Recombinant Gene Expression and 1H NMR Characteristics of the Kringle (2 + 3) Supermodule: Spectroscopic/Functional Individuality of Plasminogen Kringle Domains
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- 作者：Sabine Sö ; hndel ; Chih-Kao Hu ; Daniel Marti ; Michael Affolter ; Johann Schaller ; Miguel Lliná ; s ; and Egon E. Rickli
- 刊名：Biochemistry
- 出版年：1996
- 出版时间：February 20, 1996
- 年：1996
- 卷：35
- 期：7
- 页码：2357 - 2364
- 全文大小：461K
- 年卷期：v.35,no.7(February 20, 1996)
- ISSN：1520-4995
文摘
The plasminogen kringle 2 (K2HPg) and kringle 3(K3HPg) modules occur in tandem within thepolypeptide segment that affords the heavy chain of plasmin. TheK2HPg and K3HPg are unique amongthe plasminogen kringle domains in that they also are linked to eachother via the Cys169-Cys297(Cys4of K2HPg to Cys43 of K3HPg, kringlenumbering convention) disulfide bridge, thus generating aK2HPgK3HPg "supermodule". The kringle (2 + 3) sequenceof human plasminogen(r-EE[K2HPgK3HPg]DS)wasexpressed in Escherichia coli, using an expression vectorcontaining the phage T5 promoter/operatorN250PSN250P29 and the codons for an N-terminal hexahistidine tag toensure the isolation of therecombinant protein by affinity chromatography onNi2+-nitrilotriacetic acid/agarose underdenaturingand reducing conditions. Kringle (2 + 3) was refolded in thepresence of glutathione redox buffer. Bytaking advantage of the lysine affinity of kringle 2, the protein waspurified by affinity chromatographyon lysine-Bio-Gel. Recombinant kringle (2 + 3) was identified byamino acid composition, N-terminalsequence and mass determination. The 1H NMR spectrumshows that the intact r-K2HPgK3HPg isproperlyfolded. By reference to spectra of the individual kringles,r-K2HPg and r-K3HPg, resonances of theK2HPgand K3HPg components in the spectrum of the intactr-K2HPgK3HPg can be readily distinguished.Thestrictly conserved Leu46 residue (kringle residue numberingconvention) yields 
-methyl signals that arecharacteristic for K2HPg and K3HPg, exhibitingchemical shifts of -0.87 and -0.94 ppm, respectively,which are distinct from those of K1HPg, K4HPg,and K5HPg, (-1.04 to -1.05 ppm). Thus, thehigh-fieldLeu46 signals from K2HPg and K3HPgare well resolved from those of other kringles and can beidentifiedunambiguously in spectra of theK1HPgK2HPgK3HPgelastolytic fragment of plasminogen as well as in spectraof Glu-plasminogen. Overall,r-K2HPgK3HPg exhibits broader resonanceline widths than does the K1HPgcomponent, consistent with a lesser mobility of theK2HPgK3HPg segment within theK1HPgK2HPgK3HPgfragment, a reflection of the extra structural constraint imposed bythe disulfide bridge linking K2HPg toK3HPg. The ligand 6-aminohexanoic acid (6-AHA), whichis known to interact with r-K2HPg but not withr-K3HPg, selectively perturbs K2 aromatic signals in theintact r-K2HPgK3HPg spectrum while leavingK3resonances largely unaffected. Association constant(Ka) values for 6-AHA determined from1H NMRligand titration experiments yield Ka 
2.2± 0.3 mM-1 for the intactr-K2HPgK3HPg, comparable toKa 2.3 ± 0.2 mM-1 determined for the isolatedr-K2HPg, which demonstrates that the interactions of6-AHAwith the K2HPg ligand-binding site are not significantlyaffected by the neighboring K3HPg domainwithinthe intact r-K2HPgK3HPgsupermodule.
-methyl signals that arecharacteristic for K2HPg and K3HPg, exhibitingchemical shifts of -0.87 and -0.94 ppm, respectively,which are distinct from those of K1HPg, K4HPg,and K5HPg, (-1.04 to -1.05 ppm). Thus, thehigh-fieldLeu46 signals from K2HPg and K3HPgare well resolved from those of other kringles and can beidentifiedunambiguously in spectra of theK1HPgK2HPgK3HPgelastolytic fragment of plasminogen as well as in spectraof Glu-plasminogen. Overall,r-K2HPgK3HPg exhibits broader resonanceline widths than does the K1HPgcomponent, consistent with a lesser mobility of theK2HPgK3HPg segment within theK1HPgK2HPgK3HPgfragment, a reflection of the extra structural constraint imposed bythe disulfide bridge linking K2HPg toK3HPg. The ligand 6-aminohexanoic acid (6-AHA), whichis known to interact with r-K2HPg but not withr-K3HPg, selectively perturbs K2 aromatic signals in theintact r-K2HPgK3HPg spectrum while leavingK3resonances largely unaffected. Association constant(Ka) values for 6-AHA determined from1H NMRligand titration experiments yield Ka 2.2± 0.3 mM-1 for the intactr-K2HPgK3HPg, comparable toKa 2.3 ± 0.2 mM-1 determined for the isolatedr-K2HPg, which demonstrates that the interactions of6-AHAwith the K2HPg ligand-binding site are not significantlyaffected by the neighboring K3HPg domainwithinthe intact r-K2HPgK3HPgsupermodule.