Deregulation of allosteric response of Lactococcus lactis prolidase and its effects on enzyme activity

The allosteric behaviour of Lactococcus lactis prolidase (Xaa-Pro dipeptidase) of this proline-specific peptidase was investigated where it was hypothesized that intersubunit interactions between a loop structure and three residues near the active site contributed to this behaviour. Seven mutant prolidases were constructed, and it was observed that the loopless mutant and His303 substitution inactivated the enzyme. Ser307 substitution revealed that this residue influenced the substrate binding, as judged from its kinetic constants and substrate specificity; however, this residue did not contribute to allostery of prolidase. R293S mutation resulted in the disappearance of the allosteric behaviour yielding a Hill constant of 0.98 while the wild type had a constant of 1.58. In addition, the R293S mutation suppressed the substrate inhibition that was observed in other mutants and wild type. The K_m value of R293S was 2.9-fold larger and V_max was approximately 50% less as compared to the wild type. The results indicated that Arg293 increased the affinity for substrates while introducing allosteric behaviour and substrate inhibition. Computer modelling suggested that negative charges on the loop structure interacted with Arg293 and Ser307 to maintain these characteristics. It was, therefore, concluded that Arg293, His303, Ser307 and the loop contributed to the enzyme's allosteric characteristics.     

A heparin binding motif on the pro-domain of human procathepsin L mediates zymogen destabilization and activation

The molecular mechanism by which heparin modulates the processing of procathepsin L in the extracellular environment is proposed. We show that heparin reduces the stability of the pro form of cathepsin L at pH 5 by binding to a putative heparin binding motif (BBXB) in the pro-domain. Mutations to this motif on procathepsin L reduce heparin binding affinity and heparin-induced destabilization; in contrast, heparin only slightly destabilizes the mature cathepsin L domain. Gel analysis further shows that heparin makes procathepsin L a much better substrate for cathepsin L. Thus, heparin enhances the rate of zymogen activation by destabilization upon binding to the BBXB motif. Determining the mechanism by which procathepsin L is activated in the extracellular matrix is important to the understanding of the role that cathepsin L plays in tumour invasion.     

The role of the local environment of engineered Tyr to Trp substitutions for probing the denaturation mechanism of FIS

Factor for inversion stimulation (FIS), a 98-residue homodimeric protein, does not contain tryptophan (Trp) residues but has four tyrosine (Tyr) residues located at positions 38, 51, 69, and 95. The equilibrium denaturation of a P61A mutant of FIS appears to occur via a three-state (N₂ ⇆ I₂ ⇆ 2U) process involving a dimeric intermediate (I₂). Although it was suggested that this intermediate had a denatured C-terminus, direct evidence was lacking. Therefore, three FIS double mutants, P61A/Y38W, P61A/Y69W, and P61A/Y95W were made, and their denaturation was monitored by circular dichroism and Trp fluorescence. Surprisingly, the P61A/Y38W mutant best monitored the N₂ ⇆ I₂ transition, even though Trp38 is buried within the dimer removed from the C-terminus. In addition, although Trp69 is located on the protein surface, the P61A/Y69W FIS mutant exhibited clearly biphasic denaturation curves. In contrast, P61A/Y95W FIS was the least effective in decoupling the two transitions, exhibiting a monophasic fluorescence transition with modest concentration-dependence. When considering the local environment of the Trp residues and the effect of each mutation on protein stability, these results not only confirm that P61A FIS denatures via a dimeric intermediate involving a disrupted C-terminus but also suggest the occurrence of conformational changes near Tyr38. Thus, the P61A mutation appears to compromise the denaturation cooperativity of FIS by failing to propagate stability to those regions involved mostly in intramolecular interactions. Furthermore, our results highlight the challenge of anticipating the optimal location to engineer a Trp residue for investigating the denaturation mechanism of even small proteins.     

Compromised mutant EFEMP1 secretion associated with macular dystrophy remedied by proteostasis network alteration

An Arg345Trp (R345W) mutation in epidermal growth factor-containing, fibulin-like extracellular matrix protein 1 (EFEMP1) causes its inefficient secretion and the macular dystrophy malattia leventinese/Doyne honeycomb retinal dystrophy (ML/DHRD). To understand the influence of the protein homeostasis (or proteostasis) network in rescuing mutant EFEMP1 misfolding and inefficient secretion linked to ML/DHRD, we developed a convenient and sensitive cell-based luminescence assay to monitor secretion versus intracellular accumulation. Fusing EFEMP1 to Gaussia luciferase faithfully recapitulates mutant EFEMP1 secretion defects observed previously using more cumbersome methodology. To understand what governs mutant intracellular retention, we generated a series of R345 mutants. These mutants revealed that aromatic residue substitutions (i.e., Trp, Tyr, and Phe) at position 345 cause significant EFEMP1 secretion deficiencies. These secretion defects appear to be caused, in part, by reduced native disulfide bonding in domain 6 harboring the 345 position. Finally, we demonstrate that mutant EFEMP1 secretion and proper disulfide formation are enhanced by adaptation of the cellular environment by a reduced growth temperature and/or translational attenuation. This study highlights the mechanisms underlying the inefficient secretion of R345W EFEMP1 and demonstrates that alteration of the proteostasis network may provide a strategy to alleviate or delay the onset of this macular dystrophy.     

Mutation strategies for obtaining chitooligosaccharides with longer chains by transglycosylation reaction of family GH18 chitinase

Enhancing the transglycosylation (TG) activity of glycoside hydrolases does not always result in the production of oligosaccharides with longer chains, because the TG products are often decomposed into shorter oligosaccharides. Here, we investigated the mutation strategies for obtaining chitooligosaccharides with longer chains by means of TG reaction catalyzed by family GH18 chitinase A from Vibrio harveyi (VhChiA). HPLC analysis of the TG products from incubation of chitooligosaccharide substrates, GlcNAc_n, with several mutant VhChiAs suggested that mutant W570G (mutation of Trp570 to Gly) and mutant D392N (mutation of Asp392 to Asn) significantly enhanced TG activity, but the TG products were immediately hydrolyzed into shorter GlcNAc_n. On the other hand, the TG products obtained from mutants D313A and D313N (mutations of Asp313 to Ala and Asn, respectively) were not further hydrolyzed, leading to the accumulation of oligosaccharides with longer chains. The data obtained from the mutant VhChiAs suggested that mutations of Asp313, the middle aspartic acid residue of the DxDxE catalytic motif, to Ala and Asn are most effective for obtaining chitooligosaccharides with longer chains.     

Effects on general acid catalysis from mutations of the invariant tryptophan and arginine residues in the protein tyrosine phosphatase from Yersinia

General acid catalysis in protein tyrosine phosphatases (PTPases) is accomplished by a conserved Asp residue, which is brought into position for catalysis by movement of a flexible loop that occurs upon binding of substrate. With the PTPase from Yersinia, we have examined the effect on general acid catalysis caused by mutations to two conserved residues that are integral to this conformation change. Residue Trp354 is at a hinge of the loop, and Arg409 forms hydrogen bonding and ionic interactions with the phosphoryl group of substrates. Trp354 was mutated to Phe and to Ala, and residue Arg409 was mutated to Lys and to Ala. The four mutant enzymes were studied using steady state kinetics and heavy-atom isotope effects with the substrate p-nitrophenyl phosphate. The data indicate that mutation of the hinge residue Trp354 to Ala completely disables general acid catalysis. In the Phe mutant, general acid catalysis is partially effective, but the proton is only partially transferred in the transition state, in contrast to the native enzyme where proton transfer to the leaving group is virtually complete. Mutation of Arg409 to Lys has a minimal effect on the K(m), while this parameter is increased 30-fold in the Ala mutant. The k(cat) values for R409K and for R409A are about 4 orders of magnitude lower than that for the native enzyme. General acid catalysis is rendered inoperative by the Lys mutation, but partial proton transfer during catalysis still occurs in the Ala mutant. Structural explanations for the differential effects of these mutations on movement of the flexible loop that enables general acid catalysis are presented.     

Structural basis of the suppressed catalytic activity of wild-type human glutathione transferase T1-1 compared to its W234R mutant

The crystal structures of wild-type human theta class glutathione-S-transferase (GST) T1-1 and its W234R mutant, where Trp234 was replaced by Arg, were solved both in the presence and absence of S-hexyl-glutathione. The W234R mutant was of interest due to its previously observed enhanced catalytic activity compared to the wild-type enzyme. GST T1-1 from rat and mouse naturally contain Arg in position 234, with correspondingly high catalytic efficiency. The overall structure of GST T1-1 is similar to that of GST T2-2, as expected from their 53% sequence identity at the protein level. Wild-type GST T1-1 has the side-chain of Trp234 occupying a significant portion of the active site. This bulky residue prevents efficient binding of both glutathione and hydrophobic substrates through steric hindrance. The wild-type GST T1-1 crystal structure, obtained from co-crystallization experiments with glutathione and its derivatives, showed no electron density for the glutathione ligand. However, the structure of GST T1-1 mutant W234R showed clear electron density for S-hexyl-glutathione after co-crystallization. In contrast to Trp234 in the wild-type structure, the side-chain of Arg234 in the mutant does not occupy any part of the substrate-binding site. Instead, Arg234 is pointing in a different direction and, in addition, interacts with the carboxylate group of glutathione. These findings explain our earlier observation that the W234R mutant has a markedly improved catalytic activity with most substrates tested to date compared to the wild-type enzyme. GST T1-1 catalyzes detoxication reactions as well as reactions that result in toxic products, and our findings therefore suggest that humans have gained an evolutionary advantage by a partially disabled active site.     

Insights into the catalytic properties of bamboo vacuolar invertase through mutational analysis of active site residues

Plant acid invertases, which are either associated with the cell wall or present in vacuoles, belong to family 32 of glycoside hydrolases (GH32). Homology modeling of bamboo vacuolar invertase Boβfruct3 using Arabidopsis cell-wall invertase AtcwINV1 as a template showed that its overall structure is similar to GH32 enzymes, and that the three highly conserved motifs, NDPNG, RDP and EC, are located in the active site. This study also used site-directed mutagenesis to examine the roles of the conserved amino acid residues in these three motifs, which include Asp135, Arg259, Asp260, Glu316 and Cys317, and a conserved Trp residue (Trp159) that resides between the NDPNG and RDP motifs. The mutants W159F, W159L, E316Q and C317A retained acid invertase activity, but no invertase activity was observed for the mutant E316A or mutants with changes at Asp135, Arg259, or Asp260. The apparent K_m values of the four mutants with invertase activity were all higher than that of the wild-type enzyme. The mutants W159L and E316Q exhibited lower k_cat values than the wild-type enzyme, but an increase in the k_cat value was observed for the mutants W159F and C317A. The results of this study demonstrate that these residues have individual functions in catalyzing sucrose hydrolysis.     

Structural and mutational studies on an aldo‐keto reductase AKR5C3 from Gluconobacter oxydans

An aldo‐keto reductase AKR5C3 from Gluconobacter oxydans (designated as Gox0644) is a useful enzyme with various substrates, including aldehydes, diacetyl, keto esters, and α‐ketocarbonyl compounds. The crystal structures of AKR5C3 in apoform in complex with NADPH and the D53A mutant (AKR5C3^‐D53A) in complex with NADPH are presented herein. Structure comparison and site‐directed mutagenesis combined with biochemical kinetics analysis reveal that the conserved Asp53 in the AKR5C3 catalytic tetrad has a crucial role in securing active pocket conformation. The gain‐of‐function Asp53 to Ala mutation triggers conformational changes on the Trp30 and Trp191 side chains, improving NADPH affinity to AKR5C3, which helps increase catalytic efficiency. The highly conserved Trp30 and Trp191 residues interact with the nicotinamide moiety of NADPH and help form the NADPH‐binding pocket. The AKR5C3^‐W30A and AKR5C3^‐W191Y mutants show decreased activities, confirming that both residues facilitate catalysis. Residue Trp191 is in the loop structure, and the AKR5C3^‐W191Y mutant does not react with benzaldehyde, which might also determine substrate recognition. Arg192, which is involved in the substrate binding, is another important residue. The introduction of R192G increases substrate‐binding affinity by improving hydrophobicity in the substrate‐binding pocket. These results not only supplement the AKRs superfamily with crystal structures but also provide useful information for understanding the catalytic properties of AKR5C3 and guiding further engineering of this enzyme.     

Acquisition of photosynthetic capacity by a reaction centre that lacks the QA ubiquinone; possible insights into the evolution of reaction centres?

A photosynthetically impaired strain of Rhodobacter sphaeroides containing reaction centres with an alanine to tryptophan mutation at residue 260 of the M-polypeptide (AM260W) was incubated under photosynthetic growth conditions. This incubation produced photosynthetically competent strains containing suppressor mutations that changed residue M260 to glycine or cysteine. Spectroscopic analysis demonstrated that the loss of the Q_A ubiquinone seen in the original AM260W mutant was reversed in the suppressor mutants. In the mutant where Trp M260 was replaced by Cys, the rate of reduction of the Q_A ubiquinone by the adjacent (H_A) bacteriopheophytin was reduced by three-fold. The findings of the experiment are discussed in light of the X-ray crystal structures of the wild-type and AM260W reaction centres, and the possible implications for the evolution of reaction centres as bioenergetic complexes are considered.     

Optimization of a β‐sheet‐cap for long loop closure

Protein loops make up a large portion of the secondary structure in nature. But very little is known concerning loop closure dynamics and the effects of loop composition on fold stability. We have designed a small system with stable β‐sheet structures, including features that allow us to probe these questions. Using paired Trp residues that form aromatic clusters on folding, we are able to stabilize two β‐strands connected by varying loop lengths and composition (an example sequence: R W ITVTI – loop – KKIRV W E). Using NMR and CD, both fold stability and folding dynamics can be investigated for these systems. With the 16 residue loop peptide (sequence: R W ITVTI‐(GGGGKK)₂GGGG‐KKIRV W E) remaining folded (ΔG_U = 1.6 kJ/mol at 295K). To increase stability and extend the series to longer loops, we added an additional Trp/Trp pair in the loop flanking position. With this addition to the strands, the 16 residue loop (sequence: R W ITVRI W ‐(GGGGKK)₂GGGG‐ W KTIRV W E) supports a remarkably stable β‐sheet (ΔG_U = 6.3 kJ/mol at 295 K, T_m = ～55°C). Given the abundance of loops in binding motifs and between secondary structures, these constructs can be powerful tools for peptide chemists to study loop effects; with the Trp/Trp pair providing spectroscopic probes for assessing both stability and dynamics by NMR.     

Properties of Calmodulin Binding to NaV1.2 IQ Motif and Its Autism-Associated Mutation R1902C

Voltage-gated sodium channels (VGSCs) are fundamental to the initiation and propagation of action potentials in excitable cells. Ca²⁺/calmodulin (CaM) binds to VGSC type II (Na_V1.2) isoleucine and glutamine (IQ) motif. An autism-associated mutation in Na_V1.2 IQ motif, Arg1902Cys (R1902C), has been reported to affect the combination between CaM and the IQ motif compared to that of the wild type IQ motif. However, the detailed properties for the Ca²⁺-regulated binding of CaM to Na_V1.2 IQ (1901Lys-1927Lys, IQ_wt) and mutant IQ motif (IQ_R1902C) remains unclear. Here, the binding ability of CaM and CaM's constituent proteins including N- and C lobe to the IQ motif of Na_V1.2 and its mutant was investigated by protein pull-down experiments. We discovered that the combination between CaM and the IQ motif was U-shaped with the highest at [Ca²⁺] ≈ free and the lowest at 100 nM [Ca²⁺]. In the IQ_R1902C mutant, Ca²⁺-dependence of CaM binding was nearly lost. Consequently, the binding of CaM to IQ_R1902C at 100 and 500 nM [Ca²⁺] was increased compared to that of IQ_wt. Both N- and C lobe of CaM could bind with Na_V1.2 IQ motif and IQ_R1902C mutant, with the major effect of C lobe. Furthermore, CaMKII had no impact on the binding between CaM and Na_V1.2 IQ motif. This research offers novel insight to the regulation of Na_V1.2 IQ_wt and IQ_R1902C motif, an autism-associated mutation, by CaM.

     

Mutation in transforming growth factor beta induced protein associated with granular corneal dystrophy type 1 reduces the proteolytic susceptibility through local structural stabilization

Hereditary mutations in the transforming growth factor beta induced (TGFBI) gene cause phenotypically distinct corneal dystrophies characterized by protein deposition in cornea. We show here that the Arg555Trp mutant of the fourth fasciclin 1 (FAS1-4) domain of the protein (TGFBIp/keratoepithelin/βig-h3), associated with granular corneal dystrophy type 1, is significantly less susceptible to proteolysis by thermolysin and trypsin than the WT domain. High-resolution liquid-state NMR of the WT and Arg555Trp mutant FAS1-4 domains revealed very similar structures except for the region around position 555. The Arg555Trp substitution causes Trp555 to be buried in an otherwise empty hydrophobic cavity of the FAS1-4 domain. The first thermolysin cleavage in the core of the FAS1-4 domain occurs on the N-terminal side of Leu558 adjacent to the Arg555 mutation. MD simulations indicated that the C-terminal end of helix α3′ containing this cleavage site is less flexible in the mutant domain, explaining the observed proteolytic resistance. This structural change also alters the electrostatic properties, which may explain increased propensity of the mutant to aggregate in vitro with 2,2,2-trifluoroethanol. Based on our results we propose that the Arg555Trp mutation disrupts the normal degradation/turnover of corneal TGFBIp, leading to accumulation and increased propensity to aggregate through electrostatic interactions.     

Molecular mechanism of the selectivity between BAFF/APRIL and their receptors by molecular simulations

B-cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL) belonging to the tumour necrosis factor (TNF) ligand family can bind three unusual TNF receptors (BCMA, TACI and BR3) with various binding affinities. BAFF and APRIL are regarded as promising therapeutic targets for autoimmune diseases because of their pivotal roles in cell survival and immune regulation. In this work, we carried out molecular dynamics calculations to explore the structural and chemical features responsible for ligand recognition by extracellular functional segments of TNF receptors. We found that the conserved pocket D_cons of BAFF/APRIL contacted the DxL motif of TNF receptors, while the D_spe1–3 sub-domains were responsible for their different affinities, especially D_spe1 and D_spe2. The residues at position II–V of DxL motif were wrapped into the D_cons pocket via salt-bridge and hydrophobic interactions. The hydrophobic residues of strand3 and helix1 in TNF receptors provided remarkable contributions for the affinities to BAFF/APRIL. Additionally, Arg^VI of DxL motif played a key role in the binding selectivity via salt-bridge interaction with residue D275B in BAFF. Arg27 in BCMA contributed to the high affinity for APRIL so that BCMA showed a preference for APRIL. Our studies indicated that Arg84 and Gln95 in TACI2 played an important role in the selectivity of two cysteine-rich domain segments in TACI, leading to the higher binding affinities of TACI2 than those of TACI1. The primary cause of the disability to bind APRIL was the space conflict with the rigid conformation of the C-terminus coil of BR3. These thorough understanding of the molecular mechanism for BAFF/APRIL recognition by their receptors provides new insights for guiding inhibitor design.     

Single Arginine Mutation in Two Yeast Isocitrate Dehydrogenases: Biochemical Characterization and Functional Implication

Isocitrate dehydrogenase (IDH), a housekeeping gene, has drawn the attention of cancer experts. Mutation of the catalytic Arg132 residue of human IDH1 (HcIDH) eliminates the enzyme''s wild-type isocitrate oxidation activity, but confer the mutant an ability of reducing α-ketoglutarate (α-KG) to 2-hydroxyglutarate (2-HG). To examine whether an analogous mutation in IDHs of other eukaryotes could cause similar effects, two yeast mitochondrial IDHs, Saccharomyces cerevisiae NADP⁺-IDH1 (ScIDH1) and Yarrowia lipolytica NADP⁺-IDH (YlIDH), were studied. The analogous Arg residues (Arg148 of ScIDH1 and Arg141 of YlIDH) were mutated to His. The K _m values of ScIDH1 R148H and YlIDH R141H for isocitrate were determined to be 2.4-fold and 2.2-fold higher, respectively, than those of the corresponding wild-type enzymes. The catalytic efficiencies (k _cat/K _m) of ScIDH1 R148H and YlIDH R141H for isocitrate oxidation were drastically reduced by 227-fold and 460-fold, respectively, of those of the wild-type enzymes. As expected, both ScIDH1 R148H and YlIDH R141H acquired the neomorphic activity of catalyzing α-KG to 2-HG, and the generation of 2-HG was confirmed using gas chromatography/time of flight-mass spectrometry (GC/TOF-MS). Kinetic analysis showed that ScIDH1 R148H and YlIDH R141H displayed 5.2-fold and 3.3-fold higher affinities, respectively, for α-KG than the HcIDH R132H mutant. The catalytic efficiencies of ScIDH1 R148H and YlIDH R141H for α-KG were 5.5-fold and 4.5-fold, respectively, of that of the HcIDH R132H mutant. Since the HcIDH Arg132 mutation is associated with the tumorigenesis, this study provides fundamental information for further research on the physiological role of this IDH mutation in vivo using yeast.     

Aromatic stacking as a determinant of the thermal stability of CYP119 from Sulfolobus solfataricus

Two notable features of the thermophilic CYP119, an Arg154-Glu212 salt bridge between the F-G loop and the I helix and an extended aromatic cluster, were studied to determine their contributions to the thermal stability of the enzyme. Site-specific mutants of the salt bridge (Arg154, Glu212) and aromatic cluster (Tyr2, Trp4, Trp231, Tyr250, Trp281) were expressed and purified. The substrate-binding and kinetic constants for lauric acid hydroxylation are little affected in most mutants, but the E212D mutant is inactive and the R154Q mutant has higher K(s),K(m), and k(cat) values. The salt bridge mutants, like wild-type CYP119, melt at 91+/-1 degrees C, whereas mutation of individual residues in the extended aromatic cluster lowers the T(m) by 10-15 degrees C even though no change is observed on mutation of an unrelated aromatic residue. The extended aromatic cluster, but not the Arg154-Glu212 salt bridge, contributes to the thermal stability of CYP119.     

Long-range interaction between the enzyme active site and a distant allosteric site in the human mitochondrial NAD(P)-dependent malic enzyme

Our previous study has suggested that mutation of the amino acid residue Asp102 has a significant effect on the fumarate-mediated activation of human mitochondrial NAD(P)⁺-dependent malic enzyme (m-NAD(P)-ME). In this paper, we examine the cationic amino acid residue Arg98, which is adjacent to Asp102 and is highly conserved in most m-NAD(P)-MEs. A series of R98/D102 mutants were created to examine the possible interactions between Arg98 and Asp102 using the double-mutant cycle analysis. Kinetic analysis revealed that the catalytic efficiency of the enzyme was severely affected by mutating both Arg98 and Asp102 residues. However, the binding energy of these mutant enzymes to fumarate as determined by analysis of the K_A,Fum values, show insignificant differences, indicating that the mutation of Arg98 and Asp102 did not cause a significant decrease in the binding affinity of fumarate. The overall coupling energies for R98K/D102N as determined by analysis of the k_cat/K_m and K_A,Fum values were −2.95 and −0.32 kcal/mol, respectively. According to these results, we conclude that substitution of both Arg98 and Asp102 residues has a synergistic effect on the catalytic ability of the enzyme.     

Structure-function analysis of helix 8 of human calcitonin receptor-like receptor within the adrenomedullin 1 receptor

Adrenomedullin 1 (AM₁) receptor is a heterodimer composed of calcitonin receptor-like receptor (CLR) - a family B G protein-coupled receptor (GPCR) - and receptor activity-modifying protein 2 (RAMP2). Both family A and family B GPCRs possess an eighth helix (helix 8) in the proximal portion of their C-terminal tails; however, little is known about the function of helix 8 in family B GPCRs. We therefore investigated the structure-function relationship of human (h)CLR helix 8, which extends from Glu430 to Trp439, by separately transfecting nine point mutants into HEK-293 cells stably expressing hRAMP2. Glu430, Val431, Arg437 and Trp439 are all conserved among family B GPCRs. Flow cytometric analysis revealed that Arg437Ala or Trp438Ala mutation significantly reduced cell surface expression of the receptor complex, leading to a ∼20% reduction in specific ¹²⁵I-AM binding but little change in their IC₅₀ values. Both mutants showed 6-8-fold higher EC₅₀ values for AM-induced cAMP production and ∼50% reductions in their maximum responses. Glu430Ala mutation also reduced AM signaling by ∼45%, but surface expression and ¹²⁵I-AM binding were nearly the same as with wild-type CLR. Surprisingly, Glu430Ala and Val431Ala mutations significantly enhanced AM-induced internalization of the mutant receptor complexes. Taken together, these findings suggest that within hCLR helix 8, Glu430 is crucial for Gs coupling, and Arg437 and Trp439 are involved in both cell surface expression of the hAM₁ receptor and Gs coupling. Moreover, the Glu430-Val431 sequence may participate in the negative regulation of hAM₁ receptor internalization, which is not dependent on Gs coupling.     

Consequences of cAMP-binding site mutations on the structural stability of the type I regulatory subunit of cAMP-dependent protein kinase

The regulatory (R) subunit of cAMP-dependent protein kinase (cAPK) is a multidomain protein with two tandem cAMP-binding domains, A and B. The importance of cAMP binding on the stability of the R subunit was probed by intrinsic fluorescence and circular dichroism (CD) in the presence and absence of urea. Several mutants were characterized. The site-specific mutants R(R209K) and R(R333K) had defects in cAMP-binding sites A and B, respectively. R(M329W) had an additional tryptophan in domain B. Delta(260-379)R lacked Trp260 and domain B. The most destabilizing mutation was R209K. Both CD and fluorescence experiments carried out in the presence of urea showed a decrease in cooperativity of the unfolding, which also occurred at lower urea concentrations. Unlike native R, R(R209K) was not stabilized by excess cAMP. Additionally, CD revealed significant alterations in the secondary structure of the R209K mutant. Therefore, Arg209 is important not only as a contact site for cAMP binding but also for the intrinsic structural stability of the full-length protein. Introducing the comparable mutation into domain B, R333K, had a smaller effect on the integrity and stability of domain A. Unfolding was still cooperative; the protein was stabilized by excess cAMP, but the unfolding curve was biphasic. The R(M329W) mutant behaved functionally like the native protein. The Delta(260-379)R deletion mutant was not significantly different from wild-type RIalpha in its stability. Consequently, domain B and the interaction between Trp260 and cAMP bound to site A are not critical requirements for the structural stability of the cAPK regulatory subunit.