High-resolution structural insights on the sugar-recognition and fusion tag properties of a versatile β-trefoil lectin domain from the mushroom Laetiporus sulphureus

被引:32
作者
Angulo, Ivan [1 ]
Acebron, Ivan [1 ]
de Las Rivas, Blanca [2 ]
Munoz, Rosario [2 ]
Rodriguez-Crespo, I. [3 ]
Menendez, Margarita [4 ,5 ]
Garcia, Pedro [5 ,6 ]
Tateno, Hiroaki [7 ]
Goldstein, Irwin J. [7 ]
Perez-Agote, Begona [1 ]
Mancheno, Jose M. [1 ]
机构
[1] CSIC, Inst Quim Fis Rocasolano, Dept Cristalog & Biol Estruct, E-28006 Madrid, Spain
[2] CSIC, Inst Ciencia & Tecnol Alimentos, Grp Biotecnol Bacteriana, E-28006 Madrid, Spain
[3] Univ Complutense Madrid, Fac Quim, Dept Bioquim & Biol Mol, E-28040 Madrid, Spain
[4] CSIC, Inst Quim Fis Rocasolano, Grp Macromol, E-28006 Madrid, Spain
[5] Ctr Invest Biomed Red Enfermedades Resp, Mallorca, Illes Balears, Spain
[6] CSIC, Ctr Invest Biol, Dept Microbiol Mol & Biol Infecc, E-28040 Madrid, Spain
[7] Univ Michigan, Sch Med, Dept Biol Chem, Ann Arbor, MI 48109 USA
关键词
crystal structure; lactose; lectin; solubility tag; water rearrangement; CYSTEINE-RICH DOMAIN; CRYSTAL-STRUCTURE; CRYSTALLOGRAPHIC ANALYSIS; PROTEIN; BINDING; COMPLEX; TOXIN; CRYSTALLIZATION; SIMILARITY; ENERGETICS;
D O I
10.1093/glycob/cwr074
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
In this work, we analyzed at high resolution the sugar-binding mode of the recombinant N-terminal ricin-B domain of the hemolytic protein LSLa (LSL150) from the mushroom Laetiporus sulphureus and also provide functional in vitro evidence suggesting that, together with its putative receptor-binding role, this module may also increase the solubility of its membrane pore-forming partner. We first demonstrate that recombinant LSL150 behaves as an autonomous folding unit and an active lectin. We have determined its crystal structure at 1.47 A resolution and also that of the [LSL150:(lactose)beta, gamma)] binary complex at 1.67 A resolution. This complex reveals two lactose molecules bound to the beta and gamma sites of LSL150, respectively. Isothermal titration calorimetry indicates that LSL150 binds two lactoses in solution with highly different affinities. Also, we test the working hypothesis that LSL150 exhibits in vivo properties typical of solubility tags. With this aim, we have fused an engineered version of LSL150 (LSLt) to the N-terminal end of various recombinant proteins. All the designed LSL150-tagged fusion proteins were successfully produced at high yield, and furthermore, the target proteins were purified by a straightforward affinity procedure on agarose-based matrices due to the excellent properties of LSL150 as an affinity tag. An optimized protocol for target protein purification was devised, which involved removal of the LSL150 tag through in-column cleavage of the fusion proteins with His(6)-tagged TEV endoprotease. These results permitted to set up a novel, lectin-based system for production and purification of recombinant proteins in E. coli cells with attractive biotechnological applications.
引用
收藏
页码:1349 / 1361
页数:13
相关论文
共 55 条
[1]   Nontoxic crystal protein from Bacillus thuringiensis demonstrates a remarkable structural similarity to pore-forming toxins [J].
Akiba, T ;
Higuchi, K ;
Mizuki, E ;
Ekino, K ;
Shin, T ;
Ohba, M ;
Kanai, R ;
Harata, K .
PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS, 2006, 63 (01) :243-248
[2]   THE CCP4 SUITE - PROGRAMS FOR PROTEIN CRYSTALLOGRAPHY [J].
BAILEY, S .
ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY, 1994, 50 :760-763
[3]   A DIRECT MEASURE OF THE CONTRIBUTION OF SOLVENT REORGANIZATION TO THE ENTHALPY OF LIGAND-BINDING [J].
CHERVENAK, MC ;
TOONE, EJ .
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 1994, 116 (23) :10533-10539
[4]   Clostridium perfringens ε-toxin shows structural similarity to the pore-forming toxin aerolysin [J].
Cole, AR ;
Gibert, M ;
Popoff, M ;
Moss, DS ;
Titball, RW ;
Basak, AK .
NATURE STRUCTURAL & MOLECULAR BIOLOGY, 2004, 11 (08) :797-798
[5]   Thermodynamic studies of lectin-carbohydrate interactions by isothermal titration calorimetry [J].
Dam, TK ;
Brewer, CF .
CHEMICAL REVIEWS, 2002, 102 (02) :387-429
[6]   MolProbity: all-atom contacts and structure validation for proteins and nucleic acids [J].
Davis, Ian W. ;
Leaver-Fay, Andrew ;
Chen, Vincent B. ;
Block, Jeremy N. ;
Kapral, Gary J. ;
Wang, Xueyi ;
Murray, Laura W. ;
Arendall, W. Bryan, III ;
Snoeyink, Jack ;
Richardson, Jane S. ;
Richardson, David C. .
NUCLEIC ACIDS RESEARCH, 2007, 35 :W375-W383
[7]  
DeLano W.L., 2002, The PyMOL molecular graphics system
[8]   Structures of the Erythrina corallodendron lectin and of its complexes with mono- and disaccharides [J].
Elgavish, S ;
Shaanan, B .
JOURNAL OF MOLECULAR BIOLOGY, 1998, 277 (04) :917-932
[9]   Coot:: model-building tools for molecular graphics [J].
Emsley, P ;
Cowtan, K .
ACTA CRYSTALLOGRAPHICA SECTION D-STRUCTURAL BIOLOGY, 2004, 60 :2126-2132
[10]   The crystal structure of tetanus toxin Hc fragment complexed with a synthetic GT1b analogue suggests cross-linking between ganglioside receptors and the toxin [J].
Fotinou, C ;
Emsley, P ;
Black, I ;
Ando, H ;
Ishida, H ;
Kiso, M ;
Sinha, KA ;
Fairweather, NF ;
Isaacs, NW .
JOURNAL OF BIOLOGICAL CHEMISTRY, 2001, 276 (34) :32274-32281