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

Tsigelny, Igor F.; Grant, Bruce D.; Taylor, Susan S.; Eyck, Lynn F. Ten

doi:10.1002/(sici)1097-0282(199609)39:3<353::aid-bip7>3.0.co;2-n

Cited by 17 publications

(20 citation statements)

References 19 publications

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“…It recognizes the P-2 Arg of the peptide substrates and contributes significantly to the electrostatic properties of the peptide binding site. 7 Mutation of Glu230 to Gln (E230Q) demonstrated the importance of this residue. The E230Q mutant enzyme has reduced negative electrostatic potential on the surface of the peptide binding site.…”

Section: Introductionsupporting

confidence: 92%

“…The ionic strength was equivalent to 0.145 M sodium chloride. 7 The radii of ions were 2.0 Å , while the solvent radius was 1.4 Å . The system temperature was set at 298.15 K.…”

Section: Electrostatics Calculationsmentioning

confidence: 99%

“…The E230Q mutant enzyme has reduced negative electrostatic potential on the surface of the peptide binding site. 7 The mutant is also defective in binding peptide substrates and deficient in catalyzing the phosphoryl transfer reaction. 8 Kinetic data showed that the E230Q mutation affected the binding of ATP with only 2-fold perturbation but induced a 200-fold reduction in the binding of the peptide substrate, Kemptide.…”

Section: Introductionmentioning

confidence: 99%

“…8 Kinetic data showed that the E230Q mutation affected the binding of ATP with only 2-fold perturbation but induced a 200-fold reduction in the binding of the peptide substrate, Kemptide. 8 Another important feature of the E230Q mutant is the loss of the synergistic binding of MgATP and protein kinase inhibitor PKI (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24) to the mutant. The mutant was unable to form ternary complex crystals in the presence of MgATP and PKI.…”

Section: Introductionmentioning

confidence: 99%

“…Hence, it is very likely that the altered surface electrostatic profile of the E230Q mutant causes the observed defective kinetic properties of the enzyme. 7,8,10 We applied the relaxed-complex method described by Lin et al 11 to study the effects of the E230Q mutation on PKI binding. We obtained a qualitative estimation of the probability of PKI docking to the Csubunits.…”

Section: Introductionmentioning

confidence: 99%

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E230Q mutation of the catalytic subunit of cAMP‐dependent protein kinase affects local structure and the binding of peptide inhibitor

Ung

McCammon

2006

Biopolymers

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The active site of the mammalian cAMP-dependent protein kinase catalytic subunit (C-subunit) has a cluster of nonconserved acidic residues-Glu127, Glu170, Glu203, Glu230, and Asp241-that are crucial for substrate recognition and binding. Studies have shown that the Glu230 to Gln mutant (E230Q) of the enzyme has physical properties similar to the wild-type enzyme and has decreased affinity for a short peptide substrate, Kemptide. However, recent experiments intended to crystallize ternary complex of the E230Q mutant with MgATP and protein kinase inhibitor (PKI) could only obtain crystals of the apo-enzyme of E230Q mutant. To deduce the possible mechanism that prevented ternary complex formation, we used the relaxed-complex method (Lin, J.-H., et al. J Am Chem Soc 2002, 24, 5632-5633) to study PKI binding to the E230Q mutant C-subunit. In the E230Q mutant, we observed local structural changes of the peptide binding site that correlated closely to the reduced PKI affinity. The structural changes occurred in the F-to-G helix loop and appeared to hinder PKI binding. Reduced electrostatic potential repulsion among Asp241 from the helix loop section and the other acidic residues in the peptide binding site appear to be responsible for the structural change. # 2005 Wiley Periodicals, Inc.

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Section: Introductionsupporting

confidence: 92%

“…The ionic strength was equivalent to 0.145 M sodium chloride. 7 The radii of ions were 2.0 Å , while the solvent radius was 1.4 Å . The system temperature was set at 298.15 K.…”

Section: Electrostatics Calculationsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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E230Q mutation of the catalytic subunit of cAMP‐dependent protein kinase affects local structure and the binding of peptide inhibitor

Ung

McCammon

2006

Biopolymers

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600 ps Molecular dynamics reveals stable substructures and flexible hinge points in cAMP dependent protein kinase

Tsigelny¹,

Greenberg²,

Cox³

et al. 1999

Biopolymers

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Molecular dynamics simulations of the catalytic subunit of cAMP dependent protein kinase (cAPK) have been performed in an aqueous environment. The relations among the protein hydrogen-bonding network, secondary structural elements, and the internal motions of rigid domains were examined. The values of fluctuations of protein dihedral angles during dynamics show quite distinct maxima in the regions of loops and minima in the regions of ␣-helices and ␤-strands. Analyses of conformation snapshots throughout the run show stable subdomains and indicate that these rigid domains are constrained during the dynamics by a stable network of hydrogen bonds. The most stable subdomain during the dynamics was in the small lobe including part of the carboxy-terminal tail. The most significant flexible region was the highly conserved glycine-rich loop between ␤ strands 1 and 2 in the small lobe. Many of the main chain dihedral angle changes measured in a comparison of the crystallographic structures of "open" and "closed" conformations of cAPK correspond to the highly flexible residues found during dynamics.

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Calculating pK_a values in the cAMP‐dependent protein kinase: The effect of conformational change and ligand binding

Bjarnadottir¹

2010

Protein Science

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The conformational change observed upon ligand binding and phosphorylation for the cAMP-dependent protein kinase (protein kinase A-PKA) is of high importance for the regulation of its activity. We calculate pK a values and net charges for 18 3D structures of PKA in various conformations and liganded states to examine the role of electrostatics in ligand binding and activation. We find that the conformational change of PKA takes place without any significant net proton uptake/release at all pH values, thus indicating that PKA has evolved to reduce any pHdependent barriers to the conformational motion. We furthermore find that the binding of ligands induces large changes in the net charge of PKA at most pH values, but significantly, we find that the net charge difference at physiological pH is close to zero, thus indicating that the active-site pK a values have been preorganized for substrate binding. We are unable to unequivocally resolve the identity of the groups responsible for determining the pH-activity profile of PKA but speculate that the titration of Lys 168 or the titration of ATP itself could be responsible for the loss of activity at high pH values. Finally, we examine the effect of point mutations on the pK a values of the PKA catalytic residues and find these to be relatively insensitive to both noncharge-altering and chargealtering mutations.

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

Cited by 17 publications

References 19 publications

E230Q mutation of the catalytic subunit of cAMP‐dependent protein kinase affects local structure and the binding of peptide inhibitor

E230Q mutation of the catalytic subunit of cAMP‐dependent protein kinase affects local structure and the binding of peptide inhibitor

600 ps Molecular dynamics reveals stable substructures and flexible hinge points in cAMP dependent protein kinase

Calculating pK_a values in the cAMP‐dependent protein kinase: The effect of conformational change and ligand binding

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

Cited by 17 publications

References 19 publications

E230Q mutation of the catalytic subunit of cAMP‐dependent protein kinase affects local structure and the binding of peptide inhibitor

E230Q mutation of the catalytic subunit of cAMP‐dependent protein kinase affects local structure and the binding of peptide inhibitor

600 ps Molecular dynamics reveals stable substructures and flexible hinge points in cAMP dependent protein kinase

Calculating pKa values in the cAMP‐dependent protein kinase: The effect of conformational change and ligand binding

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Calculating pK_a values in the cAMP‐dependent protein kinase: The effect of conformational change and ligand binding