THE CRYSTAL-STRUCTURE OF THE BACILLUS-LENTUS ALKALINE PROTEASE, SUBTILISIN BL, AT 1.4 ANGSTROM RESOLUTION

被引:40
作者
GODDETTE, DW [1 ]
PAECH, C [1 ]
YANG, SS [1 ]
MIELENZ, JR [1 ]
BYSTROFF, C [1 ]
WILKE, ME [1 ]
FLETTERICK, RJ [1 ]
机构
[1] UNIV CALIF SAN FRANCISCO, DEPT BIOCHEM & BIOPHYS, SAN FRANCISCO, CA 94143 USA
关键词
CRYSTAL STRUCTURE; SUBTILISIN; PROTEASE; MULTIPLE CONFORMERS; PH STABILITY;
D O I
10.1016/0022-2836(92)90843-9
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 crystal structure of subtilisin BL, an alkaline protease from Bacillus lentus with activity at pH 11, has been determined to 1.4 Å resolution. The structure was solved by molecular replacement starting with the 2.1 Å structure of subtilisin BPN′ followed by molecular dynamics refinement using X-PLOR. A final crystallographic R-factor of 19% overall was obtained. The enzyme possesses stability at high pH, which is a result of the high pI of the protein. Almost all of the acidic side-chains are involved in some type of electrostatic interaction (ion pairs, calcium binding, etc.). Furthermore, three of seven tyrosine residues have potential partners for forming salt bridges. All of the potential partners are arginine with a pK around 12. Lysine would not function well in a salt bridge with tyrosine as it deprotonates at around the same pH as tyrosine ionizes. Stability at high pH is acquired in part from the pI of the protein, but also from the formation of salt bridges (which would affect the pI). The overall structure of the enzyme is very similar to other subtilisins and shows that the subtilisin fold is more highly conserved than would be expected from the differences in amino acid sequence. The amino acid side-chains in the hydrophobic core are not conserved, though the inter-residue interactions are. Finally, one third of the serine side-chains in the protein have multiple conformations. This presents an opportunity to correlate computer simulations with observed occupancies in the crystal structure. © 1992.
引用
收藏
页码:580 / 595
页数:16
相关论文
共 66 条
[1]   STRUCTURE OF THE PIG INSULIN DIMER IN THE CUBIC-CRYSTAL [J].
BADGER, J ;
HARRIS, MR ;
REYNOLDS, CD ;
EVANS, AC ;
DODSON, EJ ;
DODSON, GG ;
NORTH, ACT .
ACTA CRYSTALLOGRAPHICA SECTION B-STRUCTURAL SCIENCE, 1991, 47 :127-136
[2]   STRUCTURE OF ACTINIDIN, AFTER REFINEMENT AT 1.7-A RESOLUTION [J].
BAKER, EN .
JOURNAL OF MOLECULAR BIOLOGY, 1980, 141 (04) :441-484
[3]   PROTEIN DATA BANK - COMPUTER-BASED ARCHIVAL FILE FOR MACROMOLECULAR STRUCTURES [J].
BERNSTEIN, FC ;
KOETZLE, TF ;
WILLIAMS, GJB ;
MEYER, EF ;
BRICE, MD ;
RODGERS, JR ;
KENNARD, O ;
SHIMANOUCHI, T ;
TASUMI, M .
JOURNAL OF MOLECULAR BIOLOGY, 1977, 112 (03) :535-542
[4]   3-DIMENSIONAL STRUCTURE OF PROTEINASE-K AT 0.15-NM RESOLUTION [J].
BETZEL, C ;
PAL, GP ;
SAENGER, W .
EUROPEAN JOURNAL OF BIOCHEMISTRY, 1988, 178 (01) :155-171
[5]   CRYSTALLIZATION AND PRELIMINARY-X-RAY DIFFRACTION STUDIES OF AN ALKALINE PROTEASE FROM BACILLUS-LENTUS [J].
BETZEL, C ;
DAUTER, Z ;
DAUTER, M ;
INGELMAN, M ;
PAPENDORF, G ;
WILSON, KS ;
BRANNER, S .
JOURNAL OF MOLECULAR BIOLOGY, 1988, 204 (03) :803-804
[6]   CRYSTAL-STRUCTURE OF THE ALKALINE PROTEINASE SAVINASE FROM BACILLUS-LENTUS AT 1.4-A RESOLUTION [J].
BETZEL, C ;
KLUPSCH, S ;
PAPENDORF, G ;
HASTRUP, S ;
BRANNER, S ;
WILSON, KS .
JOURNAL OF MOLECULAR BIOLOGY, 1992, 223 (02) :427-445
[7]   X-RAY AND MODEL-BUILDING STUDIES ON THE SPECIFICITY OF THE ACTIVE-SITE OF PROTEINASE-K [J].
BETZEL, C ;
BELLEMANN, M ;
PAL, GP ;
BAJORATH, J ;
SAENGER, W ;
WILSON, KS .
PROTEINS-STRUCTURE FUNCTION AND GENETICS, 1988, 4 (03) :157-164
[8]   THERMITASE AND PROTEINASE-K - A COMPARISON OF THE REFINED 3-DIMENSIONAL STRUCTURES OF THE NATIVE ENZYMES [J].
BETZEL, C ;
TEPLYAKOV, AV ;
HARUTYUNYAN, EH ;
SAENGER, W ;
WILSON, KS .
PROTEIN ENGINEERING, 1990, 3 (03) :161-172
[9]   REFINED 1.2-A CRYSTAL-STRUCTURE OF THE COMPLEX FORMED BETWEEN SUBTILISIN CARLSBERG AND THE INHIBITOR EGLIN-C - MOLECULAR-STRUCTURE OF EGLIN AND ITS DETAILED INTERACTION WITH SUBTILISIN [J].
BODE, W ;
PAPAMOKOS, E ;
MUSIL, D ;
SEEMUELLER, U ;
FRITZ, H .
EMBO JOURNAL, 1986, 5 (04) :813-818
[10]   THE HIGH-RESOLUTION X-RAY CRYSTAL-STRUCTURE OF THE COMPLEX FORMED BETWEEN SUBTILISIN CARLSBERG AND EGLIN-C, AN ELASTASE INHIBITOR FROM THE LEECH HIRUDO-MEDICINALIS - STRUCTURAL-ANALYSIS, SUBTILISIN STRUCTURE AND INTERFACE GEOMETRY .2. [J].
BODE, W ;
PAPAMOKOS, E ;
MUSIL, D .
EUROPEAN JOURNAL OF BIOCHEMISTRY, 1987, 166 (03) :673-692