Modifications of bovine prothrombin fragment 1 in the presence and absence of Ca(II) ions. Loss of positive cooperativity in Ca(II) ion binding for the modified proteins.

Weber, Darin J.; Berkowitz, P; Panek, M. G.; Huh, Nam-Won; Pedersen, Lee G.; Hiskey, Richard G.

doi:10.1016/s0021-9258(18)42870-0

Cited by 14 publications

(6 citation statements)

References 32 publications

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“…Proteolysis of intact proteins has shown that the membrane contact site is expressed within residues 1-37 of factor X (26). The 3 amino-terminal residues of prothrombin can be removed with only a small impact on membrane affinity (27). Both of these properties applied at saturating calcium levels, where actual membrane contact may be studied in a manner that is independent of events such as the calcium-induced protein conformational change.…”

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confidence: 99%

Enhancing the Activity of Protein C by Mutagenesis To Improve the Membrane-Binding Site: Studies Related to Proline-10

et al. 1997

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Bovine and human protein C show high homology in the amino acids of their GLA domains (amino-terminal 44 residues), despite the about 10-fold higher membrane affinity of the human protein. A proposed membrane contact site and mechanism suggested that this difference was largely due to the presence of proline at position 10 of bovine protein C versus histidine at position 10 of human protein C [McDonald, J.F., Shah, A.M., Schwalbe, R.A., Kisiel, W., Dahlback, B., and Nelsestuen, G.L. (1997) Biochemistry, 36, 5120-5127]. This study examined the impact of replacing proline-10 in bovine protein C with histidine, and the reverse change in human protein C. In both cases, the protein containing proline-10 showed lower membrane affinity, about 10-fold lower for bovine protein C and 5-fold lower for human protein C. As expected, activated human protein C (hAPC) containing proline at position 10 showed 2.4-3.5-fold lower activity than wild type hAPC, depending on the assay used. Most interesting was that bovine APC containing histidine-10 displayed up to 15-fold higher activity than wild type bAPC. This demonstrated the ability to improve both membrane contact and activity by mutation. This general strategy should be applicable to other vitamin K-dependent proteins, providing opportunities to study function as well as to produce proteins that may find use as promoters and inhibitors of blood coagulation in pathological states.

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confidence: 99%

Enhancing the Activity of Protein C by Mutagenesis To Improve the Membrane-Binding Site: Studies Related to Proline-10

et al. 1997

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“…The acetyl methyl group of the Ace-Alai/bfl structure is tucked into the region of the Cysl8-Cys23 disulfide bond so that the turn of helix in avg-bfl/Ca (residues 15-20) is somewhat disrupted. Conversely, the TNP group is located along the surface in TNP-Alal/bfl, giving rise to potential additional calciumbinding sites observed by an equilibrium dialysis experiment (Weber et al, 1992), while the secondary structure of residues 15-20 remains similar to avg-bfl/Ca. Since the des[Ala-Asn2] /bfl demonstrated maintenance of membrane-binding capability (Weber et al, 1992), it is likely that N-acetylation alters the network that can be achieved by Alai, thus precluding the necessary conformational change which is required for membrane binding.…”

Section: Resultsmentioning

confidence: 99%

“…Effects of two different chemical modifications of the N-terminus of bfl have been studied by Welsch and Nelsestuen (1988a) and Weber et al (1992). These were N-acetylation and N-trinitrophenylation of the amino group at Alai.…”

Section: Resultsmentioning

confidence: 99%

“…Taken together, the indirect experimental evidence has suggested that the initial cooperative calcium ion binding causes a conformational change, with the binding of additional calcium 0006-2960/92/0431-8840S03.00/0 © 1992 American Chemical Society ions serving to bridge the Gla domain carboxylate side chains to the phosphate head groups of the membrane surface. An involvement of the kringle domain in membrane binding has also been implicated by recent chemical modification studies (Welsch & Nelsestuen, 1988b;Weber et al, 1992). A weak calcium ion binding site in the kringle domain has been inferred by a hydrophobic column technique (Lundblad, 1988) and by equilibrium dialysis studies employing an isolated kringle domain (Berkowitz et al, 1992).…”

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confidence: 95%

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Molecular dynamics simulation of bovine prothrombin fragment 1 in the presence of calcium ions

Hamaguchi¹,

Charifson²,

Darden³

et al. 1992

Biochemistry

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Early solvation-induced structural reorganization of calcium prothrombin fragment 1 is simulated with molecular dynamics. Initial coordinates are those of the 2.2-A resolution crystal structure [Soriano-Garcia, M., Padmanabhan, K., de Vos, A. M., & Tulinsky, A. (1992) Biochemistry 31, 2554-2556]. The molecular dynamics code AMBER, appropriately modified to include long-range (less than or equal to 22.0 A) ionic forces, was employed. The solution structure appears to equilibrate within 100 ps. Although minor changes are seen in various structural domains, the early solution structure basically maintains an intricate network of nine gamma-carboxyglutamic acid (Gla) residues encapsulating seven calcium ions. However, the Gla domain moves with respect to the kringle domain. This motion is mainly due to the movement of Ser34-Leu35 that appears to be a flexible hinge between the domains. The N-terminus of Ala 1 is in a tightly bound complex with three Gla residues that remains stable in the solution structure when the long-range electrostatic cutoff is employed and the near planar alignment of the seven calcium ions is only slightly distorted. The simulation structure is discussed in terms of experiments that studied calcium ion-induced quenching of the intrinsic fluorescence, protection of the N-terminal amino group from acetylation by calcium ions, chemical modification of the N-terminus to a trinitrophenyl derivative, and the possibility of a calcium-binding site(s) in the kringle domain.

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“…This binding mechanism appeared to be challenged by several of the earlier results. In addition, chemical modifications such as trinitrophenylation of Lys-3 and Lys-11 (16) and removal of the amino-terminal three residues of prothrombin (17) had less than a 5-fold impact on membrane affinity (<1 kcal/mol), even though they should disrupt protein structure in the region of the hydrophobic cluster. Site-directed mutation of the hydrophobic residues gave 0-5-fold impacts on membrane affinity at saturating calcium levels (11,12,14).…”

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confidence: 99%

Ionic Properties of Membrane Association by Vitamin K-Dependent Proteins: The Case for Univalency

et al. 1997

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Ionic properties of membrane interaction by prothrombin, protein Z, and other vitamin K-dependent proteins were studied to determine the relevance of a monovalent membrane contact mechanism between one phospholipid headgroup and a calcium-lined pore in the protein [McDonald, J. F., Shah, A. M., Schwalbe, R. A., Kisiel, W., Dahlback, B., and Nelsestuen, G. L. (1997) Biochemistry 36, 5120-5127]. For comparison, multivalent ionic interaction was illustrated by peptides of +3 to +5 net charge and by blood clotting factor V. As expected, the peptides were easily dissociated by salt and gave nominal charge-charge interactions (zazb values) of -13 to -17. Factor V showed much higher binding affinity despite nominal zazb values of about 9. Membrane-bound prothrombin and protein Z showed very low sensitivity to salt as long as calcium was at saturating levels (zazb values of approximately -1.3 to -1.4), appropriate for univalent ionic attraction. Prothrombin contains +3 charge groups (Lys-2, Lys-11, Arg-10) that are absent from the GLA domain (residues 1-35) of protein Z, while protein Z contains -4 charge groups (Gla-11, Asp-34, Asp-35) that are absent in prothrombin. Thus, similar zazb relationships indicated little role for these surface charges in direct membrane contact. Calcium-saturated protein Z bound to phosphatidylcholine (PC) in a manner which indicated the addition of one calcium ion, bringing the total calcium stoichiometry in the protein-membrane complex to at least 8. Protein Z bound to phosphatidic acid (PA) in a manner suggesting the need for a fully ionized phosphate headgroup, a property expected by ion pairing in an isolated environment. Electrostatic calculations showed that the proposed protein site for phosphate interaction was electropositive. The cluster of hydrophobic amino acids (Phe-5, Leu-6, and Val-9) on the surface of prothrombin was electronegative, suggesting a role in the electrostatic architecture of the GLA domain. Overall, membrane binding by vitamin K-dependent proteins appeared consistent with the formation of an ion pair in an isolated environment.

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Modifications of bovine prothrombin fragment 1 in the presence and absence of Ca(II) ions. Loss of positive cooperativity in Ca(II) ion binding for the modified proteins.

Cited by 14 publications

References 32 publications

Enhancing the Activity of Protein C by Mutagenesis To Improve the Membrane-Binding Site: Studies Related to Proline-10

Enhancing the Activity of Protein C by Mutagenesis To Improve the Membrane-Binding Site: Studies Related to Proline-10

Molecular dynamics simulation of bovine prothrombin fragment 1 in the presence of calcium ions

Ionic Properties of Membrane Association by Vitamin K-Dependent Proteins: The Case for Univalency

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