negative mutations that disrupt C-terminal sequences required for prenylation of G gamma precursors (CAAX box) and that affect residues in the N-terminal half of Ste18p. Overexpression of mutant G gamma subunits in wild-type cells blocked signal transduction; this effect was suppressed upon overexpression of G beta subunits. Mutant G gamma subunits may therefore sequester G beta subunits into nonproductive G beta gamma dimers. Because mutant G gamma subunits blocked the constitutive signal resulting from disruption of the G alpha subunit gene (GPA1), they are defective in functions required for downstream signalling. Ste18p bearing a C107Y substitution in the CAAX box displayed reduced electrophoretic mobility, consistent with a prenylation defect. G gamma subunits carrying N-terminal substitutions had normal electrophoretic mobilities, suggesting that these proteins were prenylated. G gamma subunits bearing substitutions in their N-terminal region or C-terminal CAAX box (C107Y) supported receptor-G protein coupling in vitro, whereas C-terminal truncations caused partial defects in receptor coupling.     

Rapid Uptake and Degradation of CXCL12 Depend on CXCR7 Carboxyl-terminal Serine/Threonine Residues

CXCL12 signaling through G protein-coupled CXCR4 regulates cell migration during ontogenesis and disease states including cancer and inflammation. The second CXCL12-receptor CXCR7 modulates the CXCL12/CXCR4 pathway by acting as a CXCL12 scavenger and exerts G protein-independent functions. Given the distinct properties of CXCR4 and CXCR7, we hypothesized that the distinct C-terminal domains differently regulate receptor trafficking and stability. Here, we examined epitope-tagged wild type and C-terminal mutant receptors in human embryonic kidney cells (HEK293) with respect to trafficking, stability, (125)I-CXCL12 degradation, and G protein-coupling. The 24 CXCR7 C-terminal residues were sufficient to promote rapid spontaneous internalization. Replacement of the CXCR7 C terminus with that of CXCR4 (CXCR7-4tail mutant) abolished spontaneous internalization but permitted ligand-induced internalization and phosphorylation at the heterologous domain. The reverse tail-swap caused ligand-independent internalization of the resulting CXCR4-7tail mutant. Receptor-mediated (125)I-CXCL12 uptake and release of (125)I-CXCL12 degradation products were accelerated with receptors bearing the CXCR7 C terminus and impaired after conversion of CXCR7 C-terminal serine/threonine residues into alanines. C-terminal lysine residues were dispensable for plasma membrane targeting and the CXCL12 scavenger function but involved in constitutive degradation of CXCR7. Although the CXCR7 C terminus abolished G protein coupling in the CXCR4-7tail mutant, replacement of the CXCR7 C terminus, CXCR7 second intracellular loop, or both domains with the corresponding CXCR4 domain did not result in a G protein-coupled CXCR7 chimera. Taken together, we provide evidence that the CXCR7 C terminus influences the ligand-uptake/degradation rate, G protein coupling, and receptor stability. Regulatory pathways targeting CXCR7 C-terminal serine/threonine sites may control the CXCL12 scavenger activity of CXCR7.     

Disruption of the alpha5 helix of transducin impairs rhodopsin-catalyzed nucleotide exchange

Photoactivated rhodopsin (R) catalyzes nucleotide exchange by transducin, the heterotrimeric G protein of the rod cell. Recently, we showed that certain alanine replacement mutants of the alpha5 helix of the alpha subunit of transducin (Galpha(t)) displayed very rapid nucleotide exchange rates even in the absence of R [Marin, E. P., Krishna, A. G., and Sakmar, T. P. (2001) J. Biol. Chem. 276, 27400-27405]. We suggested that R catalyzes nucleotide exchange by perturbing residues on the alpha5 helix. Here, we characterize deletion, insertion, and proline replacement mutants of amino acid residues in alpha5. In general, the proline mutants exhibited rates of uncatalyzed nucleotide exchange that were 4-8-fold greater than wild type. The proline mutants also generally displayed decreased rates of R-catalyzed activation. The degree of reduction of the activation rate correlated with the position of the residue replaced with proline. Mutants with replacement of residues at the amino terminus of alpha5 exhibited mild (<2-fold) decreases, whereas mutants with replacement of residues at the carboxyl terminus of alpha5 were completely resistant to R-catalyzed activation. In addition, insertion of a single helical turn in the form of four alanine residues following Ile339 at the carboxyl terminus of alpha5 prevented R-catalyzed activation. Together, the results provide evidence that alpha5 serves an important function in mediating R-catalyzed nucleotide exchange. In particular, the data suggest the importance of the connection between the alpha5 helix and the adjacent carboxyl-terminal region of Galpha(t).     

Complementation analysis demonstrates that insulin cross-links both alpha subunits in a truncated insulin receptor dimer

The insulin receptor is a homodimer composed of two alphabeta half receptors. Scanning mutagenesis studies have identified key residues important for insulin binding in the L1 domain (amino acids 1-150) and C-terminal region (amino acids 704-719) of the alpha subunit. However, it has not been shown whether insulin interacts with these two sites within the same alpha chain or whether it cross-links a site from each alpha subunit in the dimer to achieve high affinity binding. Here we have tested the contralateral binding mechanism by analyzing truncated insulin receptor dimers (midi-hIRs) that contain complementary mutations in each alpha subunit. Midi-hIRs containing Ala(14), Ala(64), or Gly(714) mutations were fused with Myc or FLAG epitopes at the C terminus and were expressed separately by transient transfection. Immunoblots showed that R14A+FLAG, F64A+FLAG, and F714G+Myc mutant midi-hIRs were expressed in the medium but insulin binding activity was not detected. However, after co-transfection with R14A+FLAG/F714G+Myc or F64A+FLAG/F714G+Myc, hybrid dimers were obtained with a marked increase in insulin binding activity. Competitive displacement assays revealed that the hybrid mutant receptors bound insulin with the same affinity as wild type and also displayed curvilinear Scatchard plots. In addition, when hybrid mutant midi-hIR was covalently cross-linked with (125)I(A14)-insulin and reduced, radiolabeled monomer was immunoprecipitated only with anti-FLAG, demonstrating that insulin was bound asymmetrically. These results demonstrate that a single insulin molecule can contact both alpha subunits in the insulin receptor dimer during high affinity binding and this property may be an important feature for receptor signaling.     

The 20 C-terminal amino acid residues of the chloroplast ATP synthase gamma subunit are not essential for activity.

It has been suggested that the last seven to nine amino acid residues at the C terminus of the gamma subunit of the ATP synthase act as a spindle for rotation of the gamma subunit with respect to the alpha beta subunits during catalysis (Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628). To test this hypothesis we selectively deleted C-terminal residues from the chloroplast gamma subunit, two at a time starting at the sixth residue from the end and finishing at the 20th residue from the end. The mutant gamma genes were overexpressed in Escherichia coli and assembled with a native alpha3beta3 complex. All the mutant forms of gamma assembled as effectively as the wild-type gamma. Deletion of the terminal 6 residues of gamma resulted in a significant increase (>50%) in the Ca-dependent ATPase activity when compared with the wild-type assembly. The increased activity persisted even after deletion of the C-terminal 14 residues, well beyond the seven residues proposed to form the spindle. Further deletions resulted in a decreased activity to approximately 19% of that of the wild-type enzyme after deleting all 20 C-terminal residues. The results indicate that the tip of the gammaC terminus is not essential for catalysis and raise questions about the role of the C terminus as a spindle for rotation.     

Linkage of interactions in sickle hemoglobin fiber assembly: inhibitory effect emanating from mutations in the AB region of the alpha-chain is annulled by a mutation at its EF corner

The AB and GH regions of the alpha-chain are located in spatial proximity and contain a cluster of intermolecular contact residues of the sickle hemoglobin (HbS) fiber. We have examined the role of dynamics of AB/GH region on HbS polymerization through simultaneous replacement of non-contact Ala(19) and Ala(21) of the AB corner with more flexible Gly or rigid alpha-aminoisobutyric acid (Aib) residues. The polymerization behavior of HbS with Aib substitutions was similar to the native HbS. In contrast, Gly substitutions inhibited HbS polymerization. Molecular dynamics simulation studies of alpha-chains indicated that coordinated motion of AB and GH region residues present in native (Ala) as well as in Aib mutant was disrupted in the Gly mutant. The inhibitory effect due to Gly substitutions was further explored in triple mutants that included mutation of an inter-doublestrand contact (alphaAsn(78) --> His or Gln) at the EF corner. Although the inhibitory effect of Gly substitutions in the triple mutant was unaffected in the presence of alphaGln(78), His at this site almost abrogated its inhibitory potential. The polymerization studies of point mutants (alphaGln(78) --> His) indicated that the inhibitory effect due to Gly substitutions in the triple mutant was synergistically compensated for by the polymerization-enhancing activity of His(78). Similar synergistic coupling, between alphaHis(78) and an intra-double-strand contact point (alpha16) mutation located in the AB region, was also observed. Thus, two conclusions are made: (i) Gly mutations at the AB corner inhibit HbS polymerization by perturbing the dynamics of the AB/GH region, and (ii) perturbations of AB region (through changes in dynamics of the AB/GH region or abolition of a specific fiber contact site) that influence HbS polymerization do so in concert with alpha78 site at the EF corner. The overall results provide insights about the interaction-linkage between distant regions of the HbS tetramer in fiber assembly.     

Improved autoprocessing efficiency of mutant subtilisins E with altered specificity by engineering of the pro-region.

Modification of substrate specificity of an autoprocessing enzyme is accompanied by a risk of significant failure of self-cleavage of the pro-region essential for activation. Therefore, to enhance processing, we engineered the pro-region of mutant subtilisins E of Bacillus subtilis with altered substrate specificity. A high-activity mutant subtilisin E with Ile31Leu replacement (I31L) as well as the wild-type enzyme show poor recognition of acid residues as the P1 substrate. To increase the P1 substrate preference for acid residues, Glu156Gln and Gly166Lys/Arg substitutions were introduced into the I31L gene based upon a report on subtilisin BPN' [Wells et al. (1987) Proc. Natl. Acad. Sci. USA 84, 1219-1223]. The apparent P1 specificity of four mutants (E156Q/G166K, E156Q/G166R, G166K, and G166R) was extended to acid residues, but the halo-forming activity of Escherichia coli expressing the mutant genes on skim milk-containing plates was significantly decreased due to the lower autoprocessing efficiency. A marked increase in active enzyme production occurred when Tyr(-1) in the pro-region of these mutants was then replaced by Asp or Glu. Five mutants with Glu(-2)Ala/Val/Gly or Tyr(-1)Cys/Ser substitution showing enhanced halo-forming activity were further isolated by PCR random mutagenesis in the pro-region of the E156Q/G166K mutant. These results indicated that introduction of an optimum arrangement at the cleavage site in the pro-region is an effective method for obtaining a higher yield of active enzymes.     

Asymmetric organization of the pore region of the epithelial sodium channel

Epithelial sodium channels (ENaCs) are composed of three homologous subunits that have regions preceding the second transmembrane domain (also referred as pre-M2) that form part of the channel pore. To identify residues within this region of the beta-subunit that line the pore, we systematically mutated residues Gln(523)-Ile(536) to cysteine. Wild type and mutant mouse ENaCs were expressed in Xenopus oocytes, and a two-electrode voltage clamp was used to examine the properties of mutant channels. Cysteine substitutions of 9 of 13 residues significantly altered Li(+) to Na(+) current ratios, whereas only cysteine replacement of beta Gly(529) resulted in K(+)-permeable channels. Besides beta G525C, large increases in the inhibitory constant of amiloride were observed with mutations at beta Gly(529) and beta Ser(531) within the previously identified 3-residue tract that restricts K(+) permeation. Cysteine substitution preceding (beta Phe(524) and beta Gly(525)), within (beta Gly(530)) or following (beta Leu(533)) this 3-residue tract, resulted in enhanced current inhibition by external MTSEA. External MTSET partially blocked channels with cysteine substitutions at beta Gln(523), beta Phe(524), and beta Trp(527). MTSET did not inhibit alpha beta G525C gamma, although previous studies showed that channels with cysteine substitutions at the corresponding sites within the alpha- and gamma-subunits were blocked by MTSET. Our results, placed in context with previous observations, suggest that pore regions from the three ENaC subunits have an asymmetric organization.     

Single amino acid substitutions and deletions that alter the G protein coupling properties of the V2 vasopressin receptor identified in yeast by receptor random mutagenesis

To facilitate structure-function relationship studies of the V2 vasopressin receptor, a prototypical G(s)-coupled receptor, we generated V2 receptor-expressing yeast strains (Saccharomyces cerevisiae) that required arginine vasopressin-dependent receptor/G protein coupling for cell growth. V2 receptors heterologously expressed in yeast were unable to productively interact with the endogenous yeast G protein alpha subunit, Gpa1p, or a mutant Gpa1p subunit containing the C-terminal G alpha(q) sequence (Gq5). In contrast, the V2 receptor efficiently coupled to a Gpa1p/G alpha(s) hybrid subunit containing the C-terminal G alpha(s) sequence (Gs5), indicating that the V2 receptor retained proper G protein coupling selectivity in yeast. To gain insight into the molecular basis underlying the selectivity of V2 receptor/G protein interactions, we used receptor saturation random mutagenesis to generate a yeast library expressing mutant V2 receptors containing mutations within the second intracellular loop. A subsequent yeast genetic screen of about 30,000 mutant receptors yielded four mutant receptors that, in contrast to the wild-type receptor, showed substantial coupling to Gq5. Functional analysis of these mutant receptors, followed by more detailed site-directed mutagenesis studies, indicated that single amino acid substitutions at position Met(145) in the central portion of the second intracellular loop of the V2 receptor had pronounced effects on receptor/G protein coupling selectivity. We also observed that deletion of single amino acids N-terminal of Met(145) led to misfolded receptor proteins, whereas single amino acid deletions C-terminal of Met(145) had no effect on V2 receptor function. These findings highlight the usefulness of combining receptor random mutagenesis and yeast expression technology to study mechanisms governing receptor/G protein coupling selectivity and receptor folding.     

Proline residues link the active site to transmembrane domain movements in human nucleoside triphosphate diphosphohydrolase 3 (NTPDase3)

The active sites of the membrane-bound nucleoside triphosphate diphosphohydrolases (NTPDases) regulate and are regulated by coordinated and spatially distant movements of their transmembrane helices, modulating enzyme activity, and substrate specificity. Using site-directed mutagenesis, the roles of the conserved proline residues (N-terminal: P52 and P53; C-terminal: P472, P476, P481, P484, and P485) of human NTPDase3, located in the “linker regions” that connect the N- and C-terminal transmembrane helices with the extracellular active site, were examined. Single cysteine substitutions were strategically placed in the transmembrane domain (N-terminal helix: V42C; C-terminal helix: G489C) to serve as cross-linking “sensors” of helical interactions. These “sensor” background mutant proteins (V42C and G489C NTPDase3) are enzymatically active and are cross-linked by copper phenanthroline less efficiently in the presence of adenosine triphosphate (ATP). Proline to alanine substitutions at P53, P481, P484, and P485 in the V42C background, as well as P53, P481, and P484 in the G489C background, exhibited decreased nucleotidase activities. More importantly, alanine substitutions at P53 and P481 in the V42C background and P481 in the G489C background no longer exhibited the ATP-induced decrease in transmembrane cross-linking efficiency. Interestingly, the P485A mutation abolished oxidative cross-linking at G489C both in the presence and absence of ATP. Taken together, these results suggest a role for proline residues 53 and 481 in the linker regions of human NTPDase3 for coupling nucleotide binding at the enzyme active site to movements and/or rearrangements of the transmembrane helices necessary for optimal nucleotide hydrolysis.     

Disruption of Receptor-Mediated Activation of G Protein by Mutating a Conserved Arginine Residue in the Switch II Region of the α Subunit

A naturally occurring point mutation (R231H) within one of the major 3gamma-binding surface (switch II region) on the a subunit of Gs (alpha(s)) has previously been found to disrupt receptor-mediated activation of Gs. The disruption caused by mutating this conserved residue may be a general phenomenon for all a subunits. Homologous mutants of the alpha subunit of Gz [alpha(z); a negative regulator of adenylyl cyclase (AC)] and G16 (alpha16; a stimulator of phospholipase C) were constructed and examined for receptor-mediated regulation of their corresponding effectors. The mutant alphazR209H cannot be fully activated by the delta-opioid receptor, as indicated by the impairment of the inhibition of alpha(s)-stimulated AC and betagamma-mediated stimulation of AC type II (AC2). Similarly, the mutant alpha16R216H lost the ability to mediate receptor-induced activation of phospholipase C and AC2. The receptor coupling efficacy and promiscuity of alpha16R216H were eradicated. The mutation of the conserved arginine has no observable effect on the constitutive activities of the GTPase-deficient derivatives of both alpha(z) and alpha16. The alpha subunit of Gt1 (transducin; alphat1) attenuated betagamma-mediated stimulation of AC2 by sequestrating free betagamma subunits, but the mutant alphat1R204H showed reduced ability to scavenge betagamma-mediated AC2 activation. Presumably, mutation of the conserved arginine disrupted the subunit interactions in addition to the impairment of receptor interaction.     

Recombinant collagen studies link the severe conformational changes induced by osteogenesis imperfecta mutations to the disruption of a set of interchain salt bridges

The clinical severity of Osteogenesis Imperfecta (OI), also known as the brittle bone disease, relates to the extent of conformational changes in the collagen triple helix induced by Gly substitution mutations. The lingering question is why Gly substitutions at different locations of collagen cause different disruptions of the triple helix. Here, we describe markedly different conformational changes of the triple helix induced by two Gly substitution mutations placed only 12 residues apart. The effects of the Gly substitutions were characterized using a recombinant collagen fragment modeling the 63-residue segment of the alpha1 chain of type I collagen containing no Hyp (residues 877-939) obtained from Escherichia coli. Two Gly --> Ser substitutions at Gly-901 and Gly-913 associated with, respectively, mild and severe OI variants were introduced by site-directed mutagenesis. Biophysical characterization and limited protease digestion experiments revealed that while the substitution at Gly-901 causes relatively minor destabilization of the triple helix, the substitution at Gly-913 induces large scale unfolding of an unstable region C-terminal to the mutation site. This extensive unfolding is caused by the intrinsic low stability of the C-terminal region of the helix and the mutation induced disruption of a set of salt bridges, which functions to lock this unstable region into the triple helical conformation. The extensive conformational changes associated with the loss of the salt bridges highlight the long range impact of the local interactions of triple helix and suggest a new mechanism by which OI mutations cause severe conformational damages in collagen.     

Analysis of fibrinogen A alpha-fusion proteins. Mutants which inhibit thrombin equivalently are not equally good substrates

We have examined the interaction of thrombin with fibrinogen A alpha chain residues 7-16. Using genetically engineered constructions, we have synthesized in Escherichia coli a fibrinogen A alpha 1-50 fusion protein and seven mutant proteins with single amino acid substitutions. These are: Asp7----Ala, Phe8----Tyr, Glu11----Ala, Gly12----Val, Gly13----Val, Gly14----Val, and Arg16----Leu. Competitive immunoassay of cell lysates showed that all the mutations but one, Arg16----Leu, altered the structure of the protein such that cross-reactivity with the A alpha-specific monoclonal antibody, Y18, was significantly reduced. The fusion proteins were purified and analyzed as thrombin inhibitors and substrates. All the fusion proteins are competitive inhibitors of the amidolytic hydrolysis of Spectrozyme TH, a thrombin-specific chromogenic substrate, with inhibition constants corresponding to that for fibrinogen. We conclude that these 7 amino acid substitutions do not alter thrombin binding to the fusion proteins. The fusion proteins were tested as substrates by monitoring thrombin-dependent peptide release. The natural sequence and three mutants, Asp7----Ala, Glu11----Ala, and Gly14----Val, are good substrates. The other mutants are either poor substrates or are not cleaved by thrombin within A alpha 1-50. These results indicate that residues between Asp7 and Arg16 are critical to efficient peptide hydrolysis, whereas residues outside this region are critical to thrombin binding.     

Comparison of bovine serum transferrin A and D2. I. Amino acid residue differences

A comparison is made of single components of the homozygous variants A and D2 of bovine serum transferrin by tryptic, chymotryptic and cyanogen bromide digestion. It is concluded that there are three substitutions A:D2-Glu:Asp, Lys:Arg and Asp:Gly. In the light of the recent work of Brock et al. (1980) it is concluded that all three substitutions occur in the C-terminal sequence of the chain. By homology with the sequence of human serum transferrin (MacGillivray et al., 1982) the Lys:Arg and Asp:Gly substitutions probably occur at residues 527 and 446, respectively, from the N-terminus. The Asp:Gly substitution is considered more likely than our earlier conclusion (Maeda, McKenzie & Shaw, 1977) that there is a deletion in the chain of D2 (A:D2, Asp:--). The location of the Glu:Asp substitution is not known.     

Role of the conserved DRY motif on G protein activation of rat angiotensin II receptor type 1A

To delineate the functional importance of the highly conserved triplet amino acid sequence, Asp-Arg-Tyr (DRY) among G protein-coupled receptors in the second intracellular loop, these residues of rat angiotensin II (Ang II) receptor type 1A (AT(1A)) were changed by alanine or glycine by site-directed mutagenesis. These mutant receptors were stably expressed in CHO-K1 cells, and the binding of Ang II, GTP effect, InsP(3) production, and the acidification of the medium in response to Ang II were determined. The effects of GTPgammaS on Ang II binding in the mutant receptors D125A and D125G were markedly reduced. InsP(3) production of the mutant D125A, D125G, R126A, and R126G was markedly reduced. Extracellular acidification of D125A was not distinguishable from untransfected CHO-K1 cells. Mutant Y127A was able to produce InsP(3) and acidify medium comparable with wild type AT(1A). These results indicate as follows; Asp(125) is essential for intracellular signal transduction involving G protein coupling, Arg(126) is essential for coupling of G(q) protein but not other G proteins, and Tyr(127) is not important for G protein coupling.     

Sequence of interactions in receptor-G protein coupling

Guanine nucleotide exchange in heterotrimeric G proteins catalyzed by G protein-coupled receptors (GPCRs) is a key event in many physiological processes. The crystal structures of the GPCR rhodopsin and two G proteins as well as binding sites on both catalytically interacting proteins are known, but the temporal sequence of events leading to nucleotide exchange remains to be elucidated. We employed time-resolved near infrared light scattering to study the order in which the Galpha and Ggamma C-terminal binding sites on the holo-G protein interact with the active state of the GPCR rhodopsin (R*) in native membranes. We investigated these key binding sites within mass-tagged peptides and G proteins and found that their binding to R* is mutually exclusive. The interaction of the holo-G protein with R* requires at least one of the lipid modifications of the G protein (i.e. myristoylation of the Galpha N terminus and/or farnesylation of the Ggamma C terminus). A holo-G protein with a high affinity Galpha C terminus shows a specific change of the reaction rate in the GDP release and GTP uptake steps of catalysis. We interpret the data by a sequential fit model where (i) the initial encounter between R* and the G protein occurs with the Gbetagamma subunit, and (ii) the Galpha C-terminal tail then interacts with R* to release bound GDP, thereby decreasing the affinity of R* for the Gbetagamma subunit. The mechanism limits the time in which both C-terminal binding sites of the G protein interact simultaneously with R* to a short lived transitory state.     

Comparison of bovine serum transferrin A and D2. I. Amino acid residue differences

A comparison is made of single components of the homozygous variants A and D₂ of bovine serum transferrin by tryptic, chymotryptic and cyanogen bromide digestion. It is concluded that there are three substitutions A:D₂ - Glu:Asp, Lys: Arg and Asp:Gly. In the light of the recent work of Brocket al. (1980) it is concluded that all three substitutions occur in the C-terminal sequence of the chain. By homology with the sequence of human serum transferrin (MacGillivray et al., 1982) the Lys:Arg and Asp:Gly substitutions probably occur at residues 527 and 446, respectively, from the N -terminus. The Asp:Gly substitution is considered more likely than our earlier conclusion (Maeda, McKenzie & Shaw, 1977) that there is a deletion in the chain of D₂ (A:D₂, Asp: —). The location of the Glu:Asp substitution is not known.     

Hsc70-interacting HPD loop of the J domain of polyomavirus T antigens fluctuates in ps to ns and micros to ms

The backbone dynamics of the J domain from polyomavirus T antigens have been investigated using 15N NMR relaxation and molecular dynamics simulation. Model-free relaxation analysis revealed picosecond to nanosecond motions in the N terminus, the I-II loop, the C-terminal end of helix II through the HPD loop to the beginning of helix III, and the C-terminal end of helix III to the C terminus. The backbone dynamics of the HPD loop and termini are dominated by motions with moderately large amplitudes and correlation times of the order of a nanosecond or longer. Conformational exchange on the microsecond to millisecond timescale was identified in the HPD loop, the N and C termini, and the I-II loop. A 9.7ns MD trajectory manifested concerted swings of the HPD loop. Transitions between major and minor conformations of the HPD loop featured distinct patterns of change in backbone dihedral angles and hydrogen bonds. Fraying of the C-terminal end of helix II and the N-terminal end of helix III correlated with displacements of the HPD loop. Correlation of crankshaft motions of Gly46 and Gly47 with the collective motions of the HPD loop suggested an important role of the two glycine residues in the mobility of the loop. Fluctuations of the HPD loop correlated with relative reorientation of side-chains of Lys35 and Asp44 that interact with Hsc70.