Structure-activity relationships of atrial natriuretic factor (ANF). II. Effect of chain-length modifications on vascular reactivity

The effect of the length of ANF peptides on the inhibition of the norepinephrine-induced contraction was studied. Starting from the 26 residues ANF (Arg101-Tyr126), shorter N- and/or C-terminal fragments were produced, either by N-terminal chemical cleavage or C-terminal enzymatic digestion of ANF or both respectively. The N-terminal removal of Arg101 did not modify the inhibitory response. Further N-terminal truncation up to des-Arg101-Arg102-Ser103-Ser104 ANF still produced a marked inhibitory effect on norepinephrine. In contrast C-terminal cleavage had a much more pronounced effect. Since des-Tyr126 ANF, des-Arg125-Tyr126 ANF and des-Phe124-Arg125-Tyr126 ANF exhibit much lower activities than the parent ANF. Finally, when the 5 residues C-terminal to Cys121 are removed, the resulting molecule is almost inactive. These data indicate that the C-terminal segment of ANF may modulate the binding of ANF to its receptor(s). Relatively, the N-terminal region seems to be much less important.     

Atrial granules contain an amino-terminal processing enzyme of atrial natriuretic factor

At least three enzymes have been identified in atrial tissue homogenates that are capable of processing pro-atrial natriuretic factor to active atrial peptides. The atrial peptides possess potent natriuretic, diuretic, vasorelaxant, and hemodynamic properties, and their existence has implicated the mammalian heart as an endocrine organ. We have purified and characterized a serine proteinase (Mr approximately equal to 70,000) associated with atrial granules that preferentially hydrolyzes the Arg-Ser bond in the synthetic substrates Gly-Pro-Arg-Ser-Leu-Arg, benzoyl-Gly-Pro-Arg-Ser-Leu-Arg, and benzoyl-Gly-Pro-Arg-Ser-Leu-Arg-Arg-2-naphthylamide, the Arg-2-naphthylamide bond in the substrate benzoyl-Gly-Pro-Arg-2-naphthylamide, and the Arg-Ser bond in a 31-residue substrate (Gly96-Tyr126 peptide) corresponding to residues Arg98-Ser99 in pro-atrial natriuretic factor. The Gly96-Tyr126 peptide contains the putative processing site in pro-atrial natriuretic factor and the sequence for the bioactive peptides. Our results indicate that the minimum processing site sequence is -Gly-Pro-Arg-Ser-Leu-Arg-Arg- and that the Ser99-Tyr126 natriuretic peptide is the predominant hydrolytic product. After prolonged incubation or at high enzyme concentrations, the Ser103-Tyr126 natriuretic peptide may also be formed. The Ser103-Arg125 natriuretic peptide was only a very minor product. The doublet of basic amino acids is not the primary processing site in pro-atrial natriuretic factor, but their presence may influence cleavage at the single Arg residue "upstream." Our findings are consistent with the idea that the pro-protein and the processing enzymes are packaged into the secretory granule and in response to the proper stimulus, the pro-protein is processed to the active peptides, probably during the process of secretion. The processing pathway of pro-atrial natriuretic factor is discussed.     

Communication between the two active sites of glutathione S-transferase A1-1, probed using wild-type-mutant heterodimers

To study the communication between the two active sites of dimeric glutathione S-transferase A1-1, we used heterodimers containing one wild-type (WT) active site and one active site with a single mutation at either Tyr9, Arg15, or Arg131. Tyr9 and Arg15 are part of the active site of the same subunit, while Arg131 contributes to the active site of the opposite subunit. The V(max) values of Tyr9 and Arg15 mutant enzymes were less than 2% that of WT, indicating their importance in catalysis. In contrast, V(max) values of Arg131 mutant enzymes were about 50-90% of that of WT enzyme while K(m)(GSH) values were approximately 3-8 times that of WT, suggesting that Arg131 plays a role in glutathione binding. The mutant enzyme (with a His(6) tag) and the WT enzyme (without a His(6) tag) were used to construct heterodimers (WT-Y9F, WT-Y9T, WT-R15Q, WT-R131M, WT-R131Q, and WT-R131E) by incubation of a mixture of wild-type and mutant enzyme at pH 7.5 in buffer containing 1,6-hexanediol, followed by dialysis against buffer lacking the organic solvent. The resultant heterodimers were separated from the wild-type and mutant homodimers using chromatography on nickel-nitrilotriacetic acid agarose. The V(max) values of all heterodimers were lower than expected for independent active sites. Our experiments demonstrate that mutation of an amino acid residue in one active site affects the activity in the other active site. Modeling studies show that key amino acid residues and water molecules connect the two active sites. This connectivity is responsible for the cross-talk between the active sites.     

Probing the mechanism of GSH activation in Schistosoma haematobium glutathione-S-transferase by site-directed mutagenesis and X-ray crystallography

During turnover, the catalytic tyrosine residue (Tyr10) of the sigma class Schistosoma haematobium wild-type glutathione-S-transferase is expected to switch alternately in and out of the reduced glutathione-binding site (G-site). The Tyrout10 conformer forms a pi-cation interaction with the guanidinium group of Arg21. As in other similar glutathione-S-transferases, the catalytic Tyr has a low pKa of 7.2. In order to investigate the catalytic role of Tyr10, and the structural and functional roles of Arg21, we carried out structural studies on two Arg21 mutants (R21L and R21Q) and a Tyr10 mutant, Y10F. Our crystallographic data for the two Arg21 mutants indicate that only the Tyrout10 conformation is populated, thereby excluding a role of Arg21 in the stabilisation of the out conformation. However, Arg21 was confirmed to be catalytically important and essential for the low pKa of Tyr10. Upon comparison with structural data generated for reduced glutathione-bound and inhibitor-bound wild-type enzymes, it was observed that the orientations of Tyr10 and Arg35 are concerted and that, upon ligand binding, minor rearrangements occur within conserved residues in the active site loop. These rearrangements are coupled to quaternary rigid-body movements at the dimer interface and alterations in the localisation and structural order of the C-terminal domain.     

Roles of active site residues in Pseudomonas aeruginosa phosphomannomutase/phosphoglucomutase

In Pseudomonas aeruginosa, the dual-specificity enzyme phosphomannomutase/phosphoglucomutase catalyzes the transfer of a phosphoryl group from serine 108 to the hydroxyl group at the 1-position of the substrate, either mannose 6-P or glucose 6-P. The enzyme must then catalyze transfer of the phosphoryl group on the 6-position of the substrate back to the enzyme. Each phosphoryl transfer is expected to require general acid-base catalysis, provided by amino acid residues at the enzyme active site. An extensive survey of the active site residues by site-directed mutagenesis failed to identify a single key residue that mediates the proton transfers. Mutagenesis of active site residues Arg20, Lys118, Arg247, His308, and His329 to residues that do not contain ionizable groups produced proteins for which V(max) was reduced to 4-12% of that of the wild type. The fact that no single residue decreased catalytic activity more significantly, and that several residues had similar effects on V(max), suggested that the ensemble of active site amino acids act by creating positive electrostatic potential, which serves to depress the pK of the substrate hydroxyl group so that it binds in ionized form at the active site. In this way, the necessity of positioning the reactive hydroxyl group near a specific amino acid residue is avoided, which may explain how the enzyme is able to promote catalysis of both phosphoryl transfers, even though the 1- and 6-positions do not occupy precisely the same position when the substrate binds in the two different orientations in the active site. When Ser108 is mutated, the enzyme retains a surprising amount of activity, which has led to the suggestion that an alternative residue becomes phosphorylated in the absence of Ser108. (31)P NMR spectra of the S108A protein confirm that it is phosphorylated. Although the S108A/H329N protein had no detectable catalytic activity, the (31)P NMR spectra were not consistent with a phosphohistidine residue.     

Crystal structure of a hyperthermophilic archaeal acylphosphatase from Pyrococcus horikoshii--structural insights into enzymatic catalysis, thermostability, and dimerization

Acylphosphatases catalyze the hydrolysis of the carboxyl-phosphate bond in acyl phosphates. Although acylphosphatase-like sequences are found in all three domains of life, no structure of acylphosphatase has been reported for bacteria and archaea so far. Here, we report the characterization of enzymatic activities and crystal structure of an archaeal acylphosphatase. A putative acylphosphatase gene (PhAcP) was cloned from the genomic DNA of Pyrococcus horikoshii and was expressed in Escherichia coli. Enzymatic parameters of the recombinant PhAcP were measured using benzoyl phosphate as the substrate. Our data suggest that, while PhAcP is less efficient than other mammalian homologues at 25 degrees C, the thermophilic enzyme is fully active at the optimal growth temperature (98 degrees C) of P. horikoshii. PhAcP is extremely stable; its apparent melting temperature was 111.5 degrees C and free energy of unfolding at 25 degrees C was 54 kJ mol(-)(1). The 1.5 A crystal structure of PhAcP adopts an alpha/beta sandwich fold that is common to other acylphosphatases. PhAcP forms a dimer in the crystal structure via antiparallel association of strand 4. Structural comparison to mesophilic acylphosphatases reveals significant differences in the conformation of the L5 loop connecting strands 4 and 5. The extreme thermostability of PhAcP can be attributed to an extensive ion-pair network consisting of 13 charge residues on the beta sheet of the protein. The reduced catalytic efficiency of PhAcP at 25 degrees C may be due to a less flexible active-site residue, Arg20, which forms a salt bridge to the C-terminal carboxyl group. New insights into catalysis were gained by docking acetyl phosphate to the active site of PhAcP.     

Identification of the released form of atrial natriuretic factor by the perfused rat heart

Atrial natriuretic factor (ANF), released by the isolated perfused rat heart, was extracted from the perfusates by C18 Sep-Pak cartridges and then isolated by immunoaffinity chromatography and by reverse phase HPLC. About 500 ng of immunoreactive material were so obtained and submitted to amino acid sequencing. The C-terminal Tyr was detected by radiolabelling. Identification of these residues indicated that the primary structure corresponds to ANF (Ser 99-Tyr 126) which is identical to the circulating form in the rat. These results indicate that the ANF released by the atria corresponds to a short peptide. Therefore, its maturation process may therefore take place either intracellularly or during secretion and implicates a tryptic-like cleavage after a single Arg residue in position 98.     

Structural and sequence comparisons of bacterial enoyl-CoA isomerase and enoyl-CoA hydratase

Crystal structures of enoyl-coenzyme A (CoA) isomerase from Bosea sp. PAMC 26642 (BoECI) and enoyl-CoA hydratase from Hymenobacter sp. PAMC 26628 (HyECH) were determined at 2.35 and 2.70 Å resolution, respectively. BoECI and HyECH are members of the crotonase superfamily and are enzymes known to be involved in fatty acid degradation. Structurally, these enzymes are highly similar except for the orientation of their C-terminal helix domain. Analytical ultracentrifugation was performed to determine the oligomerization states of BoECI and HyECH revealing they exist as trimers in solution. However, their putative ligand-binding sites and active site residue compositions are dissimilar. Comparative sequence and structural analysis revealed that the active site of BoECI had one glutamate residue (Glu135), this site is occupied by an aspartate in some ECIs, and the active sites of HyECH had two highly conserved glutamate residues (Glu118 and Glu138). Moreover, HyECH possesses a salt bridge interaction between Glu98 and Arg152 near the active site. This interaction may allow the catalytic Glu118 residue to have a specific conformation for the ECH enzyme reaction. This salt bridge interaction is highly conserved in known bacterial ECH structures and ECI enzymes do not have this type of interaction. Collectively, our comparative sequential and structural studies have provided useful information to distinguish and classify two similar bacterial crotonase superfamily enzymes.     

Proton nuclear magnetic resonance study of human plasma alpha-2-macroglobulin

A proton nuclear magnetic resonance (NMR) study is reported of human alpha-2-macroglobulin (alpha-2-M). It was observed that alpha-2-M, which consists of four identical subunits and has a molecular weight of 720,000, gives several sharp resonances. After cleavage of the "bait" region peptide with trypsin and subsequent removal of the peptide under a high salt condition, most of the sharp resonances disappeared, indicating that the sharp resonances observed in the native alpha-2-M originate from the amino acid residues in the bait region. Resonances due to the aromatic protons of the Tyr residue, which exists in the bait region, have been assigned on the basis of chemical shift. It was observed that the C3- and C5-H proton resonances for the Tyr residue are especially narrow, indicating that the side chain of the Tyr residue in the bait region is in a highly mobile state. Photochemically induced dynamic nuclear polarization experiments clearly show that the Tyr residue is actually exposed to the solvent. It was possible to identify resonances due to several His residues that are exposed to solvent. Other resonances, which probably originate from Arg residues in the bait region, were also observable in the conventional NMR spectra. On the basis of the present NMR data, we conclude that the bait region of the native alpha-2-M is highly flexible and exposed to solvent. On treatment of alpha-2-M with methylamine, no significant change has been detected in the NMR spectra observed in both the conventional and CIDNP mode.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heterologous expression of the vanadium-containing chloroperoxidase from Curvularia inaequalis in Saccharomyces cerevisiae and site-directed mutagenesis of the active site residues His(496), Lys(353), Arg(360), and Arg(490).

The vanadium-containing chloroperoxidase from the fungus Curvularia inaequalis is heterologously expressed to high levels in the yeast Saccharomyces cerevisiae. Characterization of the recombinant enzyme reveals that this behaves very similar to the native chloroperoxidase. Site-directed mutagenesis is performed on four highly conserved active site residues to examine their role in catalysis. When the vanadate-binding residue His(496) is changed into an alanine, the mutant enzyme loses the ability to bind vanadate covalently resulting in an inactive enzyme. The negative charges on the vanadate oxygens are compensated by hydrogen bonds with the residues Arg(360), Arg(490), and Lys(353). When these residues are changed into alanines the mutant enzymes lose the ability to effectively oxidize chloride but can still function as bromoperoxidases. A general mechanism for haloperoxidase catalysis is proposed that also correlates the kinetic properties of the mutants with the charge and the hydrogen-bonding network in the vanadate-binding site.     

Mapping the substrate-binding site of a human class mu glutathione transferase using nuclear magnetic resonance spectroscopy.

The substrate-binding site of a human muscle class mu glutathione transferase has been characterized using high-resolution nuclear magnetic resonance spectroscopy. Isotopic labeling has been used to simplify one-dimensional proton NMR spectra of the Tyr and His residues in the enzyme and two-dimensional carbon-proton spectra of the Ala and Met residues in the enzyme. The resonance lines from 8 of the 12 Tyr residues have been assigned using site-directed mutagenesis. Replacement of Tyr7 with Phe reduced the activity of the enzyme 100-fold. The proximity of His, Tyr, Ala, and Met residues to the active site has been determined using a nitroxide-labeled substrate analogue. This substrate analogue binds with high affinity (Keq = 10(6) M-1) to the enzyme and is a competitive inhibitor. None of the His residues are within 17 A of the active site. Three of the assigned Tyr residues are greater than 17 A from the active site. Quantitative measurement of paramagnetic line broadening of five additional Tyr residues places them within 13-17 A from the active site. Broadening of the Ala and Met resonance lines by the spin-labeled substrate indicates that three Ala residues are 9-16 A from the nitroxide, three Met residues are less than 9 A from the nitroxide, and two Met residues are 9-16 A from the nitroxide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of an endogenous protease that processes atrial natriuretic peptide at its amino terminus

We have partially purified a thiol-dependent protease from bovine atrial tissue that cleaves the Arg98-Ser99 bond of rat natriuretic peptide (Gly96-Tyr126) to produce the natriuretic Ser99-Tyr126 peptide (cardionatrin I). This was the only hydrolytic product we detected. The existence of the atrial natriuretic peptide system implicates the mammalian heart as an endocrine organ which participates in the hormonal regulation of extracellular fluid volume, electrolyte balance and vascular tone. This enzyme appears to be part of that system. The atrial protease also hydrolyzes the Arg-2-Napthylamide bond of natriuretic peptide stand-in substrates; on the basis of relative Vmax/Km as a measure of substrate specificity, Bz-Leu-Arg-Arg-2-Napthylamide (NA) greater than Bz-Leu-Arg-2-NA greater than Arg-2-NA. There is little or no cleavage between the Arg-Arg pair of the first substrate. Since in the Gly96-Tyr126 peptide the Arg-Arg pair is not the principle cleavage site for this enzyme, it is very unlikely that it is a principle cleavage site for this enzyme in pro-atrial natriuretic factor. It is possible that it is a cleavage site for a different enzyme or the pair may serve as a signal for cleavage at Arg98.     

Crystal structures of Staphylococcus aureus sortase A and its substrate complex

The cell wall envelope of staphylococci and other Gram-positive pathogens is coated with surface proteins that interact with human host tissues. Surface proteins of Staphylococcus aureus are covalently linked to the cell wall envelope by a mechanism requiring C-terminal sorting signals with an LPXTG motif. Sortase (SrtA) cleaves surface proteins between the threonine (T) and the glycine (G) of the LPXTG motif and catalyzes the formation of an amide bond between threonine at the C-terminal end of polypeptides and cell wall cross-bridges. The active site architecture and catalytic mechanism of sortase A has hitherto not been revealed. Here we present the crystal structures of native SrtA, of an active site mutant of SrtA, and of the mutant SrtA complexed with its substrate LPETG peptide and describe the substrate binding pocket of the enzyme. Highly conserved proline (P) and threonine (T) residues of the LPXTG motif are held in position by hydrophobic contacts, whereas the glutamic acid residue (E) at the X position points out into the solvent. The scissile T-G peptide bond is positioned between the active site Cys(184) and Arg(197) residues and at a greater distance from the imidazolium side chain of His(120). All three residues, His(120), Cys(184), and Arg(197), are conserved in sortase enzymes from Gram-positive bacteria. Comparison of the active sites of S. aureus sortase A and sortase B provides insight into substrate specificity and suggests a universal sortase-catalyzed mechanism of bacterial surface protein anchoring in Gram-positive bacteria.     

The fibrin-binding site of human plasminogen. Arginines 32 and 34 are essential for fibrin affinity of the kringle 1 domain

Kringle 1 (Tyr 79/Leu 80-His 167 and Tyr 79/Leu 80-Tyr 173), a chymotryptic fragment of human plasminogen that has high affinity for fibrin and omega-aminocarboxylic acids, has been subjected to modification with 1,2-cyclohexanedione to identify arginine residues essential for ligand binding. Reaction of 1,2-cyclohexanedione with kringle 1 was found to rapidly abolish the fibrin-Sepharose affinity of the fragment, whereas the affinity for lysine-Sepharose was lost at a significantly slower rate. Successive affinity chromatography of modified kringle 1 on fibrin- and lysine-Sepharose was used to separate kringle 1 that lost affinity for fibrin-, but retained affinity for lysine-Sepharose from kringle 1 that lost affinity for both affinants. The modified proteins were subjected to structural studies in order to locate the labeled arginine residues in kringle 1. These studies have revealed that modification of Arg 34 leads to the loss of both the fibrin- and lysine-Sepharose affinities of kringle 1, whereas reaction of Arg 32 abolishes fibrin affinity but leaves lysine-Sepharose affinity unaltered. The results suggest that Arg 32 and Arg 34 are both involved in fibrin binding and that Arg 34 is also involved in binding omega-aminocarboxylic acids. Previous NMR studies on kringles have indeed shown that the segment containing residue 34 is in the proximity of and interacts with the omega-aminocarboxylic acid-binding site. This interaction may explain the influence of omega-aminocarboxylic acids on fibrin binding by kringle 1.     

A 19F-NMR study of the membrane-binding region of D-lactate dehydrogenase of Escherichia coli.

D-Lactate dehydrogenase (D-LDH) is a membrane-associated respiratory enzyme of Escherichia coli. The protein is composed of 571 amino acid residues with a flavin adenine dinucleotide (FAD) cofactor, has a molecular weight of approximately 65,000, and requires lipids or detergents for full activity. We used NMR spectroscopy to investigate the structure of D-LDH and its interaction with phospholipids. We incorporated 5-fluorotryptophan (5F-Trp) into the native enzyme, which contains five tryptophan residues, and into mutant enzymes, where a sixth tryptophan is substituted into a specific site by oligonucleotide-directed mutagenesis, and studied the 5F-Trp-labeled enzymes using 19F-NMR spectroscopy. In this way, information was obtained about the local environment at each native and substituted tryptophan site. Using a nitroxide spin-labeled fatty acid, which broadens the resonance from any residue within 15 A, we have established that the membrane-binding area of the protein includes the region between Tyr 228 and Phe 369, but is not continuous within this region. This conclusion is strengthened by the results of 19F-NMR spectroscopy of wild-type enzyme labeled with fluorotyrosine or fluorophenylalanine in the presence and absence of a nitroxide spin-labeled fatty acid. These experiments indicate that 9-10 Phe and 3-4 Tyr residues are located near the lipid phase.     

Metabolism of bradykinin analogs by angiotensin I converting enzyme and carboxypeptidase N.

Bradykinin (BK) analogs such as Lys-Lys-BK, des-Arg9-BK and [Leu8]des-Arg9-BK were poor substrates for angiotensin I converting enzyme (ACE), and analogs containing D-Phe7 residues, or a pseudopeptide C-terminal bond, were completely resistant. However, many of these analogs were metabolized by carboxypeptidase N (CPN) including Lys-Lys-BK, [Tyr8(OMe)]BK and D-Phe7-containing analogs, with Km and Vmax values comparable to those for BK. The only analogs completely resistant to both ACE and CPN were the B2 agonist [Phe8 psi(CH2NH)Arg9]BK, the B2 agonist D-Arg[Hyp3,D-Phe7,Phe8 psi(CH2NH)Arg9]BK, and the B1 agonist [D-Phe8]des-Arg9-BK. These data indicate an important role for plasma CPN and vascular CPN-like activity in the metabolism of the widely used ACE-resistant/D-Phe7-containing antagonists of B2 kinin receptors.     

Dimeric Crystal Structure of Rabbit l-Gulonate 3-Dehydrogenase/λ-Crystallin: Insights into the Catalytic Mechanism

l-Gulonate 3-dehydrogenase (GDH) is a bifunctional dimeric protein that functions not only as an NAD⁺-dependent enzyme in the uronate cycle but also as a taxon-specific λ-crystallin in rabbit lens. Here we report the first crystal structure of GDH in both apo form and NADH-bound holo form. The GDH protomer consists of two structural domains: the N-terminal domain with a Rossmann fold and the C-terminal domain with a novel helical fold. In the N-terminal domain of the NADH-bound structure, we identified 11 coenzyme-binding residues and found 2 distinct side-chain conformers of Ser124, which is a putative coenzyme/substrate-binding residue. A structural comparison between apo form and holo form and a mutagenesis study with E97Q mutant suggest an induced-fit mechanism upon coenzyme binding; coenzyme binding induces a conformational change in the coenzyme-binding residues Glu97 and Ser124 to switch their activation state from resting to active, which is required for the subsequent substrate recruitment. Subunit dimerization is mediated by numerous intersubunit interactions, including 22 hydrogen bonds and 104 residue pairs of van der Waals interactions, of which those between two cognate C-terminal domains are predominant. From a structure/sequence comparison within GDH homologues, a much greater degree of interprotomer interactions (both polar and hydrophobic) in the rabbit GDH would contribute to its higher thermostability, which may be relevant to the other function of this enzyme as λ-crystallin, a constitutive structural protein in rabbit lens. The present crystal structures and amino acid mutagenesis studies assigned the role of active-site residues: catalytic base for His145 and substrate binding for Ser124, Cys125, Asn196, and Arg231. Notably, Arg231 participates in substrate binding from the other subunit of the GDH dimer, indicating the functional significance of the dimeric state. Proper orientation of the substrate-binding residues for catalysis is likely to be maintained by an interprotomer hydrogen-bonding network of residues Asn196, Gln199, and Arg231, suggesting a network-based substrate recognition of GDH.     

Use of a double-headed peptide substrate to study the specificity of cAMP-dependent protein kinase and posphorylase kinase.

A peptide containing 2 seryl residues, (1)Leu(2)Ser(3)Tyr(4)Arg(5)Aly(6)Tyr(7)Ser(8)Leu, was chemically synthesized and used as a substrate for phosphorylase kinase and cyclic AMP-dependent protein kinase. The sequence, TryArgGlyTyr, makes up a beta turn in the native protein. Phosphorylase kinase was found to phosphorylate specifically seryl residue2 and protein kinase seryl residue7. Km and Vmax values were obtained and compared with natural substrates. The differences in the specificity of the two enzymes might be explained by a different requirement for organized structure. As a working hypothesis, it is suggested the results could be explained if the two enzymes interacted with seryl residues at different sides of a beta turn.     

Conformational basis for SH2-Tyr(P)527 binding in Src inactivation

Src protein-tyrosine kinase contains a myristoylation motif, a unique region, an Src homology (SH) 3 domain, an SH2 domain, a catalytic domain, and a C-terminal tail. The C-terminal tail contains a Tyr residue, Tyr527. Phosphorylation of Tyr527 triggers Src inactivation, caused by Tyr(P)527 binding to the SH2 domain. In this study, we demonstrated that a conformational contribution, not affinity, is the predominant force for the intramolecular SH2-Tyr(P)527 binding, and we characterized the structural basis for this conformational contribution. First, a phosphopeptide mimicking the C-terminal tail is an 80-fold weaker ligand than the optimal phosphopeptide, pYEEI, and similar to a phosphopeptide containing three Ala residues following Tyr(P) in binding to the Src SH2 domain. Second, the SH2-Tyr(P)527 binding is largely independent of the amino acid sequence surrounding Tyr(P)527, and only slightly decreased by an inactivating mutation in the SH2 domain. Furthermore, even the unphosphorylated C-terminal tail with the sequence of YEEI suppresses Src activity by binding to the SH2 domain. These experiments demonstrate that very weak affinity is sufficient for the SH2-Tyr(P)527 binding in Src inactivation. Third, the effective intramolecular SH2-Tyr(P)527 binding is attributed to a conformational contribution that requires residues Trp260 and Leu255. Although the SH3 domain is essential for Src inactivation by Tyr(P)527, it does not contribute to the SH2-Tyr(P)527 binding. These findings suggest a conformation-based Src inactivation model, which provides a unifying framework for understanding Src activation by a variety of mechanisms.     

The contribution of acidic residues to the conformational stability of common-type acylphosphatase

Common-type acylphosphatase is a small cytosolic enzyme whose catalytic properties and three-dimensional structure are known in detail. All the acidic residues of the enzyme have been replaced by noncharged residues in order to assess their contributions to the conformational stability of acylphosphatase. The enzymatic activity parameters and the conformational free energy of each mutant were determined by enzymatic activity assays and chemically induced unfolding, respectively. Some mutants exhibit very similar conformational stability, DeltaG(H2O), and specific activity values as compared to the wild-type enzyme. By contrast, six mutants show a significant reduction of conformational stability and two mutants are more stable than the wild-type protein. Although none of the mutated acidic residues is directly involved in the catalytic mechanism of the enzyme, our results indicate that mutations of residues located on the surface of the protein are responsible for a structural distortion which propagate up to the active site. We found a good correlation between the free energy of unfolding and the enzymatic activity of acylphosphatase. This suggests that enzymatic activity measurements can provide valuable indications on the conformational stability of acylphosphatase mutants, provided the mutated residue lies far apart from the active site. Moreover, our results indicate that the distortion of hydrogen bonds rather than the loss of electrostatic interactions, contributes to the decrease of the conformational stability of the protein.