Role of cysteine residues in structural stability and function of a transmembrane helix bundle

被引:37
作者
Karim, CB [1 ]
Paterlini, MG
Reddy, LG
Hunter, GW
Barany, G
Thomas, DD
机构
[1] Univ Minnesota, Dept Biochem Mol Biol & Biophys, Minneapolis, MN 55455 USA
[2] Univ Minnesota, Dept Med Chem, Minneapolis, MN 55455 USA
[3] Univ Minnesota, Dept Chem, Minneapolis, MN 55455 USA
关键词
D O I
10.1074/jbc.M104006200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
To study the structural and functional roles of the cysteine residues at positions 36, 41, and 46 in the transmembrane domain of phospholamban (PLB), we have used Fmoc (N-(9-fluorenyl)methoxycarbonyl) solid-phase peptide synthesis to prepare alpha -amino-n-butyric acid (Abu)-PLB, the analogue in which all three cysteine residues are replaced by Abu. Whereas previous studies have shown that replacement of the three Cys residues by Ala (producing Ala-PLB) greatly destabilizes the pentameric structure, we hypothesized that replacement of Cys with Abu, which is isosteric to Cys, might preserve the pentameric stability. Therefore, we compared the oligomeric structure (from SDS-polyacrylamide gel electrophoresis) and function (inhibition of the Ca-ATPase in reconstituted membranes) of Abu-PLB with those of synthetic wild-type PLB and Ala-PLB. Molecular modeling provides structural and energetic insight into the different oligomeric stabilities of these molecules. We conclude that 1) the Cys residues of PLB are not necessary for pentamer formation or inhibitory function; 2) the steric properties of cysteine residues in the PLB transmembrane domain contribute substantially to pentameric stability, whereas the polar or chemical properties of the sulfhydryl group play only a minor role; 3) the functional potency of these PLB variants does not correlate with oligomeric stability; and 4) acetylation of the N-terminal methionine has neither a functional nor a structural effect in full-length PLB.
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收藏
页码:38814 / 38819
页数:6
相关论文
共 44 条
[1]   Acetylation at the N-terminus of actin strengthens weak interaction between actin and myosin [J].
Abe, A ;
Saeki, K ;
Yasunaga, T ;
Wakabayashi, T .
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 2000, 268 (01) :14-19
[2]   COMPUTATIONAL SEARCHING AND MUTAGENESIS SUGGEST A STRUCTURE FOR THE PENTAMERIC TRANSMEMBRANE DOMAIN OF PHOSPHOLAMBAN [J].
ADAMS, PD ;
ARKIN, IT ;
ENGELMAN, DM ;
BRUNGER, AT .
NATURE STRUCTURAL BIOLOGY, 1995, 2 (02) :154-162
[3]   Structure of the transmembrane cysteine residues in phospholamban [J].
Arkin, IT ;
Adams, PD ;
Brunger, AT ;
Aimoto, S ;
Engelman, DM ;
Smith, SO .
JOURNAL OF MEMBRANE BIOLOGY, 1997, 155 (03) :199-206
[4]   STRUCTURAL ORGANIZATION OF THE PENTAMERIC TRANSMEMBRANE ALPHA-HELICES OF PHOSPHOLAMBAN, A CARDIAC ION-CHANNEL [J].
ARKIN, IT ;
ADAMS, PD ;
MACKENZIE, KR ;
LEMMON, MA ;
BRUNGER, AT ;
ENGELMAN, DM .
EMBO JOURNAL, 1994, 13 (20) :4757-4764
[5]   Functional co-expression of the canine cardiac Ca2+ pump and phospholamban in Spodoptera frugiperda (Sf21) cells reveals new insights on ATPase regulation [J].
Autry, JM ;
Jones, LR .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1997, 272 (25) :15872-15880
[6]  
BARANY G, 1996, TECHNIQUES PROTEIN C, V7, P503
[7]  
Brinckerhoff LH, 1999, INT J CANCER, V83, P326, DOI 10.1002/(SICI)1097-0215(19991029)83:3<326::AID-IJC7>3.0.CO
[8]  
2-X
[9]  
CASE DA, 1997, AMBER5
[10]   Mutation and phosphorylation change the oligomeric structure of phospholamban in lipid bilayers [J].
Cornea, RL ;
Jones, LR ;
Autry, JM ;
Thomas, DD .
BIOCHEMISTRY, 1997, 36 (10) :2960-2967