Examination of an active-site electrostatic node in the cAMP-dependent protein kinase catalytic subunit.

The active site of the cAMP-dependent protein kinase catalytic subunit harbors a cluster of acidic residues-Asp 127, Glu 170, Glu 203, Glu 230, and Asp 241-that are not conserved throughout the protein kinase family. Based on crystal structures of the catalytic subunit, these amino acids are removed from the site of phosphoryl transfer and are implicated in substrate recognition. Glu 230, the most buried of these acidic residues, was mutated to Ala (rC[E230A]) and Gln (rC[E230Q]) and overexpressed in Escherichia coli. In contrast to the mostly insoluble and destabilized rC[E230A], rC[E230Q] is largely soluble, purifies like wild-type enzyme, and displays wild-type-like thermal stability. The mutation in rC[E230Q] causes an order of magnitude decrease in the affinity for a heptapeptide substrate, Kemptide. In addition, two independent kinetic techniques were used to dissect phosphoryl transfer and product release steps in the reaction pathway. Viscosometric and pre-steady-state quench-flow analyses revealed that the phosphoryl transfer rate constant decreases by an order of magnitude, whereas the product release rate constant remains unperturbed. Electrostatic alterations in the rC[E230Q] active site were assessed using modeling techniques that provide molecular interpretations for the substrate affinity and phosphoryl transfer rate decreases observed experimentally. These observations indicate that subsite recognition elements in the catalytic subunit make electrostatic contributions that are important not only for peptide affinity, but also for catalysis. Protein kinases may, therefore, discriminate substrates by not only binding them tightly, but also by only turning over ones that complement the electrostatic character of the active site.     

Crystal structure of the E230Q mutant of cAMP-dependent protein kinase reveals an unexpected apoenzyme conformation and an extended N-terminal A helix

Glu230, one of the acidic residues that cluster around the active site of the catalytic subunit of cAMP-dependent protein kinase, plays an important role in substrate recognition. Specifically, its side chain forms a direct salt-bridge interaction with the substrate's P-2 Arg. Previous studies showed that mutation of Glu230 to Gln (E230Q) caused significant decreases not only in substrate binding but also in the rate of phosphoryl transfer. To better understand the importance of Glu230 for structure and function, we solved the crystal structure of the E230Q mutant at 2.8 A resolution. Surprisingly, the mutant preferred an open conformation with no bound ligands observed, even though the crystals were grown in the presence of MgATP and the inhibitor peptide, IP20. This is in contrast to the wild-type protein that, under the same conditions, prefers the closed conformation of a ternary complex. The structure highlights the importance of the electrostatic surface not only for substrate binding and catalysis, but also for the mechanism for closing the active site cleft. This surface mutation clearly disrupts the recognition and binding of substrate peptide so that the enzyme prefers an open conformation that cannot trap ATP. This is consistent with the reinforcing concepts of conformational dynamics and the synergistic binding of ATP and substrate peptide. Another unusual feature of the structure is the observation of the entire N terminus (Gly1-Thr32) assumes an extended alpha-helix conformation. Finally, based on temperature factors, this mutant structure is more stable than the wild-type C-subunit in the apo state.     

Allosteric network of cAMP-dependent protein kinase revealed by mutation of Tyr204 in the P+1 loop

Previous studies on the catalytic subunit of cAMP-dependent protein kinase (PKA) identified a conserved interaction pair comprised of Tyr204 from the P+1 loop and Glu230 at the end of the alphaF-helix. Single-point mutations of Tyr204 to Ala (Y204A) and Glu230 to Gln (E230Q) both resulted in alterations in enzymatic kinetics. To understand further the molecular basis for the altered kinetics and the structural role of each residue, we analyzed the Y204A and the E230Q mutants using hydrogen/deuterium (H/D) exchange coupled with mass spectrometry and other biophysical techniques. The fact that the mutants exhibit distinct molecular properties, supports previous hypotheses that these two residues, although in the same interaction node, contribute to the same enzymatic functions through different molecular pathways. The Tyr204 mutation appears to affect the dynamic properties, while the Glu230 mutation affects the surface electrostatic profile of the enzyme. Furthermore, H/D exchange analysis defines the dynamic allosteric range of Tyr204 to include the catalytic loop and three additional distant surface regions, which exhibit increased deuterium exchange in the Y204A but not the E230Q mutant. Interestingly, these are the exact regions that previously showed decreased deuterium exchange upon binding of the RIalpha regulatory subunit of PKA. We propose that these sites, coupled with the P+1 loop through Tyr204, represent one of the major allosteric networks in the kinase. This coupling provides a coordinated response for substrate binding and enzyme catalysis. H/D exchange analysis also further defines the stable core of the catalytic subunit to include the alphaE, alphaF and alphaH-helix. All these observations lead to an interesting new way to view the structural architecture and allosteric conformational regulation of the protein kinase molecule.     

Catalytic subunit of cAMP-dependent protein kinase: Electrostatic features and peptide recognition

The electrostatic field was calculated for the mammalian cAMP-dependent protein kinase (PKA) catalytic subunit (C-subunit) complexed with a 20-residue peptide from a heat stable protein kinase inhibitor (PKI: 5–24). The electrostatic field was also calculated for the C-subunit complexed with a modeled heptapeptide substrate that has been used extensively in structure/function studies for the C-subunit. Perturbations in the electrostatic free energy were calculated when single ionizable active site residues were mutated to alanine. These perturbations in electrostatic free energy were correlated to changes in the binding energy measured in a charge-to-alanine scan of the homologous yeast C-subunit by M. J. Zoller and C. S. Gibbs [(1991) Journal of Biological Chemistry, Vol. 266, pp. 8923–8931; C. S. Gibbs and M. J. Zoller (1991) Biochemistry, Vol. 30, p. 22]. This analysis indicated that the substrate binding parameters primarily depend on electrostatic interactions between a substrate or inhibitor and the C-subunit. Amino acid replacements that led to large perturbations in the electrostatic field are listed in the text. pK_a shifts were also calculated for the substrate's phosphate accepting atom, the serine hydroxyl oxygen, when the active site ionizable residues were changed to structurally similar uncharged amino acids. The theoretical mutation of three active site residues caused large shifts in this parameter: E91Q, D166N, and D184N. The calculated pK_a shifts for these mutants indicate that the rate of phosphotransfer should be markedly reduced in these cases. This prediction has been experimentally confirmed for the D166N mutant. The correlation between calculated electrostatic free energy changes and measured binding energy, and pK_a shifts with phosphotransfer for C-subunit mutants were within experimental error of the measurements. The calculations of electrostatic energy and ΔpK_a have identified previously unconsidered active site residues in the mammalian C-subunit that contribute to binding energy and phosphotransfer. © 1996 John Wiley & Sons, Inc.     

The α-Helix of Ribonuclease T1as an Independent Stability Unit: Direct Comparison of Peptide and Protein Stability

Measurements of the change in conformational stability, Δ(ΔG), upon mutation of two acidic residues at the C terminus of the helix of ribonuclease T₁have recently been reported. Here, we investigate peptides based on the sequence of the helix with the same mutations: Glu28 replaced with Gln, Asp29 replaced with Asn, and the double mutant. In addition, the mutant Lys25 to Gln was studied. Changes in helix content of the peptides with pH confirm the conclusion found in the intact protein, that the charged forms of the acidic residues destabilize the protein by destabilizing the helix. The pH-dependence of the change in confor mational free energy for the peptides and mutant proteins show fair correspondence for D29N and the double mutant. The mutants E28Q and K25Q, on the other hand, give striking agreement between the protein and peptide systems. This agreement suggests that the helix of ribonuclease T₁behaves as an independently stabilized structural unit of the intact protein and that stabilization of the helical form of the peptide is mirrored in the protein.     

Effects of metal-binding loop mutations on ligand binding to calcium- and integrin-binding protein 1. Evolution of the EF-hand?

Calcium- and integrin-binding protein 1 (CIB1) is a ubiquitous, multifunctional regulatory protein consisting of four helix-loop-helix EF-hand motifs. Neither EF-I nor EF-II binds divalent metal ions; however, EF-III is a mixed Mg2+/Ca2+-binding site, and EF-IV is a higher-affinity Ca2+-specific site. Through the generation of several CIB1 mutant proteins, we have investigated the importance of the last (-Z) metal-coordinating position of EF-III (D127) and EF-IV (E172) with respect to the binding of CIB1 to Mg2+, Ca2+, and its biological target, the cytoplasmic domain of the platelet alphaIIb integrin. A D127N mutant had reduced Mg2+ and Ca2+ affinity at EF-III but retained affinity for the alphaIIb domain. A D127E mutant had increased Mg2+ and Ca2+ affinity at EF-III, but unexpectedly, the affinity for the alphaIIb domain was too low for binding to be observed. E172Q and E172D mutants showed no and weak Mg2+ binding at EF-IV, respectively, and each mutant had reduced Ca2+ affinity at EF-IV and showed moderate metal-dependent differences in affinity for the alphaIIb domain. Finally, a D127Q mutant bound Mg2+ and Ca2+ in a manner similar to that of D127N, but like that of D127E, the affinity for the alphaIIb domain was reduced below the detection limit. These data, combined with a NMR-based structural comparison of the Mg2+- and Ca2+-loaded CIB1-alphaIIb peptide complexes, suggest that the D127E and D127Q mutations have a disruptive effect on alphaIIb binding since they expand the metal-binding loop and change the alpha-helix positions in EF-III. Conversely, upon replacement of the ancestral Glu with Asp at the -Z position of EF-III, CIB1 gained affinity for alphaIIb, and the Ca2+ affinity of CIB1 shifted into a range where the protein is able to act as an intracellular Ca2+ sensor.     

Role of N-Terminal Myristylation in the Structure and Regulation of cAMP-Dependent Protein Kinase

The catalytic (C) subunit of cAMP-dependent protein kinase [protein kinase A (PKA)] is a major target of cAMP signaling, and its regulation is of fundamental importance to biological processes. One mode of regulation is N-myristylation, which has eluded structural and functional characterization so far because most crystal structures are of the non-myristylated enzyme, are phosphorylated on Ser10, and generally lack electron density for the first 13 residues. We crystallized myristylated wild-type (WT) PKA and a K7C mutant as binary (bound to a substrate peptide) and ternary [bound to a substrate peptide and adenosine-5′-(β,γ-imido)triphosphate] complexes. There was clear electron density for the entire N-terminus in the binary complexes, both refined to 2.0 Å, and K7C ternary complex, refined to 1.35 Å. The N-termini in these three structures display a novel conformation with a previously unseen helix from residues 1 to 7. The K7C mutant appears to have a more stable N-terminus, and this correlated with a significant decrease in the B-factors for the N-terminus in the myr-K7C complexes compared to the WT binary complex. The N-terminus of the myristylated WT ternary complex, refined to 2.0 Å, was disordered as in previous structures. In addition to a more ordered N-terminus, the myristylated K7C mutant exhibited a 53% increase in k_cat. The effect of nucleotide binding on the structure of the N-terminus in the WT protein and the kinetic changes in the K7C protein suggest that myristylation or occupancy of the myristyl binding pocket may serve as a site for allosteric regulation in the C-subunit.     

X-ray absorption and molecular dynamics study of cation binding sites in the purple membrane

The present work describes the results of a study aimed at identifying candidate cation binding sites on the extracellular region of bacteriorhodopsin, including a site near the retinal pocket. The approach used is a combined effort involving computational chemistry methods (computation of cation affinity maps and molecular dynamics) together with the Extended X-Ray Absorption Fine Structure (EXAFS) technique to obtain relevant information about the local structure of the protein in the neighborhood of Mn(2+) ions in different affinity binding sites. The results permit the identification of a high-affinity binding site where the ion is coordinated simultaneously to Asp212(-) and Asp85(-). Comparison of EXAFS data of the wild type protein with the quadruple mutant E9Q/E74Q/E194Q/E204Q at pH 7.0 and 10.0 demonstrate that extracellular glutamic acid residues are involved in cation binding.     

Manipulating conformational equilibria in the lactose permease of Escherichia coli.

A mechanism proposed for lactose/H(+) symport by the lactose permease of Escherichia coli indicates that lactose permease is protonated prior to ligand binding. Moreover, in the ground state, the symported H(+) is shared between His322 (helix X) and Glu269 (helix VIII), while Glu325 (helix X) is charge-paired with Arg302 (helix IX). Substrate binding at the outer surface between helices IV (Glu126) and V (Arg144, Cys148) induces a conformational change that leads to transfer of the H(+) to Glu325 and reorientation of the binding site to the inner surface. After release of substrate, Glu325 is deprotonated on the inside due to re-juxtapositioning with Arg302. The conservative mutation Glu269-->Asp causes a 50-100-fold decrease in substrate binding affinity and markedly reduced active lactose transport, as well as decreased rates of equilibrium exchange and efflux. Gly-scanning mutagenesis of helix VIII was employed systematically with mutant Glu269-->Asp in an attempt to rescue function, and two mutants with increased activity are identified and characterized. Mutant Thr266-->Gly/Met267-->Gly/Glu269-->Asp binds ligand with increased affinity and catalyzes active lactose transport with a marked increase in rate; however, little improvement in efflux or equilibrium exchange is observed. In contrast, mutant Gly262-->Ala/Glu269-->Asp exhibits no improvement in ligand binding but a small increase in the rate of active transport; however, an increase in the steady-state level of accumulation, as well as efflux and equilibrium exchange is observed. Remarkably, when the two sets of mutations are combined, all translocation reactions are rescued to levels approximating those of wild-type permease. The findings support the contention that Glu269 plays a pivotal role in the mechanism of lactose/H(+) symport. Moreover, the results suggest that the two classes of mutants rescue activity by altering the equilibrium between outwardly and inwardly facing conformations of the permease such that impaired protonation and/or H(+) transfer is enhanced from one side of the membrane or the other. When the two sets of mutants are combined, the equilibrium between outwardly and inwardly facing conformations and thus protonation and H(+) transfer are restored.     

Dissection of the energetic coupling across the Src SH2 domain-tyrosyl phosphopeptide interface

Src Homology (SH2) domains play critical roles in signaling pathways by binding to phosphotyrosine (pTyr)-containing sequences, thereby recruiting SH2 domain-containing proteins to tyrosine-phosphorylated sites on receptor molecules. Investigations of the peptide binding specificity of the SH2 domain of the Src kinase (Src SH2 domain) have defined the EEI motif C-terminal to the phosphotyrosine as the preferential binding sequence. A subsequent study that probed the importance of eight specificity-determining residues of the Src SH2 domain found two residues which when mutated to Ala had significant effects on binding: Tyr beta D5 and Lys beta D3. The mutation of Lys beta D3 to Ala was particularly intriguing, since a Glu to Ala mutation at the first (+1) position of the EEI motif (the residue interacting with Lys beta D3) did not significantly affect binding. Hence, the interaction between Lys beta D3 and +1 Glu is energetically coupled. This study is focused on the dissection of the energetic coupling observed across the SH2 domain-phosphopeptide interface at and around the +1 position of the peptide. It was found that three residues of the SH2 domain, Lys beta D3, Asp beta C8 and AspCD2 (altogether forming the so-called +1 binding region) contribute to the selection of Glu at the +1 position of the ligand. A double (Asp beta C8Ala, AspCD2Ala) mutant does not exhibit energetic coupling between Lys beta D3 and +1 Glu, and binds to the pYEEI sequence 0.3 kcal/mol tighter than the wild-type Src SH2 domain. These results suggest that Lys beta D3 in the double mutant is now free to interact with the +1 Glu and that the role of Lys beta D3 in the wild-type is to neutralize the acidic patch formed by Asp beta C8 and AspCD2 rather than specifically select for a Glu at the +1 position as it had been hypothesized previously. A triple mutant (Lys beta D3Ala, Asp beta C8Ala, AspCD2Ala) has reduced binding affinity compared to the double (Asp beta C8Ala, AspCD2Ala) mutant, yet binds the pYEEI peptide as well as the wild-type Src SH2 domain. The structural basis for such high affinity interaction was investigated crystallographically by determining the structure of the triple (Lys beta D3Ala, Asp beta C8Ala, AspCD2Ala) mutant bound to the octapeptide PQpYEEIPI (where pY indicates a phosphotyrosine). This structure reveals for the first time contacts between the SH2 domain and the -1 and -2 positions of the peptide (i.e. the two residues N-terminal to pY). Thus, unexpectedly, mutations in the +1 binding region affect binding of other regions of the peptide. Such additional contacts may account for the high affinity interaction of the triple mutant for the pYEEI-containing peptide.     

Substrate recognition mechanism of a glycosyltrehalose trehalohydrolase from Sulfolobus solfataricus KM1

Glycosyltrehalose trehalohydrolase (GTHase) is an α-amylase that cleaves the α-1,4 bond adjacent to the α-1,1 bond of maltooligosyltrehalose to release trehalose. To investigate the catalytic and substrate recognition mechanisms of GTHase, two residues, Asp252 (nucleophile) and Glu283 (general acid/base), located at the catalytic site of GTHase were mutated (Asp252→Ser (D252S), Glu (D252E) and Glu283→Gln (E283Q)), and the activity and structure of the enzyme were investigated. The E283Q, D252E, and D252S mutants showed only 0.04, 0.03, and 0.6% of enzymatic activity against the wild-type, respectively. The crystal structure of the E283Q mutant GTHase in complex with the substrate, maltotriosyltrehalose (G3-Tre), was determined to 2.6-Å resolution. The structure with G3-Tre indicated that GTHase has at least five substrate binding subsites and that Glu283 is the catalytic acid, and Asp252 is the nucleophile that attacks the C1 carbon in the glycosidic linkage of G3-Tre. The complex structure also revealed a scheme for substrate recognition by GTHase. Substrate recognition involves two unique interactions: stacking of Tyr325 with the terminal glucose ring of the trehalose moiety and perpendicularly placement of Trp215 to the pyranose rings at the subsites −1 and +1 glucose.     

Identification of electrostatic interaction sites between the regulatory and catalytic subunits of cyclic AMP-dependent protein kinase

Two classes of molecules inhibit the catalytic subunit (C) of the cyclic AMP-dependent protein kinase (cAPK), the heat-stable protein kinase inhibitors (PKIs) and the regulatory (R) subunits. Basic sites on C, previously identified as important for R/C interaction in yeast TPKI and corresponding to Lys213, Lys217, and Lys189 in murine Cα, were replaced with either Ala or Thr and characterized for their kinetic properties and ability to interact with RI and PKI. rC(K213A) and rC(K217A) were both defective in forming holoenzyme with RI but were inhibited readily with PKI. This contrasts with rC(R133A), which is defective in binding PKI but not RI (Wen & Taylor, 1994). Thus, the C-subunit employs two distinct electrostatic surfaces to achieve high-affinity binding with these two types of inhibitory molecules even though all inhibitors share a common consensus site that occupies the active site cleft. Unlike TPK1, mutation of Lys189 had no effect. The mutant C subunits that were defective in binding RI, rC(K213A) and rC(K217A), were then paired with three RI mutants, rRI(D140A), rRI(E143A), and rRI(D258A), shown previously to be defective in recognition of C. Although the mutations at Asp140 and Asp258 in RI were additive with respect to the C mutations, rC(K213A) and rRI(E143A) were compensatory, thus identifying a specific electrostatic interaction site between RI and C. The results are discussed in terms of the RI and C crystal structures and the sequence homology between the yeast and mammalian enzymes.     

Crystal structure of a hyperactive Escherichia coli glycerol kinase mutant Gly230 --> Asp obtained using microfluidic crystallization devices

The crystal structure of an Escherichia coli glycerol kinase mutant Gly230 --> Asp (GKG230D) was determined to 2.0 A resolution using a microfluidics based crystallization platform. The crystallization strategy involved a suite of microfluidic devices that characterized the solubility trends of GKG230D, performed nanoliter volume free interface diffusion crystallization experiments, and produced diffraction-quality crystals for in situ data collection. GKG230D displays increased enzymatic activity and decreased allosteric regulation by the glycolytic pathway intermediate fructose 1,6-bisphosphate (FBP) compared to wild-type GK (GKWT). Structural analysis revealed that the decreased allosteric regulation is a result of the altered FBP binding loop conformations in GKG230D that interfere with the wild-type FBP binding site. The altered FBP binding loop conformations in GKG230D are supported through a series of intramolecular loop interactions. The appearance of Asp230 in the FBP binding loops also repositions the wild-type FBP binding residues away from the FBP binding site. Light scattering analysis confirmed GKG230D is a dimer and is resistant to tetramer formation in the presence of FBP, whereas GKWT dimers are converted into putatively inactive tetramers in the presence of FBP. GKG230D also provides the first structural evidence for multiple GK monomer conformations in the presence of glycerol and in the absence of a nucleotide substrate and verifies that glycerol binding is not responsible for locking GK into the closed conformation necessary for GK activity.     

Structural basis for the low affinities of yeast cAMP-dependent and mammalian cGMP-dependent protein kinases for protein kinase inhibitor peptides.

Affinities of the catalytic subunit (C1) of Saccharomyces cerevisiae cAMP-dependent protein kinase and of mammalian cGMP-dependent protein kinase were determined for the protein kinase inhibitor (PKI) peptide PKI(6-22)amide and seven analogues. These analogues contained structural alterations in the N-terminal alpha-helix, the C-terminal pseudosubstrate portion, or the central connecting region of the PKI peptide. In all cases, the PKI peptides were appreciably less active as inhibitors of yeast C1 than of mammalian C alpha subunit. Ki values ranged from 5- to 290-fold higher for the yeast enzyme than for its mammalian counterpart. Consistent with these results, yeast C1 exhibited a higher Km for the peptide substrate Kemptide. All of the PKI peptides were even less active against the mammalian cGMP-dependent protein kinase than toward yeast cAMP-dependent protein kinase, and Kemptide was a poorer substrate for the former enzyme. Alignment of amino acid sequences of these homologous protein kinases around residues in the active site of mammalian C alpha subunit known to interact with determinants in the PKI peptide [Knighton, D. R., Zheng, J., Ten Eyck, L. F., Xuong, N-h, Taylor, S. S., & Sowadski, J. M. (1991) Science 253, 414-420] provides a structural basis for the inherently lower affinities of yeast C1 and cGMP-dependent protein kinase for binding peptide inhibitors and substrates. Both yeast cAMP-dependent and mammalian cGMP-dependent protein kinases are missing two of the three acidic residues that interact with arginine-18 in the pseudosubstrate portion of PKI. Further, the cGMP-dependent protein kinase appears to completely lack the hydrophobic/aromatic pocket that recognizes the important phenylalanine-10 residue in the N-terminus of the PKI peptide, and binding of the inhibitor by the yeast protein kinase at this site appears to be partially compromised.     

Mutagenesis of folylpolyglutamate synthetase indicates that dihydropteroate and tetrahydrofolate bind to the same site

The folylpolyglutamate synthetase (FPGS) enzyme of Escherichia coli differs from that of Lactobacillus casei in having dihydrofolate synthetase activity, which catalyzes the production of dihydrofolate from dihydropteroate. The present study undertook mutagenesis to identify structural elements that are directly responsible for the functional differences between the two enzymes. The amino terminal domain (residues 1-287) of the E. coli FPGS was found to bind tetrahydrofolate and dihydropteroate with the same affinity as the intact enzyme. The domain-swap chimera proteins between the E. coli and the L. casei enzymes possess both folate or pteroate binding properties and enzymatic activities of their amino terminal portion, suggesting that the N-terminal domain determines the folate substrate specificity. Recent structural studies have identified two unique folate binding sites, the omega loop in L. casei FPGS and the dihydropteroate binding loop in the E. coli enzyme. Mutants with swapped omega loops retained the activities and folate or pteroate binding properties of the rest of the enzyme. Mutating L. casei FPGS to contain an E. coli FPGS dihydropteroate binding loop did not alter its substrate specificity to using dihydropteroate as a substrate. The mutant D154A, a residue specific for the dihydropteroate binding site in E. coli FPGS, and D151A, the corresponding mutant in the L. casei enzyme, were both defective in using tetrahydrofolate as their substrate, suggesting that the binding site corresponding to the E. coli pteroate binding site is also the tetrahydrofolate binding site for both enzymes. Tetrahydrofolate diglutamate was a slightly less effective substrate than the monoglutamate with the wild-type enzyme but was a 40-fold more effective substrate with the D151A mutant. This suggests that the 5,10-methylenetetrahydrofolate binding site identified in the L. casei ternary structure may bind diglutamate and polyglutamate folate derivatives.     

Identification of an aspartic residue in the P-loop of the vanilloid receptor that modulates pore properties

Vanilloid receptor subunit 1 (VR1) is a nonselective cation channel that integrates multiple pain-producing stimuli. VR1 channels are blocked with high efficacy by the well established noncompetitive antagonist ruthenium red and exhibit high permeability to divalent cations. The molecular determinants that define these functional properties remain elusive. We have addressed this question and evaluated by site-specific neutralization the contribution on pore properties of acidic residues located in the putative VR1 pore region. Mutant receptors expressed in Xenopus oocytes exhibited capsaicin-operated ionic currents akin to those of wild type channels. Incorporation of glutamine residues at Glu(648) and Glu(651) rendered minor effects on VR1 pore attributes, while Glu(636) slightly modulated pore blockade. In contrast, replacement of Asp(646) by asparagine decreased 10-fold ruthenium red blockade efficacy and reduced 4-fold the relative permeability of the divalent cation Mg(2+) with respect to Na(+) without changing the selectivity of monovalent cations. At variance with wild type channels and E636Q, E648Q, and E651Q mutant receptors, ruthenium red blockade of D646N mutants was weakly sensitive to extracellular pH acidification. Collectively, our results suggest that Asp(646) is a molecular determinant of VR1 pore properties and imply that this residue may form a ring of negative charges that structures a high affinity binding site for cationic molecules at the extracellular entryway.     

Study on structure and assembly of the third transmembrane domain of Slc11a1

The structure and self‐assembly of the peptide corresponding to the third transmembrane domain (TMD3) of Slc11a1 and its E139A mutant are studied in 1,1,1,3,3,3‐hexafluoro‐2‐propanol (HFIP) aqueous solution by NMR and CD experiments. Slc11a1 is an integral membrane protein with 12 putative TMDs and functions as a pH‐coupled divalent metal cation transporter. Glu139 of Slc11a1 is highly conserved within predicted TMD3 of the Slc11 protein family and function‐associated. Here, we provide the first direct experimental evidence for the structural features of two 24‐residue peptides corresponding to TMD3 of Slc11a1 and its E139A mutant in 60% HFIP‐d₂ aqueous solution using CD and NMR spectroscopies. Our study shows that the membrane‐spanning peptide folds as a typical amphipathic α‐helix structure from Ile5 to Met20 with hydrophilic residues Glu12 (Glu139 in Slc11a1) and Asp19 lying on the same side of the helix. The substitution of Glu139 by an alanine residue has little effect on the structure of the peptide, but increases hydrophobicity and facilitates self‐assembly of the peptide. Although the wildtype peptide is monomeric in HFIP aqueous solution, the E139A mutant forms a dimer. The increase in hydrophobicity of the membrane‐spanning peptide and/or change in the interactions between transmembrane segments induced by E139A mutation may affect the metal ion transport of the protein. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Ca2+ occlusion and gating function of Glu309 in the ADP-fluoroaluminate analog of the Ca2+-ATPase phosphoenzyme intermediate

In the absence of ATP the sarcoplasmic reticulum ATPase (SERCA) binds two Ca(2+) with high affinity. The two bound Ca(2+) rapidly undergo reverse dissociation upon addition of EGTA, but can be distinguished by isotopic exchange indicating fast exchange at a superficial site (site II), and retardation of exchange at a deeper site (site I) by occupancy of site II. Site II mutations that allow high affinity binding to site I, but only low affinity binding to site II, show that retardation of isotopic exchange requires higher Ca(2+) concentrations with the N796A mutant, and is not observed with the E309Q mutant even at millimolar Ca(2+). Fluoroaluminate forms a complex at the catalytic site yielding stable analogs of the phosphoenzyme intermediate, with properties similar to E2-P or E1-P.Ca(2). Mutational analysis indicates that Asp(351), Lys(352), Thr(353), Asp(703), Asn(706), Asp(707), Thr(625), and Lys(684) participate in stabilization of fluoroaluminate and Mg(2+) at the phosphorylation site. In the presence of fluoroaluminate and Ca(2+), ADP (or AMP-PCP) favors formation of a stable ADP.E1-P.Ca(2) analog. This produces strong occlusion of Ca(2+) bound to both sites (I and II), whereby dissociation occurs very slowly even following addition of EGTA. Occlusion by fluoraluminate and ADP is not observed with the E309Q mutant, suggesting a gating function of Glu(309) at the mouth of a binding cavity with a single path of entry. This phenomenon corresponds to the earliest step of the catalytic cycle following utilization of ATP. Experiments on limited proteolysis reveal that a long range conformational change, involving displacement of headpiece domains and transmembrane helices, plays a mechanistic role.     

Glutamic acid 71 and aspartic acid 66 control the binding of the second calcium ion in porcine pancreatic phospholipase A2

In addition to the Ca2+ ion at the active site, porcine pancreatic phospholipase A2 (PLA) is known to bind a second calcium ion with a lower affinity at alkaline pH. The second calcium-binding site has been held responsible for effective interaction of phospholipase with organized lipid/water interfaces [van Dam-Mieras, M. C. E., Slotboom, A. J., Pieterson, W. A. and de Haas, G. H. (1975) Biochemistry 14, 5387-5394]. To study the identity of the acidic amino acid residues involved in liganding the second calcium ion in detail, we used site-directed mutagenesis to specifically alter the cDNA encoding porcine pancreatic phospholipase. Three mutant phospholipase species were constructed, each of which lacked one of the potentially important carboxylates: Asp66----Asn, Glu71----Asn and Glu92----Gln. The Gln92 mutant PLA displayed the same properties as native phospholipase indicating that Glu92 is not important for binding the second metal ion. However, Glu71 and, to a lesser extent, Asp66 are both directly involved in the low-affinity calcium binding.     

Serine-53 at the tip of the glycine-rich loop of cAMP-dependent protein kinase: role in catalysis, P-site specificity, and interaction with inhibitors

The glycine-rich loop, one of the most important motifs in the conserved protein kinase catalytic core, embraces the entire nucleotide, is very mobile, and is exquisitely sensitive to what occupies the active site cleft. Of the three conserved glycines [G(50)TG(52)SFG(55) in cAMP-dependent protein kinase (cAPK)], Gly(52) is the most important for catalysis because it allows the backbone amide of Ser(53) at the tip of the loop to hydrogen bond to the gamma-phosphate of ATP [Grant, B. D. et al. (1998) Biochemistry 37, 7708]. The structural model of the catalytic subunit:ATP:PKI((5)(-)(24)) (heat-stable protein kinase inhibitor) ternary complex in the closed conformation suggests that Ser(53) also might be essential for stabilization of the peptide substrate-enzyme complex via a hydrogen bond between the P-site carbonyl in PKI and the Ser(53) side-chain hydroxyl [Bossemeyer, D. et al. (1993) EMBO J. 12, 849]. To address the importance of the Ser(53) side chain in catalysis, inhibition, and P-site specificity, Ser(53) was replaced with threonine, glycine, and proline. Removal of the side chain (i.e., mutation to glycine) had no effect on the steady-state phosphorylation of a peptide substrate (LRRASLG) or on the interaction with physiological inhibitors, including the type-I and -II regulatory subunits and PKI. However, this mutation did affect the P-site specificity; the glycine mutant can more readily phosphorylate a P-site threonine in a peptide substrate (5-6-fold better than wild-type). The proline mutant is compromised catalytically with altered k(cat) and K(m) for both peptide and ATP and with altered sensitivity to both regulatory subunits and PKI. Steric constraints as well as restricted flexibility could account for these effects. These combined results demonstrate that while the backbone amide of Ser(53) may be required for efficient catalysis, the side chain is not.