Module-module interactions in the cell binding region of fibronectin: Stability, flexibility and specificity

被引:87
作者
Spitzfaden, C
Grant, RP
Mardon, HJ
Campbell, ID
机构
[1] UNIV OXFORD, DEPT BIOCHEM, OXFORD OX1 3QU, ENGLAND
[2] OXFORD CTR MOL SCI, OXFORD OX1 3QU, ENGLAND
[3] JOHN RADCLIFFE HOSP, NUFFIELD DEPT OBSTET & GYNAECOL, OXFORD OX3 9DU, ENGLAND
关键词
protein folding; nuclear magnetic resonance; fluorescence; relaxation;
D O I
10.1006/jmbi.1996.0736
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
The structure of mosaic proteins depends on the nature and strength of interactions between individual modules. Here we investigated the structural significance of module-module interactions in the RGD-dependent cell binding region of human fibronectin, comprising the ninth and tenth fibronectin type III. A combination of protein engineering, thermodynamics and nuclear magnetic resonance methods was employed to establish a relationship between intermodular protein-protein interactions and the structural properties of the module pair. A poly(glycine) peptide link connecting the C terminus of the ninth and the N terminus of the tenth module was introduced to probe the range of the interaction. NMR studies (Chemical shifts and N-15 relaxation) together with equilibrium and kinetic unfolding experiments were carried out on five different single and double module constructs. The results show that non-specific protein-protein interactions provide the bulk of the thermodynamic stabilization and the motional constraint of the two modules. Specific interactions between the two modules are restricted to the wild-type module pair and decline very rapidly with the insertion of additional linker residues. This low level of specificity is nonetheless sufficient to fine-tune the precise module-module orientation and to provide the full biological activity of the wild-type pair. This suggests that individual modules in mosaic proteins can achieve a high degree of motional constraint and mutual stabilization without the requirement for intricate and specific interactions in the module-module interfaces. (C) 1997 Academic Press Limited.
引用
收藏
页码:565 / 579
页数:15
相关论文
共 59 条
[51]   IMMUNOGLOBULIN-TYPE DOMAINS OF TITIN ARE STABILIZED BY AMINO-TERMINAL EXTENSION [J].
POLITOU, AS ;
GAUTEL, M ;
JOSEPH, C ;
PASTORE, A .
FEBS LETTERS, 1994, 352 (01) :27-31
[52]   FIBRONECTIN STRUCTURE AND ASSEMBLY [J].
POTTS, JR ;
CAMPBELL, ID .
CURRENT OPINION IN CELL BIOLOGY, 1994, 6 (05) :648-655
[53]  
SCHLEUCHER J, 1994, J BIOMOL NMR, V4, P301
[54]   THE SOLUTION STRUCTURE AND BACKBONE DYNAMICS OF THE FIBRONECTIN TYPE-I AND EPIDERMAL GROWTH FACTOR-LIKE PAIR OF MODULES OF TISSUE-TYPE PLASMINOGEN-ACTIVATOR [J].
SMITH, BO ;
DOWNING, AK ;
DRISCOLL, PC ;
DUDGEON, TJ ;
CAMPBELL, ID .
STRUCTURE, 1995, 3 (08) :823-833
[55]   SINGLE-STEP PURIFICATION OF POLYPEPTIDES EXPRESSED IN ESCHERICHIA-COLI AS FUSIONS WITH GLUTATHIONE S-TRANSFERASE [J].
SMITH, DB ;
JOHNSON, KS .
GENE, 1988, 67 (01) :31-40
[56]   Rotational diffusion anisotropy of human ubiquitin from N-15 NMR relaxation [J].
Tjandra, N ;
Feller, SE ;
Pastor, RW ;
Bax, A .
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 1995, 117 (50) :12562-12566
[57]   ROTATIONAL-DYNAMICS OF CALCIUM-FREE CALMODULIN STUDIED BY N-15-NMR RELAXATION MEASUREMENTS [J].
TJANDRA, N ;
KUBONIWA, H ;
REN, H ;
BAX, A .
EUROPEAN JOURNAL OF BIOCHEMISTRY, 1995, 230 (03) :1014-1024
[58]   ATOMIC-STRUCTURE OF A FRAGMENT OF HUMAN CD4 CONTAINING 2 IMMUNOGLOBULIN-LIKE DOMAINS [J].
WANG, JH ;
YAN, YW ;
GARRETT, TPJ ;
LIU, JH ;
RODGERS, DW ;
GARLICK, RL ;
TARR, GE ;
HUSAIN, Y ;
REINHERZ, EL ;
HARRISON, SC .
NATURE, 1990, 348 (6300) :411-418
[59]   SOLUTION STRUCTURE OF A PAIR OF FIBRONECTIN TYPE-1 MODULES WITH FIBRIN BINDING-ACTIVITY [J].
WILLIAMS, MJ ;
PHAN, I ;
HARVEY, TS ;
ROSTAGNO, A ;
GOLD, LI ;
CAMPBELL, ID .
JOURNAL OF MOLECULAR BIOLOGY, 1994, 235 (04) :1302-1311